Human granulocyte colony stimulating factor (hG-CSF): cloning, overexpression, purification and characterization

Background: Biopharmaceutical drugs are mainly recombinant proteins produced by biotechnological tools. The patents of many biopharmaceuticals have expired, and biosimilars are thus currently being developed. Human granulocyte colony stimulating factor (hG-CSF) is a hematopoietic cytokine that acts on cells of the neutrophil lineage causing proliferation and differentiation of committed precursor cells and activation of mature neutrophils. Recombinant hG-CSF has been produced in genetically engineered Escherichia coli ( Filgrastim) and successfully used to treat cancer patients suffering from chemotherapy-induced neutropenia. Filgrastim is a 175 amino acid protein, containing an extra N-terminal methionine, which is needed for expression in E. coli. Here we describe a simple and low-cost process that is amenable to scaling-up for the production and purification of homogeneous and active recombinant hG-CSF expressed in E. coli cells.Results: Here we describe cloning of the human granulocyte colony-stimulating factor coding DNA sequence, protein expression in E. coli BL21(DE3) host cells in the absence of isopropyl-beta-D-thiogalactopyranoside ( IPTG) induction, efficient isolation and solubilization of inclusion bodies by a multi-step washing procedure, and a purification protocol using a single cationic exchange column. Characterization of homogeneous rhG-CSF by size exclusion and reverse phase chromatography showed similar yields to the standard. The immunoassay and N-terminal sequencing confirmed the identity of rhG-CSF. The biological activity assay, in vivo, showed an equivalent biological effect (109.4%) to the standard reference rhG-CSF. The homogeneous rhG-CSF protein yield was 3.2 mg of bioactive protein per liter of cell culture.Conclusion: The recombinant protein expression in the absence of IPTG induction is advantageous since cost is reduced, and the protein purification protocol using a single chromatographic step should reduce cost even further for large scale production. The physicochemical, immunological and biological analyses showed that this protocol can be useful to develop therapeutic bioproducts. In summary, the combination of different experimental strategies presented here allowed an efficient and cost-effective protocol for rhG-CSF production. These data may be of interest to biopharmaceutical companies interested in developing biosimilars and healthcare community.

Species composition and plant use in old urban homegardens in Rio Claro, Southeast of Brazil

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Rabies laboratory diagnosis: peculiar features of samples from equine origin

O diagnóstico laboratorial da raiva é realizado através de métodos de pesquisa do corpúsculo de Negri, imunofluorescência direta e inoculação em camundongos. Na maioria dos casos, quando a amostra é bem coletada, bem conservada e o profissional responsável possui experiência, verifica-se concordância entre as técnicas utilizadas. A Seção de Raiva e Encefalomielite do Instituto Biológico ao comparar a sensibilidade das três técnicas diagnósticas, em 3713 amostras (córtex cerebral, cerebelo e hipocampo) recebidas no período de 1980-1994, sendo 3010 da espécie bovina (983 positivas) e 703 da espécie eqüina (111 positivas), observou que, no caso da raiva eqüina, esta concordância não é mantida. Verificou-se, nesta espécie, que somente em algumas oportunidades foi possível identificar, pelo método histopatológico, o corpúsculo de Negri. em relação à prova de imunofluorescência pode-se afirmar que a mesma detectou uma porcentagem menor de amostras positivas, provenientes da espécie equina, em compração com as da espécie bovina, sendo esta diferença estatisticamente significativa. A prova biológica foi a mais sensível, porém houve uma diferença, também significativa, entre o período de incubação em camundongos das amostras de origem bovina e das de origem eqüina. A presença pouco frequente de corpúsculos de Negri e o período de incubação em camundongos mais prolongado, das amostras de origem eqüina, sugerem que devem ser intensificados os estudos da patogenia da raiva nesta espécie.

