Identification of archaeal proteins that affect the exosome function in vitro

Background: The archaeal exosome is formed by a hexameric RNase PH ring and three RNA binding subunits and has been shown to bind and degrade RNA in vitro. Despite extensive studies on the eukaryotic exosome and on the proteins interacting with this complex, little information is yet available on the identification and function of archaeal exosome regulatory factors. Results: Here, we show that the proteins PaSBDS and PaNip7, which bind preferentially to poly-A and AU-rich RNAs, respectively, affect the Pyrococcus abyssi exosome activity in vitro. PaSBDS inhibits slightly degradation of a poly-rA substrate, while PaNip7 strongly inhibits the degradation of poly-A and poly-AU by the exosome. The exosome inhibition by PaNip7 appears to depend at least partially on its interaction with RNA, since mutants of PaNip7 that no longer bind RNA, inhibit the exosome less strongly. We also show that FITC-labeled PaNip7 associates with the exosome in the absence of substrate RNA. Conclusions: Given the high structural homology between the archaeal and eukaryotic proteins, the effect of archaeal Nip7 and SBDS on the exosome provides a model for an evolutionarily conserved exosome control mechanism.

Identification of genes differentially expressed in a strain of the mold Aspergillus nidulans carrying a loss-of-function mutation in the palA gene

To identify genes differentially expressed in a strain of the mold Aspergillus nidulans carrying a loss-of-function mutation in palA, a gene in the pH-responsive signal transduction pathway, suppression subtractive hybridization was performed between RNA isolated from the biA1 and biA1 palA1 strains grown under limiting inorganic phosphate at pH 5.0. We have identified several genes upregulated in the biA1 palA1 mutant strain that play important roles in mitotic fidelity, stress responses, enzyme secretion, signal transduction mechanisms, development, genome stability, phosphate sensing, and transcriptional regulation among others. The upregulation of eight of these transcripts was also validated by Northern blot. Moreover, we show that a loss of function mutation in the palA gene drastically reduced the neutral sugar content of the acid phosphatase PacA secreted by the fungus A. nidulans grown at pH 5.0 compared with a control strain.

Chemical profile of rums as a function of their origin. The use of chemometric techniques for their identification

To identify chemical descriptors to distinguish Cuban from non-Cuban rums, analyses of 44 samples of rum from 15 different countries are described. To provide the chemical descriptors, analyses of the the mineral fraction, phenolic compounds, caramel, alcohols, acetic acid, ethyl acetate, ketones, and aldehydes were carried out. The analytical data were treated through the following chemometric methods: principal component analysis (PCA), partial least square-discriminate analysis (PLS-DA), and linear discriminate analysis (LDA). These analyses indicated 23 analytes as relevant chemical descriptors for the separation of rums into two distinct groups. The possibility of clustering the rum samples investigated through PCA analysis led to an accumulative percentage of 70.4% in the first three principal components, and isoamyl alcohol, n-propyl alcohol, copper, iron, 2-furfuraldehyde (furfuraldehyde), phenylmethanal (benzaldehyde), epicatechin, and vanillin were used as chemical descriptors. By applying the PLS-DA technique to the whole set of analytical data, the following analytes have been selected as descriptors: acetone, sec-butyl alcohol, isobutyl alcohol, ethyl acetate, methanol, isoamyl alcohol, magnesium, sodium, lead, iron, manganese, copper, zinc, 4-hydroxy3,5-dimethoxybenzaldehyde (syringaldehyde), methaldehyde (formaldehyde), 5-hydroxymethyl-2furfuraldehyde (5-HMF), acetalclehyde, 2-furfuraldehyde, 2-butenal (crotonaldehyde), n-pentanal (valeraldehyde), iso-pentanal (isovaleraldehyde), benzaldehyde, 2,3-butanodione monoxime, acetylacetone, epicatechin, and vanillin. By applying the LIDA technique, a model was developed, and the following analytes were selected as descriptors: ethyl acetate, sec-butyl alcohol, n-propyl alcohol, n-butyl alcohol, isoamyl alcohol, isobutyl alcohol, caramel, catechin, vanillin, epicatechin, manganese, acetalclehyde, 4-hydroxy-3-methoxybenzoic acid, 2-butenal, 4-hydroxy-3,5-dimethoxybenzoic acid, cyclopentanone, acetone, lead, zinc, calcium, barium, strontium, and sodium. This model allowed the discrimination of Cuban rums from the others with 88.2% accuracy.

