The AF-1 Domain of the Orphan Nuclear Receptor NOR-1 Mediates Trans-activation, Coactivator Recruitment, and Activation by the Purine Anti-metabolite 6-Mercaptopurine

NOR-1/NR4A3 is an orphan member of the nuclear hormone receptor superfamily. NOR-1 and its close relatives Nurr1 and Nur77 are members of the NR4A subgroup of nuclear receptors. Members of the NR4A subgroup are induced through multiple signal transduction pathways. They have been implicated in cell proliferation, differentiation, T-cell apoptosis, chondrosarcomas, neurological disorders, inflammation, and atherogenesis. However, the mechanism of transcriptional activation, coactivator recruitment, and agonist-mediated activation remain obscure. Hence, we examined the molecular basis of NOR-1-mediated activation. We observed that NOR-1 trans-activates gene expression in a cell- and target-specific manner; moreover, it operates in an activation function (AF)-1-dependent manner. The N-terminal AF-1 domain delimited to between amino acids 1 and 112, preferentially recruits the steroid receptor coactivator (SRC). Furthermore, SRC-2 modulates the activity of the AF-1 domain but not the C-terminal ligand binding domain (LBD). Homology modeling indicated that the NOR-1 LBD was substantially different from that of hRORbeta, a closely related AF-2-dependent receptor. In particular, the hydrophobic cleft characteristic of nuclear receptors was replaced with a very hydrophilic surface with a distinct topology. This observation may account for the inability of this nuclear receptor LBD to efficiently mediate cofactor recruitment and transcriptional activation. In contrast, the N-terminal AF-1 is necessary for cofactor recruitment and can independently conscript coactivators. Finally, we demonstrate that the purine anti-metabolite 6-mercaptopurine, a widely used antineoplastic and anti-inflammatory drug, activates NOR-1 in an AF-1-dependent manner. Additional 6-mercaptopurine analogs all efficiently activated NOR-1, suggesting that the signaling pathways that modulate proliferation via inhibition of de novo purine and/or nucleic acid biosynthesis are involved in the regulation NR4A activity. We hypothesize that the NR4A subgroup mediates the genotoxic stress response and suggest that this subgroup may function as sensors that respond to genotoxicity.

Development and biological evaluation of API-ILs based on anti-bacterial and anti-fungal drugs

In recent years Ionic Liquids (ILs) are being applied in life sciences. ILs are being produce with active pharmaceutical drugs (API) as they can reduce polymorphism and drug solubility problems [1] Also ILs are being applied as a drug delivery device in innovative therapies What is appealing in ILs is the ILs building up platform, the counter-ion can be carefully chosen in order to avoid undesirable side effects or to give innovative therapies in which two active ions are paired. This work shows ILs based on ampicillin (an anti-bacterial agent) and ILs based on Amphotericin B. Also we show studies that indicate that ILs based on Ampicillin could reverse resistance in some bacteria. The ILs produced in this work were synthetized by the neutralization method described in Ferraz et. al. [2] Ampicillin anion was combined with the following organic cations 1-ethyl-3-methylimidazolium, [EMIM]; 1-hydroxy-ethyl-3-methylimidazolium, [C2OHMIM]; choline, [cholin]; tetraethylammonium, [TEA]; cetylpyridinium, [C16pyr] and trihexyltetradecylphosphonium, [P6,6,6,14]. Amphotericin B was combined with [C16pyr], [cholin] and 1-metohyethyl-3-methylimidazolium, [C3OMIM]. The ILs-APIs based on ampicillin[2] were tested against sensitive Gram-negative bacteria Escherichia coli ATCC 25922 and Klebsiella pneumonia (clinical isolated), as well as on Gram positive Staphylococcus Aureus ATCC 25923, Staphylococcus epidermidis and Enterococcus faecalis. The arising resistance developed by bacteria to antibiotics is a serious public health threat and needs new and urgent measures. We study the bacterial activity of these compounds against a panel of resistant bacteria (clinical isolated strains): E. coli CTX M9, E. coli TEM CTX M9, E. coli TEM1, E. coli CTX M2, E. coli AmpC Mox2. In this work we demonstrate that is possible to produce ILs from anti-bacterial and anti-fungal compounds. We show here that the new ILs can reverse the bacteria resistance. With the careful choice of the organic cation, it is possible to create important biological and physic-chemical properties. This work also shows that the ion-pair is fundamental in ampicillin mechanism of action.

