The use of electrospray ionization tandem mass spectrometry on the structural characterization of novel asymmetric metallo-organic supermolecules, based on pentafluorophenylporphyrins and ruthenium complexes

The novel asymmetric metallo-organic triads cis- and trans-[B(4-py)BPFPH(2){Ru(3)O(Ac)(6)(py)(2)}(Ru(bpy)(2)Cl}](PF(6))(2) (5a,b) for which cis- and trans-B(4-py)BPFPH(2)=5,10-bis(pentafluorophenyl)-15,20-bis(4-pyridyl)porphyrin and 5,15-bis(pentafluorophenyl)-10,20-bis(4-pyridyl)porphyrin, respectively; Ac = acetate; py = pyridine and bpy = 2,2`-bipyridine, as well as their corresponding monosubstituted dyads cis- and trans-[B(4-py)BPFPH(2){Ru(3)O(Ac)(6)(py)(2)}]PF(6) (4a,b) have been structurally characterized via electrospray ionization mass spectrometry (ESI-MS and ESI-MS/MS). The ESI-MS of dyads 4a,b display two characteristic Ru-multicomponent clusters of isotopologue ions corresponding to singly charged ions 4a,b(+) of m/z 1629 and doubly charged ions [4a,b+H](2+) of m/z 815 and the triads 5a,b are detected by ESI-MS as the intact doubly charged cluster of isotopologue ions of m/z 1039 [5a,b](2+). The ESI-MS/MS of 4a,b(+), [4a,b+H](2+) and [5a,b](2+) reveal characteristic dissociation pathways, which confirm the structural assignments providing additional information on the intrinsic binding strengths of the gaseous ions. Although the gas-phase behavior of each pair of isomers was rather similar, the less symmetric dyads 4a,b are distinguished via the (1)H NMR spectral profile of the pyrrolic signals. Exploratory photophysical assays have shown that both modifying motifs alter the porphyrinic core emission profile, opening the possibility to use these asymmetric systems as photophysical devices. (C) 2008 Elsevier Ltd. All rights reserved.

Molecular adaptability of nucleoside diphosphate kinase b from trypanosomatid parasites: stability, oligomerization and structural determinants of nucleotide binding

Nucleoside diphosphate kinases play a crucial role in the purine-salvage pathway of trypanosomatid protozoa and have been found in the secretome of Leishmania sp., suggesting a function related to host-cell integrity for the benefit of the parasite. Due to their importance for housekeeping functions in the parasite and by prolonging the life of host cells in infection, they become an attractive target for drug discovery and design. In this work, we describe the first structural characterization of nucleoside diphosphate kinases b from trypanosomatid parasites (tNDKbs) providing insights into their oligomerization, stability and structural determinants for nucleotide binding. Crystallographic studies of LmNDKb when complexed with phosphate, AMP and ADP showed that the crucial hydrogen-bonding residues involved in the nucleotide interaction are fully conserved in tNDKbs. Depending on the nature of the ligand, the nucleotide-binding pocket undergoes conformational changes, which leads to different cavity volumes. SAXS experiments showed that tNDKbs, like other eukaryotic NDKs, form a hexamer in solution and their oligomeric state does not rely on the presence of nucleotides or mimetics. Fluorescence-based thermal-shift assays demonstrated slightly higher stability of tNDKbs compared to human NDKb (HsNDKb), which is in agreement with the fact that tNDKbs are secreted and subjected to variations of temperature in the host cells during infection and disease development. Moreover, tNDKbs were stabilized upon nucleotide binding, whereas HsNDKb was not influenced. Contrasts on the surface electrostatic potential around the nucleotide-binding pocket might be a determinant for nucleotide affinity and protein stability differentiation. All these together demonstrated the molecular adaptation of parasite NDKbs in order to exert their biological functions intra-parasite and when secreted by regulating ATP levels of host cells.

