SEARCH · Search PubMed

Results for “Yield stability”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

SourceResearch connectionFrom yearThrough year

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 343 records · Page 19Linked to original sources

PubMed2002

Feasibility of generating adeno-associated virus packaging cell lines containing inducible adenovirus helper genes.

The adeno-associated virus (AAV) vector system is based on nonpathogenic and helper-virus-dependent parvoviruses. The vector system offers safe, efficient, and long-term in vivo gene transfer in numerous tissues. Clinical trials using AAV vectors have demonstrated vector safety as well as efficiency. The increasing interest in the use of AAV for clinical studies demands large quantities of vectors and hence a need for improvement in vector production. The commonly used transient-transfection method, although versatile and free of adenovirus (Ad), is not cost-effective for large-scale production. While the wild-type-Ad-dependent AAV producer cell lines seem to be cost-effective, this method faces the problem of wild-type Ad contamination. To overcome these shortcomings, we have explored the feasibility of creating inducible AAV packaging cell lines that require neither transfection nor helper virus infection. As a first step toward that goal, we have created a cell line containing highly inducible Ad E1A and E1B genes, which are essential for AAV production. Subsequently, the AAV Rep and Cap genes and an AAV vector containing a green fluorescent protein (GFP) reporter gene were stably introduced into the E1A-E1B cell line, generating inducible AAV-GFP packaging cell lines. Upon induction of E1A and E1B genes and infection with replication-defective Ad with E1A, E1B, and E3 deleted, the packaging cells yielded high-titer AAV-GFP vectors. Finally, the E2, E4, and VA genes of Ad, under the control of their endogenous promoters, were also introduced into these cells. A few producer cell lines were obtained, which could produce AAV-GFP vectors upon simple drug induction. Although future improvement is necessary to increase the stability and vector yield of the cells, our study has nonetheless demonstrated the feasibility of generating helper-virus-free inducible AAV producer cell lines.

Adenovirus E1A Proteins↗

PubMed2005

Enzyme stabilization by glutaraldehyde crosslinking of adsorbed proteins on aminated supports.

The stabilization achieved by different immobilization protocols have been compared using three different enzymes (glutaryl acylase (GAC), D-aminoacid oxidase (DAAO), and glucose oxidase (GOX)): adsorption on aminated supports, treatment of this adsorbed enzymes with glutaraldehyde, and immobilization on glutaraldehyde pre-activated supports. In all cases, the treatment of adsorbed enzymes on amino-supports with glutaraldehyde yielded the higher stabilizations: in the case of GOX, a stabilization over 400-fold was achieved. After this treatment, the enzymes could no longer be desorbed from the supports using high ionic strength (suggesting the support-protein reaction). Modification of the enzymes immobilized on supports that did not offer the possibility of react with glutaraldehyde showed the same stability that the non modified preparations demonstrating that the mere chemical modification did not have effect on the enzyme stability. This simple strategy seems to permit very good results in terms of immobilization rate and stability, offering some advantages when compared to the immobilization on glutaraldehyde pre-activated supports.

Adsorption↗

PubMed2006

A classification-based framework for predicting and analyzing gene regulatory response.

