The Lectin ArtinM Induces Recruitment of Rat Mast Cells from the Bone Marrow to the Peritoneal Cavity

Background: The D-mannose binding lectin ArtinM is known to recruit neutrophils, to degranulate mast cells and may have potential therapeutic applications. However, the effect of ArtinM on mast cell recruitment has not been investigated. Methodology: Male Wistar rats were injected i.p. with ArtinM or ConA (control). The ability of the lectin to degranulate peritoneal and mesenteric mast cells was examined. Recruitment of mast cells to the peritoneal cavity and mesentery after ArtinM injection was examined with or without depletion of peritoneal mast cells by distilled water. Results: ArtinM degranulated both peritoneal and mesentery mast cells in vitro. Three days after i.p. injection of the lectin there were reduced numbers of mast cells in the peritoneal lavage, while at 7 days post injection of ArtinM, the number of peritoneal mast cells was close to control values. Since immature mast cells are recruited from the bone marrow, the effect of the lectin on bone marrow mast cells was examined. Injection of ArtinM resulted in an increased number of mast cells in the bone marrow. To determine if degranulation of mast cells in the peritoneal cavity was required for the increase in bone marrow mast cells, the peritoneal cavity was depleted of mast cells with ultrapure water. Exposure to ArtinM increased the number of mast cells in the bone marrow of rats depleted of peritoneal mast cells. Conclusions: The ArtinM induced recruitment of mast cells from the bone marrow to the peritoneal cavity may partially explain the therapeutic actions of ArtinM.

Endothelial Nitric Oxide Genotypes and Haplotypes Are Not Associated with End-Stage Renal Disease

The identification of genetic markers associated with chronic kidney disease (CKD) may help to predict its development. Because reduced nitric oxide (NO) bioavailability and endothelial dysfunction are involved in CKD, genetic polymorphisms in the gene encoding the enzyme involved in NO synthesis (endothelial NO synthase [eNos]) may affect the susceptibility to CKD and the development of end-stage renal disease (ESRD). We compared genotype and haplotype distributions of three relevant eNOS polymorphisms (T(-786) C in the promoter region, Glu298Asp in exon 7, and 4b/4a in intron 4) in 110 healthy control subjects and 127 ESRD patients. Genotypes for the T(-786) C and Glu298Asp polymorphisms were determined by TaqMan (R) Allele Discrimination assay and real-time polymerase chain reaction. Genotypes for the intron 4 polymorphism were determined by polymerase chain reaction and fragment separation by electrophoresis. The software program PHASE 2.1 was used to estimate the haplotypes frequencies. We considered significant a probability value of p < 0.05/number of haplotypes (p < 0.05/8 = 0.0063). We found no significant differences between groups with respect to age, ethnicity, and gender. CKD patients had higher blood pressure, total cholesterol, and creatinine levels than healthy control subjects (all p < 0.05). Genotype and allele distributions for the three eNOS polymorphisms were similar in both groups (p > 0.05). We found no significant differences in haplotype distribution between groups (p > 0.05). The lack of significant associations between eNOS polymorphisms and ESRD suggests that eNOS polymorphisms may not be relevant to the genetic component of CKD that leads to ESRD.

Genetic polymorphisms in TLR4, CR1 and Duffy genes are not associated with malaria resistance in patients from Baixo Amazonas region, Brazil

