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81.
82.
Gil DF García-Fernández R Alonso-del-Rivero M Lamazares E Pérez M Varas L Díaz J Chávez MA González-González Y Mansur M 《FEMS yeast research》2011,11(7):575-586
Pichia pastoris is a highly successful system for the large-scale expression of heterologous proteins, with the added capability of performing most eukaryotic post-translational modifications. However, this system has one significant disadvantage - frequent proteolytic degradation by P. pastoris proteases of heterologously expressed proteins. Several methods have been proposed to address this problem, but none has proven fully effective. We tested the effectiveness of a broad specificity protease inhibitor to control proteolysis. A recombinant variant of the BPTI-Kunitz protease inhibitor ShPI-1 isolated from the sea anemone Stichodactyla helianthus, was expressed in P. pastoris. The recombinant inhibitor (rShPI-1A), containing four additional amino acids (EAEA) at the N-terminus, was folded similarly to the natural inhibitor, as assessed by circular dichroism. rShPI-1A had broad protease specificity, inhibiting serine, aspartic, and cysteine proteases similarly to the natural inhibitor. rShPI-1A protected a model protein, recombinant human miniproinsulin (rhMPI), from proteolytic degradation during expression in P. pastoris. The addition of purified rShPI-1A at the beginning of the induction phase significantly protected rhMPI from proteolysis in culture broth. The results suggest that a broad specificity protease inhibitor such as rShPI-1A can be used to improve the yield of recombinant proteins secreted from P. pastoris. 相似文献
83.
Gambarino S Costa C Astegiano S Piasentin EA Segoloni GP Cavallo R Bergallo M 《Molecular biotechnology》2011,49(2):151-158
Polyomavirus BK latently persist in different sites, including the renourinary tract, and may reactivate causing nephropathy
in renal transplant recipients or hemorrhagic cystitis in bone marrow recipients. Based on the sequence of the VP1 gene, four
genotypes have been described, corresponding to the four serologically differentiated subtypes I–IV, with different prevalence
and geographic distribution. In this study, the development and clinical validation of four different Real-Time PCR assays
for the detection and discrimination of BKV genotypes as a substitute of DNA sequencing are described. 379 BK VP1 sequences,
belonging to the main four genotypes, were aligned and “hot spots” of mutation specific for all the strains or isolates were
identified. Specific primers and probes for the detection and discrimination of each genotype by four Real-Time PCR assays
were designed and technically validated. Subsequently, the four Real-Time PCR assays were used to test 20 BK-positive urine
specimens from renal transplant patients, and evidenced a prevalence of BK genotype I, as previously reported in Europe. Results
were confirmed by sequencing. The availability of a rapid and simple genotyping method could be useful for the evaluation
of BK genotypes prevalence and studies on the impact of the infecting genotype on viral biological behavior, pathogenic role,
and immune evasion strategies. 相似文献
84.
Arroyo M García-Hidalgo J Villalón M de Eugenio L Hormigo D Acebal C García JL Prieto MA de la Mata I 《Journal of industrial microbiology & biotechnology》2011,38(9):1203-1209
Purification and matrix-assisted refolding of recombinant His-tagged polyhydroxyalkanoate (PhaZ) depolymerase from Pseudomonas putida KT2442 was carried out. His-tagged enzyme was overproduced as inclusion bodies in recombinant E. coli M15 (pREP4, pPAZ3), which were denatured by 8 M urea, immobilized on Ni2+-nitrilotriacetate-agarose matrix, and refolded by gradual removal of the chaotropic agent. The refolded enzyme could not
be eluted with 1 M imidazole buffer, leading to an immobilized biocatalyst where PhaZ depolymerase was homogeneously distributed
in the agarose support as shown by confocal scanning microscopy. Polyhydroxyoctanoate could not be hydrolyzed by this novel
immobilized biocatalyst, whereas the attached enzyme was active in the hydrolysis of p-nitrophenyl alkanoate esters, which differed in their alkyl chain length. Taking advantage of the observed esterase activity
on p-nitrophenylacetate, functional characterization of immobilized PhaZ depolymerase was carried out. The immobilized enzyme
was more stable than its soluble counterpart and showed optimal hydrolytic activity at 37°C and 50 mM phosphate buffer pH
8.0. Kinetic parameters were obtained with both p-nitrophenylacetate and p-nitrophenyloctanoate, which had not been described so far for the soluble enzyme, representing an attractive and alternative
chromogenic assay for the study of this paradigmatic enzyme. 相似文献
85.