Prevalence of pre-frailty for the component of gait speed in older adults

OBJETIVO: investigar a pré-fragilidade e os fatores associados a essa condição, considerando as medidas de velocidade da marcha dos idosos. MÉTODO: a seleção dos participantes ocorreu por meio de critérios de inclusão/exclusão e teste de rastreamento cognitivo. A amostra foi calculada com base na estimativa da proporção populacional e constituída por 195 idosos, usuários de uma Unidade Básica de Saúde de Curitiba, PR. Os dados foram coletados mediante questionário sociodemográfico/clínico e teste de velocidade da marcha. RESULTADOS: a pré-fragilidade para velocidade da marcha possui moderada prevalência (27,3%) e associou-se à faixa etária entre 60 e 69 anos, baixa escolaridade, não se sentir solitário, utilizar anti-hipertensivo, apresentar doença cardiovascular e sobrepeso. CONCLUSÃO: considera-se relevante identificar os idosos na condição de pré-fragilidade, pois, dessa maneira, existe a possibilidade de intervenção imediata com a finalidade de estacionamento do quadro. É significativo o déficit de estudos sobre a síndrome da fragilidade em idosos brasileiros, principalmente aqueles que se referem a um componente isolado. Visto que a enfermagem gerontológica se encontra nos primeiros passos referentes à temática, entende-se que a identificação da prevalência deve ser o ponto primordial das pesquisas sobre o tema.

Effects of nutritional factors on growth of Lactobacillus fermentum mixed with Saccharomyces cerevisiae in alcoholic fermentation

The growth of Lactobacillus fermentum was studied in mixed culture with Saccharomyces cerevisiae during alcoholic fermentation of high test molasses (HTM). Yeast extract or a group of 17 amino acids caused a strong and fast decrease in yeast viability due to the strong increase of acidity produced by bacteria. Pure culture of Lactobacillus fermentum in dry sugar cane broth confirmed amino acids as the main nutrients needed to stimulate the growth of bacterial contaminant during alcoholic fermentation. The absence of L. fermentum growth was obtained when leucine: isoleucine or valine were not added to the medium. Phenylalanine, alanine, glutamic acid, cystine, proline, histidine, arginine, threonine, tryptophane, serine and methionine inhibited the bacterial growth at least in one of the cultures of L. fermentum tested.

The use of extruded feed in fish food

In our country, the intensive culture of fish has been making progress due to the availability of extruded rations, which are characterized by the way they have been formulated to assist the nutritional demands of the tropical species and for facilitating a better practice of alimentary handling. This process checks the extruded product density and the desirable physical/chemical aspects to a diet that should be handled in the water, the critical factor of this activity. It is important to emphasize that this product stays to the surface of the water, facilitates the consumption control and the observation of the general aspects of the school of fish, as well as guaranteeing that no deterioration of the physical/chemical characteristics of the water occurs. The floating characteristic of the extruded ration is preferable for farmers because most of the tropical species feed at the surface of the water. In some cases, in the function of the alimentary habit, the thermal conditions of the water and the handling system produce granules with different densities. This is due to the humidity and the applied cooking heat, which modifies the time of hydration, and consequently the ability to float. To establish the physical presentation idea for a ration for tropical fishes, it is necessary that the pellets come in sizes, textures, humidities and densities in order to allow for the different phases of the lives of the fishes. This ration should be the result of characteristics of the species, such as the anatomophysiologics particularities, alimentary habits, digestive capacity and alimentary behavior. Only in this way is it possible to offer to those fishes a ration whose nutritious value and physical presentation facilitate the best biological and economic answers.Those aspects need to be reviewed because the tropical species that are cultivated in our country present selective behavior in relation to the size of the pellet. Generally, this has access to the digestive system without waste; the digestibility, therefore, is dependent upon the size of the particles and the physical aspects of the pellets. They should still consider that those granules could be less abrasive and softer. The extruded rations available in the national market present a qualitative reference to its proteic level, whose consequence allows us to infer that those are overestimated for the final phase of those fishes. Thus, the final cost of production is made unfeasible. In this point of view of the development of the national fish culture, and the growing adoption of the intensive system of production, it can link that such progress is a function of the availability of the rations in the extruded form. Although this technique of processing of rations has revolutionized the market in such a way that has been seen as preferable for farmers, some feel that there is a lack of products best suited for the initial phases of fingerlings production.

