Hyperhydricity in micropropagated carnation shoots: the role of oxidative stress

The physiology of hyperhydricity in relation to oxidative stress, mineral nutrients, antioxidant enzymes and ethylene has been studied in three micropropagated carnation cultivars under experimentally induced hyperhydricity. A marked increase in Fe content in comparison with normal tissues was observed in the hyperhydric tissues from the three cultivars. The levels of ethylene, solute leakage and malondialdehyde content were also significantly higher in the hyperhydric tissues. In relation to the time course of H₂O₂ production measured by fluorescence quenching, a similar trend could be observed for the three cultivars, with a clear increase in the generation of hydrogen peroxide in hyperhydric tissues. The activities of all the antioxidative enzymes studied, except lipoxygenase, were higher in the hyperhydric shoots. Phenylalanine ammonia-lyase (PAL) showed a significant decrease in activity in the hyperhydric tissues in comparison with the controls for the three cultivars. Soluble guaiacol peroxidase had a strong increase in activity in hyperhydric shoots of the three cultivars. These results provide, for the first time, direct evidence of H₂O₂ generation in hyperhydric tissues, characterize the response of the antioxidant system to an oxidative stress during hyperhydricity in carnation leaves and point to the accumulation of toxic forms of oxygen as the inducer of some of the abnormalities observed.     

NADH Fluorescence Imaging of Isolated Biventricular Working Rabbit Hearts

Since its inception by Langendorff¹, the isolated perfused heart remains a prominent tool for studying cardiac physiology². However, it is not well-suited for studies of cardiac metabolism, which require the heart to perform work within the context of physiologic preload and afterload pressures. Neely introduced modifications to the Langendorff technique to establish appropriate left ventricular (LV) preload and afterload pressures³. The model is known as the isolated LV working heart model and has been used extensively to study LV performance and metabolism^4-6. This model, however, does not provide a properly loaded right ventricle (RV). Demmy et al. first reported a biventricular model as a modification of the LV working heart model^{7, 8}. They found that stroke volume, cardiac output, and pressure development improved in hearts converted from working LV mode to biventricular working mode⁸. A properly loaded RV also diminishes abnormal pressure gradients across the septum to improve septal function. Biventricular working hearts have been shown to maintain aortic output, pulmonary flow, mean aortic pressure, heart rate, and myocardial ATP levels for up to 3 hours⁸.When studying the metabolic effects of myocardial injury, such as ischemia, it is often necessary to identify the location of the affected tissue. This can be done by imaging the fluorescence of NADH (the reduced form of nicotinamide adenine dinucleotide)^9-11, a coenzyme found in large quantities in the mitochondria. NADH fluorescence (fNADH) displays a near linearly inverse relationship with local oxygen concentration¹² and provides a measure of mitochondrial redox state¹³. fNADH imaging during hypoxic and ischemic conditions has been used as a dye-free method to identify hypoxic regions^{14, 15} and to monitor the progression of hypoxic conditions over time¹⁰.The objective of the method is to monitor the mitochondrial redox state of biventricular working hearts during protocols that alter the rate of myocyte metabolism or induce hypoxia or create a combination of the two. Hearts from New Zealand white rabbits were connected to a biventricular working heart system (Hugo Sachs Elektronik) and perfused with modified Krebs-Henseleit solution¹⁶ at 37 °C. Aortic, LV, pulmonary artery, and left & right atrial pressures were recorded. Electrical activity was measured using a monophasic action potential electrode. To image fNADH, light from a mercury lamp was filtered (350±25 nm) and used to illuminate the epicardium. Emitted light was filtered (460±20 nm) and imaged using a CCD camera. Changes in the epicardial fNADH of biventricular working hearts during different pacing rates are presented. The combination of the heart model and fNADH imaging provides a new and valuable experimental tool for studying acute cardiac pathologies within the context of realistic physiological conditions.     

Interactions between Diatoms and Bacteria

Summary: Diatoms and bacteria have cooccurred in common habitats for hundreds of millions of years, thus fostering specific associations and interactions with global biogeochemical consequences. Diatoms are responsible for one-fifth of the photosynthesis on Earth, while bacteria remineralize a large portion of this fixed carbon in the oceans. Through their coexistence, diatoms and bacteria cycle nutrients between oxidized and reduced states, impacting bioavailability and ultimately feeding higher trophic levels. Here we present an overview of how diatoms and bacteria interact and the implications of these interactions. We emphasize that heterotrophic bacteria in the oceans that are consistently associated with diatoms are confined to two phyla. These consistent bacterial associations result from encounter mechanisms that occur within a microscale environment surrounding a diatom cell. We review signaling mechanisms that occur in this microenvironment to pave the way for specific interactions. Finally, we discuss known interactions between diatoms and bacteria and exciting new directions and research opportunities in this field. Throughout the review, we emphasize new technological advances that will help in the discovery of new interactions. Deciphering the languages of diatoms and bacteria and how they interact will inform our understanding of the role these organisms have in shaping the ocean and how these interactions may change in future oceans.     