The main purpose of this research was to analyze the relation of the genetic polymorphisms frequently expressed by antigen-presenting cells, erythrocytes and malaria susceptibility/resistance with the human malaria infection cases. The sample used consisted of 23 Plasmodium vivax ( Pv)- and P. falciparum ( Pf)-infected patients, and 21 healthy individuals as a control group, from the Baixo Amazonas population in Para, Brazil. The Asp299Gly polymorphisms in the Toll-like receptor 4 ( TLR4), and Gly42Asp, Arg89Cys, Ala100Thr, and T-33C in the Duffy gene ( FY) were analyzed by restriction fragment length polymorphism-polymerase chain reaction. The Lys1590Glu and Arg1601Gly polymorphisms in the complement receptor type 1 (CR1) were analyzed by DNA sequencing. According to the results obtained and statistical analysis considering a significance level or alpha = 0.01, we conclude that the low heterozygote frequency (2.27%) for the Asp299Gly mutation, detected in the TLR4 gene, is not related to the Pv and Pf infections in the patients analyzed. Also, the promoter region GATA-1 analysis of the FY gene in the Pv-infected patients showed that the heterozygote frequency for the T-33C mutation (11.36% of the infected patients and 20.45% of the control patients) is not related to infection resistance. Regarding the CR1 gene, the observed heterozygote frequency (9.09%) for the Arg1601Gly mutation in Pf-infected patients when compared to heterozygote frequency in the control group (18.18%) suggests that there is no correlation with infection resistance.

Postural changes in women with chronic pelvic pain: a case control study

Background: Chronic pelvic pain (CPP) is a lower abdominal pain lasting at least 6 months, occurring continuously or intermittently and not associated exclusively with menstruation or intercourse. Although the musculoskeletal system has been found to be involved in CPP, few studies have assessed the contribution of posture in women with CPP. We aimed to determine if the frequency of postural changes was higher in women with CPP than healthy subjects. Methods: A case-control study included 108 women with CPP of more than 6 months' duration (CPP group) who consecutively attended at the Hospital of the University of Sao Paulo and 48 healthy female volunteers (control group). Postural assessment was noninvasive and performed in the standing position, with the reference points of Kendall used as normal parameters. Factors associated with CPP were assessed by logistic regression analysis. Results: Logistic regression showed that the independent factors associated with CPP were postural changes in the cervical spine (OR 4.1; 95% CI 1.6-10.7; p < 0.01) and scapulae (OR 2.9; 95% CI 1.1-7.6; p < 0.05). Conclusion: Musculoskeletal changes were associated with CPP in 34% of women. These findings suggest that a more detailed assessment of women with CPP is necessary for better diagnosis and for more effective treatment.

Selection of suitable housekeeping genes for expression analysis in glioblastoma using quantitative RT-PCR

Background: Considering the broad variation in the expression of housekeeping genes among tissues and experimental situations, studies using quantitative RT-PCR require strict definition of adequate endogenous controls. For glioblastoma, the most common type of tumor in the central nervous system, there was no previous report regarding this issue. Results: Here we show that amongst seven frequently used housekeeping genes TBP and HPRT1 are adequate references for glioblastoma gene expression analysis. Evaluation of the expression levels of 12 target genes utilizing different endogenous controls revealed that the normalization method applied might introduce errors in the estimation of relative quantities. Genes presenting expression levels which do not significantly differ between tumor and normal tissues can be considered either increased or decreased if unsuitable reference genes are applied. Most importantly, genes showing significant differences in expression levels between tumor and normal tissues can be missed. We also demonstrated that the Holliday Junction Recognizing Protein, a novel DNA repair protein over expressed in lung cancer, is extremely over-expressed in glioblastoma, with a median change of about 134 fold. Conclusion: Altogether, our data show the relevance of previous validation of candidate control genes for each experimental model and indicate TBP plus HPRT1 as suitable references for studies on glioblastoma gene expression.

In Vitro Identification of Novel Plasminogen-Binding Receptors of the Pathogen Leptospira interrogans

