SEQUENCE OF A CDNA-ENCODING BOTHROPSTOXIN-I, A MYOTOXIN FROM THE VENOM OF BOTHROPS-JARARACUSSU

With the aim of further understanding the structure/function relationships in the membrane-damaging activity of the Lys(49) phospholipase A(2) (Lys(49)-PLA(2)) sub-family, we used PCR (polymerase chain reaction) on total venom gland cDNAs from Bothrops jararacussu with degenerate oligodeoxyribonucleotides encoding the N- and C-termini of myotoxin II, a Lys(49)-PLA(2) from Bothrops asper. A 350-bp cDNA coding for bothropstoxin I (BtxtxI) was amplified. Sequencing of the amplified fragment shows that BtxtxI has a Lys(49), and comparison with the known structure of myotoxin II showed that the amino acids involved in the formation of a novel dimeric structure in this protein were also conserved.

Single dose of a vaccine based on DNA encoding mycobacterial hsp65 protein plus TDM-loaded PLGA microspheres protects mice against a virulent strain of Mycobacterium tuberculosis

The high incidence of tuberculosis around the world and the inability of BCG to protect certain populations clearly indicate that an improved vaccine against tuberculosis is needed. A single antigen, the mycobacterial heat shock protein hsp65, is sufficient to protect BALB/c mice against challenge infection when administered as DNA vaccine in a three-dose-based schedule. In order to simplify the vaccination schedule, we coencapsulated hsp65-DNA and trehalose dimicolate (TDM) into biodegradable poly(DL-lactide-co-glycolide) (PLGA) microspheres. BALB/c mice immunized with a single dose of DNA-hsp65/TDM-1oaded microspheres produced high levels of IgG2a subtype antibody and high amounts of IFN-gamma in the supernatant of spleen cell cultures. DNA-hsp65/TDM-loaded microspheres were also able to induce high IFN-gamma production in bulk lung cells from challenged mice and confer protection as effective as that attained after three doses of naked DNA administration. This new formulation also allowed a ten-fold reduction in the DNA dose when compared to naked DNA. Thus, this combination of DNA vaccine and adjuvants with immunomodulatory and carrier properties holds the potential for an improved vaccine against tuberculosis.

Cloning and expression of the cDNA encoding rat granulocyte colony-stimulating factor

Granulocyte colony-stimulating factor (G-CSF) acts on precursor hematopoietic cells to control the production and maintenance of neutrophils. Recombinant G-CSF (re-G-CSF)is used clinically to treat patients with neutropenia and has greatly reduced the infection risk associated with bone marrow transplantation. Cyclic hematopoiesis, a stem cell defect characterized by severe recurrent neutropenia, occurs in man and grey collie dogs, and can be treated by administration of re-G-CSF. Availability of the rat G-CSF cDNA would benefit the use of rats as models of gene therapy for the treatment of cyclic hematopoiesis. In preliminary rat experiments, retroviral-mediated expression of canine G-CSF caused neutralizing antibody formation which precluded long-term increases in neutrophil counts. To overcome this problem we cloned the rat G-CSF cDNA from RNA isolated from skin fibroblasts. The rat G-CSF sequence shared a high degree of identity in both the coding and non-coding regions with both the murine G-CSF (85%) and human G-CSF (74%). The signal peptides of murine and human G-CSF both contained 30 amino acids (aa), whereas the deduced signal sequence for rat G-CSF possessed 21 aa. A retrovirus encoding the rat G-CSF cDNA synthesized bioactive G-CSF from transduced vascular smooth muscle cells.

Tissue distribution of a plasmid DNA encoding Hsp65 gene is dependent on the dose administered through intramuscular delivery

In order to assess a new strategy of DNA vaccine for a more complete understanding of its action in immune response, it is important to determine the in vivo biodistribution fate and antigen expression. In previous studies, our group focused on the prophylactic and therapeutic use of a plasmid DNA encoding the Mycobacterium leprae 65-kDa heat shock protein (Hsp65) and achieved an efficient immune response induction as well as protection against virulent M. tuberculosis challenge. In the present study, we examined in vivo tissue distribution of naked DNA-Hsp65 vaccine, the Hsp65 message, genome integration and methylation status of plasmid DNA. The DNA-Hsp65 was detectable in several tissue types, indicating that DNA-Hsp65 disseminates widely throughout the body. The biodistribution was dose-dependent. In contrast, RT-PCR detected the Hsp65 message for at least 15 days in muscle or liver tissue from immunized mice. We also analyzed the methylation status and integration of the injected plasmid DNA into the host cellular genome. The bacterial methylation pattern persisted for at least 6 months, indicating that the plasmid DNA-Hsp65 does not replicate in mammalian tissue, and Southern blot analysis showed that plasmid DNA was not integrated. These results have important implications for the use of DNA-Hsp65 vaccine in a clinical setting and open new perspectives for DNA vaccines and new considerations about the inoculation site and delivery system. © 2006 Coelho-Castelo et al; licensee BioMed Central Ltd.

