Probing protein-chromophore interactions in Cph1 phytochrome by mutagenesis

We have investigated mutants of phytochrome Cph1 from the cyanobacterium Synechocystis PCC6803 in order to study chromophore-protein interactions. Cph1Delta2, the 514-residue N-terminal sensor module produced as a recombinant His6-tagged apoprotein in Escherichia coli, autoassembles in vitro to form a holoprotein photochemically indistinguishable from the full-length product. We generated 12 site-directed mutants of Cph1Delta2, focusing on conserved residues which might be involved in chromophore-protein autoassembly and photoconversion. Folding, phycocyanobilin-binding and Pr-->Pfr photoconversion were analysed using CD and UV-visible spectroscopy. MALDI-TOF-MS confirmed C259 as the chromophore attachment site. C259L is unable to attach the chromophore covalently but still autoassembles to form a red-shifted photochromic holoprotein. H260Q shows UV-visible properties similar to the wild-type at pH 7.0 but both Pr and Pfr (reversibly) bleach at pH 9.0, indicating that the imidazole side chain buffers chromophore protonation. Mutations at E189 disturbed folding but the residue is not essential for chromophore-protein autoassembly. In D207A, whereas red irradiation of the ground state leads to bleaching of the red Pr band as in the wild-type, a Pfr-like peak does not arise, implicating D207 as a proton donor for a deprotonated intermediate prior to Pfr. UV-Vis spectra of both H260Q under alkaline conditions and D207A point to a particular significance of protonation in the Pfr state, possibly implying proton migration (release and re-uptake) during Pr-->Pfr photoconversion. The findings are discussed in relation to the recently published 3D structure of a bacteriophytochrome fragment.     

ACE inhibition and atherogenesis

Recent clinical studies such as HOPE, SECURE, and APRES show that angiotensin-converting enzyme (ACE) inhibitors like ramipril improve the prognosis of patients with a high risk of atherothrombotic cardiovascular events. Atherosclerosis, as a chronic inflammatory condition of the vascular system, can turn into an acute clinical event through the rupture of a vulnerable atherosclerotic plaque followed by thrombosis. ACE inhibition has a beneficial effect on the atherogenic setting and on fibrinolysis. Endothelial dysfunction is the end of a common process in which cardiovascular risk factors contribute to inflammation and atherogenesis. By inhibiting the formation of angiotensin II, ACE inhibitors prevent any damaging effects on endothelial function, vascular smooth muscle cells, and inflammatory vascular processes. An increase in the release of NO under ACE inhibition has a protective effect. Local renin-angiotensin systems in the tissue are involved in the inflammatory processes in the atherosclerotic plaque. Circulating ACE-containing monocytes, which adhere to endothelial cell lesions, differentiate within the vascular wall to ACE-containing macrophages or foam cells with increased local synthesis of ACE and angiotensin II. Within the vascular wall, angiotensin II decisively contributes to the instability of the plaque by stimulating growth factors, adhesion molecules, chemotactic proteins, cytokines, oxidized LDL, and matrix metalloproteinases. Suppression of the increased ACE activity within the plaque can lead to the stabilization and deactivation of the plaque by reducing inflammation in the vascular wall, thus lessening the risk of rupture and thrombosis and the resultant acute clinical cardiovascular events. The remarkable improvement in the long-term prognosis of atherosclerotic patients with increased cardiovascular risk might be the clinical result of the contribution made by ACE inhibition in the vascular wall.     

Evaluation of two commercial methods for the detection of Listeria sp. and Listeria monocytogenes in a chicken nugget processing plant

This study measures the detection performances of two rapid test systems (Listeria Rapid Test Clearview and Bax system) for the screening of Listeria sp. and Listeria monocytogenes, respectively. A total of 413 samples from different sources (product from (i) different stages of processing, (ii) different environments, and (iii) different food handlers), collected from a chicken nugget processing plant, were analysed by both rapid methods and a cultural method consisting of pre-enrichment, enrichment, and isolation onto selective agars (PALCAM, LPM, and HCLA). Overall, results showed an excellent correlation between data obtained using Clearview and the cultural method, with Clearview presenting an efficiency of 99%. Bax showed a lower correlation using the cultural method, with an efficiency of 71.1%. The type of sample did not affect the efficiency of Clearview, which varied from 98.1% for product samples to 100% for environmental and food handler samples, while for Bax it had a marked influence. Efficiency of Bax varied from as high as 100% for food handlers to 37.9% for product samples.     

