Nitric oxide during ischemia attenuates oxidant stress and cell death during ischemia and reperfusion in cardiomyocytes

Nitric oxide (NO) has been implicated as a cardioprotective agent during ischemia/reperfusion (I/R), but the mechanism of protection is unknown. Oxidant stress contributes to cell death in I/R, so we tested whether NO protects by attenuating oxidant stress. Cardiomyocytes and murine embryonic fibroblasts were administered NO (10-1200 nM) during simulated ischemia, and cell death was assessed during reperfusion without NO. In each case, NO abrogated cell death during reperfusion. Cells overexpressing endothelial NO synthase (NOS) exhibited a similar protection, which was abolished by the NOS inhibitor N(omega)-nitro-l-arginine methyl ester. Protection was not mediated by guanylate cyclase or the mitochondrial K(ATP) channel, as inhibitors of these systems failed to abolish protection. NO did not prevent decreases in mitochondrial potential, but cells protected with NO demonstrated recovery of potential at reperfusion. Measurements using C11-BODIPY reveal that NO attenuates lipid peroxidation during ischemia and reperfusion. Measurements of oxidant stress using the ratiometric redox sensor HSP-FRET demonstrate that NO attenuates protein oxidation during ischemia. These findings reveal that physiological levels of NO during ischemia can attenuate oxidant stress both during ischemia and during reperfusion. This response is associated with a remarkable attenuation of cell death, suggesting that ischemic cell death may be a regulated event.     

Mitochondrial complex III ROS regulate adipocyte differentiation

Adipocyte differentiation is characterized by an increase in mitochondrial metabolism. However, it is not known whether the increase in mitochondrial metabolism is essential for differentiation or a byproduct of the differentiation process. Here, we report that primary human mesenchymal stem cells undergoing differentiation into adipocytes display an early increase in mitochondrial metabolism, biogenesis, and reactive oxygen species (ROS) generation. This early increase in mitochondrial metabolism and ROS generation was dependent on mTORC1 signaling. Mitochondrial-targeted antioxidants inhibited adipocyte differentiation, which was rescued by the addition of exogenous hydrogen peroxide. Genetic manipulation of mitochondrial complex III revealed that ROS generated from this complex is required to initiate adipocyte differentiation. These results indicate that mitochondrial metabolism and ROS generation are not simply a consequence of differentiation but are a causal factor in promoting adipocyte differentiation.     

The worker honeybee fat body proteome is extensively remodeled preceding a major life-history transition

Honeybee workers are essentially sterile female helpers that make up the majority of individuals in a colony. Workers display a marked change in physiology when they transition from in-nest tasks to foraging. Recent technological advances have made it possible to unravel the metabolic modifications associated with this transition. Previous studies have revealed extensive remodeling of brain, thorax, and hypopharyngeal gland biochemistry. However, data on changes in the abdomen is scarce. To narrow this gap we investigated the proteomic composition of abdominal tissue in the days typically preceding the onset of foraging in honeybee workers. In order to get a broader representation of possible protein dynamics, we used workers of two genotypes with differences in the age at which they initiate foraging. This approach was combined with RNA interference-mediated downregulation of an insulin/insulin-like signaling component that is central to foraging behavior, the insulin receptor substrate (irs), and with measurements of glucose and lipid levels. Our data provide new insight into the molecular underpinnings of phenotypic plasticity in the honeybee, invoke parallels with vertebrate metabolism, and support an integrated and irs-dependent association of carbohydrate and lipid metabolism with the transition from in-nest tasks to foraging.     

Amphiphilic conjugates of human brain natriuretic peptide designed for oral delivery: in vitro activity screening

Congestive heart failure (CHF) is a complex syndrome involving altered neurohormonal levels and impaired cardiac and renal function. In recent years, intravenous administration of exogenous human brain-type natriuretic peptide (hBNP) has become an important therapy in treating patients with acutely decompensated CHF. However, reports during the past year suggest that hBNP could play a prominent role in the chronic treatment of CHF patients as well. We are currently developing conjugates of hBNP suitable for oral delivery to provide a patient-friendly treatment option for chronic heart failure patients. In this report, we present in vitro activity results obtained from hBNP conjugates featuring a variety of rationally designed amphiphilic oligomers. Mapping studies revealed that the hydrophobic/hydrophilic balance of the oligomer impacted the regioselectivity of conjugation. Additionally, the regiochemistry and extent of conjugation had a significant impact on activity. Many monoconjugates retained activity comparable to native peptide and are currently under evaluation in subsequent in vivo screens.     

