L-Asparaginase from E. chrysanthemi expressed in glycoswitch®: effect of His-Tag fusion on the extracellular expression

Abstract

L-Asparaginase (L-ASNase) is an important enzyme used to treat acute lymphoblastic leukemia, recombinantly produced in a prokaryotic expression system. Exploration of alternatives production systems like as extracellular expression in microorganisms generally recognized as safe (such as Pichia pastoris Glycoswitch^®) could be advantageous, in particular, if this system is able to produce homogeneous glycosylation. Here, we evaluated extracellular expression into Glycoswitch^® using two different strains constructions containing the asnB gene coding for Erwinia chrysanthemi L-ASNase (with and without His-tag), in order to find the best system for producing the extracellular and biologically active protein. When the His-tag was absent, both cell expression and protein secretion processes were considerably improved. Three-dimensional modeling of the protein suggests that additional structures (His-tag) could adversely affect native conformation and folding from L-ASNase and therefore the expression and cell secretion of this enzyme.     

Digestive capacities allow the Mexican long-nosed bat (Leptonycteris nivalis) to live in cold environments

Digestive capabilities of nectar-feeding vertebrates to assimilate sugars affect their ability to acquire and store energy and could determine the minimal temperatures at which these animals can survive. Here, we described the sugar digestive capability of Leptonycteris nivalis and related it with its capacity to live in cold environments. We measured the enzymatic activity, food intake rate and changes in body mass of bats feeding at four different sucrose concentrations (from 5 to 35% wt./vol.). Additionally, we used a mathematical model to predict food intake and compared it with the food intake of bats. L. nivalis was able to obtain ~ 111.3 kJ of energy regardless of the sugar concentration of their food. Also, bats gained ~ 2.57 g of mass during the experimental trials and this gain was independent of sugar concentration. The affinity (1 / K_m) of sucrase (EC 3.2.1.48) was one order of magnitude higher relative to that reported for its sister species Leptonycteris yerbabuenae (0.250 and 0.0189 mmol^? 1 L, respectively), allowing this species to have a higher energy intake rate. We propose that the high ability to acquire energy conferred L. nivalis the faculty to invade cold environments, avoiding in this way the ecological competition with its sympatric species L. yerbabuenae.     

Association between Gene Polymorphism of Manganese Superoxide Dismutase and Prostate Cancer Risk

Manganese superoxide dismutase (MnSOD) is the most effective antioxidant enzyme in mitochondria and protects cells from reactive oxygen species‐induced oxidative damage. The aim of this study was to investigate the association between MnSOD Ala‐9Val gene polymorphism and prostate cancer (PCa) risk in Turkish men with prostate cancer. 33 patients with PCa and 81 control individuals were included in the study. We observed an association between MnSOD Ala/Ala frequency and a higher PCa risk. In addition, we found that the increased risk of early‐onset PCa (under age of 65) in the men homozygous for Ala allele was higher than the men homozygous for Val allele. However, we determined that MnSOD Ala‐9Val genotype was not associated with the aggressiveness of the disease. The results of our study suggest that MnSOD Ala/Ala genotype may influence on early‐onset of PCa patients, but no effect on subsequent development of the disease in Turkish men. However, our study has a limitation that is small numbers of individuals for cases and controls. Therefore, the presented study limited our statistical power to fully investigate the gene polymorphism on cancer risk. © 2013 Wiley Periodicals, Inc. J BiochemMol Toxicol 27:213‐218, 2013; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.21472     

Major plant communities of North America's most arid region: The San Felipe Desert,Baja California,México

Abstract

This vegetation study describes the landscape of the San Felipe Desert (Baja California, México) based on the phytosociological analysis of its major plant associations, as determined by the Braun-Blanquet method and supported by cluster analysis. Four geomorphological or phytotopographical units were identified in the landscape of the San Felipe Desert (salt marshes, playas or sandy flats, bajadas or alluvial fans, and malpaíses or badlands), each characterized by its own plant communities. Two communities and eleven associations were identified, four of which are described and typified for the first time: Atriplici linearis-Frankenietum palmeri, Parkinsonio microphyllae-Olneyetum tesotae, Errazurizio megacarpae-Ephedretum trifurcae and Pachycereo schottii-Prosopidetum torreyanae. Ecological, phytogeographical, bioclimatic, syntaxonomical and floristic data are provided for each of these new associations.     

Conceptual design of integrated microfluidic system for magnetic cell separation,electroporation, and transfection

For the purposes of a successful ex vivo gene therapy we have proposed and analyzed a new concept of an integrated microfluidic system for combined magnetic cell separation, electroporation, and magnetofection. For the analysis of magnetic and electric field distribution (given by Maxwell equations) as well as dynamics of magnetically labeled cell and transfection complex, we have used finite element method directly interfaced to the Matlab routine solving Newton dynamical equations of motion. Microfluidic chamber has been modeled as a channel with height and length 1 mm and 1 cm, respectively. Bottom electrode consisted of 100 parallel ferromagnetic straps and the upper electrode was plate of diamagnetic copper. From the dynamics of magnetic particle motion we have found that the characteristic time-scales for the motion of cells (mean capture time ～ 4 s) and gene complexes (mean capture time ～ 3 min), when permanent magnets are used, are in the range suitable for efficient cell separation and gene delivery. The largest electric field intensity (～10 kV/m) was observed at the edges of the microelectrodes, in the close proximity of magnetically separated cells, which is optimal for subsequent cell electroporation.     