BACKGROUND: We have recently introduced a predictive framework for studying gene transcriptional regulation in simpler organisms using a novel supervised learning algorithm called GeneClass. GeneClass is motivated by the hypothesis that in model organisms such as Saccharomyces cerevisiae, we can learn a decision rule for predicting whether a gene is up- or down-regulated in a particular microarray experiment based on the presence of binding site subsequences ("motifs") in the gene's regulatory region and the expression levels of regulators such as transcription factors in the experiment ("parents"). GeneClass formulates the learning task as a classification problem--predicting +1 and -1 labels corresponding to up- and down-regulation beyond the levels of biological and measurement noise in microarray measurements. Using the Adaboost algorithm, GeneClass learns a prediction function in the form of an alternating decision tree, a margin-based generalization of a decision tree. METHODS: In the current work, we introduce a new, robust version of the GeneClass algorithm that increases stability and computational efficiency, yielding a more scalable and reliable predictive model. The improved stability of the prediction tree enables us to introduce a detailed post-processing framework for biological interpretation, including individual and group target gene analysis to reveal condition-specific regulation programs and to suggest signaling pathways. Robust GeneClass uses a novel stabilized variant of boosting that allows a set of correlated features, rather than single features, to be included at nodes of the tree; in this way, biologically important features that are correlated with the single best feature are retained rather than decorrelated and lost in the next round of boosting. Other computational developments include fast matrix computation of the loss function for all features, allowing scalability to large datasets, and the use of abstaining weak rules, which results in a more shallow and interpretable tree. We also show how to incorporate genome-wide protein-DNA binding data from ChIP chip experiments into the GeneClass algorithm, and we use an improved noise model for gene expression data. RESULTS: Using the improved scalability of Robust GeneClass, we present larger scale experiments on a yeast environmental stress dataset, training and testing on all genes and using a comprehensive set of potential regulators. We demonstrate the improved stability of the features in the learned prediction tree, and we show the utility of the post-processing framework by analyzing two groups of genes in yeast--the protein chaperones and a set of putative targets of the Nrg1 and Nrg2 transcription factors--and suggesting novel hypotheses about their transcriptional and post-transcriptional regulation. Detailed results and Robust GeneClass source code is available for download from http://www.cs.columbia.edu/compbio/robust-geneclass.

Algorithms↗

PubMedDate not supplied

Stoichiometric network analysis.

Stoichiometric network analysis is a systematic, general approach to the qualitative, nonlinear dynamics of chemical reaction mechanisms and other systems with stoichiometry. The advantage of a qualitative approach is that no rate constants are needed to determine qualitative features of the dynamics. If one is interested in stability, the approach yields inequalities among the steady-state concentrations and the rate of flow through sequences of important reactions. These parameters are often the ones most easily measured experimentally. By comparing such experiments with the inequalities derived from stoichiometric network analysis, one can often prove that certain mechanisms cannot account for oscillations or other types of observed dynamics. The approach covers far more than stability. The existence of steady states of zero concentration has an interesting mathematics and applies to chemical evolution. The folding of the manifold of steady states can be found by direct calculation and plays a role in switching enzymes on and off. The approach leads to theorems showing that some steady states are globally attracting or, possibly, that a region containing chaos or an oscillation is globally attracting. The subject of sensitivity analysis has been reformulated in this context. Algorithms that apply many of the theoretical results to chemical networks have been developed and combined into a computer program package.

Chemical Phenomena↗

PubMed1988

Studies on the stability of VIII:C during the manufacture of a factor VIII concentrate for clinical use.

The stability of VIII:C was investigated by monitoring samples taken at different points from a routine process for the manufacture of factor VIII concentrate and by examining the stabilising influence of a number of product formulations. Loss of VIII:C over process-finishing procedures (formulation, 0.22 micron filtration, dispensing) was associated with a citrate-induced inactivation which could be prevented by controlling the ionised calcium concentration of the solution. These results were obtained using a one-stage clotting assay but were not observed using a two-stage assay. No evidence for activation was found in vitro (e.g. by FPA generation and VIII:C stability) and the yield increase suggested by the one-stage assay was supported by results from a controlled clinical evaluation.

Antigens↗

PubMed1997

Improvement of the refolding yield and solubility of hen egg-white lysozyme by altering the Met residue attached to its N-terminus to Ser.