Sol-gel preparation of near-infrared broadband emitting Er(3+)-doped SiO(2)-Ta(2)O(5) nanocomposite films

This article reports a study on the preparation, densification process, and structural and optical properties of SiO(2)-Ta(2)O(5) nanocomposite films obtained by the sol-gel process. The films were doped with Er(3+) and the Si:Ta molar ratio was 90:10. Values of refractive index, thickness and vibrational modes in terms of the number of layers and thermal annealing time are described for the films. The densification process is accompanied by OH group elimination, increase in the refractive index, and changes in film thickness. Full densification of the film is acquired after 90 min of annealing at 900 degrees C. The onset of crystallization and devitrification, with the growth of Ta(2)O(5) nanocrystals occurs with film densification, evidenced by high-resolution transmission electron microscopy. The Er(3+)-doped nanocomposite annealed at 900 degrees C consists of Ta(2)O(5) nanoparticles, with sizes around 2 nm, dispersed in the SiO(2) amorphous phase. The main emission peak of the film is detected at around 1532 nm, which can be assigned to the (4)I(13/2)->(4)I(15/2) transition of the Er(3+) ions present in the nanocomposites. This band has a full width at half medium of 64 nm, and the lifetime measured for the (4)I(13/2) levels is 5.4 ms, which is broader compared to those of other silicate systems. In conclusion, the films obtained in this work are excellent candidates for use as active planar waveguide. (C) 2010 Elsevier B.V. All rights reserved.

Shared structural determinants for the calcium-independent liposome membrane permeabilization and sarcolemma depolarization in Bothropstoxin-I, a LYS49-PLA(2) from the venom of Bothrops jararacussu

The structural determinants of myotoxicity of bothropstoxin-I (BthTX-I), a Lys49 phospholipase A(2) from Bothrops jararacussu venom, were studied by measuring the resting membrane potential in the mouse phrenic nerve-diaphragm preparation. This method proved to be around 100-fold more sensitive than the creatine kinase release assay, and was used to evaluate a total of 31 site-directed BthTX-I alanine scanning mutants. Mutants that reduced the resting membrane potential were located in a surface patch defined by residues in the C-terminal loop (residues 115-129), positions 37-39 in the membrane interfacial recognition surface (Y46 and K54), and residue K93. These results expand the known structural determinants of the biological activity as evaluated by previous creatine kinase release experiments. Furthermore, a strong correlation is observed between the structural determinants of sarcolemma depolarization and calcium-independent disruption of liposome membranes, suggesting that a common mechanism of action underlies the permeabilization of the biological and model membranes. (C) 2009 Elsevier Ltd. All rights reserved.

Fluorescence spectroscopy of small peptides interacting with microheterogeneous micelles

Many peptides containing tryptophan have therapeutic uses and can be studied by their fluorescent properties. The biological activity of these peptides involves interactions with many cellular components and micelles can function as carriers inside organisms. We report results from the interaction of small peptides containing tryptophan with several microheterogeneous systems: sodium dodecyl sulphate (SDS) micelles; sodium dodecyl sulphate-poly(ethylene oxide) (SDS-PEO) aggregates; and neutral polymeric micelles. We observed that specific parameters, such as wavelength of maximum emission and fluorescence anisotropy, could be used to ascertain the occurrence of interactions. Affinity constants were determined from changes in the intensity of emission while structural modifications in rotameric conformations were verified from time-resolved measurements. Information about the location and diffusion of peptides in the microheterogeneous systems were obtained from tryptophan emission quenching experiments using N-alkylpyridinium ions. The results show the importance of electrostatic and hydrophobic effects, and of the ionization state of charged residues, in the presence of anionic and amphiphilic SDS in the microheterogeneous systems. Conformational stability of peptides is best preserved in the interaction with the neutral polymeric micelles. (C) 2009 Elsevier B.V. All rights reserved.

Structural and Biochemical Correlates of Na(+), K(+)-ATPase Driven Ion Uptake Across the Posterior Gill Epithelium of the True Freshwater Crab, Dilocarcinus pagei (Brachyura, Trichodactylidae)