Methods and tools for encoding the WordNet.Br sentences, concept glosses, and conceptual-semantic relations

This paper presents the overall methodology that has been used to encode both the Brazilian Portuguese WordNet (WordNet.Br) standard language-independent conceptual-semantic relations (hyponymy, co-hyponymy, meronymy, cause, and entailment) and the so-called cross-lingual conceptual-semantic relations between different wordnets. Accordingly, after contextualizing the project and outlining the current lexical database structure and statistics, it describes the WordNet.Br editing GUI that was designed to aid the linguist in carrying out the tasks of building synsets, selecting sample sentences from corpora, writing synset concept glosses, and encoding both language-independent conceptual-semantic relations and cross-lingual conceptual-semantic relations between WordNet.Br and Princeton WordNet © Springer-Verlag Berlin Heidelberg 2006.

Dietary restriction abrogates antibody production induced by a DNA vaccine encoding the mycobacterial 65 kDa heat shock protein

Background: Protein-calorie malnutrition (PCM) is the most common type of malnutrition. PCM leads to immunodeficiency and consequent increased susceptibility to infectious agents. In addition, responses to prophylactic vaccines depend on nutritional status. This study aims to evaluate the ability of undernourished mice to mount an immune response to a genetic vaccine (pVAXhsp65) against tuberculosis, containing the gene coding for the heat shock protein 65 from mycobacteria. Methods: Young adult female BALB/c mice were fed ad libitum or with 80% of the amount of food consumed by a normal diet group. We initially characterized a mice model of dietary restriction by determining body and spleen weights, hematological parameters and histopathological changes in lymphoid organs. The ability of splenic cells to produce IFN-gamma and IL-4 upon in vitro stimulation with LPS or S. aureus and the serum titer of specific IgG1 and IgG2a anti-hsp65 antibodies after intramuscular immunization with pVAXhsp65 was then tested. Results: Dietary restriction significantly decreased body and spleen weights and also the total lymphocyte count in blood. This restriction also determined a striking atrophy in lymphoid organs as spleen, thymus and lymphoid tissue associated with the small intestine. Specific antibodies were not detected in mice submitted to dietary restriction whereas the well nourished animals produced significant levels of both, IgG1 and IgG2a anti-hsp65. Conclusion: 20% restriction in food intake deeply compromised humoral immunity induced by a genetic vaccine, alerting, therefore, for the relevance of the nutritional condition in vaccination programs based on these kinds of constructs. © 2009 Ishikawa et al; licensee BioMed Central Ltd.

Labour flexibility and worker security: an Argentine perspective

Includes bibliography

Flexibility and security in the labour market: improving social protection for Brazilian workers

Includes bibliography

Flexibility, protection and active policies in Chile

Includes bibliography

The influence of the flexibility of the chair seat on pressure peak and distribution of the contact area in individuals with cerebral palsy during the execution of a task

This study aimed to determine the influence of flexibility of the chair seat surface on the pressure peak and on the contact area during the execution of a task of handling an object on the seated position by individuals with spastic cerebral palsy. Ten individuals of both genders with diagnosis of spastic cerebral palsy, who had some control to voluntarily move the body and the upper limbs, participated in this study. Quantification of data was carried out in two experimental situations: (1) execution of a task of fitting with upper limbs, and with the individual placed on an adapted canvas seat; (2) execution of a task of fitting with the participant positioned on an adapted wooden seat. Data obtained were submitted to a non-parametric and descriptive statistical analysis using the Wilcoxon test. Results indicated that the use of canvas seat increased the contact area and decreased the pressure peak and the medio-lateral displacement of centre pressure on the seated posture. © 2011 Informa UK, Ltd.

Flexibility and torsional resistance of three nickel-titanium retreatment instrument systems

Aim To assess the dimensional characteristics, flexibility and torsional behaviour of nickel-titanium retreatment instruments. Methodology Using image analysis software and high-resolution digital images, the instrument length, tip angle, diameter at 3mm from the tip and the distance between the blades (pitch length) of the following eight instruments were measured (n=12 for each measurement parameter): the ProTaper Universal retreatment (PTU-R) D1, D2 and D3 instruments; the R-Endo R1, R2 and R3 retreatment instruments; and the Mtwo retreatment (Mtwo-R) sizes 25 and 15 retreatment instruments. Maximum torque and the angular deflection at fracture as well as the bending moment at 45° were measured (n=12) according to the International Standards Organisation (ISO) specification number 3630-1. Data were analysed using the analysis of variance (α=0.05). Results The length of the active part of the instruments was found to vary according to the depth of the canal into which they were designed to reach. The pitch length also increased along the active length. The PTU-R D1 and the Mtwo-R instruments had active tips. Measurements of the bending moment at 45° revealed that the Mtwo-R 15 instrument was the most flexible, whereas the PTU-R D1 was the least flexible. The maximum torque tended to increase as the instrument diameter at 3mm from the tip increased, whereas the angular deflection at fracture varied in the opposite direction. Conclusions The geometrical characteristics of the retreatment instruments and their flexibility and torsion behaviour were consistent with their intended clinical application. © 2011 International Endodontic Journal.

Encoding through generalized polynomial codes

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Labour market flexibility: what does it really mean?

Includes bibliography