Importance of adapted digestion conditions to simulate in vitro lipid digestion of broilers in different life stages.

In vitro digestion studies demonstrate large potential to gain more and quicker insights into the underlying mechanisms of feed additives, allowing the optimization of feed design. Unfortunately, current in vitro digestion models relevant for broiler chickens lack sufficient description in terms of protocols and standardisation used. Furthermore, no distinction is made between the different life phases of these animals (starter, grower, and finisher). Hence, our research aimed to establish adapted in vitro digestion conditions, corresponding to the 3 life phases in broilers, with specific focus on lipid digestion. The effect of 3 different bile salt concentrations of 2, 10, and 20 mM, and 3 different lipase activities of 5, 20, and 100 U/mL, on in vitro lipid digestion kinetics were evaluated using a full factorial design. These values were selected to represent starter, grower, and finisher birds, respectively. Our findings showed that the extent of lipid digestion was mainly influenced by lipase activity. The rate of lipid digestion was affected by an interplay between bile salt concentration and lipase activity, due to possible lipase inhibition at certain bile salt concentrations. Overall, this work resulted in 3 in vitro lipid digestion models representative for starter, grower, and finisher birds. In conclusion, this research showed the impact of adapted in vitro digestion conditions on lipid digestion kinetics and thus the need for these conditions relevant for each life phase of broilers.

The heme pocket of the globin domain of the globin-coupled sensor of Geobacter sulfurreducens--an EPR study.

The globin-coupled sensor (GCS) of Geobacter sulfurreducens is unique amongst GCSs in that its signalling domain is a transmembrane domain with yet unknown function. In the present work we use X-band continuous-wave and pulsed electron paramagnetic resonance (EPR) to investigate the ferric form of the globin domain of the G. sulfurreducens GCS (GsGCS(162)) at pH 8.5. This form shows a unique bis-histidine coordination of the heme with the F8His and E11His. In contrast with previous crystal structure data, where three conformers of the heme structure were identified, ferric GsGCS(162) assumes only one conformation in frozen solution. The EPR data of ferric GsGCS162 are compared in detail with those of other bis-histidine coordinated globins, including other GCS systems.

HisE11 and HisF8 provide bis-histidyl heme hexa-coordination in the globin domain of Geobacter sulfurreducens globin-coupled sensor.

Among heme-based sensors, recent phylogenomic and sequence analyses have identified 34 globin coupled sensors (GCS), to which an aerotactic or gene-regulating function has been tentatively ascribed. Here, the structural and biochemical characterization of the globin domain of the GCS from Geobacter sulfurreducens (GsGCS(162)) is reported. A combination of X-ray crystallography (crystal structure at 1.5 A resolution), UV-vis and resonance Raman spectroscopy reveals the ferric GsGCS(162) as an example of bis-histidyl hexa-coordinated GCS. In contrast to the known hexa-coordinated globins, the distal heme-coordination in ferric GsGCS(162) is provided by a His residue unexpectedly located at the E11 topological site. Furthermore, UV-vis and resonance Raman spectroscopy indicated that ferrous deoxygenated GsGCS(162) is a penta-/hexa-coordinated mixture, and the heme hexa-to-penta-coordination transition does not represent a rate-limiting step for carbonylation kinetics. Lastly, electron paramagnetic resonance indicates that ferrous nitrosylated GsGCS(162) is a penta-coordinated species, where the proximal HisF8-Fe bond is severed.

Archaeal protoglobin structure indicates new ligand diffusion paths and modulation of haem-reactivity.

The structural adaptability of the globin fold has been highlighted by the recent discovery of the 2-on-2 haemoglobins, of neuroglobin and cytoglobin. Protoglobin from Methanosarcina acetivorans C2A-a strictly anaerobic methanogenic Archaea-is, to the best of our knowledge, the latest entry adding new variability and functional complexity to the haemoglobin (Hb) superfamily. Here, we report the 1.3 A crystal structure of oxygenated M. acetivorans protoglobin, together with the first insight into its ligand-binding properties. We show that, contrary to all known globins, protoglobin-specific loops and an amino-terminal extension completely bury the haem within the protein matrix. Access of O(2), CO and NO to the haem is granted by the protoglobin-specific apolar tunnels reaching the haem distal site from locations at the B/G and B/E helix interfaces. Functionally, M. acetivorans dimeric protoglobin shows a selectivity ratio for O(2)/CO binding to the haem that favours O(2) ligation and anticooperativity in ligand binding. Both properties are exceptional within the Hb superfamily.

Characterization of a globin-coupled oxygen sensor with a gene-regulating function.

