Kounis syndrome following allergy injection in a paediatric patient.

Kounis syndrome is the concurrence of acute coronary syndrome or coronary vasospasm with conditions associated with the release of inflammatory cytokines through mast cell activation in the setting of allergic or anaphylactic reactions. Many identified triggers have been identified in paediatric patients including exposures, drugs, and immunisations; however, to our knowledge this is the first case report of Kounis syndrome linked to immunotherapy. We present a case of a 9-year-old with seasonal allergies presenting with clinical symptoms of Kounis syndrome following her weekly subcutaneous injection of allergens. Clinicians need a high index of suspicion for Kounis syndrome in patients who develop systemic signs of anaphylaxis with clinical, laboratory, electrocardiographic, and echocardiographic findings of acute coronary syndrome to help direct therapy and improve outcomes.

Development of an effective method of human DNA extraction from soil as forensic evidence.

In forensic investigations, it is crucial to detect a suspect's DNA from the available evidence. In an outdoor crime scene, the evidence may be mixed with the soil. However, soil is speculated to inhibit DNA extraction from forensic science samples. In the field of soil microbiology, it is necessary to extract DNA directly from soil to analyse its microbial composition. In this study, we investigated whether skim milk used in procedure of DNA extraction from soil samples could be applied to forensic science to enhance human DNA extraction efficiency from soil mixed with forensic evidence, such as blood, buccal cells, and skin cells. The use of additive reagents, skim milk and bovine serum albumin (BSA), are known to blocking reagent. In the selection of additive reagents experiment, about blood sample, using the skim milk and BSA were found to increase the DNA yield. Therefore, we observed extracted DNA yield from blood, buccal cells, and skin cells when skim milk and BSA were used as additive reagents. The DNA recovery rate was high across all samples upon addition of skim milk. However, in STR analysis, a non-specific peak was detected in the extracted DNA in the presence of skim milk, which was not detected in the presence of BSA, indicating its suitability in forensic analysis. Our study suggests that addition of BSA can efficiently aid the extraction of DNA from forensic evidence mixed with soil.

Sensitized by a sea slug: Site-specific short-term and general long-term sensitization in Aplysia following Navanax attack.

Neurobiological studies of the model species, Aplysia californica (Mollusca, Gastropoda, Euopisthobranchia), have helped advance our knowledge of the neural bases of different forms of learning, including sensitization, a non-associative increase in withdrawal behaviors in response to mild innocuous stimuli. However, our understanding of the natural context for this learning has lagged behind the mechanistic studies. Previous studies, which exclusively used artificial stimuli, such as electric shock, to produce sensitization, left open the question of which stimuli might cause sensitization in nature. Our laboratory first addressed this question by testing for short and long-term sensitization after predatory attack by a natural predator, the spiny lobster. In the present study, we tested for sensitization after attack by a very different predator, the predacious sea-slug, Navanax inermis (Mollusca, Gastropoda, Euopisthobranchia). Unlike the biting and prodding action of lobster attack, Navanax uses a rapid strike that sucks and squeezes its prey in an attempt to swallow it whole. We found that Navanax attack to the head of Aplysia caused strong immediate sensitization of head withdrawal, and weaker, delayed, sensitization of tail-mantle withdrawal. By contrast, attack to the tail of Aplysia resulted in no sensitization of either reflex. We also developed an artificial attack stimulus that allowed us to mimick a more consistently strong attack. This artificial attack produced stronger but qualitatively similar sensitization: Strong immediate sensitization of head withdrawal and weaker sensitization of tail-mantle withdrawal after head attack, immediate sensitization in tail-mantle withdrawal, but no sensitization of head withdrawal after tail attack. We conclude that Navanax attack causes robust site-specific sensitization (enhanced sensitization near the site of attack), and weaker general sensitization (sensitization of responses to stimuli distal to the attack site). We also tested for long-term sensitization (lasting longer than 24 h) after temporally-spaced delivery of four natural Navanax attacks to the head of subject Aplysia. Surprisingly, these head attacks, any one of which strongly sensitizes head withdrawal in the short term, failed to sensitize head-withdrawal in the long term. Paradoxically, these repeated head attacks produced long-term sensitization in tail-mantle withdrawal. These experiments and observations confirm that Navanax attack causes short, and long-term sensitization of withdrawal reflexes of Aplysia. They add site-specific sensitization as well as paradoxical long-term sensitization of tail-mantle withdrawal to a short list of naturally induced learning phenotypes in this model species. Together with previous observations of sensitization after lobster attack, these data strongly support the premise that sensitization in Aplysia is an adaptive response to sub-lethal predator attack.

