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At least 631 records · Page 35Linked to original sources
PubMed2003

Modelling thin-film dewetting on structured substrates and templates: bifurcation analysis and numerical simulations.

We study the dewetting process of a thin liquid film on a chemically patterned solid substrate (template) by means of a thin-film evolution equation incorporating a space-dependent disjoining pressure. Dewetting of a thin film on a homogeneous substrate leads to fluid patterns with a typical length scale, that increases monotonously in time (coarsening). Conditions are identified for the amplitude and periodicity of the heterogeneity that allow to transfer the template pattern onto the liquid structure ("pinning") emerging from the dewetting process. A bifurcation and stability analysis of the possible liquid ridge solutions on a periodically striped substrate reveal parameter ranges where pinning or coarsening ultimately prevail. We obtain an extended parameter range of multistability of the pinning and coarsening morphologies. In this regime, the selected pattern depends sensitively on the initial conditions and potential finite perturbations (noise) in the system as we illustrate with numerical integrations in time. Finally, we discuss the instability to transversal modes leading to a decay of the ridges into rows of drops and show that it may diminish the size of the parameter range where the pinning of the thin film to the template is successful.

Biomedical Engineering↗
PubMed2004

Impact of engineering flow conditions on plasmid DNA yield and purity in chemical cell lysis operations.

Chemical lysis of bacterial cells using an alkaline solution containing a detergent may provide an efficient scalable means for selectively removing covalently closed circular plasmid DNA from high-molecular-weight contaminating cellular components including chromosomal DNA. In this article we assess the chemical lysis of E. coli cells by SDS in a NaOH solution and determine the impact of pH environment and shear on the supercoiled plasmid and chromosomal DNA obtained. Experiments using a range of plasmids from 6 kb to 113 kb determined that in an unfavorable alkaline environment, where the NaOH concentration during lysis is greater than 0.15 +/- 0.03 M (pH 12.9 +/- 0.2), irreversible denaturation of the supercoiled plasmid DNA occurs. The extent of denaturation is shown to increase with time of exposure and NaOH concentration. Experiments using stirred vessels show that, depending on NaOH concentration, moderate to high mixing rates are necessary to maximize plasmid yield. While NaOH concentration does not significantly affect chromosomal DNA contamination, a high NaOH concentration is necessary to ensure complete conversion of chromosomal DNA to single-stranded form. In a mechanically agitated lysis reactor the correct mixing strategy must balance the need for sufficient mixing to eliminate potential regions of high NaOH concentrations and the need to avoid excessive breakage of the shear sensitive chromosomal DNA. The effect of shear on chromosomal DNA is examined over a wide range of shear rates (10(1)-10(5) s(-1)) demonstrating that, while increasing shear leads to fragmentation of chromosomal DNA to smaller sizes, it does not lead to significantly increased chromosomal DNA contamination except at very high shear rates (about 10(4)-10(5) s(-1)). The consequences of these effects on the choice of lysis reactor and scale-up are discussed.

Biomedical Engineering↗
PubMed2006

Engineering Escherichia coli for xylitol production from glucose-xylose mixtures.

The range of value-added chemicals produced by Escherichia coli from simple sugars has been expanded to include xylitol. This was accomplished by screening the in vivo activity of a number of heterologous xylitol-producing enzymes. Xylose reductases from Candida boidinii (CbXR), Candida tenuis (CtXR), Pichia stipitis (PsXR), and Saccharmoyces cerivisiae (ScXR), and xylitol dehydrogenases from Gluconobacter oxydans (GoXDH) and Pichia stipitis (PsXDH) were all functional in E. coli to varying extents. Replacement of E. coli's native cyclic AMP receptor protein (CRP) with a cyclic AMP-independent mutant (CRP*) facilitated xylose uptake and xylitol production from mixtures of glucose and xylose, with glucose serving as the growth substrate and source of reducing equivalents. Of the enzymes tested, overexpression of NADPH-dependent CbXR produced the highest concentrations of xylitol in shake-flask cultures (approximately 275 mM in LB cultures, approximately 180 mM using minimal medium). Expression of CbXR in strain PC09 (crp*, DeltaxylB) in a 10-L controlled fermentation containing minimal medium resulted in production of approximately 250 mM xylitol (38 g/L), with concomitant utilization of approximately 150 mM glucose. The ratio of moles xylitol produced (from xylose) per mole glucose consumed was improved to > 3.7:1 using metabolically active "resting" cells.

Bacterial Proteins↗
PubMed2000

Enhancement of catalytic efficiency of enzymes through exposure to anhydrous organic solvent at 70 degrees C. Three-dimensional structure of a treated serine proteinase at 2.2 A resolution.

