Selective, competitive and mechanism-based inhibitors of human cytochrome P450 2J2

Twenty five derivatives of the drugs terfenadine and ebastine have been designed, synthesized and evaluated as inhibitors of recombinant human CYP2J2. Compound 14, which has an imidazole substituent, is a good non-competitive inhibitor of CYP2J2 (IC(50)=400nM). It is not selective towards CYP2J2 as it also efficiently inhibits the other main vascular CYPs, such as CYP2B6, 2C8, 2C9 and 3A4; however, it could be an interesting tool to inhibit all these vascular CYPs. Compounds 4, 5 and 13, which have a propyl, allyl and benzo-1,3-dioxole terminal group, respectively, are selective CYP2J2 inhibitors. Compound 4 is a high-affinity, competitive inhibitor and alternative substrate of CYP2J2 (K(i)=160+/-50nM). Compounds 5 and 13 are efficient mechanism-based inhibitors of CYP2J2 (k(inact)/K(i) values approximately 3000Lmol(-1)s(-1)). Inactivation of CYP2J2 by 13 is due to the formation of a stable iron-carbene bond which occurs upon CYP2J2-catalyzed oxidation of 13 with a partition ratio of 18+/-3. These new selective inhibitors should be interesting tools to study the biological roles of CYP2J2.     

Cytochrome P-450 epoxygenases protect endothelial cells from apoptosis induced by tumor necrosis factor-alpha via MAPK and PI3K/Akt signaling pathways

Endothelial cells play a vital role in the maintenance of cardiovascular homeostasis. Epoxyeicosatrienoic acids (EETs), cytochrome P-450 (CYP) epoxygenase metabolites of arachidonic acid in endothelial cells, possess potent and diverse biological effects within the vasculature. We evaluated the effects of overexpression of CYP epoxygenases on tumor necrosis factor-alpha (TNF-alpha)-induced apoptosis in bovine aortic endothelial cells. CYP epoxygenase overexpression significantly increased endothelial cell viability and inhibited TNF-alpha induction of endothelial cell apoptosis as evaluated by morphological analysis of nuclear condensation, DNA laddering, and fluorescent-activated cell sorting (FACS) analysis. CYP epoxygenase overexpression also significantly inhibited caspase-3 activity and downregulation of Bcl-2 expression induced by TNF-alpha. The antiapoptotic effects of CYP epoxygenase overexpression were significantly attenuated by inhibition of the phosphatidylinositol 3-kinase (PI3K)/Akt and MAPK signaling pathways; however, inhibition of endothelial nitric oxide synthase activity had no effect. Furthermore, CYP epoxygenase overexpression significantly attenuated the extent of TNF-alpha-induced ERK1/2 dephosphorylation in a time-dependent manner and significantly increased PI3K expression and Akt phosphorylation in both the presence and absence of TNF-alpha. Collectively, these results suggest that CYP epoxygenase overexpression, which is known to increase EET biosynthesis, significantly protects endothelial cells from apoptosis induced by TNF-alpha. This effect is mediated, at least in part, through inhibition of ERK dephosphorylation and activation of PI3K/Akt signaling.     

