Search

Background: Mean platelet volume has been associated with stroke in patients with atrial fibrillation. However, its role as a predictor of left atrial stasis, assessed by transesophageal echocardiography, in patients with non-valvular atrial fibrillation has not yet been clarified., Methods: Single center cross-sectional study comprising 427 patients admitted to the emergency department due to symptomatic atrial fibrillation and undergoing transesophageal echocardiogram evaluation for exclusion of left atrial appendage thrombus before cardioversion. All patients had a complete blood count performed in the 12 hours prior to transesophageal echocardiogram. Markers of left atrial stasis were sought: left atrial appendage thrombus, dense spontaneous echocardiographic contrast and low flow velocities in the left atrial appendage. The presence of at least one of the former markers of left atrial stasis was designated left atrial abnormality. Binary logistic multivariate analysis was used for obtaining models for the prediction of transesophageal echocardiogram endpoints., Results: Left atrial appendage thrombus was found in 12.2%, dense spontaneous echocardiographic contrast in 29.7%, low flow velocities in 15.3% and left atrial abnormality in 34.2%. Mean platelet volume (exp β = 3.41 p = 0.048) alongside with previous stroke or transient ischemic attack (exp β = 5.35 p = 0.005) and troponin I (exp β = 5.07 p = 0.041) were independent predictors of left atrial appendage thrombus. Mean platelet volume was also incorporated in the predictive models of dense spontaneous echocardiographic contrast, low flow velocities and left atrial abnormality, adding predictive value to clinical, echocardiographic and laboratory variables., Conclusions: These findings suggest that mean platelet volume may be associated with the presence of markers of left atrial stasis, reinforcing a likely cardioembolic mechanism for its association with stroke in patients with non-valvular atrial fibrillation.

Background: The role of erythrocyte indexes for the prediction of left atrial stasis, assessed by transesophageal echocardiography in patients with non-valvular atrial fibrillation, has not been previously clarified., Methods: Single center cross-sectional study comprising 247 consecutive patients admitted to the emergency department due to symptomatic atrial fibrillation and undergoing transesophageal echocardiogram evaluation for exclusion of left atrial appendage thrombus (LAAT) before cardioversion. All patients had a complete blood count performed up to 12 hours prior to the transesophageal echocardiogram. Markers of left atrial stasis were sought: LAAT, dense spontaneous echocardiographic contrast (DSEC) and low flow velocities (LFV) in the left atrial appendage. Erythrocyte indexes' accuracy for detecting transesophageal echocardiogram changes was evaluated through receiver operating curve analysis. Binary logistic multivariate analysis, using solely erythrocyte indexes and in combination with other variables (i.e. CHADS2, CHA2DS2VASc classifications and left ventricle ejection fraction), was used for transesophageal echocardiogram endpoints prediction., Results: LAAT was found in 8.5%, DSEC in 26.1% and LFV in 12.1%. Mean corpuscular volume and red cell distribution width were independent predictors of LAAT and DSEC. Despite adding incremental predictive value to each other, when clinical risk factors from CHADS2 and CHA2DS2VASc classifications and left ventricle ejection fraction were added to the models, only mean corpuscular volume remained an independent predictor of LAAT and DSEC., Conclusions: These findings suggest that mean corpuscular volume and red cell distribution width may be linked to left atrial stasis markers.