Influence of flooding and soil properties on the genetic diversity and distribution of indigenous soybean-nodulating bradyrhizobia in the Philippines.

One of the strategies that is commonly used in the Philippines to improve the production of soybean is by inoculation. However, this technique often fails mainly due to the lack of information about the indigenous soybean rhizobia in the Philippines soil. In this study, the diversity of indigenous bradyrhizobia collected from the non-flooded and flooded soil conditions at 11 locations in the country was investigated using a local soybean cultivar as the host plant. The genetic variation among the 424 isolates was detected through Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) treatment and sequence analysis for 16S rRNA gene, 16S-23S rRNA internal transcribed spacer (ITS) region and rpoB housekeeping gene. All the isolates were classified under the Bradyrhizobium species namely B. elkanii, B. diazoefficiens, B. japonicum, B. yuanmingense and a considerable proportion of the isolates were clustered under Bradyrhizobium sp. The isolates which were classified under Bradyrhizobium sp. were thought to be endemic to Philippines soil as evidenced by their nucleotide divergence against the known rhizobia and the historical absence of rhizobia inoculation in the collection sites. The major influence on the distribution and diversity of soybean bradyrhizobia is attributed to the difference in the flooding period, followed by soil properties such as pH, soil type, and nutrient content. As determined, it is proposed that the major micro-symbiont of soybean in the Philippines are B. elkanii for non-flooded soils, then B. diazoefficiens and B. japonicum for flooded soils.

Effect of Flooding and the nosZ Gene in Bradyrhizobia on Bradyrhizobial Community Structure in the Soil.

We investigated the effects of the water status (flooded or non-flooded) and presence of the nosZ gene in bradyrhizobia on the bradyrhizobial community structure in a factorial experiment that examined three temperature levels (20°C, 25°C, and 30°C) and two soil types (andosol and gray lowland soil) using microcosm incubations. All microcosms were inoculated with Bradyrhizobium japonicum USDA6T, B. japonicum USDA123, and B. elkanii USDA76T, which do not possess the nosZ gene, and then half received B. diazoefficiens USDA110Twt (wt for the wild-type) and the other half received B. diazoefficiens USDA110ΔnosZ. USDA110Twt possesses the nosZ gene, which encodes N2O reductase; 110ΔnosZ, a mutant variant, does not. Changes in the community structure after 30- and 60-d incubations were investigated by denaturing-gradient gel electrophoresis and an image analysis. USDA6T and 76T strains slightly increased in non-flooded soil regardless of which USDA110T strain was present. In flooded microcosms with the USDA110Twt strain, USDA110Twt became dominant, whereas in microcosms with the USDA110ΔnosZ, a similar change in the community structure occurred to that in non-flooded microcosms. These results suggest that possession of the nosZ gene confers a competitive advantage to B. diazoefficiens USDA110T in flooded soil. We herein demonstrated that the dominance of B. diazoefficiens USDA110Twt within the soil bradyrhizobial population may be enhanced by periods of flooding or waterlogging systems such as paddy-soybean rotations because it appears to have the ability to thrive in moderately anaerobic soil.

Kinetic studies on the oxidation of semiquinone and hydroquinone forms of Arabidopsis cryptochrome by molecular oxygen.

Cryptochromes (crys) are flavoprotein photoreceptors present throughout the biological kingdom that play important roles in plant development and entrainment of the circadian clock in several organisms. Crys non-covalently bind flavin adenine dinucleotide (FAD) which undergoes photoreduction from the oxidised state to a radical form suggested to be active in signalling in vivo. Although the photoreduction reactions have been well characterised by a number of approaches, little is known of the oxidation reactions of crys and their mechanisms. In this work, a stopped-flow kinetics approach is used to investigate the mechanism of cry oxidation in the presence and absence of an external electron donor. This in vitro study extends earlier investigations of the oxidation of Arabidopsis cryptochrome1 by molecular oxygen and demonstrates that, under some conditions, a more complex model for oxidation of the flavin than was previously proposed is required to accommodate the spectral evidence (see P. Müller and M. Ahmad (2011) J. Biol. Chem. 286, 21033-21040 [1]). In the absence of an electron donor, photoreduction leads predominantly to the formation of the radical FADH(•). Dark recovery most likely forms flavin hydroperoxide (FADHOOH) requiring superoxide. In the presence of reductant (DTT), illumination yields the fully reduced flavin species (FADH(-)). Reaction of this with dioxygen leads to transient radical (FADH(•)) and simultaneous accumulation of oxidised species (FAD), possibly governed by interplay between different cryptochrome molecules or cooperativity effects within the cry homodimer.

