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At least 883 records · Page 49Linked to original sources
PubMed1998

Effects of metal binding affinity on the chemical and thermal stability of site-directed mutants of rat oncomodulin.

Tryptophan fluorescence was used to study the stability and unfolding behavior of several single tryptophan mutants of the metal-binding protein rat oncomodulin (OM); F102W, Y57W, Y65W and the engineered protein CDOM33 which had the 12 residues of the CD loop replaced with a more potent metal binding site. Both the thermal and the chemical stability were improved upon binding of metal ions with the order apo < Ca2+ < Tb3+. During thermal denaturation, the transition midpoints (T(un)) of Y65W was the lowest, followed by Y57W and F102W. The placement of the Trp residue in the F-helix in F102W made the protein slightly more thermostable, although the fluorescence response was readily affected by chemical denaturants, which acted through the disruption of hydrogen bonds at the C-terminal end of the F-helix. Under both thermal and chemical denaturation, the engineered protein showed the highest stability. This indicated that increasing the number of metal ligating oxygens in the binding site, either by using a metal ion with a higher coordinate number (i.e., Tb3+) which binds more carboxylate ligands, or by providing more ligating groups, as in the CDOM33 replacement, produces notable improvements in protein stability.

Acrylamide↗
PubMed1993

Characterization of particle- and vapor-phase organic fraction emissions from a heavy-duty diesel engine equipped with a particle trap and regeneration controls.

The effects of a ceramic particle trap on the chemical and biological character of the exhaust from a heavy-duty diesel engine have been studied during steady-state operation and during periods of trap regeneration. Phase I of this project involved developing and refining the methods using a Caterpillar 3208 engine, and Phase II involved more detailed experiments with a Cummins LTA10-300 engine, which met Federal 1988 particulate matter standards, and a ceramic particle trap with built-in regeneration controls. During the Phase I experiments, samples wee collected at the Environmental Protection Agency (EPA)* steady-state mode 4 (50% load at intermediate speed). Varying the dilution ratio to obtain a constant filter-face temperature resulted in less variability in total particulate matter (TPM), particle-associated soluble organic fraction (SOF), solids (SOL), and polynuclear aromatic hydrocarbon (PAH) levels than sampling with a constant dilution ratio and allowing filter-face temperature to vary. A modified microsuspension Ames assay detected mutagenicity in the SOF samples, and in the semivolatile organic fraction extracted from XAD-2 resin (XAD-2 resin organic component, XOC) with at least 10 times less sample mass than the standard plate incorporation assay. Measurement techniques for PAH and nitro-PAH in the SOF and XOC also were developed during this portion of the project. For the Phase II work, two EPA steady-state rated speed modes were selected: mode 11 (25% load) and mode 9 (75% load). With or without the trap, filter-face temperatures were kept at 45 degrees +/- 2 degrees C, nitrogen dioxide (NO2) levels less than 5 parts per million (ppm), and sampling times less than 60 minutes. Particle sizes were determined using an electrical aerosol analyzer. Similar sampling methods were used when the trap was regenerated, except that a separate dilution tunnel and sampling system was designed and built to collect all of the regeneration emissions. The SOF and XOC were extracted from their collection media with dichloromethane. Levels of 12 PAH and nitro-PAH compounds with known biological effects were determined using high-pressure liquid chromatography with fluorescence detection. Two additional dinitro-PAHs were analyzed semi-quantitatively, and several representative SOF and XOC samples were screened for three additional PAH compounds. Mutagenicity was assessed using the modified Ames microsuspension assay.(ABSTRACT TRUNCATED AT 400 WORDS)

Air Pollutants↗
PubMedDate not supplied

A Genetically engineered cell-based system for detecting metabolism-mediated toxicity.

Xenobiotics undergoing bioactivation by CYP450 enzymes form reactive metabolites that may exert direct metabolism-mediated toxicity. An in vitro model was developed to study the direct toxic effects that follow the metabolic activation of chemicals. The model uses monolayer cultures of genetically engineered NIH-3T3 or V79 cells that express individual human or rat CYP450 isoforms, respectively. Following exposure to 1,3-dichloropropanol or cyclophosphamide, basal cytotoxicity endpoints, including neutral red uptake and Alamar Blue( reduction were used to assess changes in cell number and functional viability resulting from the formation of metabolites. Cell lines that express cytochrome P450 enzymes metabolised the test compounds, leading to increased toxicity compared with that observed in the control cell line. The use of specific inhibitors confirmed that the formation of reactive metabolites was CYP450-isoform dependent. These results indicate that a panel of genetically engineered cell lines expressing various cytochrome P450 enzyme isoforms can be used to reveal measurable metabolising capabilities, and could become a useful tool for the detection and possible determination of CYP450 isoforms in human liver metabolism-mediated toxicity.