Background: It is currently unknown if the increased risk of stroke in subjects with chronic kidney disease and atrial fibrillation (AF) is due to the presence of left atrial stasis or to any other vascular or systemic conditions., Methods: This was a retrospective study of 372 subjects undergoing evaluation during an AF episode. The following markers of left atrial stasis were sought on transesophageal echocardiogram: left atrial or left atrial appendage thrombus (LAAT), dense spontaneous echocardiographic contrast (DSEC), and low flow velocities (LFV) in the left atrial appendage. Subgroup comparisons were performed according to the level of estimated glomerular filtration rate (eGFR) using the Chronic Kidney Disease Epidemiology Collaboration equation as follows: ≥90, 45-89.9, and <45 ml/min/1.73 m2., Results: LAAT was found in 11.6%, DSEC in 29.0%, and LFV in 14.9% of cases. A significant increase in the prevalence of DSEC was observed in the lower categories of eGFR: 37.8% in eGFR <45 ml/min, 30.7% in eGFR 45-89.9 ml/min, and 17.0% in eGFR ≥90 ml/min (p = 0.009; γ for trend = 0.297, p = 0.002). The same was observed when assessing left atrial abnormality, i.e. the presence of at least one of the former transesophageal echocardiogram changes. On multivariate analysis, clinical parameters from CHADS2 (congestive heart failure, hypertension, age ≥75, diabetes mellitus and stroke) and CHA2DS2-VASc (age 65-74, history of vascular disease, and female gender along with the clinical variables from CHADS2) were predictors of transesophageal echocardiogram changes and an additive predictive value was found for eGFR., Conclusions: Our results suggest an association between compromised renal function as assessed through eGFR and markers of left atrial stasis in patients with AF. The increased risk of stroke in this population may be due to thromboembolism., (Copyright © 2012 S. Karger AG, Basel.)

This work aimed at describing the first biochemical and structural data of a lectin belonging to Swartzieae, a primitive Legume Taxa. A lactose-binding seed lectin (SLL) was purified by affinity chromatography of crude saline extracts of Swartzia laevicarpa on immobilized lactose. The SLL agglutinated rabbit erythrocytes but not rat or human (A, B, O) erythrocytes. Lectin activity was retained after heating at 100 �C for 15 min and was best inhibited by Nacetylgalactosamine, lactose and galactose. The lectin exhibited a single electrophoretic pattern that corresponded to a molecular mass of 29,000 Da, which was confirmed by MS analysis. In addition, the lectin reacted positively with Schiff's reagent. The unique N-terminal amino acid sequence (39 residues) and the internal peptide sequence were determined by Edman degradation and MS/MS, respectively. The sequencing revealed complete homology of the SLL with legume lectins belonging to primitive groups (Dalbergieae and Sophoreae). The SLL (at 1 mg/ml) did not exhibit antifungal activity against various phytopathogens or cytotoxicity (at 100 μg/ml) towards different cancer cell lines.

Introduction: When accurately diagnosed, non-massive Pulmonary embolism (PE) has a low mortality rate. However, some patients initially considered to be low risk show progressive deterioration. This research aims at developing a preliminary score that allows detection of low risk patients potentially eligible for outpatient treatment., Materials and Methods: Retrospective cohort study involving 142 asymptomatic/mildly symptomatic and hemodynamically stable patients with PE and no clinical/echocardiographic signs of right ventricular dysfunction. Collected data: risk factors, analytic/gasometric parameters, admission echocardiogram, thoracic CT angiography. Patients followed for 6months. Primary endpoint: 1-month all-cause mortality. Secondary endpoints: Intrahospital and 6-month all-cause mortality. A score designed for identification of very low risk patients eligible for outpatient treatment was developed and its prognostic accuracy compared to that of the Geneva and simplified PESI models., Results: A score for predicting 1-month mortality (Low Risk Pulmonary Embolism Decision [LR-PED] rule) was obtained using Binary Logistic Regression, including: age, atrial fibrillation at admission, previous heart failure, admission heart rate, creatinine, glycaemia, troponin I and C-reactive protein at admission. ROC curve analysis assessed its overall accuracy for predicting 1-month, intrahospital and 6-month mortality (AUC=0.756, 0.763 and 0.854, respectively). Compared to Geneva and simplified PESI, the LR-PED rule showed higher sensitivity and negative predictive value for the detection of the lowest risk patients. The net reclassification improvement index revealed significant successful upward risk reclassification by the LR-PED model of patients reaching primary or secondary outcomes., Conclusions: LR-PED rule seems more attractive than Geneva or simplified PESI in its ability to identify patients at very low mortality risk who would be potentially eligible for outpatient treatment. Prospective validation of this score in larger cohorts is mandatory before its potential implementation., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

