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PubMed1994

[Gentecnically engineered V79 cell lines in combination with analytical-chemical procedures for replacing and refining animal experimentations]

Since 1986 V79 Chinese hamster cells are being genetically engineered for rat and human enzymes for studies on metabolism dependent effects in toxicology and pharmacology under defined conditions. The metabolic activation of potentially harmful chemicals is of interest in toxicology, because metabolites may have their own toxic potency. Anabolic and catabolic pathways of drugs are of interest in pharmacology, because metabolism has a decisive impact on the efficacy of drugs. As an example for the usefulness of V79 cells genetically engineered for metabolic competence, and their advantage over animal experimentation, metabolic activation of polycyclic aromatic hydrocarbons has been studied. Comparative studies using V79 cells expressing rat and human enzymes revealed striking differences in the metabolite profile, which made evident, that human beings are at more risk than the rat, when exposed to polycyclic aromatic hydrocarbons. This suggests at the same time, that results obtained from animal experimentation are to be treated with caution, if they form the basis for risk assessment.

Journal Article↗
PubMed1995

Mapping the folding intermediate of human carbonic anhydrase II. Probing substructure by chemical reactivity and spin and fluorescence labeling of engineered cysteine residues.

Several conformation-sensitive parameters have shown that human carbonic anhydrase II exists as a stable and compact equilibrium folding intermediate of molten globule type. In this study we have continued a previously initiated mapping of the intermediate structure. Cys residues were engineered, one at a time, into various regions of the protein structure, so as to obtain chemically reactive probes and handles for spectroscopic probes. These probes were used to specifically report on conformational changes accompanying the folding process. Thus, the accessibility of the introduced Cys residues to specific chemical labeling by radioactive iodoacetate was used to monitor the stability and compactness of the substructure surrounding each Cys residue. In addition, a spin-label (nitroxide radical) and a fluorescent probe (IAEDANS) were attached to the inserted SH-groups to give complementary information. The mobility of the spin-label was used to indicate local changes in structure, and the fluorophore was used to probe local changes in polarity at various stages of unfolding. Much of the predominant beta-structure, consisting of 10 beta-strands extending throughout the entire molecule, appears to be compact and largely intact in the intermediate. Thus, beta-strands 3-7, probed at positions 68, 97, 118, 123, 206, and 245, seem to have a native-like structure in the folding intermediate. In contrast, a more flexible structure is found around positions 56, 176, and 256 in the peripheral beta-strands 1, 2, and 9, showing that the stability of the secondary structure in the intermediate state is less in the outer parts of the protein. A hydrophobic region, containing beta-strands 3-5, seems to be remarkably stable and is not ruptured until strong denaturing conditions (5 M GuHCl) are applied. The stability of this hydrophobic beta-core appears to increase toward the center. This stable region is contained in the middle of a sequentially continuous antiparallel structure that spans beta-strands 2-6, suggesting that this part might represent a site where folding is initiated.

Carbonic Anhydrases↗
PubMed1995

Use of non-radioactive methods for the determination of the expression, the sequence and the copy-number of transgene in mice.

Marshall's observation that "toxicology goes molecular", is turning out to be more true than ever; namely as it is observed that toxicological endpoints are the point of interaction between proteins and genes following the administration of toxicants. Transgenic mice represent a valuable tool for studying the adverse effects of chemicals in genetically engineered animals such as p53 deficient mice, or mice carrying the v-Ha-ras oncogene. The latter were used in our laboratory for such toxicological assessments of chemicals. In order to verify that the transgene was expressed in normal, as well as in tumor cells, the transgene was detected in different tissues fixed with various solutions using in situ hybridization. It was also specifically retrotranscribed from paraffin-embedded tissues and consequently sequenced using a Taq polymerase reaction. We found that the transgene was expressed in various organs. It carries a specific mutation of codon 12 leading to the activation of its encoded product (transducin: p21v-Ha-ras). Moreover using a laser scanning densitometer, it has been demonstrated that 2 to 3 copies of the transgene were present per genome-equivalent in some tissues. All experiments were realized using non-radioactive labelling and detection (chemiluminescent or colorigenic) methods. Indeed, the screening of such animals was realized in a easier and a safer manner using the methods described in this paper than the usual methods based on the use of radiolabelled precursors.

Animals↗
PubMed2004

Genetically engineered mouse models for drug discovery: new chemical genetic approaches.