Corona C Frazzini V Silvestri E Lattanzio R La Sorda R Piantelli M Canzoniero LM Ciavardelli D Rizzarelli E Sensi SL 《PloS one》2011,6(3):e17971
Background
The pathogenic road map leading to Alzheimer''s disease (AD) is still not completely understood; however, a large body of studies in the last few years supports the idea that beside the classic hallmarks of the disease, namely the accumulation of amyloid-β (Aβ) and neurofibrillary tangles, other factors significantly contribute to the initiation and the progression of the disease. Among them, mitochondria failure, an unbalanced neuronal redox state, and the dyshomeostasis of endogenous metals like copper, iron, and zinc have all been reported to play an important role in exacerbating AD pathology. Given these factors, the endogenous peptide carnosine may be potentially beneficial in the treatment of AD because of its free-radical scavenger and metal chelating properties.Methodology
In this study, we explored the effect of L-carnosine supplementation in the 3xTg-AD mouse, an animal model of AD that shows both Aβ- and tau-dependent pathology.Principal Findings
We found that carnosine supplementation in 3xTg-AD mice promotes a strong reduction in the hippocampal intraneuronal accumulation of Aβ and completely rescues AD and aging-related mitochondrial dysfunctions. No effects were found on tau pathology and we only observed a trend toward the amelioration of cognitive deficits.Conclusions and Significance
Our data indicate that carnosine can be part of a combined therapeutic approach for the treatment of AD. 相似文献86.
R.P. Bateman E. Hidalgo J. García C. Arroyo G.M. ten Hoopen V. Adonijah & U. Krauss 《The Annals of applied biology》2005,147(2):129-138
This article describes two field trials carried out at La Lola, Costa Rica, to assess control measures against frosty pod rot of cocoa (Theobroma cacao) caused by Moniliophthora (Crinipellis) roreri. In the first, factorial, trial the control agents were applied using motorised mistblowers (MMs) and hydraulic sprayers fitted with a narrow angle cone nozzle. There was an interaction between agents and application methods; together with previous application data for the most active fungicide (copper hydroxide), these trials indicate that best yields are achieved with sprays that maximise deposits on pods. We describe the droplet size spectra produced by a Stihl SR400 MM under a range of conditions because this has become the standard method of fungicide application in this series of trials at La Lola. The factor that had the largest effect on droplet size spectrum was the presence or the absence of a detachable baffle plate in front of the air‐shear nozzle. In both trials described here, MMs were fitted with baffle plates, a formulation pump and restrictor transmitting 550 mL min?1 to deliver an estimated equivalent of 190 L ha?1. Copper hydroxide as prophylactic applications at 1500 g a.i. ha?1 have, to date, shown the most consistent (but incomplete) improvement in healthy pod yield. Use of copper fungicides may be cost effective when farm‐gate cocoa prices exceed approximately $1.25 kg?1. In these trials, isolates of the hyperparasitic fungi Clonostachys byssicola and Trichoderma asperellum and two off‐patent triazole fungicides (bitertanol and triadimenol) made no significant improvement to healthy yields. The systemic oxathiin fungicide flutolanil, at a dosage of 300 g a.i. ha?1, appears to protect pods substantially at early stages but gives proportionately less control of M. roreri than copper at later stages of pod development. 相似文献
87.
Henry Rivera Begoña Merinero Mercedes Martinez-Pardo Ignacio Arroyo Pedro Ruiz-Sala Belen Bornstein Clara Serra-Suhe Esther Gallardo Ramon Marti Maria J. Moran Cristina Ugalde Luis A. Perez-Jurado Antoni L. Andreu Rafael Garesse Magdalena Ugarte Joaquin Arenas Miguel A. Martin 《Mitochondrion》2010,10(4):362-368
The aim of this study was to identify the causative genetic lesion in two apparently unrelated newborns having lethal lactic acidosis, multi-organ failure and congenital malformations including interrupted aortic arch, who exhibited mild methylmalonic aciduria, combined mitochondrial respiratory chain deficiency, and marked muscle mitochondrial DNA depletion. A novel mutation in the SUCLG1 gene was identified. Phenotype severity in Succinate-CoA ligase dysfunction appears to be more correlated to the muscle mtDNA content than to the tissue distribution of the heterodimer subunits. Prominent impairment of mitochondrial respiratory chain may result in deep ravages in developmental tissues leading to multiple organ failure and malformations. 相似文献
88.