An analysis of the time-dependent genetic response to the death-inducer staurosporine was performed in Neurospora crassa by transcriptional profiling. Staurosporine induced two major genes encoding an ABC transporter and a protein with similarity to regulatory subunits of potassium channels. The transcriptional response is dependent on the activity of a novel transcription factor. Deletion mutants in differentially expressed genes displayed altered sensitivity to staurosporine, underscoring significant proteins involved in the response to the drug. A null-mutant of the ABC transporter (abc3) is extremely sensitive to staurosporine, accumulates more staurosporine than the wild type strain and is defective in energy-dependent export of the drug, indicating that the ABC3 protein is the first described staurosporine transporter. It was located in the plasma membrane by immunofluorescence microscopy. The combination of inhibitors of ABC transporters or of potassium channels with staurosporine leads to an enhanced activity against N. crassa and pathogenic fungi paving the way to the development of more potent and specific antifungals. Our results highlight the general use of transcriptional profiling for the identification of novel proteins involved in cell death and their potential use as drug targets., (Copyright © 2011 Elsevier Inc. All rights reserved.)

We studied staurosporine-induced cell death in the filamentous fungus Neurospora crassa. The generation of reactive oxygen species during the process appears to be an important signaling event, since addition of the antioxidant glutathione prevents the effects of staurosporine on fungal growth. Selected mutants with mutations in respiratory chain complex I are extremely sensitive to the drug, stressing the involvement of complex I in programmed cell death. Following this finding, we determined that the complex I-specific inhibitor rotenone used in combination with staurosporine results in a synergistic and specific antifungal activity, likely through a concerted action on intracellular glutathione depletion. Paradoxically, the synergistic antifungal activity of rotenone and staurosporine is observed in N. crassa complex I mutants and in Saccharomyces cerevisiae, which lacks complex I. In addition, it is not observed when other complex I inhibitors are used instead of rotenone. These results indicate that the rotenone effect is independent of complex I inhibition. The combination of rotenone and staurosporine is effective against N. crassa as well as against the common pathogens Aspergillus fumigatus and Candida albicans, pointing to its usefulness as an antifungal agent.

A sodium ion efflux, together with a proton influx and an inside-positive DeltaPsi, was observed during NADH-respiration by Rhodothermus marinus membrane vesicles. Proton translocation was monitored by fluorescence spectroscopy and sodium ion transport by (23)Na-NMR spectroscopy. Specific inhibitors of complex I (rotenone) and of the dioxygen reductase (KCN) inhibited the proton and the sodium ion transport, but the KCN effect was totally reverted by the addition of menaquinone analogues, indicating that both transports were catalyzed by complex I. We concluded that the coupling ion of the system is the proton and that neither the catalytic reaction nor the establishment of the delta-pH are dependent on sodium, but the presence of sodium increases proton transport. Moreover, studies of NADH oxidation at different sodium concentrations and of proton and sodium transport activities allowed us to propose a model for the mechanism of complex I in which the presence of two different energy coupling sites is suggested.

A miniaturized and low-cost algal growth-inhibition assay, with Pseudokirchneriella subcapitata, based on the standard ISO 8692 and using 96-well microplates, was tested and optimized in this work, to be used as a useful tool for pollutant phytotoxicity screening. For validation, the performance of the microplate algal growth-inhibition assay was first compared with the standard flask assay for the toxicity testing of five reference toxicants (copper(II) sulfate, zinc sulfate, potassium permanganate, potassium dichromate and 3,5-Dichlorophenol) and six wastewater samples. Statistical evaluation of EC(50) results from both methods demonstrated a good agreement between microplate and flask assays either in testing chemicals (r (2) = 0.975, p < 0.0017) or environmental samples toxicity (r (2) = 0.984, p < 0.0001). In addition, the performance of this algal microplate bioassay was also evaluated in comparison with Lemna test, ISO 20079, for phytotoxicity assessment of 27 wastewater samples from industries and treatment plants. The results showed that the algal test was more sensitive for most of the samples, but a significant agreement between both tests was observed (r (2) = 0.644, p < 0.0001). In conclusion, this miniaturized test can be a good tool to include in a battery of tests for phytotoxicity screening of a wide range of chemicals and environmental samples, with the advantage of requiring low sample volumes for the test, allowing large numbers of samples to be tested, and generating low volumes of waste.