Caspase-Cleaved Tau Co-Localizes with Early Tangle Markers in the Human Vascular Dementia Brain.

Vascular dementia (VaD) is the second most common form of dementia in the United States and is characterized as a cerebral vessel vascular disease that leads to ischemic episodes. Whereas the relationship between caspase-cleaved tau and neurofibrillary tangles (NFTs) in Alzheimer's disease (AD) has been previously described, whether caspase activation and cleavage of tau occurs in VaD is presently unknown. To investigate a potential role for caspase-cleaved tau in VaD, we analyzed seven confirmed cases of VaD by immunohistochemistry utilizing a well-characterized antibody that specifically detects caspase-cleaved tau truncated at Asp421. Application of this antibody (TauC3) revealed consistent labeling within NFTs, dystrophic neurites within plaque-rich regions and corpora amylacea (CA) in the human VaD brain. Labeling of CA by the TauC3 antibody was widespread throughout the hippocampus proper, was significantly higher compared to age matched controls, and co-localized with ubiquitin. Staining of the TauC3 antibody co-localized with MC-1, AT8, and PHF-1 within NFTs. Quantitative analysis indicated that roughly 90% of PHF-1-labeled NFTs contained caspase-cleaved tau. In addition, we documented the presence of active caspase-3 within plaques, blood vessels and pretangle neurons that co-localized with TauC3. Collectively, these data support a role for the activation of caspase-3 and proteolytic cleavage of TauC3 in VaD providing further support for the involvement of this family of proteases in NFT pathology.

Re-evaluation of the near infrared spectra of mitochondrial cytochrome c oxidase: Implications for non invasive in vivo monitoring of tissues.

We re-determined the near infrared (NIR) spectral signatures (650-980nm) of the different cytochrome c oxidase redox centres, in the process separating them into their component species. We confirm that the primary contributor to the oxidase NIR spectrum between 700 and 980nm is cupric CuA, which in the beef heart enzyme has a maximum at 835nm. The 655nm band characterises the fully oxidised haem a3/CuB binuclear centre; it is bleached either when one or more electrons are added to the binuclear centre or when the latter is modified by ligands. The resulting 'perturbed' binuclear centre is also characterised by a previously unreported broad 715-920nm band. The NIR spectra of certain stable liganded species (formate and CO), and the unstable oxygen reaction compounds P and F, are similar, suggesting that the latter may resemble the stable species electronically. Oxidoreduction of haem a makes no contribution either to the 835nm maximum or the 715nm band. Our results confirm the ability of NIRS to monitor the CuA centre of cytochrome oxidase activity in vivo, although noting some difficulties in precise quantitative interpretations in the presence of perturbations of the haem a3/CuB binuclear centre.

Connecting model species to nature: predator-induced long-term sensitization in Aplysia californica.

Previous research on sensitization in Aplysia was based entirely on unnatural noxious stimuli, usually electric shock, until our laboratory found that a natural noxious stimulus, a single sublethal lobster attack, causes short-term sensitization. We here extend that finding by demonstrating that multiple lobster attacks induce long-term sensitization (≥24 h) as well as similar, although not identical, neuronal correlates as observed after electric shock. Together these findings establish long- and short-term sensitization caused by sublethal predator attack as a natural equivalent to sensitization caused by artificial stimuli.

Nitrogen dioxide oxidizes mitochondrial cytochrome c.

We previously reported that high micromolar concentrations of nitric oxide were able to oxidize mitochondrial cytochrome c at physiological pH, producing nitroxyl anion (Sharpe and Cooper, 1998 Biochem. J. 332, 9-19). However, the subsequent re-evaluation of the redox potential of the NO/NO(-) couple suggests that this reaction is thermodynamically unfavored. We now show that the oxidation is oxygen-concentration dependent and non stoichiometric. We conclude that the effect is due to an oxidant species produced during the aerobic decay of nitric oxide to nitrite and nitrate. The species is most probably nitrogen dioxide, NO(2)(•) a well-known biologically active oxidant. A simple kinetic model of NO autoxidation is able to explain the extent of cytochrome c oxidation assuming a rate constant of 3×10(6)M(-1)s(-1) for the reaction of NO(2)(•) with ferrocytochrome c. The importance of NO(2)(•) was confirmed by the addition of scavengers such as urate and ferrocyanide. These convert NO(2)(•) into products (urate radical and ferricyanide) that rapidly oxidize cytochrome c and hence greatly enhance the extent of oxidation observed. The present study does not support the previous hypothesis that NO and cytochrome c can generate appreciable amounts of nitroxyl ions (NO(-) or HNO) or of peroxynitrite.