When hen egg-white lysozyme was produced in Escherichia coli, it possessed an extra methionine residue at the N-terminus (Met(-1)-lysozyme). The Met(-1)-lysozyme showed a decreased refolding yield and solubility compared with the native hen egg-white lysozyme, as the methionine is a hydrophobic amino acid. A Met(-2)Pro(-1) or Met(-2)Ser(-1) sequence was introduced at the N-terminus of hen egg-white lysozyme. The methionine residue in these hen egg-white lysozymes was completely removed by methionine aminopeptidase, as expected, since the penultimate residue was proline or serine. From the analyses of solubility, stability and refolding yield, it was found that an extra Ser residue attached to the N-terminus of hen egg-white lysozyme (Ser(-1)-lysozyme) showed closer characteristics to the native hen egg-white lysozyme than did Met(-1) or an extra Pro residue attached to the N-terminus of hen egg-white lysozyme (Pro(-1)-lysozyme). Moreover, the tertiary conformation of Ser(-1)-lysozyme examined by NMR spectroscopy and its activity were almost identical with those of native hen egg-white lysozyme.

Animals↗

PubMedDate not supplied

Analysis of pharmaceutical dosage forms for oxfendazole: I. Reverse phase liquid chromatographic determination of oxfendazole in swine premix.

A reverse phase liquid chromatographic (LC) procedure is described for quantitating oxfendazole (2-(methoxycarbonylamino)-5-phenylsulfinylbenzimidazole] in swine premix. Sample preparation consists of extracting oxfendazole with an acetone-methanol mixture. An aliquot of the extract is then centrifuged to separate undissolved premix excipients. Internal standard is added to the supernate and the sample is further diluted with water-acetonitrile-phosphoric acid (80 + 20 + 1). Oxfendazole is quantitatively determined using a Partisil-5-ODS-3 column with acetonitrile-0.01 M phosphate buffer (pH 6.0) as the mobile phase. The method is stability specific and yields a mean recovery of 101.1 +/- 0.4% for the 1.35% premix formulation. The dependence of chromatographic performance characteristics on mobile phase organic content, pH, and buffer concentration is also reported.

Animal Feed↗

PubMed1993

Stability and activity of immobilized hydrolytic enzymes in two-liquid-phase systems: acid phosphatase, beta-glucosidase, and beta-fructofuranosidase entrapped in poly(2-hydroxyethyl methacrylate) matrices.

Enzyme storage stability and hydrolysis yield were measured in experiments carried out with three model hydrolytic enzymes: acid phosphatase (EC 3.1.3.2), beta-glucosidase (EC 3.2.1.4), and beta-fructofuranosidase (EC 3.2.1.26) entrapped in hydrogels of poly(2-hydroxyethyl methacrylate). Runs were performed at 30 degrees C, under intensive stirring (500 rev min-1), in 50% v/v biphasic media prepared with buffer and organic solvents, whose log P value varied from 0.68 to 8.8. Storage stability was also monitored in the pure solvents. The small average particle size (125-210 microns) and the intensive stirring eliminate hindrances of intra- and interphase mass transfer resistances. The hydrophilic matrix protects the enzymes against thermal and chemical deactivation, thus allowing good production per unit weight of biocatalyst. In biphasic media, storage stability, with the exception of acid phosphatase, was not dependent on solvent polarity. On the contrary, a significant trend was observed when the enzymes were stored in neat organic solvents.

Acid Phosphatase↗

PubMed2002

Improvement of recombinant protein yield by a combination of transcriptional amplification and stabilization of gene expression.

We explored the use of a cascade circuit for heterologous gene expression that consists of a regulatory module with a salicylate-inducible system that controls the expression of a second regulator, xylS2, whose product is activated by common inducers. Activation and increasing the concentration of the second regulator synergistically induced heterologous genes downstream of the Pm promoter in the expression module. This module can be placed in multicopy vectors or in the chromosome of a host strain by means of minitransposons. Using reporter genes, we evaluated gene regulation capacity and gross production of the system with different configurations. The highest yield was obtained when the expression module was in a multicopy plasmid after a 6-h induction. However, expression modules in plasmids showed low stability after induction even with selective pressure. The chromosomal configuration had the lowest basal levels and induced levels comparable to those of plasmid configurations, resulting in accumulation of more than 10% of the total protein. Unlike the configurations in plasmids, the yield was maintained for at least 3 days even without selective pressure. In conclusion, the cascade system in the chromosome configuration is more efficient for long-term fermentation because of the great stability of the overexpressing phenotype in spite of the high levels of expression.