Identification of Novel Immunoregulatory Molecules in Human Thymic Regulatory CD4(+)CD25(+) T Cells by Phage Display

Thymic CD4(+)CD25(+) cells play an important role in immune regulation and are continuously developed in the thymus as an independent lineage. How these cells are generated, what are their multiple pathways of suppressive activity and which are their specific markers are questions that remain unanswered. To identify molecules involved in the function and development of human CD4(+)CD25(+) T regulatory cells we targeted thymic CD4(+)CD25(+) cells by peptide phage display. A phage library containing random peptides was screened ex vivo for binding to human thymic CD4(+)CD25(+) T cells. After four rounds of selection on CD4(+)CD25(+) enriched populations of thymocytes, we sequenced several phage displayed peptides and selected one with identity to the Vitamin D Receptor (VDR). We confirmed the binding of the VDR phage to active Vitamin D in vitro, as well as the higher expression of VDR in CD4(+)CD25(+) cells. We suggest that differential expression of VDR on natural Tregs may be related to the relevance of Vitamin D in function and ontogeny of these cells.

A hybrid Particle Swarm Optimization - Simplex algorithm (PSOS) for structural damage identification

This study proposes a new PSOS-model based damage identification procedure using frequency domain data. The formulation of the objective function for the minimization problem is based on the Frequency Response Functions (FRFs) of the system. A novel strategy for the control of the Particle Swarm Optimization (PSO) parameters based on the Nelder-Mead algorithm (Simplex method) is presented; consequently, the convergence of the PSOS becomes independent of the heuristic constants and its stability and confidence are enhanced. The formulated hybrid method performs better in different benchmark functions than the Simulated Annealing (SA) and the basic PSO (PSO(b)). Two damage identification problems, taking into consideration the effects of noisy and incomplete data, were studied: first, a 10-bar truss and second, a cracked free-free beam, both modeled with finite elements. In these cases, the damage location and extent were successfully determined. Finally, a non-linear oscillator (Duffing oscillator) was identified by PSOS providing good results. (C) 2009 Elsevier Ltd. All rights reserved

Fast experimental identification of suspension models for control design

This paper presents both the theoretical and the experimental approaches of the development of a mathematical model to be used in multi-variable control system designs of an active suspension for a sport utility vehicle (SUV), in this case a light pickup truck. A complete seven-degree-of-freedom model is successfully quickly identified, with very satisfactory results in simulations and in real experiments conducted with the pickup truth. The novelty of the proposed methodology is the use of commercial software in the early stages of the identification to speed up the process and to minimize the need for a large number of costly experiments. The paper also presents major contributions to the identification of uncertainties in vehicle suspension models and in the development of identification methods using the sequential quadratic programming, where an innovation regarding the calculation of the objective function is proposed and implemented. Results from simulations of and practical experiments with the real SUV are presented, analysed, and compared, showing the potential of the method.

Identification of a juvenile hormone esterase-like gene in the honey bee, Apis mellifera L. - Expression analysis and functional assays

Tight control over circulating juvenile hormone (JH) levels is of prime importance in an insect`s life cycle. Consequently, enzymes involved in JH metabolism, especially juvenile hormone esterases (JHEs), play major roles during metamorphosis and reproduction. In the highly eusocial Hymenoptera, JH has been co-opted into additional functions, primarily in the development of the queen and worker castes and in age-related behavioral development of workers. Within a set of 21 carboxylesterases predicted in the honey bee genome we identified one gene (Amjhe-like) that contained the main functional motifs of insect JHEs. Its transcript levels during larval development showed a maximum at the switch from feeding to spinning behavior, coinciding with a JH titer minimum. In adult workers, the highest levels were observed in nurse bees, where a low JH titer is required to prevent the switch to foraging. Functional assays showed that Amjhe-like expression is induced by JH-III and suppressed by 20-hydroxyecdysone. RNAi-mediated silencing of Amjhe-like gene function resulted in a six-fold increase in the JH titer in adult worker bees. The temporal profile of Amjhe-like expression in larval and adult workers, the pattern of hormonal regulation and the knockdown phenotype are consistent with the function of this gene as an authentic JHE. (C) 2008 Elsevier Inc. All rights reserved.