Globin-coupled sensors (GCSs) are multiple-domain transducers, consisting of a regulatory globin-like heme-binding domain and a linked transducer domain(s). GCSs have been described in both Archaea and bacteria. They are generally assumed to bind O(2) (and perhaps other gaseous ligands) and to transmit a conformational change signal through the transducer domain in response to fluctuating O(2) levels. In this study, the heme-binding domain, AvGReg178, and the full protein, AvGReg of the Azotobacter vinelandii GCS, were cloned, expressed, and purified. After purification, the heme iron of AvGReg178 was found to bind O(2). This form was stable over many hours. In contrast, the predominant presence of a bis-histidine coordinate heme in ferric AvGReg was revealed. Differences in the heme pocket structure were also observed for the deoxygenated ferrous state of these proteins. The spectra showed that the deoxygenated ferrous derivatives of AvGReg178 and AvGReg are characterized by a penta-coordinate and hexa-coordinate heme iron, respectively. O(2) binding isotherms indicate that AvGReg178 and AvGReg show a high affinity for O(2) with P(50) values at 20 degrees C of 0.04 and 0.15 torr, respectively. Kinetics of CO binding indicate that AvGReg178 carbonylation conforms to a monophasic process, comparable with that of myoglobin, whereas AvGReg carbonylation conforms to a three-phasic reaction, as observed for several proteins with bis-histidine heme iron coordination. Besides sensing ligands, in vitro data suggest that AvGReg(178) may have a role in O(2)-mediated NO-detoxification, yielding metAvGReg(178) and nitrate.

Anoxia or oxygen and glucose deprivation in SH-SY5Y cells: a step closer to the unraveling of neuroglobin and cytoglobin functions.

Several studies support the hypothesis that neuroglobin and cytoglobin play a protective role against cell death when cellular oxygen supply is critical. Although the underlying molecular mechanisms are unknown, previous reports suggest that this protection can be realised by the fact that they act as ROS scavengers. In this study, expression of neuroglobin and cytoglobin was evaluated in a human neuroblastoma cell line (SH-SY5Y) under conditions of anoxia or oxygen and glucose deprivation (OGD). The cells could survive prolonged anoxia without significant loss of viability. They became anoxia sensitive when deprived of glucose. OGD induced significant cell death after 16 h resulting in 54% dead cells after 32 h. Necrosis was the main process involved in OGD-induced cell death. After reoxygenation, apoptotic neurons became more abundant. Real-time quantitative PCR and Western blotting revealed that neuroglobin and cytoglobin were upregulated, the former under OGD and the latter under anoxic conditions. Under OGD, cell survival was significantly reduced after inhibiting cytoglobin expression by transfection with antisense ODN. Moreover, cell survival was significantly enhanced by neuroglobin or cytoglobin overexpression. When neuroglobin or cytoglobin protein expression increased or decreased, the H(2)O(2) level was found to be lower or higher, respectively. We conclude that neuroglobin or cytoglobin act as ROS scavengers under ischemic conditions.

Neuroglobin and cytoglobin expression in mice. Evidence for a correlation with reactive oxygen species scavenging.

Although essentially unknown, several functions are hypothesized for neuroglobin and cytoglobin, two new members of the globin family. In this article, we try to shed more light on their possible roles in hypoxia and detoxification of reactive oxygen species in vivo. The relative transcriptional changes of neuroglobin and cytoglobin in a situation of chronic hypoxia in mice were examined using real-time quantitative PCR. The kinetics of the hypoxic expression of neuroglobin (brain, eyes) and cytoglobin (brain, eyes, liver, heart, skeletal muscle) is organ-specific. Moreover, reactive oxygen species production is higher in liver than in the other tissues. In eyes, the negative correlation, after reoxygenation, between neuroglobin protein level and H(2)O(2) concentration is a first proof of a reactive oxygen species-scavenging function for neuroglobin. In addition, apoptotic cell death after hypoxia is for the first time demonstrated in heart and liver.

Neuroglobin and cytoglobin overexpression protects human SH-SY5Y neuroblastoma cells against oxidative stress-induced cell death.

Although reactive oxygen species (ROS) at physiological concentrations are required for normal cell function, excessive production of ROS is detrimental to cells. Neuroglobin and cytoglobin are two globins, whose functions are still a matter of debate. A potential role in the detoxification of ROS is suggested. The influence of neuroglobin and cytoglobin on cell death after oxidative stress in human neuroblastoma SH-SY5Y cells was evaluated. Exposure of SH-SY5Y cells to paraquat or H(2)O(2) resulted in a concentration- and time-dependent induction of apoptotic and necrotic cell death. H(2)O(2) was 16 times more potent to induce cell death as compared to paraquat. SH-SY5Y cells transfected with plasmid DNA containing the neuroglobin or cytoglobin sequence showed enhanced survival after exposure to 300 microM H(2)O(2) for 24h as compared to untransfected controls. This finding suggests that neuroglobin and cytoglobin protect SH-SY5Y cells against oxidative stress-induced cell death.