To better comprehend the structural and biochemical underpinnings of ion uptake across the gills of true freshwater crabs, we performed an ultrastructural, ultracytochemical and morphometric investigation, and kinetically characterized the Na(+), K(+)-ATPase, in posterior gill lamellae of Dilocarcinus pagei. Ultrastructurally, the lamellar epithelia are markedly asymmetrical: the thick, mushroom-shaped, proximal ionocytes contain elongate mitochondria (41% cell volume) associated with numerous (approximate to 14 mu m(2) membrane per mu m(3) cytoplasm), deep invaginations that house the Na(+), K(+)-ATPase, revealed ultracytochemically. Their apical surface is amplified (7.5 mu m(2) mu m(-2)) by stubby evaginations whose bases adjoin mitochondria below the subcuticular space. The apical membrane of the thin, distal ionocytes shows few evaginations (1.6 mu m(2) mu m(-2)), each surrounding a mitochondrion, abundant in the cytoplasm below the subcuticular space; basolateral invaginations and mitochondria are few. Fine basal cytoplasmic bridges project across the hemolymph space, penetrating into the thick ionocytes, suggesting ion movement between the epithelia. Microsomal Na(+), K(+)-ATPase specific activity resembles marine crabs but is approximate to 5-fold less than in species from fluctuating salinities, and freshwater shrimps, suggesting ion loss compensation by strategies other than Na(+) uptake. Enzyme apparent K(+) affinity attains 14-fold that of marine crabs, emphasizing the relevance of elevated K(+) affinity to the conquest of fresh water. Western blotting and biphasic ouabain inhibition disclose two alpha-subunit isoforms comprising distinct functional isoenzymes. While enzyme activity is not synergistically stimulated by NH(4)(+) and K(+), each increases affinity for the other, possibly assuring appropriate intracellular K(+) concentrations. These findings reveal specific structural and biochemical adaptations that may have allowed the establishment of the Brachyura in fresh water. J. Exp. Zool. 313A:508-523, 2010. (C) 2010 Wiley-Liss, Inc.

Hematological parameters and anaerobic threshold in Brazilian soccer players throughout a training program

We assessed the responses of hematological parameters and their relationship to the anaerobic threshold of Brazilian soccer players during a training program. Twelve athletes were evaluated at the beginning (week 0, T1), in the middle (week 6, T2), and at the end (week 12, T3) of the soccer training program. On the first day at 7:30 AM, before collecting the blood sample at rest for the determination of the hematological parameters, the athletes were conducted to the anthropometric evaluation. On the second day at 8:30 AM, the athletes had their anaerobic threshold measured. Analysis of variance with Newman-Keuls`post hoc was used for statistical comparisons between the parameters measured during the soccer training program. Correlations between the parameters analyzed were determined using the Pearson`s correlation coefficient. Erythrocytes concentration, hemoglobin, and hematocrit were significantly increased from T1 to T2. The specific soccer training program led to a rise in erythrocytes, hemoglobin, and hematocrit from T1 to T2. We assumed that these results occurred due to the plasma volume reduction and may be explained by the soccer training program characteristics. Furthermore, we did not observe any correlation between the anaerobic threshold and the hematological parameters.

Oculometric parameters of hyperopia in children with esotropic amblyopia

Purpose: To study the oculometric parameters of hyperopia in children with esotropic amblyopia, comparing amblyopic eyes with fellow eyes. Methods: Thirty-seven patients (5-8 years old) with bilateral hyperopia and esotropic amblyopia underwent a comprehensive ophthalmic examination, including cycloplegic refraction, keratometry and A-scan ultrasonography. Anterior chamber depth, lens thickness, vitreous chamber depth and total axial length were recorded. The refractive power of the crystalline lens was calculated using Bennett`s equations. Paired Student`s t-tests were used to compare ocular biometric measurements between amblyopic eyes and their fellow eyes. The associations of biometric parameters with refractive errors were assessed using Pearson correlation coefficients and linear regression. Multivariable models including axial length, corneal power and lens power were also constructed. Results: Amblyopic eyes were found to have significantly more hyperopic refraction, less corneal power, greater lens power, shorter vitreous chamber depth and shorter axial length, despite similar anterior chamber depth and lens thickness. The strongest correlation with refractive error was observed for the axial length/corneal radius ratio (r(36) = -0.92, p < 0.001 for amblyopic and r(36) = 0.87, p < 0.001 for fellow eyes). Axial length accounted for 39.2% (R(2)) of the refractive error variance in amblyopic eyes and 35.5% in fellow eyes. Adding corneal power to the model increased R(2) to 85.7% and 79.6%, respectively. A statistically significant correlation was found between axial length and corneal power, indicating decreasing corneal power with increasing axial length, and they were similar for amblyopic eyes (r(36) = 0.53,p < 0.001) and fellow eyes (r(36) = -0.57, p < 0.001). A statistically significant correlation was also found between axial length and lens power, indicating decreasing lens power with increasing axial length (r(36) = -0.72, p < 0.001 for amblyopic eyes and r(36) = -0.69, p < 0.001 for fellow eyes). Conclusions: We observed that the correlation among the major oculometric parameters and their individual contribution to hyperopia in esotropic children were similar in amblyopic and non-amblyopic eyes. This finding suggests that the counterbalancing effect of greater corneal and lens power associated with shorter axial length is similar in both eyes of patients with esotropic amblyopia.