PRISM: a data management system for high-throughput proteomics

Advanced proteomic research efforts involving areas such as systems biology or biomarker discovery are enabled by the use of high level informatics tools that allow the effective analysis of large quantities of differing types of data originating from various studies. Performing such analyses on a large scale is not feasible without a computational platform that performs data processing and management tasks. Such a platform must be able to provide high-throughput operation while having sufficient flexibility to accommodate evolving data analysis tools and methodologies. The Proteomics Research Information Storage and Management system (PRISM) provides a platform that serves the needs of the accurate mass and time tag approach developed at Pacific Northwest National Laboratory. PRISM incorporates a diverse set of analysis tools and allows a wide range of operations to be incorporated by using a state machine that is accessible to independent, distributed computational nodes. The system has scaled well as data volume has increased over several years, while allowing adaptability for incorporating new and improved data analysis tools for more effective proteomics research.     

Auditory sensitivity and ecological relevance: the functional audiogram as modelled by the bat detecting moth ear

Auditory sensitivity has often been measured by identifying neural threshold in real-time (online) which can introduce bias in the audiograms that are produced. We tested this by recording auditory nerve activity of the notodontid moth Nadata gibbosa elicited by bat-like ultrasound and analysing the response offline. We compared this audiogram with a published online audiogram showing that the bias introduced can result in a difference in the audiogram shape. In the second part of our study we compared offline audiograms using spike number as threshold with others that used spike period and stimulus/spike latency, variables that have been suggested as providing behaviourally functional criteria. These comparisons reveal that functional audiograms are more flatly tuned than simple spike audiograms. The shapes of behavioural audiograms are discussed in the context of the selection pressure that maintains their shape, bat predation. Finally, we make predictions on the distance from bats at which notodontid moths use negative phonotaxis or the acoustic startle response.     

Safety and whole-body antioxidant potential of a novel anthocyanin-rich formulation of edible berries

Edible berry extracts rich in anthocyanins possess a broad spectrum of therapeutic, pharmacologic and anti-carcinogenic properties. Six berry extracts (wild blueberry, bilberry, cranberry, elderberry, raspberry seeds and strawberry), singly and in combination, were studied in our laboratories for antioxidant efficacy, cytotoxic potential, cellular uptake and anti-angiogenic properties. Combinations of edible berry extracts were evaluated to develop a synergistic formula, OptiBerry, which exhibited high oxygen radical absorbance capacity (ORAC) value, low cytotoxicity and superior anti-angiogenic properties compared to the other combinations tested. The current study sought to determine the broad spectrum safety and antioxidant potential of OptiBerry in vivo. Acute oral LD₅₀ of OptiBerry was greater than 5 g/kg in rats. Acute dermal LD₅₀ of OptiBerry was greater than 2 g/kg. No changes in the body weight or adverse effects were observed following necropsy. Primary skin and eye irritation studies were conducted in New Zealand albino rabbits. OptiBerry was classified as slightly irritating to the skin (primary skin irritation index 0.3) and minimally irritating to the eye (maximum mean total score 6.0). The antioxidant potential of OptiBerry was investigated in rats and mice by assessing GSH redox status in tissues as well as by a unique state-of-the-art electron paramagnetic resonance (EPR) imaging of whole-body redox status. A clinically relevant hyperbaric oxygen (HBO) exposure system (2 atm, 2 h) was employed to study the antioxidant properties of OptiBerry. OptiBerry feeding (8 weeks) significantly prevented HBO-induced GSH oxidation in the lung and liver of vitamin E-deficient Sprague Dawley rats. Furthermore, OptiBerry-fed mice, when exposed to HBO, demonstrated significant protection in whole-body HBO-induced oxidation compared to the unfed controls by EPR imaging. Taken together, these results indicate that OptiBerry is reasonably safe and possess antioxidant properties.     

Application of the accurate mass and time tag approach in studies of the human blood lipidome

We report a preliminary demonstration of the accurate mass and time (AMT) tag approach for lipidomics. Initial data-dependent LC-MS/MS analyses of human plasma, erythrocyte, and lymphocyte lipids were performed in order to identify lipid molecular species in conjunction with complementary accurate mass and isotopic distribution information. Identified lipids were used to populate initial lipid AMT tag databases containing 250 and 45 entries for those species detected in positive and negative electrospray ionization (ESI) modes, respectively. The positive ESI database was then utilized to identify human plasma, erythrocyte, and lymphocyte lipids in high-throughput LC-MS analyses based on the AMT tag approach. We were able to define the lipid profiles of human plasma, erythrocytes, and lymphocytes based on qualitative and quantitative differences in lipid abundance.