Acute Hepatitis B in a patient previously positive for antibody to surface antigen (anti-HBs) determined by radioimmunoassay: case report and review of the literature

The determination of anti-HBs as a screening test before vaccination has been advisable in order to encounter immune individuals that don't need to receive vaccine protection. A case-report is presented and three other cases are reviewed from the literature. Anti-HBs was positive in these health-care personnels that developped typical acute B hepatitis. Different subtyping involving the d/y determinants were found in the first case, but false-positive anti-HBs even with high titres, determined by RIA, were found in the other cases. Concomitant determination of anti-HBc or absence of screening tests seem to be more reasonable policies until a low-cost and risk-free vaccine is produced.

Prevalence of anti-p: Falciparum sporozoite antibodies in adults in the amapa region of Brazil

17 of 20 adult sera from the Amapa region of Brazil were active in the inhibition of P. falciparum sporozoite invasion (ISI) assay which has been correlated with protective antibodies. In contrast 11 sera were positive in IFA tests and 6 were positive in CSP tests. These results suggest that the ISI assay will be useful for evaluating naturally acquired protective anti-sporozoite antibodies in endemic areas, particularly during vaccine efficacy studies using sporozoite-based vaccines.

Passive haemagglutination test for human neurocysticercosis immunodiagnosis: I. Standardization and evaluation of the passive haemagglutination test for the detection of anti-Cysticercus cellulosae antibodies

A passive haemagglutination test (PHA) for human neurocysticercosis was standardized and evaluated for the detection of specific antibodies to Cysticercus cellulosae in cerebrospinal fluid (CSF). For the assay, formaldehyde-treated group O Rh-human red cells coated with the cysticerci crude total saline extract (TS) antigen were employed. A total of 115 CSF samples from patients with neurocysticercosis was analysed, of these 94 presented reactivity, corresponding to 81.7% sensitivity, in which confidence limit of 95% probability (CL95%) ranged from 74.5% to 88.9%. Eighty-nine CSF samples derived from individuals of control group presented as nonreactive in 94.4% (CL95% from 89.6% to 99.2%). The positive and negative predictive values were 1.4% and 99.9%, respectively, considering the mean rate of that this assay provide a rapid, highly reproducible, and moderately sensitive mean of detecting specific antibodies in CSF samples.

Anti-vibration gloves and the forearm efforts during tools operations

The objective of this paper is to analyze the forearm muscular contraction levels associated to the use of anti-vibration gloves, by comparing the contraction levels with gloves and without gloves. Two different vibration tools were used in a simulated work environment: (1) A compact Duty Multi-Cutter Bosch and (2) and a Percussion Drill with a drill bit Ø20 mm. Standard operations were performed by each subject in the following materials: (1) Performing cross- sectional cuts in 80x40 mm pine section and (2) performing 20 mm diameter holes in a concrete slab 2 x 2 m, 70 mm thick. The forearm contraction level were measured by surface electromyography in four different muscles: Flexor Digitorum Superficialis (FDS), Flexor Carpi Ulnaris (FCU), Extensor Carpi Radialis Longus (ECRL) e Extensor Carpi Ulnaris (ECU). For the flexor muscles (FDS, FCU), an increase tendency in muscular contraction was observed when the operations are performed without gloves (2-5% MVE increase in the FDS and 3-9% MVE increase in the FCU). For the extensor muscles ECU a decrease tendency in muscular contraction was observed when the operations are performed without gloves (1-10% MVE decrease). Any tendency was found in the ECRL muscle. ECU was the muscle with the highest contraction level for 79% and 71% of the operators, during the operations respectively with the multi-cutter (P50= 27-30%MVE) and with the percussion drill (P50=46-55%MVE). As a final conclusion from this study, anti- vibration gloves may increase the forearm fatigue in the posterior region of the forearm (ECU muscle) during operations with the mentioned tools