The Use of Smell Identification Tests in the Diagnosis of Parkinson`s Disease in Brazil

Smell identification tests may be of routine clinical value in the differential diagnosis of PD but are subject to cultural variation and have not been systematically evaluated in the Brazilian population. We have applied culturally adapted translations of the University of Pennsylvania 40-item smell identification test (UPSIT-40) and the 16-item identification test from Sniffin` Sticks (SS-16) to nondemented Brazilian PD patients and controls. Pearson`s correlation coefficient between the test scores was 0.76 (95% CI 0.70-0.81, n = 204, P < 0.001). To calculate reliability measures for each test we used the diagnosis (either PD or control) as outcome variable for separate logistic regression analyses using the score in the UPSIT-40 or the SS-16 as a covariate. The SS-16 specificity was 89.0% with a sensitivity of 81.1% (106 PD and 118 controls). The UPSIT-40 specificity was 83.5% and its sensitivity 82.1% (95 PD and 109 controls). Regression curves were used to associate an individual`s smell test score with the probability of belonging to the PD, as opposed to the control group. Our data provide support for the use of the UPSIT-40 and SS-16 to help distinguish early PD from controls. (c) 2008 Movement Disorder Society

New SMS mutation leads to a striking reduction in spermine synthase protein function and a severe form of Snyder-Robinson X-linked recessive mental retardation syndrome

We report the identification of a novel mutation at a highly conserved residue within the N-terminal region of spermine synthase (SMS) in a second family with Snyder-Robinson X-linked mental retardation syndrome ( OMIM 309583). This missense mutation, p.G56S, greatly reduces SMS activity and leads to severe epilepsy and cognitive impairment. Our findings contribute to a better delineation and expansion of the clinical spectrum of Snyder-Robinson syndrome, support the important role of the N-terminus in the function of the SMS protein, and provide further evidence for the importance of SMS activity in the development of intellectual processing and other aspects of human development.

Perception of dysphagia: lack of correlation with objective measurements of esophageal function

Background The mechanism underlying increased perception of food bolus passage in the absence of esophageal mechanical obstruction has not been completely elucidated. A correlation between the intensity of the symptom and the severity of esophageal dysfunction, either motility (manometry) or bolus transit (impedance) has not been clearly demonstrated. The aim of this study was to analyze the correlation between objective esophageal function assessment (with manometry and impedance) and perception of bolus passage in healthy volunteers (HV) with normal and pharmacologically-induced esophageal hypocontractility, and in patients with gastro-esophageal reflux disease (GERD) with and without ineffective esophageal motility (IEM). Methods Combined manometry-impedance was performed in 10 HV, 19 GERD patients without IEM and nine patients with IEM. Additionally, nine HV were studied after 50 mg sildenafil, which induced esophageal peristaltic failure. Perception of each 5 mL viscous swallow was evaluated using a 5-point scale. Manometry identified hypocontractility (contractions lower than 30 mmHg) and impedance identified incomplete bolus clearance. Key Results In HV and in GERD patients with and without IEM, there was no association between either manometry or impedance and perception on per swallow analysis (OR: 0.842 and OR: 2.017, respectively), as well as on per subject analysis (P = 0.44 and P = 0.16, respectively). Lack of correlation was also found in HV with esophageal hypocontractility induced by sildenafil. Conclusions & Inferences There is no agreement between objective measurements of esophageal function and subjective perception of bolus passage. These results suggest that increased bolus passage perception in patients without mechanical obstruction might be due to esophageal hypersensitivity.

AMP hydrolysis in soluble and microsomal rat cardiac cell fractions: kinetic characterization and molecular identification of 5 `-nucleotidase

The present study describes the enzymatic properties and molecular identification of 5`-nucleotidase in soluble and microsomal fractions from rat cardiac ventricles. Using AMP as a substrate, the results showed that the cation and the concentration required for maximal activity in the two fractions was magnesium at a final concentration of 1 mM. The pH optimum for both fractions was 9.5. The apparent K-m (Michaelis constant) values calculated from the Eadie-Hofstee plot were 59.7 +/- 10.4 mu M and 134.8 +/- 32.1 mu M, with V-max values of 6.7 +/- 0.4 and 143.8 +/- 23.8 nmol P-i/min/mg of protein (means +/- S.D., n = 4) from soluble and microsomal fractions respectively. Western blotting analysis of ecto-5`-nucleotidase revealed a 70 kDa protein in both fractions, with the major proportion present in the microsomal fraction. The presence of these enzymes in the heart probably has a physiological function in adenosine signalling. Furthermore, the presence of ecto-5`-nucleotidase in the microsomal fraction could have a role in the modulation of the excitation-contraction-coupling process through involvement of the Ca2+ influx into the sarcoplasmic reticulum. The measurement of maximal enzyme activities in the two fractions highlights the potential capacity of the different pathways of purine metabolism in the heart.