Pediatric inpatient hospital resource use for congenital heart defects

Background: Congenital heart defects (CHDs) occur in approximately 8 per 1000 live births. Improvements in detection and treatment have increased survival. Few national estimates of the healthcare costs for infants, children and adolescents with CHDs are available. Methods: We estimated hospital costs for hospitalizations using pediatric (0–20 years) hospital discharge data from the 2009 Healthcare Cost and Utilization Project Kids' Inpatient Database (KID) for hospitalizations with CHD diagnoses. Estimates were up‐weighted to be nationally representative. Mean costs were compared by demographic factors and presence of critical CHDs (CCHDs). Results: Up‐weighting of the KID generated an estimated 4,461,615 pediatric hospitalizations nationwide, excluding normal newborn births. The 163,980 (3.7%) pediatric hospitalizations with CHDs accounted for approximately $5.6 billion in hospital costs, representing 15.1% of costs for all pediatric hospitalizations in 2009. Approximately 17% of CHD hospitalizations had a CCHD, but it varied by age: approximately 14% of hospitalizations of infants, 30% of hospitalizations of patients aged 1 to 10 years, and 25% of hospitalizations of patients aged 11 to 20 years. Mean costs of CHD hospitalizations were higher in infancy ($36,601) than at older ages and were higher for hospitalizations with a CCHD diagnosis ($52,899). Hospitalizations with CCHDs accounted for 26.7% of all costs for CHD hospitalizations, with hypoplastic left heart syndrome, coarctation of the aorta, and tetralogy of Fallot having the highest total costs. Conclusion: Hospitalizations for children with CHDs have disproportionately high hospital costs compared with other pediatric hospitalizations, and the 17% of hospitalizations with CCHD diagnoses accounted for 27% of CHD hospital costs. Birth Defects Research (Part A) 100:934–943, 2014. © 2014 Wiley Periodicals, Inc.     

Foodborne Transmission of Nipah Virus in Syrian Hamsters

Since 2001, outbreaks of Nipah virus have occurred almost every year in Bangladesh with high case-fatality rates. Epidemiological data suggest that in Bangladesh, Nipah virus is transmitted from the natural reservoir, fruit bats, to humans via consumption of date palm sap contaminated by bats, with subsequent human-to-human transmission. To experimentally investigate this epidemiological association between drinking of date palm sap and human cases of Nipah virus infection, we determined the viability of Nipah virus (strain Bangladesh/200401066) in artificial palm sap. At 22°C virus titers remained stable for at least 7 days, thus potentially allowing food-borne transmission. Next, we modeled food-borne Nipah virus infection by supplying Syrian hamsters with artificial palm sap containing Nipah virus. Drinking of 5×10⁸ TCID₅₀ of Nipah virus resulted in neurological disease in 5 out of 8 hamsters, indicating that food-borne transmission of Nipah virus can indeed occur. In comparison, intranasal (i.n.) inoculation with the same dose of Nipah virus resulted in lethal respiratory disease in all animals. In animals infected with Nipah virus via drinking, virus was detected in respiratory tissues rather than in the intestinal tract. Using fluorescently labeled Nipah virus particles, we showed that during drinking, a substantial amount of virus is deposited in the lungs, explaining the replication of Nipah virus in the respiratory tract of these hamsters. Besides the ability of Nipah virus to infect hamsters via the drinking route, Syrian hamsters infected via that route transmitted the virus through direct contact with naïve hamsters in 2 out of 24 transmission pairs. Although these findings do not directly prove that date palm sap contaminated with Nipah virus by bats is the origin of Nipah virus outbreaks in Bangladesh, they provide the first experimental support for this hypothesis. Understanding the Nipah virus transmission cycle is essential for preventing and mitigating future outbreaks.     

Analogues of 3-Hydroxyisoxazole-Containing Glutamate Receptor Ligands Based on the 3-Hydroxypyrazole-Moiety: Design,Synthesis and Pharmacological Characterization

A series of analogues of the glutamate receptor ligands (S)-2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propionic acid (AMPA) and AMOA were synthesized in which the 3-hydroxyisoxazole moiety was exchanged for a 3-hydroxypyrazole moiety. This exchange enables further substitution at the additional nitrogen atom in the heterocyclic core. Several of the analogues have activity at AMPA receptors equipotent to the antagonist ATPO, demonstrating that additional substitution can be accommodated in the antagonist binding site. Modelling studies offer an explanation for the pharmacological pattern observed for the compounds and suggest that this scaffold may be developed further to obtain subtype selective antagonists.     

Comparison of hierarchical and six degrees-of-freedom marker sets in analyzing gait kinematics

The objective of this study was to determine how marker spacing, noise, and joint translations affect joint angle calculations using both a hierarchical and a six degrees-of-freedom (6DoF) marker set. A simple two-segment model demonstrates that a hierarchical marker set produces biased joint rotation estimates when sagittal joint translations occur whereas a 6DoF marker set mitigates these bias errors with precision improving with increased marker spacing. These effects were evident in gait simulations where the 6DoF marker set was shown to be more accurate at tracking axial rotation angles at the hip, knee, and ankle.     