The fresh water crustacean Daphnia magna is widely used as a test organism in aquatic toxicology to assess the adverse effects of individual substances or complex mixtures, e.g. industrial wastewaters. Cultures are held in several European testing laboratories and testing is typically carried out according to internationally standardised protocols. However, despite accounting for many potential confounding factors these guidelines do not currently take into account any specification related to the use of a specific clone. Cultures from seven laboratories were used to assess genetic variability by random-amplified polymorphic DNA polymerase chain reaction. Results pointed out the existence of two main clone clusters Responses in the acute Daphnia immobilisation test showed no direct correlation with genetic clusters resulting from random genetic markers (random-amplified polymorphic DNA) analysis. Considering that genetic differences are the most probable cause for the ecotoxicological test data, further analysis concerning gene expression and genetic stability should be performed.

Rhodothermus marinus, a thermohalophilic gram negative bacterium, contains a type I NADH/quinone oxidoreductase (complex I). Its purification was optimized, yielding large amounts of pure and active protein. Furthermore, the stoichiometry of NADH oxidation and quinone reduction was shown to be 1:1. The large amounts of protein enabled a thorough characterization by electron paramagnetic resonance (EPR) spectroscopy at different temperatures and microwave powers, using NADH, NADPH, and dithionite as reducing agents. A minimum of two [2Fe-2S](2+/1+) and four [4Fe-4S](2+/1+) centers were observed in the purified complex. Redox titrations monitored by EPR spectroscopy made possible the determination of the reduction potentials of the iron-sulfur centers; with the exception of one of the [4Fe-4S](2+/1+) centers, which has a lower reduction potential, all the other centers have reduction potentials of -240 +/- 20 mV, pH 7.5.

The NADH:menaquinone oxidoreductase (Nqo) is one of the enzymes present in the respiratory chain of the thermohalophilic bacterium Rhodothermus marinus. The genes coding for the R. marinus Nqo subunits were isolated and sequenced, clustering in two operons [nqo1 to nqo7 (nqoA) and nqo10 to nqo14 (nqoB)] and two independent genes (nqo8 and nqo9). Unexpectedly, two genes encoding homologues of a NhaD Na+/H+ antiporter (NhaD) and of a pterin-4alpha-carbinolamine dehydratase (PCD) were identified within nqoB, flanked by nqo13 and nqo14. Eight conserved motives to harbour iron-sulphur centres are identified in the deduced primary structures, as well as two consensus sequences to bind nucleotides, in this case NADH and FMN. Moreover, the open-reading-frames of the putative NhaD and PCD were shown to be co-transcribed with the other complex I genes encoded by nqoB. The possible role of these two genes in R. marinus complex I is discussed.

Succinate:quinone oxidoreductase (SQR), a di-haem enzyme purified from Rhodothermus marinus, reveals an HQNO-sensitive succinate:quinone oxidoreductase activity with several menaquinone analogues as electron acceptors that decreases with lowering the redox midpoint potential of the quinones. A turnover with the low-potential 2,3-dimethyl-1,4-naphthoquinone that is the closest analogue of menaquinone, although low, can be detected in liposome-reconstituted SQR. Reduction of the quinone is not stimulated by an imposed K+-diffusion membrane potential of a physiological sign (positive inside the vesicles). Nor does the imposed membrane potential increase the reduction level of the haems in R. marinus SQR poised with the succinate/fumarate redox couple. The data do not support a widely discussed hypothesis on the electrogenic transmembrane electron transfer from succinate to menaquinone catalysed by di-haem SQRs. The role of the membrane potential in regulation of the SQR activity is discussed.