Cloning, Molecular↗

PubMed1988

External skeletal fixation: choosing a system based on biomechanical stability.

Due to the increased popularity of external fixators for treating long-bone fractures, many devices are being introduced to the market. The choice of a particular fixation device depends on the anticipated loading conditions or the demands the fixator might encounter during the healing process. This study compares the biomechanical stability (rigidity, yield-load, failure-load) and load to produce 1 mm of fracture-gap displacement of various half-frames of five systems tested in axial compression, torsion, and both anterior-posterior and medial-lateral bending; the slippage tolerances of various interfaces of the universal joints or clamps were also analyzed. The frames were mounted on acrylic rods, with a midshaft transverse saw-cut, displaced by 10 mm, and set to standardized dimensions and tightened at set torques. In terms of stability, the Brooker and Hoffman systems are, in general, less stable than the RxFx, AO, and Orthofix fixators. The single half-frames of all systems, except the Orthofix, were particularly weak, and the double and stacked half-frames of each system were more stable.

Biomechanical Phenomena↗

PubMed2001

Selection of stabilizing additive for lipase immobilization on controlled pore silica by factorial design.

Candida rugosa lipase was covalently immobilized on silanized controlled pore silica (CPS) previously activated with glutaraldehyde in the presence of several additives to improve the performance of the immobilized form in long-term operation. Proteins (albumin and lecithin) and organic molecules (beta-cyclodextrin and polyethylene glycol [PEG]-1500) were added during the immobilization procedure, and their effects are reported and compared to the behavior of the immobilized biocatalyst in the absence (lacking) of additive. The selection of the most efficient additive at different lipase loadings (150-450 U/g of dry support) was performed by experimental design. Two 22 full factorial designs with two repetitions at the center point were employed to evaluate the immobilization yield. A better stabilizing effect was found when small amounts of albumin or PEG-1500 were added simultaneously to the lipase onto the support. The catalytic activity had a maximum (193 U/mg) for lipase loading of 150 U/g of dry support using PEG-1500 as the stabilizing additive. This immobilized system was used to perform esterification reactions under repeated batch cycles (for the synthesis of butyl butyrate as a model). The half-life of the lipase immobilized on CPS in the presence of PEG-1500 was found to increase fivefold compared with the control (immobilized lipase on CPS without additive).

Albumins↗

PubMed2002

Structural parameterization of the binding enthalpy of small ligands.

A major goal in ligand and drug design is the optimization of the binding affinity of selected lead molecules. However, the binding affinity is defined by the free energy of binding, which, in turn, is determined by the enthalpy and entropy changes. Because the binding enthalpy is the term that predominantly reflects the strength of the interactions of the ligand with its target relative to those with the solvent, it is desirable to develop ways of predicting enthalpy changes from structural considerations. The application of structure/enthalpy correlations derived from protein stability data has yielded inconsistent results when applied to small ligands of pharmaceutical interest (MW < 800). Here we present a first attempt at an empirical parameterization of the binding enthalpy for small ligands in terms of structural information. We find that at least three terms need to be considered: (1) the intrinsic enthalpy change that reflects the nature of the interactions between ligand, target, and solvent; (2) the enthalpy associated with any possible conformational change in the protein or ligand upon binding; and, (3) the enthalpy associated with protonation/deprotonation events, if present. As in the case of protein stability, the intrinsic binding enthalpy scales with changes in solvent accessible surface areas. However, an accurate estimation of the intrinsic binding enthalpy requires explicit consideration of long-lived water molecules at the binding interface. The best statistical structure/enthalpy correlation is obtained when buried water molecules within 5-7 A of the ligand are included in the calculations. For all seven protein systems considered (HIV-1 protease, dihydrodipicolinate reductase, Rnase T1, streptavidin, pp60c-Src SH2 domain, Hsp90 molecular chaperone, and bovine beta-trypsin) the binding enthalpy of 25 small molecular weight peptide and nonpeptide ligands can be accounted for with a standard error of +/-0.3 kcal x mol(-1).