3T3 Cells↗
PubMed1993

[Influence of the running conditions of a diesel engine on the mutagenic effects of its emissions].

A direct exposure method to detect mutagenicity of car exhausts was applied to study the influence of the running conditions of a diesel engine functioning on an assay platform. Biological and physico-chemical approaches were carried out together to appreciate the quality of diesel emissions. Exhausts, emitted by a diesel engine (Renault, 2068 cm3) were sampled from a dilution tube connected to a constant volume sampler. Regulated pollutants (CO, NOx, HC and particles) and some non-regulated pollutants (monoaromatic hydrocarbons (MAH) and polyaromatic hydrocarbons (PAH), aldehydes) were analysed when the engine ran at different operating conditions of load (full, 3/4, 2/4 and 1/4) and of engine speed (from 1000 to 4000 rev./min). The mutagenicity (Ames test) of diesel exhaust was assessed by a direct exposure method which does not need extraction of the particles. Bacteria are pre-seeded onto the agar plate and exposed directly to the diesel exhausts parallelly to the measurements of pollutants. The engine speed and the load influence the level, the composition and the induced mutagenicity of the diesel emissions and the results show that: when engine speed increases all the gaz phase indicators increase independently of the load, those of the particulate phase and mutagenicity increase at low load but decrease at full load; when load increase, all the indicators of both phases decrease except NOx which increases.

Carbon Monoxide↗
PubMedDate not supplied

A new graph descriptor for molecules containing cycles. Application as screening criterion for searching molecular structures within large databases of organic compounds.

The search of molecular structures inside a large database of chemical compounds is a critical step for many computer programs used in several domains of chemistry. During the last years, the size of many chemical databases has dramatically increased, hence in the meantime, search engines needed to be more and more powerful. The speed and the efficiency of screening processes of the chemical compounds are thus essential. Looking forward for algorithms dedicated to structure and substructure search, we have developed a new graph descriptor for structures containing cycles in order to find efficient indexation and classification criteria of molecular structures. This graph descriptor can be used as a screening criteria for structure and substructure search in large databases of organic compounds.

Journal Article↗
PubMedDate not supplied

Swelling and mechanical behaviors of chemically cross-linked hydrogels of elastin-like polypeptides.

Genetically engineered elastin-like polypeptides consisting of Val-Pro-Gly-X-Gly repeats, where X was chosen to be Lys every 7 or 17 pentapeptides (otherwise X was Val), were synthesized and expressed in E. coli, purified, and chemically cross-linked using tris-succinimidyl aminotriacetate to produce hydrogels. Swelling experiments indicate hydrogel mass decreases by 80-90% gradually over an approximate 50 degrees C temperature range. Gels ranged in stiffness from 0.24 to 3.7 kPa at 7 degrees C and from 1.6 to 15 kPa at 37 degrees C depending on protein concentration, lysine content, and molecular weight. Changes in gel stiffness and loss angle with cross-linking formulation suggest a low-temperature gel structure that is nearly completely elastic, where force is transmitted almost exclusively through fully extended polypeptide chains and chemical cross-links, and a high-temperature gel structure, where ELP chains are contracted and force is transmitted through chemical cross-links as well as frictional contact between polypeptide chains.

Cross-Linking Reagents↗
PubMedDate not supplied

Enhanced production of (R)-1,2-propanediol by metabolically engineered Escherichia coli.

1,2-Propanediol (1,2-PD) is a major commodity chemical currently derived from propylene. Previously, we have demonstrated the production of enantiomerically pure (R)-1,2-propanediol from glucose by an engineered E. coli expressing genes for NADH-linked glycerol dehydrogenase and methylglyoxal synthase. In this work, we investigate three methods to improve 1,2-PD in E. coli. First, we investigated improving the host by eliminating production of a byproduct, lactate. To do this, we constructed strains with mutations in two enzymes involved in lactate production, lactate dehydrogenase and glyoxalase I. (Surprisingly, when mutations were made in its ability to produce lactate, one strain of E. coli [MM294], produced a small amount of 1,2-PD without any added genes.) Second, we constructed a complete pathway to 1,2-PD from the glycolytic intermediate, dihydroxyacetone phosphate. Our previous 1, 2-PD producing strains relied on at least one endogenous E. coli activity and only produced 0.7 g/L of 1,2-PD. The complete pathway involved the coexpression of methylglyoxal synthase (mgs), glycerol dehydrogenase (gldA), and either yeast alcohol dehydrogenase (adhI) or E. coli 1,2-propanediol oxidoreductase (fucO). Third, we investigated bioprocessing improvements by carrying out a fed-batch fermentation with the best engineered strain (expressing mgs, gldA, and fucO). A final titer of 4.5 g/L of (R)-1,2-PD was produced, with a final yield of 0.19 g of 1,2-PD per gram of glucose consumed. This work provides a basis for further strain and process improvement.

