Identification of Regions Involved in Substrate Binding and Dimer Stabilization within the Central Domains of Yeast Hsp40 Sis1

Protein folding, refolding and degradation are essential for cellular life and are regulated by protein homeostatic processes such those that involve the molecular chaperone DnaK/Hsp70 and its co-chaperone DnaJ. Hsp70 action is initiated when proteins from the DnaJ family bind an unfolded protein for delivery purposes. In eukaryotes, the DnaJ family can be divided into two main groups, Type I and Type II, represented by yeast cytosolic Ydj1 and Sis1, respectively. Although sharing some unique features both members of the DnaJ family, Ydj1 and Sis1 are structurally and functionally distinct as deemed by previous studies, including the observation that their central domains carry the structural and functional information even in switched chimeras. In this study, we combined several biophysical tools for evaluating the stability of Sis1 and mutants that had the central domains (named Gly/Met rich domain and C-terminal Domain I) deleted or switched to those of Ydj1 to gain insight into the role of these regions in the structure and function of Sis1. The mutants retained some functions similar to full length wild-type Sis1, however they were defective in others. We found that: 1) Sis1 unfolds in at least two steps as follows: folded dimer to partially folded monomer and then to an unfolded monomer. 2) The Gly/Met rich domain had intrinsically disordered characteristics and its deletion had no effect on the conformational stability of the protein. 3) The deletion of the C-terminal Domain I perturbed the stability of the dimer. 4) Exchanging the central domains perturbed the conformational stability of the protein. Altogether, our results suggest the existence of two similar subdomains in the C-terminal domain of DnaJ that could be important for stabilizing each other in order to maintain a folded substrate-binding site as well as the dimeric state of the protein.

Characterization of Opsin Gene Alleles Affecting Color Vision in a Wild Population of Titi Monkeys (Callicebus brunneus)

The color vision of most platyrrhine primates is determined by alleles at the polymorphic X-linked locus coding for the opsin responsible for the middle- to long-wavelength (M/L) cone photopigment. Females who are heterozygous at the locus have trichromatic vision, whereas homozygous females and all males are dichromatic. This study characterized the opsin alleles in a wild population of the socially monogamous platyrrhine monkey Callicebus brunneus (the brown titi monkey), a primate that an earlier study suggests may possess an unusual number of alleles at this locus and thus may be a subject of special interest in the study of primate color vision. Direct sequencing of regions of the M/L opsin gene using feces-, blood-, and saliva-derived DNA obtained from 14 individuals yielded evidence for the presence of three functionally distinct alleles, corresponding to the most common M/L photopigment variants inferred from a physiological study of cone spectral sensitivity in captive Callicebus. Am. J. Primatol. 73:189-196, 2011. (C) 2010 Wiley-Liss, Inc.

Ambient levels of air pollution induce clinical worsening of blepharitis

Background: Even though air pollutants exposure is associated with changes in the ocular surface and tear film, its relationship to the clinical course of blepharitis, a common eyelid disease, had not yet been investigated. Our objective was to investigate the correlation between air pollution and acute manifestations of blepharitis. Method: We recorded all cases of changes in the eyelids and ocular surface, and rated clinical findings on a scale from zero (normal) to two (severe alterations). Daily values of carbon monoxide, particulate matter smaller than 10 mu m in diameter and nitrogen dioxide concentrations and meteorological variables (temperature and relative humidity) in the vicinity of the medical service were obtained. Specific linear regression models for each outcome were constructed including pollutants as independent variables (single pollutant models). Temperature and humidity were included as confounding variables. Results: increases of 28.8 mu g/m(3) in the concentration of particulate matter and 1.1 ppm in the concentration of CO were associated with increases in cases of blepharitis on the day of exposure (5 cases, 95% CI: 1-10 and 6 cases, 95% CI: 1-12, respectively). Conclusion: Exposure to usual air pollutants concentrations present in large cities affects, in a consistent manner, the eyes of residents contributing to the increasing incidence of diseases of the eyelid margin. (C) 2011 Elsevier Inc. All rights reserved.