Comparison of serum hepatitis B virus replication markers in patients with chronic hepatitis B: studies on HBeAg/Anti-HBe system, viral dna polymerase and HBV-DNA

The detection of HBV-DNA in serum by molecular hybridization is the most sensitive and specific marker of replication and infectivity of hepatitis B virus and currently is proposed as a routine diagnostic technique in the follow-up of HBV - related diseases. Comparing different techniques already described, we found that direct spotting of serum samples on nitrocellulose membranes under vacuum filtration, followed by denaturing and neutralizing washes is more practical, simple, sensible and reproducible. DNA polymerase assay using phosphonoformic acid as specific viral inhibitor has shown 86.8% of concordance with HBV-DNA detection, and so, it is an useful alternative in the follow-up of hepatitis B chronic patients. We found 19.2% HBeAg positive samples with no other markers of viral replication and no anti-HBe positive sample had detectable HBV-DNA. Discordance between the 2 systems have been extensively described, and we confirm this for the first time in our country. Molecular biological techniques are essential to determine the replication status of chronic hepatitis B patients.

Enzyme linked immunosorbent assay: determination of anti-adenovirus antibodies in an infant population

In order to define an accurate assay for anti-adenovirus antibody detection, a recently developed ELISA was compared with IFA and CF. On 58 sera, the ELISA was more sensitive than both CF and IFA, which showed relative sensitivities of 63% and 94%, respectively. It was not possible to determine the exact specificity of the tests because of the lack of a gold standard. Furthermore, the ELISA was used to define the prevalence of adenovirus antibodies in 116 infants between 1 and 24 months old (mean 7.28). The data showed that maternal antibodies waned by the age of 5 to 6 months and that more than 80% of the children had been infected by adenoviruses by the age of 10 months.

Seasonal variation of anti-resa/Pf155 Plasmodium falciparum antibodies in three localities from the state of Amapá, Brazil

Anti-RESA/Pf155 antibodies were assayed in sera of individuals from three localities (Laranjal do Jari, Vila Padaria and Vila Paraíso) in the State of Amapá, Brazil, during the long-rains and short-rains seasons. All of these had negative blood smears for malaria. Most of the sera collected were positive in Indirect Fluorescent Antibody (IFA) with P. falciparum parasites, with no seasonal variation. A high percentage of these sera (62% to 100%) was RESA positive by Modified Indirect Fluorescent Antibody (MIFA), with a significant (p < 0.05) increase of geometric mean titers during the short-rains season, when the transmission of the disease is highest. ELISA with three repetitive RESA peptides (EENV)3 (4x3), (EENVEHDA)2 (8x2) and (DDEHVEEPTVA)2(11x2) did not reveal statistically significant seasonal variations, although a small enhancement of positivity was observed in V. Padaria (15.3 to 38.8%) in the short-rains season with the 8x2 peptides, and with 4x3 and 8x2 peptides in V. Paraíso, with a decrease in 11x2. MIFA titers appeared to be correlated mainly to the peptide 4x3 and it was the immunodominant in the three localities.

Anti-HCV related to HCV PCR and risk factors analysis in a blood donor population of Central Brazil

Data concerning HCV infection in Central Brazil are rare. Upon testing 2,350 voluntary blood donors from this region, we found anti-HCV prevalence rates of 2.2% by a second generation ELISA and 1.4% after confirmation by a line immunoassay. Antibodies against core, NS4, and NS5 antigens of HCV were detected in 81.8%, 72.7%, and 57.5%, respectively, of the positive samples in the line immunoassay. HCV viremia was present in 76.6% of the anti-HCV-positive blood donors. A relation was observed between PCR positivity and serum reactivity in recognizing different HCV antigens in the line immunoassay. The majority of the positive donors had history of previous parenteral exposure. While the combination of ALT>50 IU/l and anti-HBc positivity do not appear to be good surrogate markers for HCV infection, the use of both ALT anti-HCV tests is indicated in the screening of Brazilian blood donors.