Escherichia coli↗
PubMed2001

Characterisation of the transition states for protein folding: towards a new level of mechanistic detail in protein engineering analysis.

The field of protein folding now offers considerable excitement. Comparative studies of the transition-state structures for a series of protein families with analogous structures have helped to uncover the overall rules for protein folding. In addition, new protein engineering experiments that continuously follow the growth of the folding nucleus have started to fill in the missing details.

Models, Chemical↗
PubMed2005

Micro- and nanoscale modification of poly(2-hydroxyethyl methacrylate) hydrogels by AFM lithography and nanoparticle incorporation.

Poly(2-hydroxyethyl methacrylate) (PHEMA) hydrogels are widely used as biomaterials. Due to their unique combination of biocompatibility and good mechanical properties, they have potential as scaffolds for tissue engineering applications. To this purpose, topographic and chemical patterning at the nano- to the mesoscale is crucial in order to favor and to characterize cell adhesion and proliferation. Here we report the characterization of as-prepared and patterned PHEMA hydrogels, produced by conventional radical polymerization in water and dimethylformamide. We have obtained chemical and morphological micro- and nanoscale patterning by atomic force microscopy based lithography. We also demonstrate that it is possible to incorporate carbon nanoparticles in the hydrogel matrix by supersonic cluster beam deposition.

Biocompatible Materials↗
PubMedDate not supplied

Cellular and metabolic engineering. An overview.

Metabolic engineering is defined as the purposeful modification of intermediary metabolism using recombinant DNA techniques. Cellular engineering, a more inclusive term, is defined as the purposeful modification of cell properties using the same techniques. Examples of cellular and metabolic engineering are divided into five categories: 1. Improved production of chemicals already produced by the host organism; 2. Extended substrate range for growth and product formation; 3. Addition of new catabolic activities for degradation of toxic chemicals; 4. Production of chemicals new to the host organism; and 5. Modification of cell properties. Over 100 examples of cellular and metabolic engineering are summarized. Several molecular biological, analytical chemistry, and mathematical and computational tools of relevance to cellular and metabolic engineering are reviewed. The importance of host selection and gene selection is emphasized. Finally, some future directions and emerging areas are presented.

Bacteria↗
PubMedDate not supplied

Knock prediction for dual fuel engines by using a simplified combustion model.

The present work used a methane-air mixture chemical kinetics scheme consisting of 119 elementary reaction steps and 41 chemical species to develop a simplified combustion model for prediction of the knock in dual fuel engines. Calculated values by the model for natural gas operation showed good agreement with corresponding experimental values over a broad range of operating conditions.

Carbon Monoxide↗
PubMed2004

Guided cell patterning on gold-silicon dioxide substrates by surface molecular engineering.

We report an effective approach to patterning cells on gold-silicon dioxide substrates with high precision, selectivity, stability, and reproducibility. This technique is based on photolithography and surface molecular engineering and requires no cell positioning or delivery devices, thus significantly reducing the potential damage to cells. The cell patterning was achieved by activating the gold regions of the substrate with functionalized thiols that covalently bind proteins onto the gold regions to guide subsequent cell adhesion while passivating the silicon dioxide background with polyethylene glycol to resist cell adhesion. Fourier transform infrared reflectance spectroscopy verified the successful immobilization of proteins on gold surfaces. Protein patterns were visualized by tagging proteins with Rhodamine fluorescent probes. Time-of-flight secondary ion mass spectrometry was used to characterize the chemistry of both the cell-adhesive and cell-resistant regions of surfaces after each key chemical reaction occurring during the molecular surface engineering. The ability of the engineered surfaces to guide cell adhesion was illustrated by differential interference contrast (DIC) reflectance microscopy. The cell patterning technique introduced in this study is compatible with micro- and photo-electronics, and may have many medical, environmental, and defense applications.

Animals↗
PubMedDate not supplied

[Biosynthesis and isolation of a recombinant protein for producing genetically-engineered human proinsulin].