While standard transgenic and knockout mouse technologies have provided a wealth of information for target selection and validation, there have been great advances in using more sophisticated modeling techniques to achieve temporal and spatial regulation of individual genes in adult animals. Recent developments in RNA interference (RNAi) technology in in vivo models promise to further improve upon the static and irreversible features of gene knockouts. Chemical genetic approaches create novel functional alleles of targets and allow fine modulation of protein function in vivo by small molecules, providing the most pharmacologically relevant target validation. Using these advanced models, one can not only ask whether the function of the target is critical for the initiation and maintenance of the disease, but also whether therapies designed to alter the function of the target would be safe and efficacious. In this review, we describe various in vivo tools for target validation in mouse models, discuss advantages and disadvantages of each approach, and give examples of their impact on drug discovery.

Animals↗
PubMed2004

Directed enzyme evolution and selections for catalysis based on product formation.

Enzyme engineering by molecular modelling and site-directed mutagenesis can be remarkably efficient. Directed enzyme evolution appears as a more general strategy for the isolation of catalysts as it can be applied to most chemical reactions in aqueous solutions. Selections, as opposed to screening, allow the simultaneous analysis of protein properties for sets of up to about 10(14) different proteins. These approaches for the parallel processing of molecular information 'Is the protein a catalyst?' are reviewed here in the case of selections based on the formation of a specific reaction product. Several questions are addressed about in vivo and in vitro selections for catalysis reported in the literature. Can the selection system be extended to other types of enzymes? Does the selection control regio- and stereo-selectivity? Does the selection allow the isolation of enzymes with an efficient turnover? How should substrates be substituted or mimicked for the design of efficient selections while minimising the number of chemical synthesis steps? Engineering sections provide also some clues to design selections or to circumvent selection biases. A special emphasis is put on the comparison of in vivo and in vitro selections for catalysis.

Biotechnology↗
PubMed2002

Tissue engineering: advances in in vitro cartilage generation.

Damaged or diseased articular cartilage frequently leads to progressive debilitation resulting in a marked decrease in the quality of life. Tissue engineering, a budding field in modern biomedical sciences, promises creation of viable substitutes for failing organs or tissues. It represents the amalgamation of rapid developments in cellular and molecular biology on the one hand and material, chemical and mechanical engineering on the other. Current tissue engineering approaches are mainly focused on the restoration of pathologically altered tissue structure based on the transplantation of cells in combination with supportive matrices and biomolecules. The ability to manipulate and reconstitute tissue structure and function in vitro has tremendous clinical implications and is likely to have a key role in cell and gene therapies in coming years.

Biocompatible Materials↗
PubMed1993

Arthrobacter D-xylose isomerase: chemical modification of carboxy groups and protein engineering of pH optimum.

To try to lower the pH optimum, the carboxy groups of Arthrobacter D-xylose isomerase were coupled to glycinamide using a water-soluble carbodi-imide. In conditions that substituted all of the 59 carboxy groups in the denatured monomer, a maximum of 30 groups/monomer reacted in the native enzyme, whether in presence or absence of ligands, and the enzyme remained fully active and tetrameric throughout the coupling reaction. Purification by f.p.l.c. ion-exchange chromatography gave broad symmetrical peaks with increased pI, suggesting that the modified enzymes are essentially homogeneous. However, they are less stable than native enzyme in 8 M urea or on heating ('melting points' of 59 degrees versus 73 degrees C for the apoenzymes and 67 degrees versus 81.5 degrees C for the Mg(2+)-enzymes). Kinetic studies of the D-fructose isomerase activity at 30 degrees C showed that the glycinamidylated enzyme had unaltered activation constant for Mg2+, and Km was also similar to that of the native enzyme at pH 7.3, but increased rapidly at higher pH rather than remaining constant. Vmax. was constant from pH 6.2 to 8.0, suggesting a reduced pKa for His-219, which controls Vmax. in the native enzyme (normally 6.0). Three mutants were constructed by protein engineering with a view to reducing the pH optimum of enzyme activity. Two of these, Glu140-->Lys and Asp189-->Lys, could be detected in crude extracts of Escherichia coli by SDS/PAGE, but could not be purified, whereas mutant Trp136-->Glu was produced as a tetramer in amounts similar to the wild-type enzyme. However, it did not show any enzyme activity and was less stable in 0-9 M urea gradient PAGE.

Aldose-Ketose Isomerases↗
PubMedDate not supplied

Chemical technology for the toxic gas flow control through process water system.