Background: Leptospirosis is a multisystem disease caused by pathogenic strains of the genus Leptospira. We have reported that Leptospira are able to bind plasminogen (PLG), to generate active plasmin in the presence of activator, and to degrade purified extracellular matrix fibronectin. Methodology/Principal Findings: We have now cloned, expressed and purified 14 leptospiral recombinant proteins. The proteins were confirmed to be surface exposed by immunofluorescence microscopy and were evaluated for their ability to bind plasminogen (PLG). We identified eight as PLG-binding proteins, including the major outer membrane protein LipL32, the previously published rLIC12730, rLIC10494, Lp29, Lp49, LipL40 and MPL36, and one novel leptospiral protein, rLIC12238. Bound PLG could be converted to plasmin by the addition of urokinase-type PLG activator (uPA), showing specific proteolytic activity, as assessed by its reaction with the chromogenic plasmin substrate, D-Val-Leu-Lys 4-nitroanilide dihydrochloride. The addition of the lysine analog 6-aminocaproic acid (ACA) inhibited the protein-PLG interaction, thus strongly suggesting the involvement of lysine residues in plasminogen binding. The binding of leptospiral surface proteins to PLG was specific, dose-dependent and saturable. PLG and collagen type IV competed with LipL32 protein for the same binding site, whereas separate binding sites were observed for plasma fibronectin. Conclusions/Significance: PLG-binding/activation through the proteins/receptors on the surface of Leptospira could help the bacteria to specifically overcome tissue barriers, facilitating its spread throughout the host.

VIABILITY OF SPORULATED OOCYSTS OF NEOSPORA CANINUM AFTER EXPOSURE TO DIFFERENT PHYSICAL AND CHEMICAL TREATMENTS

The aim of the present study was to evaluate the viability of Neospora caninum sporulated oocysts after various chemical and physical treatments. Bioassays in gerbils and molecular techniques (PCR-RFLP) were used for identification of the oocysts shed by experimentally infected dogs. Sporulated oocysts were purified and divided into 11 treatment groups as follows: absolute ethanol for 1 hr; 20 C for 6 hr; 4 C for 6 hr; 60 C for 1 min; 100 C for 1 min; 10% formaldehyde for 1 hr; 10% ammonia for 1 hr; 2% iodine for 1 hr; 10% sodium hypochlorite for I hr; 70% ethanol for I hr; and one group was left untreated and kept as a positive control. All chemical treatments were performed at room temperature (37 C). A total of 33 gerbils, or 3 gerbils per treatment, were used for bioassays. After treatment, the oocysts were divided into aliquots of 1,000 oocysts and orally administered to gerbils. After 63 days, the gerbils were anesthetized and killed with 0.2 ml of T61; blood and tissue samples were collected for serological (IFAT and western blotting), molecular (real-time PCR), histopathology, and immunohistochemical tests. Treatments were considered effective only if all 5 detection techniques tested negative. High temperatures at 100 C for 1 min and 10% sodium hypochlorite for 1 hr were the only treatments that met this condition, effectively inactivating all oocysts.

Revisiting clinical trials on glycemic control and cardiovascular risk

The most relevant clinical trials, assessing the role of glycemic control in reducing cardiovascular risk, are examined. The UKPDS was the first to address this issue. More recent trials (ACCORD, ADVANCE and VADT) are controversial and evidences did not support that strict glycemic control (reflected by normal glycated hemoglobin) exclusively is sufficient to reduce cardiovascular risk in complicated individuals with long-term type 2 diabetes mellitus. Some possible reasons for controversies are included.

Identification of three distinguishable phenotypes in golden retriever muscular dystrophy

Duchenne muscular dystrophy (DMD) is a human disease characterized by progressive and irreversible skeletal muscle degeneration caused by mutations in genes coding for important muscle proteins. Unfortunately, there is no efficient treatment for this disease; it causes progressive loss of motor and muscular ability until death. The canine model (golden retriever muscular dystrophy) is similar to DMD, showing similar clinical signs. Fifteen dogs were followed from birth and closely observed for clinical signs. Dogs had their disease status confirmed by polymerase chain reaction analysis and genotyping. Clinical observations of musculoskeletal, morphological, gastrointestinal, respiratory, cardiovascular, and renal features allowed us to identify three distinguishable phenotypes in dystrophic dogs: mild (grade I), moderate (grade II) and severe (grade III). These three groups showed no difference in dystrophic alterations of muscle morphology and creatine kinase levels. This information will be useful for therapeutic trials, because DMD also shows significant, inter- and intra-familiar clinical variability. Additionally, being aware of phenotypic differences in this animal model is essential for correct interpretation and understanding of results obtained in pre-clinical trials.

