Recombinant glucagon: a differential biological activity.

In Brazil, there is a growing demand for specialised pharmaceuticals, and the high cost of their importation results in increasing costs, reaching US$ 1.34 billion in 2012 and US$ 1.61 billion in 2013. Worldwide expenses related to drugs could reach US$ 1.3 trillion in 2018, especially due to new treatments for hepatitis C and cancer. Specialised or high-cost pharmaceutical drugs used for the treatment of viral hepatitis, multiple sclerosis, HIV and diabetes are distributed free of charge by the Brazilian government. The glucagon peptide was included in this group of high-cost biopharmaceuticals in 2008. Although its main application is the treatment of hypoglycaemia in diabetic patients, it can also be used with patients in an alcoholic coma, for those patients with biliary tract pain, and as a bronchodilator. Therefore, in order to reduce biopharmaceutical production costs, the Brazilian government passed laws focusing on the development and increase of a National Pharmaceutical Industrial Centre, including the demand for the national production of glucagon. For that reason and given the importance and high cost of recombinant glucagon, the purpose of this study was to develop methods to improve production, purification and performance of the biological activity of recombinant glucagon. Glucagon was recombined into a plasmid vector containing a Glutathione S-transferase tag, and the peptide was expressed in a heterologous Escherichia coli system. After purification procedures and molecular analyses, the biological activity of this recombinant glucagon was examined using in vivo assays and showed a highly significant (p < 0.00001) and prolonged effect on glucose levels when compared with the standard glucagon. The experimental procedure described here facilitates the high level production of recombinant glucagon with an extended biological activity.

Characterization of JBURE-IIb isoform of Canavalia ensiformis (L.) DC urease.

Ureases, nickel-dependent enzymes that catalyze the hydrolysis of urea into ammonia and bicarbonate, are widespread in plants, bacteria, and fungi. Previously, we cloned a cDNA encoding a Canavalia ensiformis urease isoform named JBURE-II, corresponding to a putative smaller urease protein (78kDa) when compared to other plant ureases. Aiming to produce the recombinant protein, we obtained jbure-IIb, with different 3' and 5' ends, encoding a 90kDa urease. Three peptides unique to the JBURE-II/-IIb protein were detected by mass spectrometry in seed extracts, indicating that jbure-II/-IIb is a functional gene. Comparative modeling indicates that JBURE-IIb urease has an overall shape almost identical to C. ensiformis major urease JBURE-I with all residues critical for urease activity. The cDNA was cloned into the pET101 vector and the recombinant protein was produced in Escherichia coli. The JBURE-IIb protein, although enzymatically inactive presumably due to the absence of Ni atoms in its active site, impaired the growth of a phytopathogenic fungus and showed entomotoxic properties, inhibiting diuresis of Rhodnius prolixus isolated Malpighian tubules, in concentrations similar to those reported for JBURE-I and canatoxin. The antifungal and entomotoxic properties of the recombinant JBURE-IIb apourease are consistent with a protective role of ureases in plants.

Métodos de diagnóstico para detecção de prostatopatias caninas / Canine prostatic disease: diagnosis methods

As doenças prostáticas são comuns em cães, desenvolvendo-se após a maturidade sexual. Entre as afecções que acometem a próstata desses animais, a mais frequente é a hiperplasia prostática benigna (HPB). As neoplasias prostáticas, apesar de menos frequentes, são altamente malignas e um diagnóstico tardio está associado a um prognóstico ruim. Os principais métodos de diagnóstico das enfermidades prostáticas em cães incluem um exame clínico detalhado, ultrassonografia, punção aspirativa e biópsia. A utilização de biomarcadores séricos vem sendo estudada como uma maneira de se detectar a doença precocemente, evitando possíveis complicações no quadro clínico dos pacientes. Assim, estudos visando ao diagnóstico precoce de prostatopatias caninas, especialmente o câncer prostático, são cruciais para um tratamento eficaz e melhora da qualidade de vida do animal acometido.

Prostatic diseases are common in older dogs, developing after sexual maturity. The most common affecting canine prostate is benign prostatic hyperplasia (BPH). Although less frequent, prostatic neoplasias are highly malignant, and a late diagnosis is closely associated with a poor prognosis. The main methods of diagnosis of the canine prostatic disease include a detailed clinical exam, ultrasound, prostatic fine-needle aspirate and biopsy. Studies towards early diagnosis of this condition are crucial for effective treatment and improvement of quality of life of affected animals. The use of biomarkers has been studied for early detection of the disease, avoiding possible complications in clinical patients.

Short-term evaluation in growing rats of diet containing Bacillus thuringiensis Cry1Ia12 entomotoxin: nutritional responses and some safety aspects.

The Cry1Ia12 entomotoxin from a Brazilian Bacillus thuringiensis strain is currently being expressed in cotton cultivars to confer resistance to insect-pests. The present study aimed to assess the effects of a diet containing Cry1Ia12 protein on growing rats. A test diet containing egg white and Cry1Ia12 (0.1% of total protein) as a protein source was offered to rats for ten days. In addition, an acute toxicity bioassay was performed in rats with a single oral dose of the entomotoxin (12 mg/animal). No adverse effects were observed in the animals receiving the test diet when compared to those receiving a control diet (egg white). The analysed parameters included relative dry weight of internal organs, duodenum histology, blood biochemistry, and nutritional parameters. The results of the acute toxicity test showed no mortality or behaviour alteration. Thus, Cry1Ia12 toxin at the tested concentration does not cause deleterious effects on growing rats when incorporated in the diet for 10 days.