Hyperfine correlation spectroscopy and electron spin echo envelope modulation spectroscopy study of the two coexisting forms of the hemeprotein cytochrome c6 from Anabaena Pcc7119

Oxidized cytochrome c₆ from Anabaena PCC 7119 was studied by electron spin echo envelope modulation spectroscopy. Hyperfine couplings of the unpaired electron with several nuclei were detected, in particular those of the nitrogens bound to the iron atom. Combining the experimental information here presented and previous continuous wave-electron paramagnetic resonance and electron nuclear double resonance results, some details on the electronic structure of the heme center in the protein are obtained. These results are discussed on the basis of a molecular model that considers one unpaired electron localized mainly in the iron d orbitals and propagation of the spin density within the heme center via spin polarization of the nitrogen σ-orbitals. The coexistence of two heme forms at physiological pH values in this c-type cytochrome is also discussed taking into account the experimental evidence.     

Evaluation of cardiovascular risk and oxidative stress parameters in hypercholesterolemic subjects on a standard healthy diet including low-fat milk enriched with plant sterols

A healthy diet and plant sterols (PS) are recommended for reducing low-density lipoprotein (LDL) cholesterol and, subsequently, the risk of premature cardiovascular disease. PS mediate a decrease in fat-soluble vitamin concentration, which can lead to a general impairment of antioxidative defenses and an increase in oxidative stress. Thus, we evaluated the effects of a healthy diet, including PS-enriched low-fat milk, on cardiovascular risk and oxidative stress parameters in hypercholesterolemic subjects.This was a randomized parallel trial employing 40 subjects and consisting of two 3-month intervention phases. After 3 months on a standard healthy diet, subjects were divided into two intervention groups: a diet group and a diet+PS group (2 g/day). Lipid profile, apolipoproteins, high-sensitivity C-reactive protein and oxidative stress parameters were analyzed. Diet significantly reduced total and LDL cholesterol (4.0% and 4.7%, respectively), produced an increase in the level of β-carotene (23%) and improved the antioxidant capacity of LDL cholesterol particles (4.6%). PS induced a significant decrease in total cholesterol (6.4%), LDL (9.9%) and the apolipoprotein B100/apolipoprotein A1 ratio (4.9%), but led to a decrease in cryptoxanthin level (29%) without any change being observed in the antioxidant capacity of LDL cholesterol particles, total antioxidant status or lipid peroxidation. After 3 months, we observed the positive effect of including a PS supplement in dietary measures, as the lipoprotein-mediated risk of cardiovascular disease was reduced. Despite a decrease in the concentration of cryptoxanthin, no evidence of a global impairment of antioxidative defenses or an enhancement of oxidative stress parameters was found.     

Lotus endemic to the Canary Islands are nodulated by diverse and novel rhizobial species and symbiotypes

Genetic and symbiotic characterization of 34 isolates from several Lotus species endemic to the Canary Islands showed extraordinary diversity, with bacteria belonging to different species of the genera Mesorhizobium (17 isolates), Sinorhizobium (12 isolates) and Rhizobium/Agrobacterium (5 isolates). In a previous report, we showed that the Sinorhizobium isolates mostly belonged to S. meliloti. Here, we focused on the remaining isolates. The Lotus mesorhizobial strains were distributed in the rrs tree within six poorly resolved branches. Partial sequences from atpD and recA genes produced much better resolved phylogenies that were, with some exceptions, congruent with the ribosomal phylogeny. Thus, up to six different mesorhizobial species were detected, which matched with or were sister species of M. ciceri, M. alhagi, M. plurifarium or M. caraganae, and two represented new lineages that did not correspond to any of the currently recognized species. Neither M. loti nor Bradyrhizobium sp. (Lotus), recognized as the typical Lotus-symbionts, were identified among the Canarian Lotus isolates, although their nodulation genes were closely related to M. loti. However, several subbranches of mesorhizobia nodulating Lotus spp. could be differentiated in a nodC tree, with the isolates from the islands distributed in two of them (A1 and A3). Subbranch A1 included reference strains of M. loti and a group of isolates with a host range compatible with biovar loti, whereas A3 represented a more divergent exclusive subbranch of isolates with a host range almost restricted to endemic Lotus and it could represent a new biovar among the Lotus rhizobia.     