The leptospiral antigen Lp49 is a two-domain protein with putative protein binding function

Pathogenic Leptospira is the etiological agent of leptospirosis, a life-threatening disease that affects populations worldwide. Currently available vaccines have limited effectiveness and therapeutic interventions are complicated by the difficulty in making an early diagnosis of leptospirosis. The genome of Leptospira interrogans was recently sequenced and comparative genomic analysis contributed to the identification of surface antigens, potential candidates for development of new vaccines and serodiagnosis. Lp49 is a membrane-associated protein recognized by antibodies present in sera from early and convalescent phases of leptospirosis patients. Its crystal structure was determined by single-wavelength anomalous diffraction using selenomethionine-labelled crystals and refined at 2.0 angstrom resolution. Lp49 is composed of two domains and belongs to the all-beta-proteins class. The N-terminal domain folds in an immunoglobulin-like beta-sandwich structure, whereas the C-terminal domain presents a seven-bladed beta-propeller fold. Structural analysis of Lp49 indicates putative protein-protein binding sites, suggesting a role in Leptospira-host interaction. This is the first crystal structure of a leptospiral antigen described to date. (C) 2008 Elsevier Inc. All rights reserved.

Identification of an Operon, Pil-Chp, That Controls Twitching Motility and Virulence in Xylella fastidiosa

Xylella fastidiosa is an important phytopathogenic bacterium that causes many serious plant diseases, including Pierce`s disease of grapevines. Disease manifestation by X. fastidiosa is associated with the expression of several factors, including the type IV pili that are required for twitching motility. We provide evidence that an operon, named Pil-Chp, with genes homologous to those found in chemotaxis systems, regulates twitching motility. Transposon insertion into the pilL gene of the operon resulted in loss of twitching motility (pilL is homologous to cheA genes encoding kinases). The X. fastidiosa mutant maintained the type IV pili, indicating that the disrupted pilL or downstream operon genes are involved in pili function, and not biogenesis. The mutated X. fastidiosa produced less biofilm than wild-type cells, indicating that the operon contributes to biofilm formation. Finally, in planta the mutant produced delayed and less severe disease, indicating that the Pil-Chp operon contributes to the virulence of X. fastidiosa, presumably through its role in twitching motility.

Quality evaluation of DNA obtained from stored human saliva and its applicability to identification in Forensic Dentistry

PURPOSE: This study evaluated the quality of DNA obtained from stored human saliva and its applicability to human identification. METHODS: The saliva samples of 20 subjects, collected in the form of saliva in natura and from mouth swabs and stored at -20ºC, were analyzed. After 7 days, the DNA was extracted from the 40 saliva samples and subjected to PCR and electrophoresis. After 180 days, the technique was repeated with the 20 swab samples. RESULTS: The first-stage results indicated that DNA was successfully extracted in 97.5% of reactions, 95% of saliva in natura and 100% of swab saliva samples, with no statistically significant difference between the forms of saliva. In the second phase, the result was positive for all 20 analyzed samples (100%). Subsequently, in order to analyze the quality of the DNA obtained from human saliva, the SIX3-2 gene was tested on the 20 mouth swab samples, and the PCR products were digested using the MbO1 restriction enzyme to evaluate polymorphisms in the ADRA-2 gene, with positive results for most samples. CONCLUSION: It was concluded that the quantity and quality of DNA from saliva and the techniques employed are adequate for forensic analysis of DNA.

Self-perception and malocclusion and their relation to oral appearance and function

The aim of this study was to evaluate the relationship between malocclusion and self-perception of oral appearance/function, in 12/15-year-old Brazilian adolescents. The cluster sample consisted of 717 teenagers attending 24 urban public (n=611) and 5 rural public (n=107) schools in Maringá/PR. Malocclusion was measured using the Dental Aesthetic Index (DAI), in accordance with WHO recommendations. A parental questionnaire was applied to collect information on esthetic perception level and oral variables related to oral health. Univariate and multiple logistic regression analyses were performed. Multiple logistic regression confirmed that for 12-year-old, missing teeth (OR=2.865) and presence of openbite (open occlusal relationship) (OR=2.865) were risk indicators for speech capability. With regard to 15-year-old, presence of mandibular overjet (horizontal overlap) (OR=4.016) was a risk indicator for speech capability and molar relationship (OR=1.661) was a risk indicator for chewing capability. The impact of malocclusion on adolescents' life was confirmed in this study. Speech and chewing capability were associated with orthodontic deviations, which should be taken into consideration in oral health planning, to identify risk groups and improve community health services.