Production of a Rhizopus oryzae lipase from Pichia pastoris using alternative operational strategies

Different cultivation strategies have been compared for the production of Rhizopus oryzae lipase (ROL) from Pichia pastoris. Several drawbacks have been found using a methanol non-limited fed-batch. On the one hand, oxygen limitation appeared at early cell dry weights and, on the other hand, high cell death was observed. A temperature limited fed-batch has been proposed to solve both problems. However, in our case study a methanol non-limited fed-batch results in better productivities. Finally, a lower salt medium were used to overcome cell death problems and a temperature limited fed-batch was applied thereafter to solve oxygen transfer limitations. This combined strategy has resulted in lower productivities when compared to a methanol non-limited fed-batch. However the culture could be longer prolonged and a 1.3-fold purer final product was obtained mainly due to cell death reduction.     

Transient depletion of Ku70 and Xrcc4 by RNAi as a means to manipulate the non-homologous end-joining pathway

Non-homologous end joining (NHEJ) is the major DNA double-strand break (DSB) repair pathway in mammalian cells and is likely responsible for the non-homologous integration of transgenes. In higher eukaryotes, this pathway predominates over the homologous recombination (HR) pathway and therefore may account for the low level of HR events that occur in mammalian cells. We evaluated the effects of transient RNAi-induced down-regulation of key components of the NHEJ pathway in human HCT116 cells. Treatment with siRNA targeting Ku70 and Xrcc4 reduced corresponding protein levels by 80-90% 48h after transfection, with a return to normal levels by 96h. Additionally, down-regulation of Ku70 and Xrcc4 resulted in a concomitant depletion of both Ku70 and Ku86 proteins. Biological consequences of transient RNAi-mediated depletion of Ku70 and Xrcc4 included sensitization to gamma radiation and a significant decrease in the expression of a linear GFP reporter gene. The results highlight the possibility of a successful means to manipulate the NHEJ pathway by RNAi.     

Antioxidant responses and lipid peroxidation following intranasal 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration in rats: increased susceptibility of olfactory bulb

We evaluated an alternative method to investigate a possible involvement of environmental toxins in the pathology of Parkinson's disease (PD). There is considerable evidence supporting the role of oxidative stress in the toxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a neurotoxin largely used to modeling PD in primates and rodents. We have recently demonstrated that rats treated with intranasal (i.n.) infusion of MPTP suffer from progressive signs of PD that are correlated with time-dependent degeneration in dopaminergic neurons. In the present study, we investigated the time-dependent (2 h to 7 days) effect of a single i.n. administration of MPTP (0.1 mg/nostril) on the glutathione-related antioxidant status and lipid peroxidation (TBARS) in the adult Wistar rat brain. The effects were more pronounced in the olfactory bulb at 6 h after i.n. MPTP administration, as indicated by an increase in TBARS and total glutathione (GSH-t) levels, and also in the gamma-glutamyl transpeptidase (GGT) activity. Increased levels of TBARS, GSH-t and GGT activity were also observed at 6 h post-MPTP infusion in some structures (e.g. striatum, hippocampus and prefrontal cortex). No difference regarding glutathione reductase activity was observed in any of the brain structures analyzed, while a marked decrease in glutathione peroxidase activity was specifically observed in the substantia nigra 7 days after MPTP treatment. These results demonstrate that a single i.n. infusion of MPTP in rats induces significant alterations in the brain antioxidant status and lipid peroxidation, reinforcing the notion that the olfactory system represents a particularly sensitive route for the transport of neurotoxins into the central nervous system that may be related to the etiology of PD.     