Selective lateral muscle activation in moderate medial knee osteoarthritis subjects does not unload medial knee condyle

There is some debate in the literature regarding the role of quadriceps-hamstrings co-contraction in the onset and progression of knee osteoarthritis. Does co-contraction during walking increase knee contact loads, thereby causing knee osteoarthritis, or might it be a compensatory mechanism to unload the medial tibial condyle? We used a detailed musculoskeletal model of the lower limb to test the hypothesis that selective activation of lateral hamstrings and quadriceps, in conjunction with inhibited medial gastrocnemius, can actually reduce the joint contact force on the medial compartment of the knee, independent of changes in kinematics or external forces. “Baseline” joint loads were computed for eight subjects with moderate medial knee osteoarthritis (OA) during level walking, using static optimization to resolve the system of muscle forces for each subject?s scaled model. Holding all external loads and kinematics constant, each subject?s model was then perturbed to represent non-optimal “OA-type” activation based on mean differences detected between electromyograms (EMG) of control and osteoarthritis subjects. Knee joint contact forces were greater for the “OA-type” than the “Baseline” distribution of muscle forces, particularly during early stance. The early-stance increase in medial contact load due to the “OA-type” perturbation could implicate this selective activation strategy as a cause of knee osteoarthritis. However, the largest increase in the contact load was found at the lateral condyle, and the “OA-type” lateral activation strategy did not increase the overall (greater of the first or second) medial peak contact load. While “OA-type” selective activation of lateral muscles does not appear to reduce the medial knee contact load, it could allow subjects to increase knee joint stiffness without any further increase to the peak medial contact load.     

Assembly of mitochondrial cytochrome c-oxidase, a complicated and highly regulated cellular process

Cytochrome c-oxidase (COX), the terminal enzyme of the mitochondrial respiratory chain, plays a key role in the regulation of aerobic production of energy. Biogenesis of eukaryotic COX involves the coordinated action of two genomes. Three mitochondrial DNA-encoded subunits form the catalytic core of the enzyme, which contains metal prosthetic groups. Another 10 subunits encoded in the nuclear DNA act as a protective shield surrounding the core. COX biogenesis requires the assistance of >20 additional nuclear-encoded factors acting at all levels of the process. Expression of the mitochondrial-encoded subunits, expression and import of the nuclear-encoded subunits, insertion of the structural subunits into the mitochondrial inner membrane, addition of prosthetic groups, assembly of the holoenzyme, further maturation to form a dimer, and additional assembly into supercomplexes are all tightly regulated processes in a nuclear-mitochondrial-coordinated fashion. Such regulation ensures the building of a highly efficient machine able to catalyze the safe transfer of electrons from cytochrome c to molecular oxygen and ultimately facilitate the aerobic production of ATP. In this review, we will focus on describing and analyzing the present knowledge about the different regulatory checkpoints in COX assembly and the dynamic relationships between the different factors involved in the process. We have used information mostly obtained from the suitable yeast model, but also from bacterial and animal systems, by means of large-scale genetic, molecular biology, and physiological approaches and by integrating information concerning individual elements into a cellular system network.     

A peripheral benzodiazepine receptor targeted agent for in vitro imaging and screening

We developed a molecular imaging agent (MIA), a conjugable form of PK11195 (conPK11195) coupled to a lissamine dye (Liss-ConPK11195), which targets the peripheral benzodiazepine receptor (PBR). To determine that our compound specifically binds to this 18 kDa protein, primarily expressed on the mitochondria, we performed classic binding studies on live MDA-MB-231 breast cancer cells and measured fluorescence in cell fractions of C6 glioma cells. We found that conPK11195 conjugated to the fluorophore retained significant binding to its target. Here we demonstrate the utility of the agent for in vitro imaging of live cells by specific binding to the protein of interest.