The aim of this work is focused on the safety and toxicological aspects due to under-pressure industrial plant management, above all in the case which the gas is very dangerous for human health and environment. Here is illustrated the safe method of control of risks through specific choices of engineering devices and chemical process: in this way we have shown the mathematical calculation regarding the case of ammonia flow gas running in the piping and plant under-pressure. In this paper the Authors show the assessment of the technological solution for falling down of a toxic gas as NH3, which lets off from safety values facilities. The under pressure industrial plants with ammonia are protected through the safety valves, settled at 20 bar pressure. The out-let gas flow is capted by a tank of a water bulk of five time theoretical water amount necessary to the complete absorption of gas. In order to prevent any health risk and carry out a safety management, it needs to verify two basic aspects, with connected specific techniques: 1. The safety valves technology through the mathematical calculation of operating device; 2. The absorption process of the toxic agent for controlling of dangerous runaway of gas.

Air Pollutants↗
PubMed2005

[Fabrication and properties of a composite chitosan/type II collagen scaffold for tissue engineering cartilage].

OBJECTIVE: To develop a novel porous three-dimensional scaffold and to investigate its physico-chemical properties for tissue engineering cartilage. METHODS: Refined 88% deacetylation degree chitosan was prepared and dissolved in 0.2 mol/L acetate acid and fully mixed with highly purified porcine type II collagen in 0.5 mol/L acetate acid solution in a ratio of 4 to 1 (wt/wt). Freeze-drying process was employed to fabricate the composite scaffold. The construct was cross-linked by use of 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS). A mechanical tester was utilized to determine the tensile strength change before and after cross-linking. The microstructure was observed via scanning electron microscopy (SEM). The lysozyme degradation was performed to evaluate the degradability of the scaffold in vitro. RESULTS: A bulk scaffold with desired configuration was obtained. The mechanical test showed that the cross-linking treatment could enhance the mechanical strength of the scaffold. The SEM results revealed that the two constituents evenly distributed in the scaffold and that the matrix was porous, sponge-like with interconnected pore sizing 100-250 microm. In vitro lysozyme degradation indicated that cross-linked or uncross-linked composite scaffolds had faster degradation rate than the chitosan matrix. CONCLUSION: Chitosan and type II collagen can be developed into a porous three-dimensional scaffold. The related physico-chemical tests suggest that the composite scaffold meets requirements for tissue engineered scaffold and may serve as an alternative cell-carrier for tissue engineering cartilage.

Cartilage↗
PubMed1999

Lymphopoietic cancer and other major causes of death among petrochemical researchers: an update.

This study updates lymphopoietic cancer (LHC) mortality statistics and other major causes of death through 1992 for 13,188 petrochemical researchers employed between 1964 and 1986. Significant deficits of deaths were observed for all causes, all cancers, ischaemic heart disease and all external causes. The subcategory of 'all other LHC' was elevated among males in an exposure class containing scientists and engineers. This finding was statistically significant based on national but not state comparison rates. Poisson regression analyses showed that increasing exposure classes were not associated with LHC, but a relationship was noted for total years worked. A non-significant increase in breast cancer among females was also observed but was concentrated among the lowest exposure class. This study and other similar investigations suggest various subcategories of LHC deaths are marginally elevated among chemical researchers and engineers. Evidence for a work-related LHC hazard for this population, however, has not been identified.

Chemical Industry↗
PubMed1997

The new era of biotech insulin analogues.

Many of the structural properties of insulin have evolved in response to the requirements of biosynthesis, processing, transport and storage in the pancreatic beta cells, properties that are not necessary for the biological action of the hormone. It is therefore not surprising that wild-type insulin has far from optimal characteristics for replacement therapy. For example, native human insulin self-associates to hexameric units, which limits the possibilities for the absorption of the molecule by various routes. During the last decade new techniques of molecular design have emerged and recombinant DNA technology offers new and exciting opportunities for rational protein drug design. This review describes examples of recent advances in insulin engineering aimed at optimizing the hormone for therapy. Such approaches focus on improvements in the pharmacokinetic properties, storage stability, and feasibility for less intrusive routes of administration.

Animals↗
PubMed2000

Cell surface engineering by a modified Staudinger reaction.

Selective chemical reactions enacted within a cellular environment can be powerful tools for elucidating biological processes or engineering novel interactions. A chemical transformation that permits the selective formation of covalent adducts among richly functionalized biopolymers within a cellular context is presented. A ligation modeled after the Staudinger reaction forms an amide bond by coupling of an azide and a specifically engineered triarylphosphine. Both reactive partners are abiotic and chemically orthogonal to native cellular components. Azides installed within cell surface glycoconjugates by metabolism of a synthetic azidosugar were reacted with a biotinylated triarylphosphine to produce stable cell-surface adducts. The tremendous selectivity of the transformation should permit its execution within a cell's interior, offering new possibilities for probing intracellular interactions.

Acetylation↗
PubMed1990

Prevention of occupational contact dermatitis.