A set of experiments, using model compounds, was carried out in order to evaluate the influence of amide linkages on acidity measurements of humic substances (HS). Three benzene-carboxylic acids (2,4-dihydroxybenzoic acid; 3,5-dihydroxybenzoic acid, and phthalic acid) and two peptides (dl-alanyl-dl-alanine, and glycil-l-leucine) were employed to simulate the major acidic functional groups present in humic material. The acidity of the samples was measured, using the Schnitzer and Gupta methods, for each compound separately, as well as for selected mixtures with different compound combinations. General results showed that, in the absence of peptides, phenolic groups with high pK(a) values are not detected in the barium hydroxide reaction and the data so obtained do not represent the real concentrations of the acidic groups. For the mixtures containing peptides, the presence of amide linkages distorts the results relative to the total acidity (TA) mainly because under the extreme conditions of the reaction with Ba(OH)(2) hydrolysis of the peptide occurs, consuming extra hydroxyl groups and increasing artificially the phenolic content. Such a condition is minimized by the reduction of the reaction time.

Thermophiles are organisms that grow optimally above 50 degrees C and up to approximately 120 degrees C. These extreme conditions must have led to specific characteristics of the cellular components. In this paper we extensively analyze the types of respiratory complexes from thermophilic aerobic prokaryotes. The different membrane-bound complexes so far characterized are described, and the genomic data available for thermophilic archaea and bacteria are analyzed. It is observed that no specific characteristics can be associated to thermophilicity as the different types of complexes I-IV are present randomly in thermophilic aerobic organisms, as well as in mesophiles. Rather, the extensive genomic analyses indicate that the differences concerning the several complexes are related to the organism phylogeny, i.e., to evolution and lateral gene transfer events.

The rotenone sensitive NADH:menaquinone oxidoreductase (NDH-I or complex I) from the thermohalophilic bacterium Rhodothermus marinus has been purified and characterized. Three of its subunits react with antibodies against 78, 51, and 21.3c kDa subunits of Neurospora crassa complex I. The optimum conditions for NADH dehydrogenase activity are 50 degrees C and pH 8.1, and the enzyme presents a KM of 9 microM for NADH. The enzyme also displays NADH:quinone oxidoreductase activity with two menaquinone analogs, 1,4-naphtoquinone (NQ) and 2,3-dimethyl-1,4-naphtoquinone (DMN), being the last one rotenone sensitive, indicating the complex integrity as purified. When incorporated in liposomes, a stimulation of the NADH:DMN oxidoreductase activity is observed by dissipation of the membrane potential, upon addition of CCCP. The purified enzyme contains 13.5 +/- 3.5 iron atoms and approximately 3.7 menaquinone per FMN. At least five iron-sulfur centers are observed by EPR spectroscopy: two [2Fe-2S](2+/1+) and three [4Fe-4S](2+/1+) centers. By fluorescence spectroscopy a still unidentified chromophore was detected in R. marinus complex I.

A comprehensive phylogenetic analysis of the core subunits of succinate:quinone oxidoreductases and quinol:fumarate oxidoreductases is performed, showing that the classification of the enzymes as type A to E based on the type of the membrane anchor fully correlates with the specific characteristics of the two core subunits. A special emphasis is given to the type E enzymes, which have an atypical association to the membrane, possibly involving anchor subunits with amphipathic helices. Furthermore, the redox properties of the SQR/QFR proteins are also reviewed, stressing out the recent observation of redox-Bohr effect upon haem reduction, observed for the Desulfovibrio gigas and Rhodothermus marinus enzymes, which indicates a direct protonation event at the haems or at a nearby residue. Finally, the possible contribution of these enzymes to the formation/dissipation of a transmembrane proton gradient is discussed, considering recent experimental and structural data.