Glucocorticoid and Estrogen Receptors Are Reduced in Mitochondria of Lung Epithelial Cells in Asthma

Mitochondrial glucocorticoid (mtGR) and estrogen (mtER) receptors participate in the coordination of the cell's energy requirement and in the mitochondrial oxidative phosphorylation enzyme (OXPHOS) biosynthesis, affecting reactive oxygen species (ROS) generation and induction of apoptosis. Although activation of mtGR and mtER is known to trigger anti-inflammatory signals, little information exists on the presence of these receptors in lung tissue and their role in respiratory physiology and disease. Using a mouse model of allergic airway inflammation disease and applying confocal microscopy, subcellular fractionation, and Western blot analysis we showed mitochondrial localization of GR alpha and ER beta in lung tissue. Allergic airway inflammation caused reduction in mtGR alpha, mtER beta, and OXPHOS enzyme biosynthesis in lung cells mitochondria and particularly in bronchial epithelial cells mitochondria, which was accompanied by decrease in lung mitochondrial mass and induction of apoptosis. Confirmation and validation of the reduction of the mitochondrial receptors in lung epithelial cells in human asthma was achieved by analyzing autopsies from fatal asthma cases. The presence of the mitochondrial GR alpha and ER beta in lung tissue cells and especially their reduction in bronchial epithelial cells during allergic airway inflammation suggests a crucial role of these receptors in the regulation of mitochondrial function in asthma, implicating their involvement in the pathophysiology of the disease.

The effects of particulate ambient air pollution on the murine umbilical cord and its vessels: A quantitative morphological and immunohistochemical study

Previous studies have shown that particulate matter (PM) compromise birth weight and placental morphology. We hypothesized that exposing mice to ambient PM would affect umbilical cord (UC) morphology. To test this, mice were kept in paired open-top exposure chambers at the same location and ambient conditions but, in one chamber, the air was filtered (F) and, in the other, it was not (NF). UCs were analysed stereologically and by immunohistochemistry to localize isoprostane and endothelin receptors. The cords of mice from NF chambers were smaller in volume due to loss of mucoid connective tissue and decrease in volume of collagen. These structural changes and in umbilical vessels were associated with greater volumes of regions immunostained for isoprostane, ETAR and ETBR. Findings indicate that the adverse effects of PM on birth weight may be mediated in part by alterations in UC structure or imbalances in the endogenous regulators of vascular tone and oxidative stress. (C) 2012 Elsevier Inc. All rights reserved.

The impact of medical education and networking on the outcome of leukemia treatment in developing countries. The experience of International Consortium on Acute Promyelocytic Leukemia (IC-APL)

Objectives: Several clinical trials conducted in Europe and US reported favorable outcomes of patients with APL treated with the combination of all trans retinoic acid (ATRA) and anthracyclines. Nevertheless, the results observed in developing countries with the same regimen was poorer, mainly due to high early mortality mainly due bleeding. The International Consortium on Acute Promyelocytic Leukemia (IC-APL) is an initiative of the International Members Committee of the ASH and the project aims to reduce this gap through the establishment of international network, which was launched in Brazil, Mexico and Uruguay. Methods: The IC-APL treatment protocol is similar to the PETHEMA 2005, but changing idarubicin to daunorubicin. All patients with a suspected diagnosis of APL were immediately started on ATRA, while bone marrow samples were shipped to a national central lab where genetic verification of the diagnosis was performed. The immunofluorescence using an anti-PML antibody allowed a rapid confirmation of the diagnosis and, the importance of supportive measures was reinforced. Results: The interim analysis of 97 patients enrolled in the IC-APL protocol showed that complete remission (CR) rate was 83% and the 2-year overall survival and disease-free survival were 80% and 90%, respectively. Of note, the early mortality rate was reduced to 7.5%. Discussion: The results of IC-APL demonstrate the impact of educational programs and networking on the improvement of the leukemia treatment outcome in developing countries.

