Copper(II) complexes with an avian prion N-terminal region and their potential SOD-like activity

Potentiometric and spectroscopic (UV-Vis, CD and EPR) studies were carried out on copper(II) complexes with chicken prion protein N-terminal fragments, Ac-(PHNPGY)₄-NH₂, and the mutated residue, Ac-(PHNPGF)₄-NH₂, to assess the role of tyrosine in the copper coordination. Both thermodynamic and spectroscopic results indicate that chicken prion fragments are not able to bind more than two copper ions and only with the involvement of side chain tyrosine groups. The prevailing complex shows one copper ion bound to four imidazole nitrogen atoms in the 1:1 metal to ligand ratio systems. The superoxide dismutase (SOD)-like activity of copper(II) complexes with the avian peptides and mammal analogue, Ac-(PHGGGWGQ)₄-NH₂, was also investigated by means of Pulse radiolysis. The copper(II) complexes with avian peptides do not display SOD-like activity, while very low activity has been detected for the copper(II) complexes with mammalian tetraoctarepeat.     

A dramatic conformational rearrangement is necessary for the activation of DNR from Pseudomonas aeruginosa. Crystal structure of wild‐type DNR

The opportunistic pathogen Pseudomonas aeruginosa can grow in low oxygen, because it is capable of anaerobic respiration using nitrate as a terminal electron acceptor (denitrification). An intermediate of the denitrification pathway is nitric oxide, a compound that may become cytotoxic at high concentration. The intracellular levels of nitric oxide are tightly controlled by regulating the expression of the enzymes responsible for its synthesis and degradation (nitrite and nitric oxide reductases). In this article, we present the crystallographic structure of the wild‐type dissimilative nitrate respiration regulator (DNR), a master regulator controlling expression of the denitrification machinery and a putative target for new therapeutic strategies. Comparison with other structures among the CRP‐FNR class of regulators reveals that DNR has crystallized in a conformation that has never been observed before. In particular, the sensing domain of DNR has undergone a rotation of more than 50° with respect to the other structures. This suggests that DNR may undergo an unexpected and very large conformational rearrangement on activation. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Inclusion of ionization states of ligands in affinity calculations

When estimating binding affinities of a ligand, which can exists in multiple forms, for a target molecule, one must consider all possible competing equilibria. Here, a method is presented that estimates the contribution of the protonation equilibria of a ligand in solution to the measured or calculated binding affinity. The method yields a correction to binding constants that are based on the total concentration of inhibitor (the sum of all ionized forms of the inhibitor in solution) to account for the complexed form of the inhibitor only. The method is applied to the calculation of the difference in binding affinity of two inhibitors, 2‐phosphoglycolate (PGA) and its phoshonate analog 3‐phosphonopropionate (3PP), for the glycolytic enzyme triosephosphate isomerase. Both inhibitors have three titrating sites and exist in solution as a mixture of different forms. In this case the form that actually binds to the enzyme is present at relative low concentrations. The contributions of the alternative forms to the difference in binding energies is estimated by means of molecular dynamics simulations and corrections. The inhibitors undergo a pK_a shift upon binding that is estimated by ab initio calculations. An interesting finding is that the affinity difference of the two inhibitors is not due to different interactions in the active site of the enzyme, but rather due to the difference in the solvation properties of the inhibitors. Protein 2009. © 2008 Wiley‐Liss, Inc.     

Characterization of juvenile maritime pine (Pinus pinaster Ait.) ectomycorrhizal fungal community using morphotyping, direct sequencing and fruitbodies sampling

Using ectomycorrhizal root tip morphotyping (anatomical and morphological identification), molecular analysis (internal transcribed spacer region amplification and sequencing), and fruitbody sampling, we assessed diversity and composition of the ectomycorrhizal fungal community colonizing juvenile Pinus pinaster Ait. under natural conditions in NW Spain. Overall, we found 15 Basidiomycetes and two Ascomycetes. Members of the family Thelephoraceae represented up to 59.4% of the samples. The most frequent species was Tomentella sublilacina followed by Thelephora terrestris, Russula drimeia, Suillus bovinus, and Paxillus involutus, while the less frequent were Pseudotomentella tristis, Lactarius subdulcis, Russula ochroleuca, and Entoloma conferendum. From October 2007 to June 2008, we sampled 208 sporocarps belonging to seven genera and nine species: Thelephora terrestris, Paxillus involutus, Suillus bovinus, Xerocomus badius, Scleroderma verrucosum, Amanita gemmata, A. rubescens, Amanita sp., and Russula sp. The species belonging to the genus Amanita, X. badius and S. verrucosum were not found on root samples. By comparing our results with a bibliographic review of papers published from 1922 to 2006, we found five genera and six species which have not been previously reported in symbiosis with P. pinaster. This is the first time that the diversity of the ectomycorrhizal fungal community associated with P. pinaster was investigated using molecular techniques. Considering that only 38% of the genera found by sequencing were found as fruitbodies, we conclude that integrating morphotyping and sporocarps surveys with molecular analysis of ectomycorrhizas is important to documenting the ectomycorrhizal fungus community. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The application of a plug-flow reactor to measure the biodegradable dissolved organic carbon (BDOC) in seawater