Characterization of natural peptide ligands from HLA-DP2: new insights into HLA-DP peptide-binding motifs 总被引:2,自引:0,他引:2
Although natural peptide ligands of HLA-DR and HLA-DQ molecules have been extensively studied, information about peptides naturally bound to HLA-DP is limited. Here we describe HLA-DP2 peptide ligands corresponding to 24 different source proteins that were identified by peptide pool elution and mass spectrometry sequencing from HLA-DP2 molecules expressed on EBV-LCLs. Sequencing analysis led to the identification of both promiscuous and allele-specific peptides. Moreover, the alignment of the natural ligands for HLA-DP2 described here, combined with previous results from our group and others concerning HLA-DP2 antigen presentation and HLA-DP molecular modelling, provide a better understanding of HLA-DP2 peptide-binding motifs. 相似文献
89.
Manuel B.?MoralesEmail author Vincent?Bretagnolle Beatriz?Arroyo 《Biodiversity and Conservation》2005,14(13):3135-3150
Stochastic computer simulations are used to evaluate the sensitivity of Little bustard population parameters, estimating the
survival probabilities of the seven endangered Little bustard populations of central-western France for which conservation
actions are currentlybeing or have been implemented. Different scenarios of parameter compensation for those nuclei to establish
parameter levels assuring population viability are discussed. Adult survival, productivity per female, initial population
size and carrying capacity were the most sensitive parameters in a hypothetical, isolated population. Juvenile survival also
affected population survival, although its sensitivity was lower. Sex ratio did not have a linear effect on population survival,
but probability of extinction increased for extreme values. Productivity per female and initial population size, varied strongly
among the populations studied, determining their average time of extinction and growth rate. When a metapopulation scenario
was simulated, the survival probabilities of each population and the metapopulations stayed close to 1.0 if no mortality was
associated to migration. When mortality during migration was included in the simulations, the metapopulation's probability
of survival significantly decreased under 90%. This approach may help managers to correctly address conservation measures
and design effective strategies, which should be directed mainly to improve productivity, enhance female survival, and minimise
mortality during migration (e.g. promoting insect-rich nesting substrates, avoiding female killing and nest destruction at
harvesting, reducing the risk of collision with powerlines, or controlling poaching). 相似文献
90.
Intragranular vesiculotubular compartments are involved in piecemeal degranulation by activated human eosinophils 总被引:1,自引:0,他引:1
Eosinophils, leukocytes involved in allergic, inflammatory and immunoregulatory responses, have a distinct capacity to rapidly secrete preformed granule-stored proteins through piecemeal degranulation (PMD), a secretion process based on vesicular transport of proteins from within granules for extracellular release. Eosinophil-specific granules contain cytokines and cationic proteins, such as major basic protein (MBP). We evaluated structural mechanisms responsible for mobilizing proteins from within eosinophil granules. Human eosinophils stimulated for 30-60 min with eotaxin, regulated on activation, normal, T-cell expressed and secreted (RANTES) or platelet activating factor exhibited ultrastructural features of PMD (e.g. losses of granule contents) and extensive vesiculotubular networks within emptying granules. Brefeldin A inhibited granule emptying and collapsed intragranular vesiculotubular networks. By immunonanogold ultrastructural labelings, CD63, a tetraspanin membrane protein, was localized within granules and on vesicles outside of granules, and mobilization of MBP into vesicles within and extending from granules was demonstrated. Electron tomography with three dimension reconstructions revealed granule internal membranes to constitute an elaborate tubular network able to sequester and relocate granule products upon stimulation. We provide new insights into PMD and identify eosinophil specific granules as organelles whose internal tubulovesicular networks are important for the capacity of eosinophils to secrete, by vesicular transport, their content of preformed and granule-stored cytokines and cationic proteins. 相似文献