Notes on the Laboulbeniales (Ascomycota) parasitic on Diptera from Portugal and other countries

Eighteen species of Stigmatomyces are reported for the first time from continental Portugal and/or from the Azores. These are Stigmatomyces asteiae W. Rossi et Cesari, Stigmatomyces athyroglossae W. Rossi et Cesari, Stigmatomycescanzonerii W. Rossi et Cesari, Stigmatomyces ceratophorus Whisler, Stigmatomyces constrictus Thaxt., Stigmatomyces crassicollis Thaxt., Stigmatomyces divergatus Thaxt., Stigmatomyces discocerinae Thaxt., Stigmatomyces ensinae Thaxt., Stigmatomyces cf. ephydrae L. Mercier et R. A. Poiss., Stigmatomycesgeomyzae W. Rossi et Cesari, Stigmatomyces limnophorae Thaxt., Stigmatomyces majewskii H. L. Dainat, Manier et Balazuc, Stigmatomyces papuanus Thaxt., Stigmatomycesplatensis Speg., Stigmatomyces ptylomyiae Thaxt. and Stigmatomyces purpureus Thaxt., Stigmatomyces rugosus Thaxt. New records of these species are also reported from Australia, Canada, Canary Islands (Spain), Costa Rica, Cuba, Ecuador, Finland, Germany, Great Britain, Greece, Kenya, Hungary, Israel, Kyrgyzstan, Lebanon, Morocco, Saudi Arabia, Sierra Leone, Taiwan, Thailand, Turkey, Uganda, United Arab Emirates, USA, Yemen and Zimbabwe. The new records increase, sometimes considerably, information about distribution of these parasites. Two synonymies are also established: Stigmatomyces autriquei Balazuc = Stigmatomyces ensinae Thaxt.; Stigmatomyces psilopae Thaxt. var. camarguensis H. L. Dainat et J. Dainat = S. rugosus Thaxt.     

Lipid and protein oxidation in hepatic homogenates and cell membranes exposed to bile acids

Cholestasis occurs in a variety of hepatic diseases and causes damage due to accumulation of bile acids in the liver. The aim was to investigate the effect of several bile acids, i.e. chenodeoxycholic, taurochenodeoxycholic, deoxycholic, taurodeoxycholic, ursodeoxycholic, lithocholic and taurolithocholic (TLC), in inducing oxidative damage. Hepatic tissue of male Sprague-Dawley rats was incubated with or without 1 mM of each bile acid, with or without 0.1 mM FeCl₃ and 0.1 mM ascorbic acid for the purpose of generating free radicals. Several bile acids increased lipid and protein oxidation, with TLC being the most pro-oxidative (657% and 175% in homogenates and 350% and 311% in membranes, respectively). TLC also enhanced iron-induced oxidative stress to lipids (21% in homogenates and 29% in membranes) and to proteins (74% in membranes). This enhancement was dose- and time-dependent and was reduced by melatonin. These results suggest that bile acids differentially mediate hepatic oxidative stress and may be involved in the physiopathology of cholestasis.     

Renoprotection by α-mangostin is related to the attenuation in renal oxidative/nitrosative stress induced by cisplatin nephrotoxicity

Cisplatin (CDDP) is a chemotherapeutic agent that produces nephrotoxicity associated with oxidative/nitrosative stress. α-Mangostin (α-M) is a xanthone extracted from mangosteen with antioxidant and anti-inflammatory properties. The purpose of this study was to evaluate the renoprotective effect of α-M on the CDDP-induced nephrotoxicity. α-M was administered (12.5 mg/kg/day, i.g.) for 10 days (7 days before and 3 days after CDDP injection). On day 7, rats were treated with a single injection of CDDP (7.5 mg/Kg, i.p.); 3 days after the rats were killed. α-M attenuated renal dysfunction, structural damage, oxidative/nitrosative stress, decrease in catalase expression and increase in mRNA levels of tumour necrosis factor alpha and transforming growth factor beta. In conclusion the renoprotective effect of α-M on CDDP-induced nephrotoxicity was associated with the attenuation in oxidative/nitrosative stress and inflammatory and fibrotic markers and preservation of catalase activity.     

Isolated respiring heart mitochondria release reactive oxygen species in states 4 and 3

Isolated mitochondria respiring on physiological substrates, both in state 4 and 3, are reported to be or not to be a source of reactive oxygen species (ROS). The cause of these discrepancies has been investigated. As protein concentration was raised in in vitro assays at 37°C, the rate of H₂O₂ release by rat heart mitochondria supplemented with pyruvate/malate or with succinate (plus rotenone) was shown to increase (0.03–0.15?mg?protein/ml), to decrease (0.2–0.5?mg?protein/ml) and to be negligible (over 0.5?mg?protein/ml). The inhibition of mitochondrial respiration (with rotenone or antimycin A) or the increase in the oxygen concentration dissolved in the assay medium allowed an enhancement of ROS production rate throughout the studied range of protein concentrations. In mitochondria respiring in state 3 on pyruvate/malate or on succinate (plus rotenone), ROS release vanished for protein concentrations over 0.5 or 0.2?mg/ml, respectively. However, ROS production rates measured with low protein concentrations (below 0.1?mg/ml) or in oxygen-enriched media were similar or even slightly higher in the active respiratory state 3 than in the resting state 4 for both substrates. Consequently, these findings indicate that isolated mitochondria, respiring in vitro under conditions of forward electron transport, release ROS with Complex I- and II-linked substrates in the resting condition (state 4) and when energy demand is maximal (state 3), provided that there is sufficient oxygen dissolved in the medium.