Baroreflex dysfunction in rats submitted to protein restriction

Earlier studies from the authors' laboratory showed that malnourishment induces alterations in the cardiovascular homeostasis increasing the basal mean arterial pressure and heart rate. In this study, the authors evaluated whether the sympathetic and parasympathetic efferent activities contribute to changes in the cardiovascular homeostasis through altered modulation of the arterial baroreflex of malnourished rats. After weaning, male Fischer rats were given 15% (Normal Protein--NP) or 6% (Low Protein--LP) protein diet for 35 d. The baroreflex gain and latency were evaluated before and after selective autonomic blockades in control and malnourished rats. It was observed that malnourishment affected the baroreflex gain in response to activation and deactivation of the arterial baroreflex. Moreover, malnourished rats showed increased baroreflex latency as compared to that of control rats. Regarding the autonomic efferent activity directed to the heart, the data showed increased sympathetic and decreased parasympathetic efferent activities in malnourished rats, and such alterations could be related to the observed changes in the arterial baroreflex gain as well as in the basal mean arterial pressure and heart rate.     

<Emphasis Type="Italic">Tuberosciponoceras</Emphasis>: a new dimorphic ammonoid genus from the Upper Albian of Patagonia,Argentina

Tuberosciponoceras insolitum n. gen., n. sp. is a single dimorphic species in the Upper Albian of the Austrat Basin, southern Patagonia. The short-lived genus was characterized by distinct ventral tubercles on all ribs and a deep, trifid internal lobe.     

Early root growth plasticity in seedlings of three Mediterranean woody species

Using a 141 F₂ population generated from maize inbred B64 × teosinte Zea nicaraguensis cross, quantitative trait loci (QTLs) controlling aerenchyma formation in roots under non-flooding drained soil conditions were identified. Seedlings of Z. nicaraguensis formed clear aerenchyma in the cortex of adventitious roots in non-flooding conditions, whereas the maize inbred line B64 did not. In the F₂ population, the capacity to develop aerenchyma exhibited wide and continuous variation, suggesting the trait was controlled by multiple genes. A linkage map was developed using 85 SSR markers, covering 1,224 cM across all ten chromosomes. Composite interval mapping analysis revealed that four QTLs for aerenchyma formation under non-flooding conditions were located to two regions of chromosome 1 (identified as Qaer1.02-3 and Qaer1.07), chromosome 5 (Qaer5.09) and chromosome 8 (Qaer8.06-7), and these explained 46.5% of the total phenotypic variance. The multiple interval mapping approach identified additional QTLs on chromosomes 1 (Qaer1.01) and 5 (Qaer5.01). Using these results, it may be possible to use SSR markers linked to aerenchyma formation in a marker assisted selection approach to introduce aerenchyma formation in drained soil conditions into maize for the eventual development of flooding tolerant maize hybrids.     

Expression of the sucrose binding protein from soybean: renaturation and stability of the recombinant protein

The sucrose binding protein (SBP) belongs to the cupin family of proteins and is structurally related to vicilin-like storage proteins. In this investigation, a SBP isoform (GmSBP2/S64) was expressed in E. coli and large amounts of the protein accumulated in the insoluble fraction as inclusion bodies. The renatured protein was studied by circular dichroism (CD), intrinsic fluorescence, and binding of the hydrophobic probes ANS and Bis-ANS. The estimated content of secondary structure of the renatured protein was consistent with that obtained by theoretical modeling with a large predominance of beta-strand structure (42%) over the alpha-helix (9.9%). The fluorescence emission maximum of 303 nm for SBP2 indicated that the fluorescent tryptophan was completely buried within a highly hydrophobic environment. We also measured the equilibrium dissociation constant (K(d)) of sucrose binding by fluorescence titration using the refolded protein. The low sucrose binding affinity (K(d)=2.79+/-0.22 mM) of the renatured protein was similar to that of the native protein purified from soybean seeds. Collectively, these results indicate that the folded structure of the renatured protein was similar to the native SBP protein. As a member of the bicupin family of proteins, which includes highly stable seed storage proteins, SBP2 was fairly stable at high temperatures. Likewise, it remained folded to a similar extent in the presence or absence of 7.6M urea or 6.7 M GdmHCl. The high stability of the renatured protein may be a reminiscent property of SBP from its evolutionary relatedness to the seed storage proteins.