Direct immobilization of gangliosides onto gold-carboxymethyldextran sensor surfaces by hydrophobic interaction: applications to antibody characterization

We describe a novel immobilization technique to investigate interactions between immobilized gangliosides (GD3, GM1, and GM2) and their respective antibodies, antibody fragments, or binding partners using an optical biosensor. Immobilization was performed by direct injection onto a carboxymethyldextran sensor chip and did not require derivatization of the sensor surface or the ganglioside. The ganglioside appeared to bind to the sensor surface by hydrophobic interaction, leaving the carbohydrate epitope available for antibody or, in the case of GM1, cholera toxin binding. The carboxyl group of the dextran chains on the sensor surface did not appear to be involved in the immobilization as evidenced by equivalent levels of immobilization following conversion of the carboxyl groups into acyl amino esters, but rather the dextran layer provided a hydrophilic coverage of the sensor chip which was essential to prevent nonspecific binding. This technique gave better reactivity and specificity for anti- ganglioside monoclonal antibodies (anti-GD3: KM871, KM641, R24; and anti-GM2: KM966) than immobilization by hydrophobic interaction onto a gold sensor surface or photoactivated cross-linking onto carboxymethydextran. This rapid immobilization procedure has facilitated detailed kinetic analysis of ganglioside/antibody interactions, with the surface remaining viable for a large number of cycles (>125). Kinetic constants were determined from the biosensor data using linear regression, nonlinear least squares and equilibrium analysis. The values of kd, ka, and KAobtained by nonlinear analysis (KAKM871 = 1.05, KM641 = 1.66, R24 = 0.14, and KM966 = 0.65 x 10(7) M- 1) were essentially independent of concentration and showed good agreement with data obtained by other analytical methods.      

The prostaglandins system and insulin release. Studies with the isolated perfused rat pancreas.

Using the isolated perfused rat pancreas PGE2 (1 MUM and 10 muM) had no effect on basal or glucose (10 and 20 mM)-induced insulin release (IR). PGF2 alpha stimulated basal IR at 1 muM and inhibited IR at 10 muM. The glucose-induced IR was unaffected by this PG. Furosemide (5 and 10 mM) led to a monophastic IR at low glucose (glu) and to a potentiation of IR at high glu. Only high indomethacin (Indo) (50 microgram/ml) inhibited glu-induced IR. The stimulatory effect of furosemide on IR could not be inhibited by indomethacin. However mepacrine (0.1 mM) abolished the furosemide effect. Also glu-induced IR was inhibited by mepacrine. Acetylsalicylic acid (30 mg/100 ml) had no significant influence on glu-induced IR. These findings provide evidence that phospholipase activation rather than increased PG synthesis might primarily be involved in the secretory process of insulin.     

Neuroendocrine and behavioral response to social confrontation: residents versus intruders, active versus passive coping styles

We investigated in the present study the neuroendocrine correlates in intruder and resident rats of a social confrontation. Adult male Wistar rats (intruders) were introduced into the home cage of a well-trained resident to induce characteristic agonistic interactions including physical attacks prior to separation by a wire mesh. The hypothalamic-pituitary-adrenal (HPA) axis activity and the intrahypothalamic release of arginine vasopressin (AVP) were monitored via chronically implanted jugular venous catheters and microdialysis probes aimed at the hypothalamic paraventricular nucleus (PVN), respectively. Based on the behavioral data collected during the 30-min confrontation, intruders and residents were additionally classified into two different subgroups: intruders which showed almost no freezing behavior (active copers) versus those showing pronounced freezing behavior (passive copers) and residents which were either predominantly aggressive or non-aggressive. The neuroendocrine data show that social confrontation caused a significantly increased secretion of the adrenocorticotropic hormone (ACTH) into plasma in both intruder subgroups, independently of their coping strategy. In contrast, plasma ACTH in residents was increased in response to social confrontation in non-aggressive animals only, whereas aggressive residents failed to mount an ACTH response. Interestingly, plasma AVP decreased in response to social confrontation in active intruders. As measured in microdialysates, the two groups of residents and passive intruders failed to show significant changes of intra-PVN release of AVP. In contrast, an increased release of this neuropeptide within the PVN could be monitored for active intruders. The data of the present study suggest that the different interpretation of an aversive encounter results in differences in the neuroendocrine response and intrahypothalamic vasopressinergic signaling in intruders versus residents.     

The extracellular domains of ErbB3 retain high ligand binding affinity at endosome pH and in the locked conformation

The extracellular, ligand binding regions of ErbB receptors consist of four domains that can assume at least two alternative conformations, extended and locked. The locked conformation, observed in several crystal structures, is held together by a noncovalent intramolecular tether and is incompatible with current models for receptor dimerization and ligand activation. Based on structures of ligand-receptor complexes in the extended conformation, the high affinity ligand binding pocket between domains I and III is disrupted in the locked conformation. Therefore the biological role of the locked conformation is not clear. To address the impact of the locked conformation on ligand binding, we compared extracellular domains of wild-type ErbB3, mutant domains in a constitutively locked or extended conformation and partial extracellular domain constructs. We found that the constitutively locked receptor domains and truncated constructs carrying only domains I-II or III-IV strongly bind ligand, albeit with reduced affinity compared to wild-type receptor. This suggests that the locked conformation cannot be discounted for ligand binding. The significant binding by both partial interfaces in domains I and III also suggests that "partial bivalency" may be the reason for the low nanomolar and high picomolar binding observed for ErbB3 in the respective "low" and high affinity states. In contrast to EGFR (ErbB1), ErbB3 retains high ligand binding affinity at an endosome-comparable pH in both the extended and locked conformations. Ligand affinity for the locked conformation even improves at low pH. For ErbB3, the contribution of domain I to ligand binding is strong and increases at low pH while its contribution is thought to be minimal for EGFR, regardless of pH. This shift in domain contribution and pH dependency provides a mechanistic explanation for some of the divergent properties of EGFR and ErbB3.