Contact dermatitis is the most frequent type of occupational skin disease. Although prevention of contact dermatitis in the workplace should ideally be accomplished through total elimination of cutaneous exposure to hazardous substances, this is often not feasible. Therefore eight basic elements of a multidimensional approach to prevention have been identified. These elements include recognition of potential cutaneous irritants and allergens, engineering controls or chemical substitution to prevent skin exposure, personal protection with appropriate clothing or barrier creams, personal and environmental hygiene, regulation of potential allergens and irritants within the workplace, educational efforts to promote awareness of potential allergens and irritants, motivational techniques to promote safe work conditions and practices, and preemployment and periodic health screening. A comprehensive prevention program based on this multidimensional approach requires the cooperative efforts of employees, employers, engineers, chemists, industrial hygienists, safety and supervisory personnel, union representatives, governmental agencies, and occupational health practitioners.

Dermatitis, Contact↗
PubMed2004

BRENDA, the enzyme database: updates and major new developments.

BRENDA (BRaunschweig ENzyme DAtabase) represents a comprehensive collection of enzyme and metabolic information, based on primary literature. The database contains data from at least 83,000 different enzymes from 9800 different organisms, classified in approximately 4200 EC numbers. BRENDA includes biochemical and molecular information on classification and nomenclature, reaction and specificity, functional parameters, occurrence, enzyme structure, application, engineering, stability, disease, isolation and preparation, links and literature references. The data are extracted and evaluated from approximately 46,000 references, which are linked to PubMed as long as the reference is cited in PubMed. In the past year BRENDA has undergone major changes including a large increase in updating speed with >50% of all data updated in 2002 or in the first half of 2003, the development of a new EC-tree browser, a taxonomy-tree browser, a chemical substructure search engine for ligand structure, the development of controlled vocabulary, an ontology for some information fields and a thesaurus for ligand names. The database is accessible free of charge to the academic community at http://www.brenda. uni-koeln.de.

Animals↗
PubMed2001

Meta-analysis of multiple myeloma and benzene exposure.

Epidemiologic studies have suggested that benzene exposure may be a risk factor of multiple myeloma (MM). We performed meta-analyses of case-control studies to assess the association between occupational exposure to benzene and the risk of MM. We divided the occupational sources of benzene exposure into 4 categories, benzene and/or organic solvents, petroleum, petroleum products, and engine exhaust, for conducting the meta-analysis. As a result, a significant positive association was indicated between exposure to engine exhaust and MM (summary odds ratio or summary OR=1.34, 95% confidence interval or 95%CI=1.14-1.57). However, no significant associations were obtained for benzene and/or organic solvents (summary OR=0.74, 95%CI=0.60-0.90), petroleum (summary OR=1.11, 95%CI=0.96-1.28) and petroleum products (summary OR=1.08, 95%CI=0.89-1.33) with risk of MM. These results suggested that benzene exposure itself was not likely to be a risk factor of MM. It is thought that several harmful chemical agents in engine exhaust, other than benzene, could be etiologically related to the risk of MM. Further case-control studies on MM are needed to obtain more information about detailed occupational exposure to toxic substances.

Air Pollutants, Occupational↗
PubMed1999

Dielectric Spectroscopic and Molecular Simulation Evidence for Aggregation of Surfactant-stabilized Calcium Carbonate Nanocolloids in Organic Media.

Dielectric spectroscopy carried out on overbased phenate micelle particles of nominal diameter approximately 2 nm containing calcium carbonate cores in hydrophobic liquids indicates that the micelles are strongly aggregated. Mean cluster sizes in excess of approximately 10(3) individual micelles are typically found in toluene. The level of association is a little higher in dodecane which is chemically closer to engine oil, the usual solvent for these systems. The mean aggregate size increases dramatically with concentration above an effective solids volume fraction of approximately 0.1 on treating the micelles as spheres. Aggregate size also depends on the level of overbasing, with lower levels of overbasing giving more micelles in the cluster. Molecular dynamics simulations of individual micelle particles reveal them to have large dipole moments originating mainly from the amorphous carbonate cores. Dipoles of magnitude approximately 20D are typical for a range of different surfactant types used in the model. The magnitude of the dipole depends somewhat on the chemical composition of the stabilizing surfactant shell. Monte Carlo simulations of two phenate nanocolloids taking into account all atom and charge pair interactions show these particles to have a strong short-range coulombic attraction of typically -25kBT at T = 298 K in the favored "side-by-side" relative arrangement. This attraction could be responsible for the strong level of association inferred from the dielectric spectroscopy experiments. Copyright 1999 Academic Press.

Journal Article↗