Structural shifts of aldehyde dehydrogenase enzymes were instrumental for the early evolution of retinoid-dependent axial patterning in metazoans

Aldehyde dehydrogenases (ALDHs) catabolize toxic aldehydes and process the vitamin A-derived retinaldehyde into retinoic acid (RA), a small diffusible molecule and a pivotal chordate morphogen. In this study, we combine phylogenetic, structural, genomic, and developmental gene expression analyses to examine the evolutionary origins of ALDH substrate preference. Structural modeling reveals that processing of small aldehydes, such as acetaldehyde, by ALDH2, versus large aldehydes, including retinaldehyde, by ALDH1A is associated with small versus large substrate entry channels (SECs), respectively. Moreover, we show that metazoan ALDH1s and ALDH2s are members of a single ALDH1/2 clade and that during evolution, eukaryote ALDH1/2s often switched between large and small SECs after gene duplication, transforming constricted channels into wide opened ones and vice versa. Ancestral sequence reconstructions suggest that during the evolutionary emergence of RA signaling, the ancestral, narrow-channeled metazoan ALDH1/2 gave rise to large ALDH1 channels capable of accommodating bulky aldehydes, such as retinaldehyde, supporting the view that retinoid-dependent signaling arose from ancestral cellular detoxification mechanisms. Our analyses also indicate that, on a more restricted evolutionary scale, ALDH1 duplicates from invertebrate chordates (amphioxus and ascidian tunicates) underwent switches to smaller and narrower SECs. When combined with alterations in gene expression, these switches led to neofunctionalization from ALDH1-like roles in embryonic patterning to systemic, ALDH2-like roles, suggesting functional shifts from signaling to detoxification.

Voiding Dysfunction in Patients With Parkinson`s Disease: Impact of Neurological Impairment and Clinical Parameters

Aims: We assessed the lower urinary tract symptoms (LUTS) of patients with Parkinson`s disease (PD) and their association with different clinical parameters. Methods: We prospectively evaluated 110 patients (84 men), with a mean age of 61.8 +/- 9.6 years. Mean duration of the disease was 12.3 +/- 7.2 years. Neurological impairment was assessed by the Hoehn-Yahr and the Unified Parkinson Disease Rating scales. LUTS were assessed by the International Continence Society questionnaire. We evaluated the impact of age, PD duration, neurological impairment, gender, and use of anti-Parkinsonian drugs on the voiding function. Results: On multivariate analysis, voiding dysfunction increased with the neurological impairment, but not with patient`s age or disease duration. Quality of life (QOL) was affected by the severity of LUTS, and the symptoms with the worst impact were frequency and nocturia. Sixty-three (57.2%) patients were symptomatic. They did not differ with the asymptomatic as to age and disease duration, but had more severe neurological impairment. No impact on LUTS was associated with the use of levodopa, anticholinergics, and dopamine receptor agonists. Men and women were similarly affected by urinary symptoms. Conclusions: The severity of the neurological disease is the only predictive factor for the occurrence of voiding dysfunction, which affects men and women alike. Neztrourol. Urodynam. 28.510-515, 2009. (C) 2009 Wiley-Liss, Inc.

Combined glucosamine and chondroitin sulfate provides functional and structural benefit in the anterior cruciate ligament transection model