Animals↗

PubMed2003

Connecting terminal carboxylate groups in nine-coordinate lanthanide podates: consequences on the thermodynamic, structural, electronic, and photophysical properties.

The hydrolysis of terminal (t)butyl-ester groups provides the novel nonadentate podand tris[2-[N-methylcarbamoyl-(6-carboxypyridine-2)-ethyl]amine] (L13) which exists as a mixture of slowly interconverting conformers in solution. At pH = 8.0 in water, its deprotonated form [L13 - 3H](3-) reacts with Ln(ClO(4))(3) to give the poorly soluble and stable podates [Ln(L13 - 3H)] (log(beta(110)) = 6.7-7.0, Ln = La-Lu). The isolated complexes [Ln(L13 - 3H)](H(2)O)(7) (Ln = Eu, 8; Tb, 9; Lu, 10) are isostructural, and their crystal structures show Ln(III) to be nine-coordinate in a pseudotricapped trigonal prismatic site defined by the donor atoms of the three helically wrapped tridentate binding units of L13. The Ln-O(carboxamide) bonds are only marginally longer than the Ln-O(carboxylate) bonds in [Ln(L13 - 3H)], thus producing a regular triple helix around Ln(III) which reverses its screw direction within the covalent Me-TREN tripod. High-resolution emission spectroscopy demonstrates that (i) the replacement of terminal carboxamides with carboxylates induces only minor electronic changes for the metallic site, (ii) the solid-state structure is maintained in water, and (iii) the metal in the podate is efficiently protected from interactions with solvent molecules. The absolute quantum yields obtained for [Eu(L13 - 3H)] (Phi(Eu)(tot)= 1.8 x 10(-3)) and [Tb(L13 - 3H)] (Phi(Eu)(tot)= 8.9 x 10(-3)) in water remain modest and strongly contrast with that obtained for the lanthanide luminescence step (Phi(Eu) = 0.28). Detailed photophysical studies assign this discrepancy to the small energy gap between the ligand-centered singlet ((1)pi pi*) and triplet ((3)pi pi*) states which limits the efficiency of the intersystem crossing process. Theoretical TDDFT calculations suggest that the connection of a carboxylate group to the central pyridine ring prevents the sizable stabilization of the triplet state required for an efficient sensitization process. The thermodynamic and electronic origins of the advantages (stability, lanthanide quantum yield) and drawbacks (solubility, sensitization) brought by the "carboxylate effect" in lanthanide complexes are evaluated for programming predetermined properties in functional devices.

Journal Article↗

PubMed2006

Human recombinant prolidase from eukaryotic and prokaryotic sources. Expression, purification, characterization and long-term stability studies.

Prolidase is a Mn(2+)-dependent dipeptidase that cleaves imidodipeptides containing C-terminal proline or hydroxyproline. In humans, a lack of prolidase activity causes prolidase deficiency, a rare autosomal recessive disease, characterized by a wide range of clinical outcomes, including severe skin lesions, mental retardation, and infections of the respiratory tract. In this study, recombinant prolidase was produced as a fusion protein with an N-terminal histidine tag in eukaryotic and prokaryotic hosts and purified in a single step using immobilized metal affinity chromatography. The enzyme was characterized in terms of activity against different substrates, in the presence of various bivalent ions, in the presence of the strong inhibitor Cbz-Pro, and at different temperatures and pHs. The recombinant enzyme with and without a tag showed properties mainly indistinguishable from those of the native prolidase from fibroblast lysate. The protein yield was higher from the prokaryotic source, and a detailed long-term stability study of this enzyme at 37 degrees C was therefore undertaken. For this analysis, an 'on-column' digestion of the N-terminal His tag by Factor Xa was performed. A positive effect of Mn(2+) and GSH in the incubation mixture and high stability of the untagged enzyme are reported. Poly(ethylene glycol) and glycerol had a stabilizing effect, the latter being the more effective. In addition, no significant degradation was detected after up to 6 days of incubation with cellular lysate. Generation of the prolidase in Escherichia coli, because of its high yield, stability, and similarity to native prolidase, appears to be the best approach for future structural studies and enzyme replacement therapy.

