John Anthony Bauer

Ritonavir: An Extraordinary Example of Conformational Polymorphism

AbstractPurpose. In the summer of 1998, Norvir semi-solid capsules supplies were threatened as a result of a new much less soluble crystal form of ritonavir. This report provides characterization of the two polymorphs and the structures and hydrogen bonding network for each form. Methods. Ritonavir polymorphism was investigated using solid state spectroscopy and microscopy techniques including solid state NMR, Near Infrared Spectroscopy, powder X-ray Diffraction and Single crystal X-ray. A sensitive seed detection test was developed. Results. Ritonavir polymorphs were thoroughly characterized and the structures determined. An unusual conformation was found for form II that results in a strong hydrogen bonding network A possible mechanism for heterogeneous nucleation of form II was investigated. Conclusions. Ritonavir was found to exhibit conformational polymorphism with two unique crystal lattices having significantly different solubility properties. Although the polymorph (form II) corresponding to the “cis” conformation is a more stable packing arrangement, nucleation, even in the presence of form II seeds, is energetically unfavored except in highly supersaturated solutions. The coincidence of a highly supersaturated solution and a probable heterogeneous nucleation by a degradation product resulted in the sudden appearance of the more stable form II polymorph.

Impaired Myofibrillar Energetics and Oxidative Injury During Human Atrial Fibrillation

Background—Atrial fibrillation (AF) is associated with severe contractile dysfunction and structural and electrophysiological remodeling. Mechanisms responsible for impaired contractility are undefined, and current therapies do not address this dysfunction. We have found that myofibrillar creatine kinase (MM-CK), an important controller of myocyte contractility, is highly sensitive to oxidative injury, and we hypothesized that increased oxidative stress and energetic impairment during AF could contribute to contractile dysfunction. Methods and Results—Right atrial appendages were obtained from AF patients undergoing the Maze procedure and from control patients who were in normal sinus rhythm and undergoing cardiac surgery. MM-CK activity was reduced in AF patients compared with controls (25.4±3.4 versus 18.2±3.8 &mgr;mol/mg of myofibrillar protein per minute; control versus AF;P <0.05). No reduction in total CK activity or myosin ATPase activity was detected. This selective reduction in MM-CK activity was associated with increased relative expression of the &bgr;-myosin isoform (25±6 versus 63±5%&bgr;, CTRL versus AF;P <0.05). Western blotting of AF myofibrillar isolates demonstrated no changes in protein composition but showed increased prevalence of protein oxidation as detected by Western blotting for 3-nitrotyrosine (peroxynitrite biomarker) and protein carbonyls (hydroxyl radical biomarker;P <0.05). Patterns of these oxidative markers were distinct, which suggests discrete chemical events and differential protein vulnerabilities in vivo. MM-CK inhibition was statistically correlated to extent of nitration (P <0.01) but not to carbonyl presence. Conclusions—The present results provide novel evidence of oxidative damage in human AF that altered myofibrillar energetics may contribute to atrial contractile dysfunction and that protein nitration may be an important participant in this condition.

MAP kinase phosphatase 1 controls innate immune responses and suppresses endotoxic shock

Septic shock is a leading cause of morbidity and mortality. However, genetic factors predisposing to septic shock are not fully understood. Excessive production of proinflammatory cytokines, particularly tumor necrosis factor (TNF)-α, and the resultant severe hypotension play a central role in the pathophysiological process. Mitogen-activated protein (MAP) kinase cascades are crucial in the biosynthesis of proinflammatory cytokines. MAP kinase phosphatase (MKP)-1 is an archetypal member of the dual specificity protein phosphatase family that dephosphorylates MAP kinase. Thus, we hypothesize that knockout of the Mkp-1 gene results in prolonged MAP kinase activation, augmented cytokine production, and increased susceptibility to endotoxic shock. Here, we show that knockout of Mkp-1 substantially sensitizes mice to endotoxic shock induced by lipopolysaccharide (LPS) challenge. We demonstrate that upon LPS challenge, Mkp-1−/− cells exhibit prolonged p38 and c-Jun NH2-terminal kinase activation as well as enhanced TNF-α and interleukin (IL)-6 production compared with wild-type cells. After LPS challenge, Mkp-1 knockout mice produce dramatically more TNF-α, IL-6, and IL-10 than do wild-type mice. Consequently, Mkp-1 knockout mice develop severe hypotension and multiple organ failure, and exhibit a remarkable increase in mortality. Our studies demonstrate that MKP-1 is a pivotal feedback control regulator of the innate immune responses and plays a critical role in suppressing endotoxin shock.

Oxidative pathways in cardiovascular disease: roles, mechanisms, and therapeutic implications

Despite some recent declines, cardiovascular disease (CVD) remains the major cause of death in the United States and worldwide. Most recent advances in the treatment of CVD states have been produced by inhibition of mechanisms involved in disease progress. Many studies conducted in the last decade have illustrated increased biological oxidative pathways during CVD in animals and humans. Thus, increased production of reactive oxygen species may be a unifying mechanism in CVD progression, and antioxidants may have therapeutic value in this setting. In this review we address the following questions: Do oxidative mechanisms play a role in CVD? Where do the oxidants come from? What are the relevant oxidative events? What are the therapeutic implications?