Most of the ambient dissolved organic carbon (DOC) is refractory to microbial degradation; bacteria can consume a minor but variable part of the DOC pool over periods of hours and days. It is important to increase our knowledge of the dynamics of the biodegradable fraction of DOC (BDOC) to understand the global carbon budget.Several methods for determining BDOC have been developed; however, the problem of most of them is the time (days/weeks) required for the colonization and/or determination.In this paper, we describe the application to seawater of a plug-flow bioreactor to measure BDOC within 3–4 h. The bioreactor was built following Søndergaard and Worm [Søndergaard, M., Worm, J., 2001. Measurement of biodegradable dissolved organic carbon (BDOC) in lake water with a bioreactor. Water Res. 35, 2505-2513.] protocols for the measurement of BDOC in lake water. We analyzed BDOC on samples collected in the Gulf of Trieste during autumn–winter and summer 2003–2004. BDOC concentrations varied from 8 to 24 μM and represented from 10.3% to 25.5% of the total DOC. To evaluate the effectiveness of this method, we compared bioreactor BDOC measurement with data obtained from batch cultures. The results indicate that BDOC in coastal seawater can be measured rapidly and reliably with this bioreactor.     

Synthesis,cannabinoid receptor affinity,molecular modeling studies and in vivo pharmacological evaluation of new substituted 1-aryl-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxamides. 2. Effect of the 3-carboxamide substituent on the affinity and selectivity profile

New substituted 1-aryl-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxamides were synthesized by replacing the 2,4-dichlorobenzyl and cyclohexyl moieties at the 3-carboxamide nitrogen of the previously reported CB₁ receptor antagonists/inverse agonists 4 and 5. Several ligands showed potent affinity for the hCB₁ receptor, with K_i concentrations comparable to the reference compounds 1, 4 and 5, and exhibited CB₁ selectivity comparable to 1 and 2. Docking experiments and molecular dynamics (MD) simulations explained the potent hCB₁ binding affinity of compounds 31 and 37. According to our previous studies, 31 and 37 formed a H-bond with K3.28(192), which accounted for the high affinity for the receptor inactive state and the inverse agonist activity. The finding of inhibition of food intake following their acute administration to rats, supported the concept that the CB₁ selective compounds 4 and 52 act as antagonists/inverse agonists.     

α-Synuclein and Its A30P Mutant Affect Actin Cytoskeletal Structure and Dynamics

The function of α-synuclein, a soluble protein abundant in the brain and concentrated at presynaptic terminals, is still undefined. Yet, α-synuclein overexpression and the expression of its A30P mutant are associated with familial Parkinson''s disease. Working in cell-free conditions, in two cell lines as well as in primary neurons we demonstrate that α-synuclein and its A30P mutant have different effects on actin polymerization. Wild-type α-synuclein binds actin, slows down its polymerization and accelerates its depolymerization, probably by monomer sequestration; A30P mutant α-synuclein increases the rate of actin polymerization and disrupts the cytoskeleton during reassembly of actin filaments. Consequently, in cells expressing mutant α-synuclein, cytoskeleton-dependent processes, such as cell migration, are inhibited, while exo- and endocytic traffic is altered. In hippocampal neurons from mice carrying a deletion of the α-synuclein gene, electroporation of wild-type α-synuclein increases actin instability during remodeling, with growth of lamellipodia-like structures and apparent cell enlargement, whereas A30P α-synuclein induces discrete actin-rich foci during cytoskeleton reassembly. In conclusion, α-synuclein appears to play a major role in actin cytoskeletal dynamics and various aspects of microfilament function. Actin cytoskeletal disruption induced by the A30P mutant might alter various cellular processes and thereby play a role in the pathogenesis of neurodegeneration.     

Low-Vision Rehabilitation by Means of MP-1 Biofeedback Examination in Patients with Different Macular Diseases: A Pilot Study

Macular disease is one of the main causes of visual impairment. We studied the efficacy of low-vision rehabilitation by means of MP-1 biofeedback examination in patients with different macular disease. Five patients were enrolled (3 female and 2 male, mean age 53.8 years) and a total of 9 eyes was examined: 2 eyes with vitelliform dystrophy, 1 with a post-traumatic macular scar, 2 with Stargardt disease, 2 with myopic macular degeneration, 2 with cone dystrophy. All the patients underwent the following tests: visual acuity, reading speed, fixation test, MP-1 microperimetry. Low-vision rehabilitation, which lasted 10 weeks, consisted of 10 training sessions of 10 min for each eye, performed once a week using the MP-1 biofeedback examination. Statistical analysis was performed using Student’s t-test. p values less than 0.05 were considered statistically significant. After training all patients displayed an improvement in visual acuity, fixation behaviour, retinal sensitivity and reading speed. Fixation behaviour within the 2° diameter circle improved and was statistically significant for reading speed (p = 0.01). Reading speed improved from a mean value of 64.3 to 92 words/min. Our results show that audio feedback can, by increasing attentional modulation, help the brain to fix the final preferred retinal locus. Audio feedback facilitates stimuli transmission between intraretinal neurons as well as between the retina and brain, which is where the highest level of stimuli processing occurs, thereby probably supporting a “remapping phenomenon”.