Animals↗

PubMed1984

Local stability involved in characterizing and controlling promoters in eukaryotes.

Eukaryotic promoters with known in vivo activities have been analysed for characteristic stability patterns. Correlation of transcription yield in promoter mutants with size and stability of individual domains of the promoter stability profile supports the conclusion that eukaryotic promoters are built up by at least three elements: a region enabling the transcription ("enhancer"), with a characteristic stability pattern; an activator domain, with high GC content, whose activator potential is controlled by the domain stability and length; a trap domain, with high AT content, setting the cap site. The activated enzyme undergoes a steady deactivation process, losing half of its activity upon moving 55 bases between the activator and the trap site.

Base Composition↗

PubMed2003

Listening to the blue: the time-resolved thermodynamics of the bacterial blue-light receptor YtvA and its isolated LOV domain.

YtvA is a bacterial flavo-protein related to plant phototropin. The photochemistry of YtvA and of its isolated LOV domain (YtvA-LOV) has been characterized by optical, mass spectrometric and photocalorimetric methods. The energy content (E390) of the FMN-C4a-thiol photoadduct (YtvA390 and YtvA-LOV390) and its structural volume change (deltaV390), with respect to the parent state, have been determined by means of Laser Induced Optoacoustic Spectroscopy (LIOAS). The high value of E390, 136 and 115 kJ mol(-1), respectively, ensures a large driving force for the dark recovery to the unphotolyzed state and points to a strained conformation of the protein or/and the chromophore in the photoadduct. The value of deltaV390 is significantly different for the two proteins, deltaV390 = -12.5 ml mol(-1) in YtvA and -17.2 ml mol(-1) in YtvA-LOV. The kinetics of the dark recovery reaction for YtvA-LOV is slower than for full-length YtvA, with taurec = 3900 and 2600 s at 25 degrees C, respectively, and shows a different temperature dependence. A similarly slow kinetics can be induced in YtvA by high ionic strength. Minor differences are observed in the fluorescence and photoadduct formation quantum yield. The overall stability is higher for YtvA than for YtvA-LOV. The data as a whole are indicative of an interaction between the two domains of YtvA, most probably mediated by electrostatic interactions that renders the full-length protein a compact and more rigid unit. The results reported here support the idea that the formation of the photoadduct changes the dynamics of the protein, depending on the conformational flexibility of the parent state. Flashing of the photoadduct induces a negligible deltaV, with 96% of the excitation energy dissipated as heat in <20 ns, indicating that the photoadduct does not undergo a photocycle on the LIOAS time scale, or that the photoinduced reactions occur with very low yield.

Acoustics↗

PubMedDate not supplied

[Production of Candida lipolytica protoplasts].

Optimal conditions were found for the production and isolation of the protoplasts of Candida lipolytica. The maximum amount of the protoplasts was produced after 90 minutes of the incubation with a crude preparation of the enzyme from Helix pomatia (100 mg/g wet biomass). Longer incubation results in lysis of the protoplasts and structural damages of the intracellular components. The yield of the protoplasts does not depend on the nature of stabilizing agent. A decrease in the stabilizer concentration increases the yield of the protoplasts four times. Preliminary treatment of the yeast cells with a 0.1 M solution of SH compounds (cysteine, beta-mercaptoethanol) does not increase the yield of the protoplasts; and 0.2 M solution of these compounds decreased the yield of the protoplasts.

Candida↗

PubMedDate not supplied

[Modification of Bacillus intermedius ribonuclease by dialdehyde dextran].

Conditions for modification of Bacillus intermedius ribonuclease with dialdehyde dextran was optimized by Box and Wilson's method to achieve the enzyme with higher activity. The generalized parameter including the yield of enzyme protein and enzyme stability during modification was used. The yield of activity and preparation characteristics depended on a number of enzyme-carrier bonds.

Bacillus↗