Effect of osmolytes on the activity of anti-cancer enzyme L-Asparaginase II from Erwinia chrysanthemi

L-asparaginase is used for the treatment of acute lymphoblastic leukaemia (ALL); however, its formulation presents drawbacks such as a lack of stability and formation of aggregates. Osmolytes are small molecules accumulated by cells in response to environmental stresses and present a protective behaviour, favouring the equilibrium of macromolecules towards the native conformation. Therefore, osmolytes are employed as excipients in pharmaceutical protein formulations. Herein, recombinant L-ASNase II from Erwinia chrysanthemi (ErA II) was analysed with respect to the effect of osmolytes on kinetic and stability of this biopharmaceutical. The aggregation profiles were analysed trough nanotracking particle analysis and dynamic light scattering. The majority of the tested osmolytes increased ErA II specific activity and stability, being more pronounced for sucrose and sorbitol, which increased almost 70% of ErA II activity. The polyol preserved total enzyme activity for 30 days while sucrose preserved 81.1 ± 5.3% total enzyme activity over this period. Each osmolyte resulted in a specific aggregation profile and the presence of sucrose or sorbitol resulted in a lower quantity of aggregates in the range of 100–300 nm. The present findings may contribute to the improvement of adjuvants in L-ASNase formulations and the optimization of other biopharmaceutical formulations.publishe

Presence of Atrazine in Water in a Recharge Area of Guarany Aquifer in Brazil

The region of Ribeirao Preto City located in Sao Paulo State, southeastern Brazil, is an important sugarcane, soybean and corn producing area. This region is also an important recharge area for groundwater of the Guarany aquifer, a water supply source of the city and region. The cultivation of grain and sugar cane in thes area demands the frequent use of the herbicide atrazine (2-chloro-4- (ethylamino)-6-(isopropylamino)-S-triazine). This research was conducted to characterize the potential contamination of groundwater with atrazine. Surface water samples were collected in the Espraiado stream in a selected watershed on the area, during the years of 1995-1998. Groundwater was also collected in wells located at the edge of the watershed during the years of 1999 to 2002. The water samples were analyzed by HPLC (High Performance Liquid Chromatography) procedure followed by GC-MS form confirmation. To predict the atrazine leaching in the area, the CMLS-94 (Chemical Movement Layered Soil) simulation model was also used. Only four atrazine detections in surface water were found, however, none of them were confirmed with GC-MS. No atrazine was detected in groundwater samples. The results obtained by the CMLS-94 simulations predicted that atrazine, after four years from the application date, would not have reached the depth of the confined aquifer (40m)

Pion-Muon Asymmetry Revisited

Long ago an unexpected and unexplainable phenomena was observed. The distribution of muons from positive pion decay at rest was anisotropic with an excess in the backward direction relative to the direction of the proton beam from which the pions were created. Although this effect was observed by several different groups with pions produced by different means, the result was not accepted by the physics community, because it is in direct conflict with a large set of other experiments indicating that the pion is a pseudoscalar particle. It is possible to satisfy both sets of experiments if helicity-zero vector particles exist and the pion is such a particle. Helicity-zero vector particles have direction but no net spin. For the neutral pion to be a vector particle requires an additional modification to conventional theory as discussed herein. An experiment is proposed which can prove that the asymmetry in the distribution of muons from pion decay is a genuine physical effect because the asymmetry can be modified in a controllable manner. A positive result will also prove that the pion is NOT a pseudoscalar particle.Comment: 9 pages, 3 figure

Optimization of clavulanic acid production by Streptomyces daufpe 3060 by response surface methodology

Clavulanic acid is a β-lactam antibiotic which has a potent β-lactamase inhibiting activity. In order to optimize its production by the new isolate Streptomyces DAUFPE 3060, the influence of two independent variables, temperature and soybean flour concentration, on clavulanic acid and biomass concentrations was investigated in 250 mL-Erlenmeyers according to a 2² central composite design. To this purpose, temperature and soybean flour (SF) concentration were varied in the ranges 26-34°C and 10-50 g/L, respectively, and the results evaluated utilizing the Response Surface Methodology. The experimental maximum production of clavulanic acid (629 mg/L) was obtained at 32°C and 40 g/L SF after 48 h, while the maximum biomass concentration (3.9 g/L) at 30°C and 50 g/L soybean flour, respectively. These values are satisfactorily close to those (640 mg/L and 3.75 g/L, respectively) predicted by the model, thereby demonstrating the validity of the mathematical approach adopted in this study.Brazilian Research Funding InstitutionsCoordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES

"Antarctic yeasts as a source of L-asparaginase: Characterization of a glutaminase-activity free L-asparaginase from psychrotolerant yeast Leucosporidium scottii L115"

"Microorganisms from extreme environments, such as the Antarctic ecosystems, have a great potential to produce enzymes with novel characteristics. Within this context, L-asparaginase (ASNase) obtained from yeast species has been poorly studied. In this study, yeasts isolated from samples collected at Admiralty Bay (King George Island, Antarctica) were tested to produce ASNase. From an initial screening of 40 strains, belonging to 13 different species, Leucosporidium scottii L115 produced an ASNase activity (LsASNase activity: 6.24 U g-1 of dry cell weight) with the lowest glutaminase activity. The LsASNase was purified 227-fold, with a specific activity of 137.01 U mg-1 at 37 ◦C, without glutaminase activity. Moreover, the maximum enzyme activity was observed at pH 7.5 and at a temperature of 55 ◦C. The enzyme is a multimer of 462 kDa, presenting a single band of 53 kDa molecular mass in reduced conditions; after PGNase F treatment, a single band of 45 kDa was observed. The enzymatic kinetic evaluation revealed an allosteric regulation of the enzyme and the kinetic parameters were determined at 37 ◦C, pH 7.0 as substrate affinity constant, K0.5 = 233 μM, kcat = 54.7 s − 1 and Hill coefficient, nH = 1.52, demonstrating positive cooperativity by the enzyme and the substrate. This is the first study to report L. scottii as a source of glutaminase-activity free L-asparaginase, an acute lymphoblastic leukemia drug feature suitable for the treatment of asparagine synthetase negative cancer cells.