The steady-state mechanism of cytochrome c oxidase: redox interactions between metal centres.

The steady-state behaviour of isolated mammalian cytochrome c oxidase was examined by increasing the rate of reduction of cytochrome c. Under these conditions the enzyme's 605 (haem a), 655 (haem a3/CuB) and 830 (CuA) nm spectral features behaved as if they were at near equilibrium with cytochrome c (550 nm). This has implications for non-invasive tissue measurements using visible (550, 605 and 655 nm) and near-IR (830 nm) light. The oxidized species represented by the 655 nm band is bleached by the presence of oxygen intermediates P and F (where P is characterized by an absorbance spectrum at 607 nm relative to the oxidized enzyme and F is characterized by an absorbance spectrum at 580 nm relative to the oxidized enzyme) or by reduction of haem a3 or CuB. However, at these ambient oxygen levels (far above the enzyme Km), the populations of reduced haem a3 and the oxygen intermediates were very low (<10%). We therefore interpret 655 nm changes as reduction of the otherwise spectrally invisible CuB centre. We present a model where small anti-cooperative redox interactions occur between haem a-CuA-CuB (steady-state potential ranges: CuA, 212-258 mV; haem a, 254-281 mV; CuB, 227-272 mV). Contrary to static equilibrium measurements, in the catalytic steady state there are no high potential redox centres (>300 mV). We find that the overall reaction is correctly described by the classical model in which the Michaelis intermediate is a ferrocytochrome c-enzyme complex. However, the oxidation of ferrocytochrome c in this complex is not the sole rate-determining step. Turnover is instead dependent upon electron transfer from haem a to haem a3, but the haem a potential closely matches cytochrome c at all times.

Steady state redox levels in cytochrome oxidase: relevance for in vivo near infrared spectroscopy (NIRS).

In the visible/NIR (600-900 nm) three different redox centres are potentially detectable in vivo in mitochondrial cytochrome c oxidase: haem a (605nm), the binuclear haem a3/CuB centre (655 nm) and CuA (830 nm). In this paper we report changes in the steady state reduction of these centres following increases in the rate of electron entry into the purified enzyme complex under conditions of saturating oxygen tension. As turnover is increased all three centres becomes progressively reduced. Analysis of the steady states indicated that all three centres remained in apparent equilibrium with cytochrome c throughout the titration. The calculated redox potentials of CuA (+224 mV) and haem a (+267 mV) were consistent with previous equilibrium data. The 655 nm band was also found to be oxygen and flux sensitive. It may be a useful additional in vivo detectable chromophore. However, it titrated with an apparent redox potential of +230 mV, far from its equilibrium value (+400 mV). The implications of these results for the interpretation of non invasive measurements of mitochondrial function are discussed.

Cytochrome bd confers nitric oxide resistance to Escherichia coli.

The aerobic respiratory chain of Escherichia coli has two terminal quinol oxidases: cytochrome bo and cytochrome bd. Cytochrome bd was thought to function solely to facilitate micro-aerobic respiration. However, it has recently been shown to be overexpressed under conditions of nitric oxide (NO) stress; we show here that cytochrome bd is crucial for protecting E. coli cells from NO-induced growth inhibition by virtue of its fast NO dissociation rate.

A dynamic model of nitric oxide inhibition of mitochondrial cytochrome c oxidase.

Nitric oxide can inhibit mitochondrial cytochrome oxidase in both oxygen competitive and uncompetitive modes. A previous model described these interactions assuming equilibrium binding to the reduced and oxidised enzyme respectively (Mason, et al. Proc. Natl. Acad. Sci. U S A 103 (2006) 708-713). Here we demonstrate that the equilibrium assumption is inappropriate as it requires unfeasibly high association constants for NO to the oxidised enzyme. Instead we develop a model which explicitly includes NO binding and its enzyme-bound conversion to nitrite. Removal of the nitrite complex requires electron transfer to the binuclear centre from haem a. This revised model fits the inhibition constants at any value of substrate concentration (ferrocytochrome c or oxygen). It predicts that the inhibited steady state should be a mixture of the reduced haem nitrosyl complex and the oxidized-nitrite complex. Unlike the previous model, binding to the oxidase is always proportional to the degree of inhibition of oxygen consumption. The model is consistent with data and models from a recent paper suggesting that the primary effect of NO binding to the oxidised enzyme is to convert NO to nitrite, rather than to inhibit enzyme activity (Antunes et al. Antioxid. Redox Signal. 9 (2007) 1569-1579).