A Lagrangian Identification of the Main Sources of Moisture Affecting Northeastern Brazil during Its Pre-Rainy and Rainy Seasons

This work examines the sources of moisture affecting the semi-arid Brazilian Northeast (NEB) during its pre-rainy and rainy season (JFMAM) through a Lagrangian diagnosis method. The FLEXPART model identifies the humidity contributions to the moisture budget over a region through the continuous computation of changes in the specific humidity along back or forward trajectories up to 10 days period. The numerical experiments were done for the period that spans between 2000 and 2004 and results were aggregated on a monthly basis. Results show that besides a minor local recycling component, the vast majority of moisture reaching NEB area is originated in the south Atlantic basin and that the nearby wet Amazon basin bears almost no impact. Moreover, although the maximum precipitation in the ""Poligono das Secas'' region (PS) occurs in March and the maximum precipitation associated with air parcels emanating from the South Atlantic towards PS is observed along January to March, the highest moisture contribution from this oceanic region occurs slightly later (April). A dynamical analysis suggests that the maximum precipitation observed in the PS sector does not coincide with the maximum moisture supply probably due to the combined effect of the Walker and Hadley cells in inhibiting the rising motions over the region in the months following April.

Optical identification of the transient supersoft X-ray source RX J0527.8-6954, in the LMC

Context. Close binary supersoft X-ray sources (CBSS) are binary systems that contain a white dwarf with stable nuclear burning on its surface. These sources, first discovered in the Magellanic Clouds, have high accretion rates and near-Eddington luminosities (10(37)-10(38) erg s(-1)) with high temperatures (T = 2-7 x 10(5) K). Aims. The total number of known objects in the MC is still small and, in our galaxy, even smaller. We observed the field of the unidentified transient supersoft X-ray source RX J0527.8-6954 in order to identify its optical counterpart. Methods. The observation was made with the IFU-GMOS on the Gemini South telescope with the purpose of identifying stars with possible He II or Balmer emission or else of observing nebular extended jets or ionization cones, features that may be expected in CBSS. Results. The X-ray source is identified with a B5e V star that is associated with subarcsecond extended H alpha emission, possibly bipolar. Conclusions. If the primary star is a white dwarf, as suggested by the supersoft X-ray spectrum, the expected orbital period exceeds 21 h; therefore, we believe that the 9.4 h period found so far is not associated to this system.

PFLEIDERER 2: IDENTIFICATION OF A NEW GLOBULAR CLUSTER IN THE GALAXY

We provide evidence that indicates the star cluster Pfleiderer 2, which is projected in a rich field, as a newly identified Galactic globular cluster. Since it is located in a crowded field, core extraction and decontamination tools were applied to reveal the cluster sequences in B, V, and I color-magnitude diagrams (CMDs). The main CMD features of Pfleiderer 2 are a tilted red giant branch and a red horizontal branch, indicating a high metallicity around solar. The reddening is E(B - V) = 1.01. The globular cluster is located at a distance of d(circle dot) = 16 +/- 2 kpc from the Sun. The cluster is located 2.7 kpc above the Galactic plane and at a distance of R(GC) = 9.7 kpc from the Galactic center, which is unusual for a metal-rich globular cluster.