Dot-ELISA for the detection of anti-Cysticercus cellulosae antibodies in cerebrospinal fluid using a new solid phase (resin-treated polyester fabric) and Cysticercus longicollis antigens

A dot-ELISA was developed for the detection of antibodies in CSF in the immunologic diagnosis of human neurocysticercosis, using antigen extracts of the membrane and scolex of Cysticercus cellulosae (M+S-Cc) and, alternately, membrane (M) and vesicular fluid (VF) of Cysticercus longicollis (Cl) covalently bound to a new solid phase consisting of polyester fabric treated with N-methylol-acrylamide resin (dot-RT). The test was performed at room temperature, with reduced incubation times and with no need for special care in the manipulation of the support. The sensitivity rates obtained were 95.1% for antigen Cc and 97.6% for antigen Cl. Specificity was 90.6% when Cc was used, and 96.9% and 100% when M-Cl and VF-Cl were used, respectively. No significant differences in titer were observed between tests carried out with homologous and heterologous antigens. The low cost and easy execution of the dot-RT test using antigen extracts of Cysticercus longicollis indicate the test for use in the immunodiagnosis of human neurocysticercosis.

ANTI M. leprae IgM ANTIBODY DETERMINATION BY ULTRAMICROIMMUNOENZYMATIC (UMELISA HANSEN) FOR THE DIAGNOSIS AND MONITORING LEPROSY

The relationship between the IgM antibody response, antigenic load as well as the clinical improvement after chemotherapy was studied in order to obtain useful data for the early diagnosis and monitoring leprosy. A level of 82% (94/115) agreement was obtained between IgM UMELISA HANSEN and slitskin smear examination. Discrepant results were observed in 16 patients who showed positive IgM response despite negative by the skin smear examination. In these patients, the IgM response was seen to be associated to the early signal for bacilli recurrence in the skin. In one of these patients the presence of bacilli was demonstrated in the skin, two months after IgM antibodies being detected by UMELISA HANSEN. Also in one of the treated patients positive by both diagnostic techniques, a remarkable decrease in the IgM antibody levels was seen, correlating with a significant clinical improvement. Moreover it was found a direct relationship between the IgM antibody response and bacterial antigenic load, regardless the time elapsed in the disease's evolution.

PREVALENCE OF ANTI- Toxocara ANTIBODIES IN A RANDOM SAMPLE OF INPATIENTS AT A CHILDREN'S HOSPITAL IN VITÓRIA, ESPÍRITO SANTO, BRAZIL

In the streets of Vitória, in the State of Espírito Santo, Brazil, are large number of stray dogs, many of which are infected with Toxocara canis, suggesting a high risk for human infection. In order to investigate the prevalence of Toxocara infection in children in Espírito Santo we studied the prevalence of anti-Toxocara antibodies in 100 random inpatients over one year of age, at the Children's Hospital N.S. da Glória, the reference children's hospital for the State.All the sera were collected during the period between October 1996 and January 1997. The mean age was 6.6±4.1 yrs. (1 to 14 yrs., median 6yrs.) and there were patients from all of the different wards of the hospital. Sixty-eigth patients came from the metropolitan area of Vitória and the other 32 from 17 other municipalities. The anti-Toxocara antibodies were investigated by ELISA-IgG using a secretory-excretory antigen obtained from second stage larvae. All sera were adsorbed with Ascaris suum antigen before the test. Thirty-nine sera (39%) were positive, predominantly from boys, but the gender difference was not statistically significant (boys:25/56 or 44.6%; girls:14/44 or 31.8%; p=0.311). The prevalence of positive sera was higher, but not statistically significant, in children from the urban periphery of metropolitan Vitória (formed by the cities of Vitória, Cariacica, Vila Velha, Serra and Viana) than in children from 17 other municipalities (44.1% and 28.1% respectively, p=0.190). Although the samples studied do not represent all children living in the State of Espírito Santo, since the Children's Hospital N.S. da Glória admits only patients from the state health system, it is probable that these results indicate a high frequency of Toxocara infection in children living in Espírito Santo. Further studies of population samples are necessary to ascertain the prevalence of Toxocara infection in our country.