Endothelial dysfunction and peroxynitrite formation are early events in angiotensin-induced cardiovascular disorders

Angiotensin II (ANG II) is a well‐established participant in many cardiovascular disorders, but the mechanisms involved are not clear. Vascular cell experiments suggest that ANG II is a potent stimulator of free radicals such as superoxide anion, an agent known to inactivate nitric oxide and promote the formation of peroxynitrite. Here we hypothesized that ANG II reduces the efficacy of NO‐mediated vascular relaxation and promotes vascular peroxynitrite formation in vivo. ANG II was infused in rats at sub‐pressor doses for 3 days. Systolic blood pressure and heart rate were unchanged on day 3 despite significant reductions in plasma renin activity. Thoracic aorta was isolated for functional and immunohistochemical evaluations. No difference in isolated vascular contractile responses to KCI (125 mM), phenylephrine, or ANG II was observed between groups. In contrast, relaxant response to acetylcholine (ACh) was decreased sixfold without a change in relaxant response to sodium nitroprusside. Extensive prevalence of 3‐nitrotyrosine (3‐NT, a stable biomarker of tissue peroxynitrite formation) immunoreactivity was observed in ANG II‐treated vascular tissues and was specifically confined to the endothelium. Digital image analysis demonstrated a significant inverse correlation between ACh relaxant response and 3‐NT immunoreactivity. These data demonstrate that ANG II selectively modifies vascular NO control at sub‐pressor exposures in vivo. Thus, endothelial dysfunction apparently precedes other established ANG II‐induced vascular pathologies, and this may be mediated by peroxynitrite formation in vivo. Wattanapitayakul, S., Weinstein, D. M., Holycross, B. J., Bauer, J. A. Endothelial dysfunction and peroxynitrite formation are early events in angiotensin‐induced cardiovascular disorders. FASEB J. 14, 271–278 (2000)

Peroxynitrite induced nitration and inactivation of myofibrillar creatine kinase in experimental heart failure

OBJECTIVE Oxidative stress is implicated in the initiation and progression of congestive heart failure, but the putative reactive species and cellular targets involved remain undefined. We have previously shown that peroxynitrite (ONOO(-), an aggressive biological oxidant and nitrating agent) potently inhibits myofibrillar creatine kinase (MM-CK), a critical controller of contractility known to be impaired during heart failure. Here we hypothesized that nitration and inhibition of MM-CK participate in cardiac failure in vivo. METHODS Heart failure was induced in rats by myocardial infarction (left coronary artery ligation) and confirmed by histological analysis at 8 weeks postinfarct (1.3+/-1.4 vs. 37.7+/-3.2% left ventricular circumference; sham control vs. CHF, n=10 each). RESULTS Immunohistochemistry demonstrated significantly increased protein nitration in failing myocardium compared to control (optical density: 0.58+/-0.06 vs. 0.93+/-0.09, sham vs. CHF, P<0.05). Significant decreases in MM-CK activity and content were observed in failing hearts (MM-CK k(cat): 6.0+/-0.4 vs. 3.0+/-0.3 micromol/nM M-CK/min, P<0.05; 6.8+/-1.3 vs. 4.7+/-1.2% myofibrillar protein, P<0.05), with no change in myosin ATPase activity. In separate experiments, isolated rat cardiac myofibrils were exposed to ONOO(-) (2-250 microM) and enzyme studies were conducted. Identical to in vivo studies, selective reductions in MM-CK were observed at ONOO(-) concentrations as low as 2 microM (IC(50)=92.5+/-6.0 microM); myosin ATPase was unaffected with ONOO(-) concentrations as high as 250 microM. Concentration dependent nitration of MM-CK occurred and extent of nitration was statistically correlated to extent of CK inhibition (P<0.001). Immunoprecipitation of MM-CK from failing left ventricle yielded significant evidence of tyrosine nitration. CONCLUSION These data demonstrate that cardiac ONOO(-) formation and perturbation of myofibrillar energetic controllers occur during experimental heart failure; MM-CK may be a critical cellular target in this setting.

Concurrent hydralazine administration prevents nitroglycerin-induced hemodynamic tolerance in experimental heart failure.