Endophytic and entomopathogenic strains of Beauveria sp to control the bovine tick Rhipicephalus (Boophilus) microplus

Pathogenicity of strains of the entomopathogenic fungus Beauveria bassiana and endophytic strains of Beauveria sp against the bovine tick Rhipicephalus (Boophilus) microplus was tested in laboratory bioassays and under field conditions. Suspensions containing 10(5), 10(7) and 10(9) conidia/mL were prepared of each fungal strain for laboratory bioassays. The ticks were maintained at 28 degrees C, 90 +/- 5% relative humidity, and the following variables were evaluated: initial female weight, egg weight, hatching percentage, reproductive efficiency, and percentage control. For tests under field conditions, a Beauveria suspension containing 10(6) conidia/mL was sprayed on tick-infested cows. After 72 h, the ticks were collected to estimate mortality under field conditions. Laboratory bioassays showed a mortality of 20 to 50% of the ticks seven days after inoculation with 10(7) Beauveria conidia/mL. Under field conditions 10(6) Beauveria conidia/mL induced 18-32% mortality. All Beauveria strains were effective in biological control of R. (Boophilus) microplus under laboratory and field test conditions. This is the first demonstration that endophytic fungi can be used for biological control of the cattle tick; this could help reduce environmental contamination by diminishing the need for chemical acaricides. Two endophytic strains were isolated from maize leaves and characterized by molecular sequencing of 5.8S rDNA ITS1 and ITS2 and morphological analyses of conidia. We found that these two endophytic Beauveria isolates, designated B95 and B157, are close to Beauveria amorpha.

Detection of polymorphisms of the mtDNA control region of Caretta caretta (Testudines: Cheloniidae) by PCR-SSCP

Marine turtles are increasingly being threatened worldwide by anthropogenic activities. Better understanding of their life cycle, behavior and population structure is imperative for the design of adequate conservation strategies. The mtDNA control region is a fast-evolving matrilineal marker that has been employed in the study of marine turtle populations. We developed and tested a simple molecular tracing system for Caretta caretta mtDNA haplotypes by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP). Using this technique, we were able to distinguish the SSCP patterns of 18 individuals of the haplotypes CC-A4, CC-A24 and CCxLO, which are commonly found in turtles sampled on the Brazilian coast. When we analyzed 15 turtles with previously unknown sequences, we detected two other haplotypes, in addition to the other four. Based on DNA sequencing, they were identified as the CC-A17 and CC-A1 haplotypes. Further analyses were made with the sea turtles, Chelonia mydas (N = 8), Lepidochelys olivacea (N = 3) and Eretmochelys imbricata (N = 1), demonstrating that the PCR-SSCP technique is able to distinguish intra-and interspecific variation in the family Cheloniidae. We found that this technique can be useful for identifying sea turtle mtDNA haplotypes, reducing the need for sequencing.

Control of the Intracellular Redox State by Glucose Participates in the Insulin Secretion Mechanism

Background: Production of reactive oxygen species (ROS) due to chronic exposure to glucose has been associated with impaired beta cell function and diabetes. However, physiologically, beta cells are well equipped to deal with episodic glucose loads, to which they respond with a fine tuned glucose-stimulated insulin secretion (GSIS). In the present study, a systematic investigation in rat pancreatic islets about the changes in the redox environment induced by acute exposure to glucose was carried out. Methodology/Principal Findings: Short term incubations were performed in isolated rat pancreatic islets. Glucose dose- and time-dependently reduced the intracellular ROS content in pancreatic islets as assayed by fluorescence in a confocal microscope. This decrease was due to activation of pentose-phosphate pathway (PPP). Inhibition of PPP blunted the redox control as well as GSIS in a dose-dependent manner. The addition of low doses of ROS scavengers at high glucose concentration acutely improved beta cell function. The ROS scavenger N-acetyl-L-cysteine increased the intracellular calcium response to glucose that was associated with a small decrease in ROS content. Additionally, the presence of the hydrogen peroxide-specific scavenger catalase, in its membrane-permeable form, nearly doubled glucose metabolism. Interestingly, though an increase in GSIS was also observed, this did not match the effect on glucose metabolism. Conclusions: The control of ROS content via PPP activation by glucose importantly contributes to the mechanisms that couple the glucose stimulus to insulin secretion. Moreover, we identified intracellular hydrogen peroxide as an inhibitor of glucose metabolism intrinsic to rat pancreatic islets. These findings suggest that the intracellular adjustment of the redox environment by glucose plays an important role in the mechanism of GSIS.