Haptoglobin Genotype-dependent Differences in Macrophage Lysosomal Oxidative Injury

The major function of the Haptoglobin (Hp) protein is to control trafficking of extracorpuscular hemoglobin (Hb) thru the macrophage CD163 receptor with degradation of the Hb in the lysosome. There is a common copy number polymorphism in the Hp gene (Hp 2 allele) that has been associated with a severalfold increased incidence of atherothrombosis in multiple longitudinal studies. Increased plaque oxidation and apoptotic markers have been observed in Hp 2-2 atherosclerotic plaques, but the mechanism responsible for this finding has not been determined. We proposed that the increased oxidative injury in Hp 2-2 plaques is due to an impaired processing of Hp 2-2-Hb complexes within macrophage lysosomes, thereby resulting in redox active iron accumulation, lysosomal membrane oxidative injury, and macrophage apoptosis. We sought to test this hypothesis in vitro using purified Hp-Hb complex and cells genetically manipulated to express CD163. CD163-mediated endocytosis and lysosomal degradation of Hp-Hb were decreased for Hp 2-2-Hb complexes. Confocal microscopy using lysotropic pH indicator dyes demonstrated that uptake of Hp 2-2-Hb complexes disrupted the lysosomal pH gradient. Cellular fractionation studies of lysosomes isolated from macrophages incubated with Hp 2-2-Hb complexes demonstrated increased lysosomal membrane oxidation and a loss of lysosomal membrane integrity leading to lysosomal enzyme leakage into the cytoplasm. Additionally, markers of apoptosis, DNA fragmentation, and active caspase 3 were increased in macrophages that had endocytosed Hp 2-2-Hb complexes. These data provide novel mechanistic insights into how the Hp genotype regulates lysosomal oxidative stress within macrophages after receptor-mediated endocytosis of Hb.     

Effects of Tobacco Smoking on the Degeneration of the Intervertebral Disc: A Finite Element Study

Tobacco smoking is associated with numerous pathological conditions. Compelling experimental evidence associates smoking to the degeneration of the intervertebral disc (IVD). In particular, it has been shown that nicotine down-regulates both the proliferation rate and glycosaminoglycan (GAG) biosynthesis of disc cells. Moreover, tobacco smoking causes the constriction of the vascular network surrounding the IVD, thus reducing the exchange of nutrients and anabolic agents from the blood vessels to the disc. It has been hypothesized that both nicotine presence in the IVD and the reduced solute exchange are responsible for the degeneration of the disc due to tobacco smoking, but their effects on tissue homeostasis have never been quantified. In this study, a previously presented computational model describing the homeostasis of the IVD was deployed to investigate the effects of impaired solute supply and nicotine-mediated down-regulation of cell proliferation and biosynthetic activity on the health of the disc. We found that the nicotine-mediated down-regulation of cell anabolism mostly affected the GAG concentration at the cartilage endplate, reducing it up to 65% of the value attained in normal physiological conditions. In contrast, the reduction of solutes exchange between blood vessels and disc tissue mostly affected the nucleus pulposus, whose cell density and GAG levels were reduced up to 50% of their normal physiological levels. The effectiveness of quitting smoking on the regeneration of a degenerated IVD was also investigated, and showed to have limited benefit on the health of the disc. A cell-based therapy in conjunction with smoke cessation provided significant improvements in disc health, suggesting that, besides quitting smoking, additional treatments should be implemented in the attempt to recover the health of an IVD degenerated by tobacco smoking.     

Impedance spectroscopy flow cytometry: on-chip label-free cell differentiation.

BACKGROUND: The microfabricated impedance spectroscopy flow cytometer used in this study permits rapid dielectric characterization of a cell population with a simple microfluidic channel. Impedance measurements over a wide frequency range provide information on cell size, membrane capacitance, and cytoplasm conductivity as a function of frequency. The amplitude, opacity, and phase information can be used for discrimination between different cell populations without the use of cell markers. METHODS: Polystyrene beads, red blood cells (RBCs), ghosts, and RBCs fixed in glutaraldehyde were passed through a microfabricated flow cytometer and measured individually by using two simultaneously applied discrete frequencies. The cells were characterized at 1,000 per minute in the frequency range of 350 kHz to 20 MHz. RESULTS: Cell size was easily measured with submicron accuracy. Polystyrene beads and RBCs were differentiated using opacity. RBCs and ghosts were differentiated using phase information, whereas RBCs and fixed RBCs were differentiated using opacity. RBCs fixed using increasing concentrations of glutaraldehyde showed increasing opacity. This increased opacity was linked to decreased cytoplasm conductivity and decreased membrane capacitance, both resulting from protein cross-linking. CONCLUSIONS: This work presents label-free differentiation of cells in an on-chip flow cytometer based on impedance spectroscopy, which will be a powerful tool for cell characterization.