An enzyme-linked immunosorbent poly(ADP-ribose) polymerase biomarker assay for clinical trials of PARP inhibitors

Many established cancer therapies involve DNA-damaging chemotherapy or radiotherapy. The DNA repair capacity of the tumor represents a common mechanism used by cancer cells to survive DNA-damaging therapy. Poly(ADP-ribose) polymerase (PARP) is a nuclear enzyme that is activated by DNA damage and has critical roles in DNA repair. Inhibition of PARP potentiates the activity of DNA-damaging agents such as temozolomide, topoisomerase inhibitors and radiation in both in vitro and in vivo preclinical models. Recently, several PARP inhibitors have entered clinical trials either as single agents or in combination with DNA-damaging chemotherapy. Because PARP inhibitors are not cytotoxic, a biomarker assay is useful to guide the selection of an optimal biological dose. We set out to develop an assay that enables us to detect 50% PAR reduction in human tumors with 80% power in a single-plate assay while assuring no more than a 10% false-positive rate. We have developed and optimized an enzyme-linked immunosorbent assay (ELISA) to measure PARP activity that meets the above-mentioned criterion. This robust assay is able to detect PAR levels of 30-2000 pg/ml in both tumor and peripheral blood monocyte samples. In a B16F10 mouse syngeneic tumor model, PARP inhibitor ABT-888 potentiates the effect of temozolomide in suppressing tumor growth, and PARP activity is greatly reduced by ABT-888 at efficacious doses. In summary, the ELISA assay described here is suitable for biomarker studies in clinical trials of PARP inhibitors.     

Novel erythropoietin-based therapeutic candidates with extra N-glycan sites that block hematopoiesis but preserve neuroplasticity

Neurological disorders affect millions of people causing behavior-cognitive disabilities. Nowadays they have no effective treatment. Human erythropoietin (hEPO) has been clinically used because of its neurotrophic and cytoprotective properties. However, the erythropoietic activity (EA) should be considered as a side effect. Some analogs like non-sialylated EPO, carbamylated EPO, or EPO peptides have been developed showing different weaknesses: erythropoiesis preservation, low stability, potential immunogenicity, or fast clearance. Herein, we used a novel strategy that blocks the EA but preserves hEPO neurobiological actions. N-glycoengineering was accomplished to add a new glycosylation site within the hEPO sequence responsible for its EA. hEPO-derivatives were produced by CHO.K1 cells, affinity-purified and functionally analyzed studying their in vitro and in vivo EA, their in vitro neuronal plasticity in hippocampal neurons and their neuroprotective action by rescuing hippocampal neurons from apoptosis. Muteins Mut 45_47 (K45 > N45 + N47 > T47), Mut 104 (S104 > N104), and Mut 151_153 (G151 > N151 + K153 > T153) lost their EA but preserved their neuroprotection activity and enhanced neuroplasticity more efficiently than hEPO. Interestingly, Mut 45_47 resulted in a promising candidate to explore as neurotherapeutic considering not only its biopotency but also its pharmacokinetic potential due to the hyperglycosylation.     

The Spanish translation and cultural adaptation of the Diagnostic Interview Schedule for Children (DISC) in Puerto Rico

This article illustrates a comprehensive cross-cultural adaptation model used to translate into Spanish and to culturally adapt the Diagnostic Interview Schedule for Children (DISC). The process strived to identify similar phenomena to those identified by the original English version in a dissimilar context. To attain cross-cultural equaivalency five important dimensions were addressed: semantic, technical, content, criterion and conceptual. To meet this challenge various steps were taken, including bilingual committee, back-translation, reliability and validity testing. The result is an instrument which could be used, not only in Puerto Rico, but also in other Spanishspeaking child and adolescent populations after appropriate cultural adaptations.     

