Enzymatic, immunological and phylogenetic characterization of Brucella suis urease

<p>Abstract</p> <p>Background</p> <p>The sequenced genomes of the <it>Brucella </it>spp. have two urease operons, <it>ure</it>-1 and <it>ure</it>-2, but there is evidence that only one is responsible for encoding an active urease. The present work describes the purification and the enzymatic and phylogenomic characterization of urease from <it>Brucella suis </it>strain 1330. Additionally, the urease reactivity of sera from patients diagnosed with brucellosis was examined.</p> <p>Results</p> <p>Urease encoded by the <it>ure</it>-1 operon of <it>Brucella suis </it>strain 1330 was purified to homogeneity using ion exchange and hydrophobic interaction chromatographies. The urease was purified 51-fold with a recovery of 12% of the enzyme activity and 0.24% of the total protein. The enzyme had an isoelectric point of 5, and showed optimal activity at pH 7.0 and 28–35°C. The purified enzyme exhibited a Michaelis-Menten saturation kinetics with a <it>K</it>_{<it>m </it>}of 5.60 ± 0.69 mM. Hydroxyurea and thiourea are competitive inhibitors of the enzyme with K_iof 1.04 ± 0.31 mM and 26.12 ± 2.30 mM, respectively. Acetohydroxamic acid also inhibits the enzyme in a competitive way. The molecular weight estimated for the native enzyme was between 130–135 kDa by gel filtration chromatography and 157 ± 7 kDa using 5–10% polyacrylamide gradient non-denaturing gel. Only three subunits in SDS-PAGE were identified: two small subunits of 14,000 Da and 15,500 Da, and a major subunit of 66,000 Da. The amino terminal sequence of the purified large subunit corresponded to the predicted amino acid sequence encoded by <it>ureC1</it>. The UreC1 subunit was recognized by sera from patients with acute and chronic brucellosis. By phylogenetic and cluster structure analyses, <it>ureC1 </it>was related to the <it>ureC </it>typically present in the <it>Rhizobiales</it>; in contrast, the <it>ureC2 </it>encoded in the <it>ure</it>-2 operon is more related to distant species.</p> <p>Conclusion</p> <p>We have for the first time purified and characterized an active urease from <it>B. suis</it>. The enzyme was characterized at the kinetic, immunological and phylogenetic levels. Our results confirm that the active urease of <it>B. suis </it>is a product of <it>ure</it>-1 operon.</p

Mesenchymal Stem Cells Genetically Modified by Lentivirus-Express Soluble TRAIL and Interleukin-12 Inhibit Growth and Reduced Metastasis-Relate Changes in Lymphoma Mice Model

Background: Cancer treatment has many side effects; therefore, more efficient treatments are needed. Mesenchymal stem cells (MSC) have immunoregulatory properties, tumor site migration and can be genetically modified. Some proteins, such as soluble TRAIL (sTRAIL) and interleukin-12 (IL-12), have shown antitumoral potential, thus its combination in solid tumors could increase their activity. Materials and Methods: Lentiviral transduction of bone marrow MSC with green fluorescent protein (GFP) and transgenes (sTRAIL and IL-12) was confirmed by fluorescence microscopy and Western blot. Soluble TRAIL levels were quantified by ELISA. Lymphoma L5178Y cells express a reporter gene (GFP/mCherry), and TRAIL receptor (DR5). Results: An in vivo model showed that combined treatment with MSC expressing sTRAIL+IL-12 or IL-12 alone significantly reduced tumor volume and increased survival in BALB/c mice (p p < 0.05). It presented less tissue dysplasia confirmed by fluorescence and hematoxylin–eosin dye; nevertheless, treatment not inhibited hepatic metastasis. Conclusions: MSC expressing IL-12, is or combination with BM-MSC expressing sTRAIL represents an antitumor strategy for lymphoma tumors since they increase survival and reduce tumor development. However, the combination did not show significative additive effect. The localized application did not inhibit metastasis but reduced morphological alterations of tissue associated with liver metastasis