A quantitative approach to nitric oxide inhibition of terminal oxidases of the respiratory chain.

Inhibition of terminal respiratory oxidases by nitric oxide (NO) plays important physiological roles in signaling and host defense. Using a bacterial quinol oxidase and mitochondrial cytochrome c oxidase, this chapter describes simple polarographic methods to quantify the kinetic characteristics of inhibition by NO. This chapter points out the inherent pitfalls of both experimental design and data analysis and compares alternative methods. Additionally, it describes a system designed to acquire polarographic and spectral data simultaneously to permit identification of spectral intermediates under defined conditions.

Nitric oxide inhibition of respiration involves both competitive (heme) and noncompetitive (copper) binding to cytochrome c oxidase.

NO reversibly inhibits mitochondrial respiration via binding to cytochrome c oxidase (CCO). This inhibition has been proposed to be a physiological control mechanism and/or to contribute to pathophysiology. Oxygen reacts with CCO at a heme iron:copper binuclear center (a(3)/Cu(B)). Reports have variously suggested that during inhibition NO can interact with the binuclear center containing zero (fully oxidized), one (singly reduced), and two (fully reduced) additional electrons. It has also been suggested that two NO molecules can interact with the enzyme simultaneously. We used steady-state and kinetic modeling techniques to reevaluate NO inhibition of CCO. At high flux and low oxygen tensions NO interacts predominantly with the fully reduced (ferrous/cuprous) center in competition with oxygen. However, as the oxygen tension is raised (or the consumption rate is decreased) the reaction with the oxidized enzyme becomes increasingly important. There is no requirement for NO to bind to the singly reduced binuclear center. NO interacts with either ferrous heme iron or oxidized copper, but not both simultaneously. The affinity (K(D)) of NO for the oxygen-binding ferrous heme site is 0.2 nM. The noncompetitive interaction with oxidized copper results in oxidation of NO to nitrite and behaves kinetically as if it had an apparent affinity of 28 nM; at low levels of NO, significant binding to copper can occur without appreciable enzyme inhibition. The combination of competitive (heme) and noncompetitive (copper) modes of binding enables NO to interact with mitochondria across the full in vivo dynamic range of oxygen tension and consumption rates.

The heme domain of cellobiose oxidoreductase: a one-electron reducing system.

Phanerochaete chrysosporium cellobiose oxidoreductase (CBOR) comprises two redox domains, one containing flavin adenine dinucleotide (FAD) and the other protoheme. It reduces both two-electron acceptors, including molecular oxygen, and one-electron acceptors, including transition metal complexes and cytochrome c. If the latter reacts with the flavin, the reduced heme b acts merely as a redox buffer, but if with the b heme, enzyme action involves a true electron transfer chain. Intact CBOR fully reduced with cellobiose, CBOR partially reduced by ascorbate, and isolated ascorbate-reduced heme domain, all transfer electrons at similar rates to cytochrome c. Reduction of cationic one-electron acceptors via the heme group supports an electron transfer chain model. Analogous reactions with natural one-electron acceptors can promote Fenton chemistry, which may explain evolutionary retention of the heme domain and the enzyme's unique character among secreted sugar dehydrogenases.

Oxygen reduction by cellobiose oxidoreductase: the role of the haem group.

We have used optical and electron paramagnetic spectroscopy to study the flavohaem enzyme cellobiose oxidoreductase (CBOR) from Phanerochaete chrysosporium. We have examined redox cycles of the enzyme in which the oxidation of cellobiose to cellobionolactone is coupled to the reduction of oxygen. During turnover flavin can reduce oxygen with one electron to produce superoxide or two electrons to produce hydrogen peroxide. Addition of superoxide dismutase significantly extended the time courses of these cycles, slowing the re-oxidation rate of both cofactors. Addition of catalase also affected the haem time course, but to a lesser extent. Experiments in which superoxide was generated in the reaction mixture showed that this radical greatly enhanced the rate of haem re-oxidation. From these results we propose a mechanism in which reactive oxygen species generation by CBOR flavin subsequently re-oxidises CBOR haem. We discuss this mechanism in relationship to the biological function of this enzyme, namely lignocellulose degradation.