Evidence that combined glucosamine sulfate and chondroitin sulfate (Gluchon) or isolated glucosamine (Glu) modifies joint damage in osteoarthritis (OA) is still lacking. We studied joint pain and cartilage damage using the anterior cruciate ligament transection (ACLT) model. Wistar rats were subjected to ACLT of the right knee ( OA) or sham operation. Groups received either Glu (500 mg/kg), Gluchon (500 mg/kg glucosamine +400 mg/kg chondroitin) or vehicle (non-treated-NT) per os starting 7 days prior to ACLT until sacrifice at 70 days. Joint pain was evaluated daily using the rat-knee joint articular incapacitation test. Structural joint damage was assessed using histology and biochemistry as the chondroitin sulfate ( CS) content of cartilage by densitometry (microgram per milligram dried cartilage), comparing to standard CS. The molar weight (Mw) of the CS samples, used as a qualitative biochemical parameter, was obtained by comparing their relative mobility on a polyacrylamide gel electrophoresis to standard CS. Gluchon, but not Glu, significantly reduced joint pain (P<0.05) compared to NT. There was an increase in CS content in the OA group (77.7 +/- 8.3 mu g/mg) compared to sham (53.5 +/- 11.2 mu g/mg) (P<0.05). The CS from OA samples had higher Mw (4:62 +/- 0:24 x 10(4) g/mol) compared to sham (4:18 +/- 0:19 x 10(4) g/mol) (P<0.05). Gluchon administration significantly reversed both the increases in CS content (54.4 +/- 12.1 mu g/mg) and Mw (4:18 +/- 0:2 x 104 g/mol) as compared to NT. Isolated Glu decreased CS content though not reaching statistical significance. Cartilage histology alterations were also significantly prevented by Gluchon administration. Gluchon provides clinical (analgesia) and structural benefits in the ACLT model. This is the first demonstration that biochemical alterations occurring in parallel to histological damage in OA are prevented by Gluchon administration.

Relationship between induced sputum cytology and inflammatory status with lung structural and functional abnormalities in asbestosis

Background Asbestosis is associated with lung cellular and immunological abnormalities. Induced sputum cytology and local and systemic markers of inflammation may be helpful to characterize disease status and progression in these patients. Methods Thirty-nine ex-workers with asbestosis on high-resolution CT (HRCT) and 21 non-exposed controls were evaluated. Sputum cytology and IL-8 in serum and sputum were related to lung function impairment. Results Subjects with asbestosis had reduced sputum cellularity but higher macrophagel neutrophil ratio and % macrophage as compared with controls. Sputum and serum IL-8 were also higher in patients with asbestosis (P < 0.05). In addition, evidence of lung architectural distorption on HRCT was associated with increased levels of serum IL-8. Interestingly, absolute macrophage number was negatively correlated with total lung capacity (r = -0.40; P = 0.04) and serum IL-8 to lung diffiusing capacity (r = -0.45; P = 0.01). Conclusions Occupationally exposed subjects with asbestosis on HRCT have cytologic abnormalities in induced sputum and increased local and systemic pro-inflammatory status which are correlated to functional impairment.

Increased Brain Membrane Fluidity in Schizophrenia

Recent findings showing significant correlations between phospholipase A2 (PLA2) activity and structural changes in schizophrenic brains contribute to the membrane hypothesis of schizophrenia, which was hampered because a clean functional link between elevated PLA2 activity and brain structure was missing (Neuroimage, 2010; 52: 1314-1327). We measured membrane fluidity parameters and found that brain membranes isolated from the prefrontal cortex of schizophrenic patients showed significantly increased flexibility of fatty acid chains. Our findings support a possible link between elevated PLA2 activity in cortical areas of schizophrenic patients and subsequent alterations of the biophysical parameters of neuronal membranes leading to structural changes in these areas.

Gamma ventral capsulotomy for treatment of resistant obsessive-compulsive disorder: A structural MRI pilot prospective study

Objective: The purpose of this study was to investigate regional structural abnormalities in the brains of five patients with refractory obsessive-compulsive disorder (OCD) submitted to gamma ventral capsulotomy. Methods: We acquired morphometric magnetic resonance imaging (MRI) data before and after 1 year of radiosurgery using a 1.5-T MRI scanner. Images were spatially normalized and segmented using optimized voxel-based morphometry (VBM) methods. Voxelwise statistical comparisons between pre- and post-surgery MRI scans were performed using a general linear model. Findings in regions predicted a priori to show volumetric changes (orbitofrontal cortex, anterior cingulate gyrus, basal ganglia and thalamus) were reported as significant if surpassing a statistical threshold of p<0.001 (uncorrected for multiple comparisons). Results: We detected a significant regional postoperative increase in gray matter volume in the right inferior frontal gyri (Brodmann area 47, BA47) when comparing all patients pre and postoperatively. Conclusions: Our results support the current theory of frontal-striatal-thalamic-cortical (FSTC) circuitry involvement in OCD pathogenesis. Gamma ventral capsulotomy is associated with neurobiological changes in the inferior orbitofrontal cortex in refractory OCD patients. (C) 2008 Elsevier Ireland Ltd. All rights reserved.