Cytotaxonomic analysis of Brazilian species of the genus Amazona (Psittacidae, Aves) and confirmation of the genus Salvatoria (Ribeiro, 1920)

The karyotypes of 12 species of Psittacidae of the genus Amazona were studied: A. aestiva, A. amazonica, A. brasiliensis, A. autumnalis, A. farinosa, A, festiva, A. kawalli, A. ochrocephala, A. pretrei, A. rhodocorytha, A. vinacea and A. xanthops. The metaphases were obtained using a short term culture of leather pulp. Eleven of the twelve analyzed species were karyotypically homogeneous, with only a few divergences in chromosomes 2 and 3. The species A. xanthops showed large karyotypic differences compared to the genus Amazona. Consequently, the genus Salvatoria (Ribeiro, Rev. Mus. Paul. 12: 1-82, 1920) was confirmed, and A. xanthops renamed Salvatoria xanthops. The study showed the chromosomic conservation of the genus Amazona and the need for further taxonomic studies of the karyotype of the Psittacidae.

Demonstration of the cellular viability and safety of Enterococcus faecium CRL 183 in long-term experiments

Enterococcus faecium CRL 183, a strain isolated from NSLAB cheese starter, has been the focus of much research on its potential probiotic capacity, although its survival through the gastrointestinal tract has not been demonstrated so far. In order to determine the capacity of E. faecium CRL 183 to survive such conditions, this strain was administered daily to rats for 30 weeks. The experimental animals were divided into Group I: those that did not receive E. faecium, Group II: those that received a pure culture of E. faecium CRL 183 and Group III: animals that received E. faecium CRL 183 in the form of a fermented soy-based product. Faecal samples were collected at the beginning and at the 50%, 75% and 100% stages of the experimental period. Isolation and counts of Enterococcus were carried out on KF selective media. To distinguish the various Enterococcus species in the faeces, biochemical (API Strep 20) and molecular (PCR) tests were performed. Initially, E. faecium was absent from the intestinal flora of the rats; however, after 15 weeks of administration, E. faecium could be recovered from the faeces of Groups II and III, demonstrating that E. faecium CRL 183 was able to survive gastrointestinal transit under the study conditions. This is further evidence of the probiotic qualities of this strain. The safety of the strain was also investigated with regard to body weight and serum biochemical analysis.

Evaluation of isopathic treatment of Salmonella enteritidis in poultry

Background. Salmonellosis is a common problem worldwide in commercially reared poultry. It is associated with human Salmonellosis. No fully satisfactory method of control is available.Method: Nosodes to an antibiotic-resistant strain of Salmonella enterica serovar Enteritidis in D30 (30X) potency were prepared. One day old chicks (N = 180) were divided into four groups: two control and two different preparations of the nosode. Treatments were administered in drinking water for 10 days. The birds were challenged by a broth culture of the same Salmonella, by mouth, on day 17. Cloacal swabs were taken twice weekly for Salmonella enterica serovar Enteritidis.Results: Birds receiving active treatment were less likely to grow the strain of Salmonella from cloacal swabs compared to control.Conclusion: Isopathy is low cost and non-toxic. It may have a role to play in the widespread problem of Salmonella in poultry. Further research should be conducted.