BackgroundOrganic nitrates such as nitroglycerin and isosorbide dinitrate are useful in the treatment of congestive heart failure (CHF), but tolerance develops rapidly during continuous administration. Because combination therapy of nitrate and hydralazine has been shown to provide both short- and long-term benefit but nitrate alone produces hemodynamic tolerance, we questioned whether hydralazine can preserve the favorable preload effects of nitroglycerin. Methods and ResultsUsing an in vivo model of nitroglycerin tolerance in the CHF rat, we examined the effects of hydralazine bolus dosing during continuous nitroglycerin infusion. Continuous infusion of nitroglycerin alone (10 jpg/mmn) produced initial reductions in left ventricular end-diastolic pressure of 40-50%, which returned to baseline by 8 hours (tolerance development). Coadministration of hydralazine (2X0.1 mg) maintained the effects of nitroglycerin infusion on left ventricular end-diastolic pressure (45% reduction at 10 hours). This hydralazine dose alone reduced left ventricular peak systolic pressure by approximately 12 ± 3% but had no effect on left ventricular end-diastolic pressure. Hydralazine dosing did not affect steady-state plasma concentrations of nitroglycerin or metabolites, and hydralazine was unable to prevent nitroglycerin tolerance induced in vitro. ConclusionsThe beneficial interaction of hydralazine on the preload effects of nitroglycerin may explain the long-term clinical efficacy of hydralazine/nitrate combination in CHF. Our results also suggest that the mechanism of in vivo nitrate tolerance in CHF may be systemic rather than vascular in origin. (Circulation 1991;84:35–39)

Pediatric Precursors of Adult Cardiovascular Disease: Noninvasive Assessment of Early Vascular Changes in Children and Adolescents

BACKGROUND. Until recently, our understanding of the childhood antecedents of adult cardiovascular disease was limited mainly to autopsy studies and pathologic findings in teens and young adults who died from accidental causes. Recent advances in the understanding of atherosclerosis and new technologies allowing detection of early events have made it possible to observe anatomic and physiologic evidence of cardiovascular disease in young adults and children. OBJECTIVES. The goal of this article was to introduce pediatricians to new methods for noninvasive measurement of cardiovascular disease and its precursors, to describe the potential application of these techniques in detecting childhood precursors of adult cardiovascular disease, and to summarize knowledge gained from this approach. METHODS. We conducted a computerized search of peer-reviewed articles listed in PubMed and Medline from 1980 to April 2006. We reviewed 63 and 84 articles from the adult and pediatric literature, respectively. RESULTS. Reviewing the research on childhood antecedents of adult cardiovascular disease is sobering. Vascular alterations in anatomy, physiology, mechanical properties, and proinflammatory and prothrombotic changes are present from a very early age of childhood and are associated with the risk factors common in adult cardiovascular disease. At the same time, this body of research supports the concept that the vascular impairment from childhood may improve over time with appropriate intervention. CONCLUSIONS. The measurement tools and concepts described in this article offer diagnostic and therapeutic opportunities for collaboration between clinical pediatricians and pediatric researchers. These partnerships will enable pediatricians to contribute in an effort to reduce the burdens of cardiovascular disease to individuals, families, and society.

Therapeutic implications of human endothelial nitric oxide synthase gene polymorphism

Vascular endothelial dysfunction is now recognized as a common phenomenon in an array of cardiovascular disorders. Production of nitric oxide via the endothelial isoform of nitric oxide synthase [eNOS (previously termed NOS3 or ecNOS)] is vital for a healthy endothelium; several polymorphic variations of the gene encoding eNOS (NOS3) are now known and have been investigated with respect to disease risk. Surprisingly, only approximately half of these studies have demonstrated significant associations between NOS3 polymorphisms and cardiovascular disease, and many reports are contradictory. Central issues include adequate statistical power, appropriateness of control cohorts, multigene interactions and plausible biological consequences. So far, the inconsistencies are not unique to the NOS3 polymorphisms, but probably represent the broad challenges in defining genetic aspects of complex disease processes.

Atrial Glutathione Content, Calcium Current, and Contractility

Atrial fibrillation (AF) is characterized by decreased L-type calcium current (ICa,L) in atrial myocytes and decreased atrial contractility. Oxidant stress and redox modulation of calcium channels are implicated in these pathologic changes. We evaluated the relationship between glutathione content (the primary cellular reducing moiety) and ICa,L in atrial specimens from AF patients undergoing cardiac surgery. Left atrial glutathione content was significantly lower in patients with either paroxysmal or persistent AF relative to control patients with no history of AF. Incubation of atrial myocytes from AF patients (but not controls) with the glutathione precursor N-acetylcysteine caused a marked increase in ICa,L. To test the hypothesis that glutathione levels were mechanistically linked with the reduction in ICa,L, dogs were treated for 48 h with buthionine sulfoximine, an inhibitor of glutathione synthesis. Buthionine sulfoximine treatment resulted in a 24% reduction in canine atrial glutathione content, a reduction in atrial contractility, and an attenuation of ICa,L in the canine atrial myocytes. Incubation of these myocytes with exogenous glutathione also restored ICa,L to normal or greater than normal levels. To probe the mechanism linking decreased glutathione levels to down-regulation of ICa, the biotin switch technique was used to evaluate S-nitrosylation of calcium channels. S-Nitrosylation was apparent in left atrial tissues from AF patients; the extent of S-nitrosylation was inversely related to tissue glutathione content. S-Nitrosylation was also detectable in HEK cells expressing recombinant human cardiac calcium channel subunits following exposure to nitrosoglutathione. S-Nitrosylation may contribute to the glutathione-sensitive attenuation of ICa,L observed in AF.