Mycorrhizal response of <Emphasis Type="Italic">Clusia pusilla</Emphasis> growing in two different soils in the field

Arbuscular mycorrhizas (AM) are important for promoting the mineral nutrition, growth and survival of plants used to rehabilitate degraded areas. Clusia pusilla is an evergreen shrub which is tolerant of high irradiance, germinates readily and can be easily reproduced by cuttings. All these characteristics make this species useful in the recovery of deforested areas. The aim of this work was to explore the response of C. pusilla to AM in the field, in two types of soil: the shrubland soil in which the species naturally grows and in a soil of a riparian forest. Eight treatments were performed in each type of soil. The treatments consisted of a non-mycorrhizal control and mycorrhizal plants colonized by one of the three AM inocula tested in the presence or absence of triple superphosphate (150 kg ha^-1). After 11 months of growth in the shrubland soil, C. pusilla seedlings showed an increase in height and dry weight in response to the fertilizer but not to mycorrhizas. In contrast, in the forest soil the arbuscular mycorrhizal fungi (AMF) effect was equivalent to the fertilizer effect, and the two effects interacted positively. The lack of response to AM in shrubland soil was caused by its high sand content, which hinders the retention of the inocula. Due to a higher clay content, the forest soil binds inocula more tightly than shrubland soil. In conclusion, C. pusilla appears to benefit greatly from the addition of AMF in forest soil, though it requires an additional P source for such benefits in shrubland soil. This P source must be organic so that phosphorus is not lost by leaching. Although the growth rate of this species is very low, its survival can be guaranteed with the application of AMF inocula together with P-fertilizer applied at a low rate.     

Tumor-associated mutations in a conserved structural motif alter physical and biochemical properties of human RAD51 recombinase

Human RAD51 protein catalyzes DNA pairing and strand exchange reactions that are central to homologous recombination and homology-directed DNA repair. Successful recombination/repair requires the formation of a presynaptic filament of RAD51 on ssDNA. Mutations in BRCA2 and other proteins that control RAD51 activity are associated with human cancer. Here we describe a set of mutations associated with human breast tumors that occur in a common structural motif of RAD51. Tumor-associated D149N, R150Q and G151D mutations map to a Schellman loop motif located on the surface of the RecA homology domain of RAD51. All three variants are proficient in DNA strand exchange, but G151D is slightly more sensitive to salt than wild-type (WT). Both G151D and R150Q exhibit markedly lower catalytic efficiency for adenosine triphosphate hydrolysis compared to WT. All three mutations alter the physical properties of RAD51 nucleoprotein filaments, with G151D showing the most dramatic changes. G151D forms mixed nucleoprotein filaments with WT RAD51 that have intermediate properties compared to unmixed filaments. These findings raise the possibility that mutations in RAD51 itself may contribute to genome instability in tumor cells, either directly through changes in recombinase properties, or indirectly through changes in interactions with regulatory proteins.     

Photosystem II Cyclic Heterogeneity and Photoactivation in the Diazotrophic, Unicellular Cyanobacterium Cyanothece Species ATCC 51142

The unicellular, diazotrophic cyanobacterium Cyanothece sp. ATCC 51142 demonstrated important modifications to photosystem II (PSII) centers when grown under light/dark N₂-fixing conditions. The properties of PSII were studied throughout the diurnal cycle using O₂-flash-yield and pulse-amplitude-modulated fluorescence techniques. Nonphotochemical quenching (q_N) of PSII increased during N₂ fixation and persisted after treatments known to induce transitions to state 1. The q_N was high in cells grown in the dark, and then disappeared progressively during the first 4 h of light growth. The photoactivation probability, ε, demonstrated interesting oscillations, with peaks near 3 h of darkness and 4 and 10 h of light. Experiments and calculations of the S-state distribution indicated that PSII displays a high level of heterogeneity, especially as the cells prepare for N₂ fixation. We conclude that the oxidizing side of PSII is strongly affected during the period before and after the peak of nitrogenase activity; changes include a lowered capacity for O₂ evolution, altered dark stability of PSII centers, and substantial changes in q_N.