Host-guest complexation of a nitroheterocyclic compound with cyclodextrins: a spectrofluorimetric and molecular modeling study

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

Evaluation of the components of production of the ricinus according of rates of calcareous rock and match

The soil of the Paraiba, in generality, are acid and with low levels of available match, seriously limiting the income of the plants. A research in vegetation house was lead, with the objective to evaluate doses of calcareous soil and match in the culture of ricinus. The treatments had been composites for five levels of match: 0.0; 80; 160; 240 and 360 kg ha(-1) of P(2)O(5) and four dosis of calcareous rock: 0.0; 2.5; 3.5 and 4.5 t ha(-1) in experimental delineation of blocks casualized with 4 factorial project x 3 (four levels of match and three doses of calcareous soil) more two treatments you add zero of match (0.0 P(2)O(5): 2,5 t ha(-1) of calcareous soil) and calcareous soil zero (240 P(2)O(5): 0.0 t ha(-1) of calcareous soil), with three repetitions. The calcareous soil reduced the acidity of the ground and effect of the application of the match (>= 80 kg ha(-1) of P(2)O(5)) in the presence of the collagen (>= 2.5 t ha(-1) of CaCO(3)) with of the calcareous soil (>= 2.5 t ha(-1) of CaCO(3)) in the presence of the match was not verified well (>= 80 kg ha(-1) of P(2)O(5)). The match applied in the absence of the calcareous soil was limited to the income of the culture of what the calcareous soil in the absence of the match.

Stimulation of polygalacturonase production in an immobilized system by Aspergillus sp.: effect of pectin and glucose

The synthesis of polygalacturonases (PG) is known to be influenced by Aspergillus growth conditions, namely, environmental factors and pectin content in the cultivation medium containing a mixed carbon source. Optimal conditions were attained at a temperature of 30 A degrees C and an initial pH of 4.5. PG activity (3.29 and 2.48 U/mL) was determined after a two-day culture of Aspergillus sp. HC1 and Aspergillus sp. CC1, respectively, in a basic medium containing 2% citrus pectin as the sole carbon source. The addition of glucose (2% w/v) to the basic medium led to a 2-fold increase in PG production. However, enzyme synthesis was repressed when a higher concentration of glucose was used in the medium containing the mixed carbon source. Spores from the two fungi were immobilized in a 3% Ca-alginate system and the mechanical strength of the gel beads allowed the use of this process system 6-fold longer (288 h) than the free culture. In the Aspergillus sp. CC1 immobilized system, PG production increased nearly 10-fold in the medium with 2% glucose added (5.95 U/mL) in comparison to the medium without sugar (0.55 U/mL). The results demonstrate that a different response in activity was produced by free and entrapped spore systems. PG production remained approximately constant throughout the six 48 h cycles in the medium containing citrus pectin (2% w/v) as the sole carbon source.

Isolation and characterization of Wharton's jelly-derived multipotent mesenchymal stromal cells obtained from bovine umbilical cord and maintained in a defined serum-free three-dimensional system

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

Antibody response to the 43 kDa glycoprotein of Paracoccidioides brasiliensis as a marker for the evaluation of patients under treatment

Sera of patients with paracoccidioidomycosis contained IgG-, IgA-, and IgM-specific antibodies to a 43 kDa antigen contained in the filtrate of a culture of Paracoccidioides brasiliensis. IgG- and IgA-specific antibodies were present in all observed patients. The IgM response was more frequent in acute cases, and the mean titers of IgG- and IgM-specific antibodies were higher in the acute forms. By the fourth month of chemotherapy, there was a decay of IgG, IgA, and IgM antibody titers to this antigen in acute cases, correlating with clinical improvement. The detection of IgG and IgA antibodies and the sequential determination of antibodies to the 43 kDa glycoprotein may be useful tools for serodiagnosis and evaluation of therapeutic efficacy.

The effects of acute hypoxia and hypercapnia on oxygen consumption of the freshwater European eel

When exposed to hypoxia, eels Anguilla anguilla were able to regulate and maintain VO2 down to a water oxygen tension (PWO2) of about 25 mmHg, a value far below those reported in other studies. When exposed to hypercapnia, eels showed a depression in VO2 as water carbon dioxide tension (PWCO2) increased. Faced with combined hypoxia-hypercapnia, eels showed an increase in their sensitivity to hypoxia, and the critical oxygen tension increased to 40-45 mmHg. The possible mechanisms underlying these responses were discussed, and the implications of such findings for extensive culture of eels were highlighted.