Performance of polymeric reinforcements in vehicle structures submitted to frontal impact

Preformed structural reinforcements have shown good performance in crash tests, where the great advantage is their weight. These reinforcements are designed with the aim of increasing the rigidity of regions with large deformations, thus stabilising sections of the vehicle that work as load path during impact. The objective of this work is to show the application of structural reinforcements made of polymeric material PA66 in the field of vehicle safety, through finite element simulations. Simulations of frontal impact at 50 km/h and in ODB (offset deformable barrier) at 57 km/h configurations (standards such as ECE R-94 and ECE R-12) were performed in the software LS-DYNA R (R) and MADYMO (R). The simulations showed that the use of polymeric reinforcements leads to a 70% reduction in A-pillar intrusion, a 65% reduction in the displacement of the steering column and a 59% reduction in the deformation in the region of the occupant legs and feet. The level of occupant injuries was analysed by MADYMO (R) software, and a reduction of 23.5% in the chest compression and 80% in the tibia compression were verified. According to the standard, such conditions lead to an improvement in the occupant safety in a vehicle collision event.

Serological detection of Plasmodium vivax malaria using recombinant proteins corresponding to the 19-kDa C-terminal region of the merozoite surface protein-1

Abstract Background Serological tests to detect antibodies specific to Plasmodium vivax could be a valuable tool for epidemiological studies, for screening blood donors in areas where the malaria is not endemic and for diagnosis of infected individuals. Because P. vivax cannot be easily obtained in vitro, ELISA assays using total or semi-purified antigens are rarely used. Based on this limitation, we tested whether recombinant proteins representing the 19 kDa C-terminal region of the merozoite surface protein-1 of P. vivax (MSP119) could be useful for serological detection of malaria infection. Methods Three purified recombinant proteins produced in Escherichia coli (GST-MSP119, His6-MSP119 and His6-MSP119-PADRE) and one in Pichia pastoris (yMSP119-PADRE) were compared for their ability to bind to IgG antibodies of individuals with patent P. vivax infection. The method was tested with 200 serum samples collected from individuals living in the north of Brazil in areas endemic for malaria, 53 serum samples from individuals exposed to Plasmodium falciparum infection and 177 serum samples from individuals never exposed to malaria. Results Overall, the sensitivity of the ELISA assessed with sera from naturally infected individuals was 95%. The proportion of serum samples that reacted with recombinant proteins GST-MSP119, His6-MSP119, His6-MSP119-PADRE and yMSP119-PADRE was 90%, 93.5%, 93.5% and 93.5%, respectively. The specificity values of the ELISA determined with sera from healthy individuals and from individuals with other infectious diseases were 98.3% (GST-MSP119), 97.7% (His6-MSP119 and His6-MSP119-PADRE) or 100% (yMSP119-PADRE). Conclusions Our study demonstrated that for the Brazilian population, an ELISA using a recombinant protein of the MSP119 can be used as the basis for the development of a valuable serological assay for the detection of P. vivax malaria.

Pathogenicity of Nc-Bahia and Nc-1 strains of Neospora caninum in experimentally infected cows and buffaloes in early pregnancy

Neospora caninum is a protozoan parasite known as an important cause of bovine abortion worldwide. Little is currently known about how different strains of N. caninum vary in their pathogenicity. In this study, we compared a Brazilian strain, Nc-Bahia, with the first isolate of this coccidian, Nc-1. Eight cows and seven buffaloes were submitted to fixed-time artificial insemination protocols for a better control of pregnancy. Group 1 was inoculated with Nc-Bahia (n=8; five cows and three buffaloes), and Group 2 was inoculated with Nc-1 (n=5; two cows and three buffaloes). One nonpregnant female of each species was left uninfected as sentinel controls for potential environmental infection. All inoculated animals received 5×108 tachyzoites of N. caninum, by intravenous route, on the 70th day of gestation. Uninfected animals remained seronegative throughout the experiment, indicating no exogenous infection, whereas all inoculated animals became seropositive to N. caninum. In Group 1, abortion was found in only one cow on 42 days postinfection (dpi; frequency of abortion=12.5 %), whilst all animals from Group 2 aborted on 35 dpi (frequency of abortion= 100 %). Parasite DNA was detected by seminested PCR in maternal, foetal and placental tissues, confirming vertical transmission in Groups 1 and 2, although histological lesions had different frequencies and degrees of severity between the groups. There was evidence of lower pathogenicity of Nc- Bahia compared to Nc-1 when used in experimental infection, as it caused fewer abortions, as well as less frequent and milder histological lesions. This was the first time Nc-Bahia has been used for experimental infection.