Isolation of the recombinant protein from a genetically engineered Escherichia coli 1854 producer for further chemical enzymatic transformation into human insulin through proinsulin was studied. Under optimal conditions, the recombinant protein formation was more than 35% of the total cell proteins. Structures of the polypeptides obtained and purified chromatographically were confirmed by amino acid analysis. Human proinsulin was derived from the recombinant protein isolated.

Amino Acid Sequence↗
PubMed2006

General, spontaneous ion replacement reaction for the synthesis of micro- and nanostructured metal oxides.

A novel spontaneous ion replacement route based on the solubility difference as the driving force to synthesize a number of metal oxides has been established. We present a comprehensive study on the ion replacement reaction for chemical synthesis of micro- and nanostructured Mn2O3, ZnO, CuO, CdO, Al2O3, and CaO samples. This novel approach described herein is derived from the solubility difference between two carbonate salts, in which a metal cation can be driven from one liquid phase into another solid phase in the solution system. The resulting metal carbonate salts are initially formed and subsequently calcined to form highly crystallined metal oxides. The variation of pH values, reaction temperature, and reagent shapes can vary the solubility of these two carbonate salts, which thus changes the final morphology of metal oxides. The present work makes a progress to simply and mildly synthesize metal oxides with various morphologies, due to the fact that materials with a desired morphology are a key engineering step toward their shape-dependent chemical and physical properties.

Journal Article↗
PubMed2002

Photopolymerizable hydrogels for tissue engineering applications.

Photopolymerized hydrogels are being investigated for a number of tissue engineering applications because of the ability to form these materials in situ in a minimally invasive manner such as by injection. In addition, hydrogels, three-dimensional networks of hydrophilic polymers that are able to swell large amounts of water, can be made to resemble the physical characteristics of soft tissues. Hydrogel materials also generally exhibit high permeability and good biocompatibility making, these materials attractive for use in cell encapsulation and tissue engineering applications. A number of hydrogel materials can be formed via photopolymerization processes mild enough to be carried out in the presence of living cells. This allows one to homogeneously seed cells throughout the scaffold material and to form hydrogels in situ. This review presents advantages of photopolymerization of hydrogels and describes the photoinitiators and materials in current use. Applications of photopolymerized hydrogels in tissue engineering that have been investigated are summarized.

Animals↗
PubMed2001

Structural adaptation of enzymes to low temperatures.

A systematic comparative analysis of 21 psychrophilic enzymes belonging to different structural families from prokaryotic and eukaryotic organisms is reported. The sequences of these enzymes were multiply aligned to 427 homologous proteins from mesophiles and thermophiles. The net flux of amino acid exchanges from meso/thermophilic to psychrophilic enzymes was measured. To assign the observed preferred exchanges to different structural environments, such as secondary structure, solvent accessibility and subunit interfaces, homology modeling was utilized to predict the secondary structure and accessibility of amino acid residues for the psychrophilic enzymes for which no experimental three-dimensional structure is available. Our results show a clear tendency for the charged residues Arg and Glu to be replaced at exposed sites on alpha-helices by Lys and Ala, respectively, in the direction from 'hot' to 'cold' enzymes. Val is replaced by Ala at buried regions in alpha-helices. Compositional analysis of psychrophilic enzymes shows a significant increase in Ala and Asn and a decrease in Arg at exposed sites. Buried sites in beta-strands tend to be depleted of VAL: Possible implications of the observed structural variations for protein stability and engineering are discussed.

Alanine↗
PubMed2003

Adhesive protein interactions with chitosan: consequences for valve endothelial cell growth on tissue-engineering materials.

Stable endothelialization of a tissue-engineered heart valve is essential for proper valve function, although adhesive characteristics of the native valve endothelial cell (VEC) have rarely been explored. This research evaluated VEC adhesive qualities and attempted to enhance VEC growth on the biopolymer chitosan, a novel tissue-engineering scaffold material with promising biological and chemical properties. Aortic VEC cultures were isolated and found to preferentially adhere to fibronectin, collagen types IV and I over laminin and osteopontin in a dose-dependent manner. Seeding of VEC onto comparison substrates revealed VEC growth and morphology to be preferential in the order: tissue culture polystyrene > gelatin, poly(DL-lactide-co-glycolide), chitosan > poly(hydroxy alkanoate). Adhesive protein precoating of chitosan did not significantly enhance VEC growth, despite equivalent protein adsorption as to polystyrene. Initial cell adhesion to protein-precoated chitosan, however, was higher than for polystyrene. Composite chitosan/collagen type IV films were investigated as an alternative to simple protein precoatings, and were shown to improve VEC growth and morphology over chitosan alone. These findings suggest potential manipulation of chitosan properties to improve amenability to valve tissue-engineering applications.

Animals↗