The enzyme behavior in anhydrous media has important applications in biotechnology. So far chemical modifications and protein engineering have been used to alter the catalytic power of the enzymes. For the first time, it is demonstrated that an exposure of enzyme to anhydrous organic solvents at optimized high temperature enhances its catalytic power through local changes at the binding region. Six enzymes: proteinase K, wheat germ acid phosphatase, alpha-amylase, beta-glucosidase, chymotrypsin and trypsin have been exposed to acetonitrile at 70 degrees C for three hours. The activities of these enzymes were found to be considerably enhanced. In order to understand the basis of this change in the activity of these enzymes, the structure of one of these treated enzymes, proteinase K has been analyzed in detail using X-ray diffraction method. The overall structure of the enzyme is similar to the native structure in aqueous environment. The hydrogen bonding system of the catalytic triad is intact after the treatment. However, the water structure in the substrate binding site undergoes some rearrangement as some of the water molecules are either displaced or completely absent. The most striking observation concerning the water structure pertains to the complete deletion of the water molecule which occupied the position at the so-called oxyanion hole in the active site of the native enzyme. Three acetonitrile molecules were found in the present structure. All the acetonitrile molecules are located in the recognition site. The sites occupied by acetonitrile molecules are independent of water molecules. The acetonitrile molecules are involved in extensive interactions with the protein atoms. All of them are interlinked through water molecules. The methyl group of one of the acetonitrile molecules (CCN1) interacts simultaneously with the hydrophobic side chains of Leu-96, Ile-107, and Leu-133. The development of such a hydrophobic environment at the recognition site introduces a striking conformation change in Ile-107 by rotating its side chain about C(alpha)--C(beta) bond by 180 degrees to bring about the delta-methyl group within the range of attractive van der Waals interactions with the methyl group of CCN1. A similar change has earlier been observed in proteinase K when it is complexed to a substrate analog lactoferrin fragment.

Acetonitriles↗
PubMed2005

"Designer acids": combined acid catalysis for asymmetric synthesis.

Lewis and Brønsted acids can be utilized as more-effective tools for chemical reactions by sophisticated engineering ("designer acids"). The ultimate goal of such "designer acids" is to form a combination of acids with higher reactivity, selectivity, and versatility than the individual acid catalysts. One possible way to take advantage of such abilities may be to apply a "combined acids system" to the catalyst design. The concept of combined acids, which can be classified into Brønsted acid assisted Lewis acid (BLA), Lewis acid assisted Lewis acid (LLA), Lewis acid assisted Brønsted acid (LBA), and Brønsted acid assisted Brønsted acid (BBA), can be a particularly useful tool for the design of asymmetric catalysis, because combining such acids will bring out their inherent reactivity by associative interaction, and also provide more-organized structures that allow an effective asymmetric environment.

Journal Article↗
PubMed2002

Modified model for the regulation of the tryptophan operon in Escherichia coli.

This article proposes a modification of the model developed by Sinha (1988) and Sen and Liu (1990) for the regulation dynamics of the tryptophan operon in E. coli based on a consistent overall balance of the agent repressing the mRNA transcription. The dynamics of the model are analyzed by means of continuation techniques and the influence of periodic fluctuations in the intracellular demand for tryptophan is addressed. The analysis provides deeper insight into the dynamics of this operon system and the results obtained may be a useful starting point for the optimization of tryptophan yield in bacterial cultures.

Bacterial Proteins↗
PubMed2001

A personal account of the role of peptide research in drug discovery: the case of hepatitis C.

Although peptides themselves are not usually the end products of a drug discovery effort, peptide research often plays a key role in many aspects of this process. This will be illustrated by reviewing the experience of peptide research carried out at IRBM in the course of our study of hepatitis C virus (HCV). The target of our work is the NS3/4A protease, which is essential for maturation of the viral polyprotein. After a thorough examination of its substrate specificity we fine-tuned several substrate-derived peptides for enzymology studies, high-throughput screening and as fluorescent probes for secondary binding assays. In the course of these studies we made the key observation: that the protease is inhibited by its own cleavage products. Single analog and combinatorial optimization then derived potent peptide inhibitors. The crucial role of the NS4A cofactor was also addressed. NS4A is a small transmembrane protein, whose central domain is the minimal region sufficient for enzyme activation. Structural studies were performed with a peptide corresponding to the minimal activation domain, with a series of product inhibitors and with both. We found that NS3/4A is an induced fit enzyme, requiring both the cofactor and the substrate to acquire its bioactive conformation; this explained some puzzling results of 'serine-trap' type inhibitors. A more complete study on NS3 activation, however, requires the availability of the full-length NS4A protein. This was prepared by native chemical ligation, after sequence engineering to enhance its solubility; structural studies are in progress. Current work is focused on the P' region of the substrate, which, at variance with the P region, is not used for ground state binding to the enzyme and might give rise to inhibitors showing novel interactions with the enzyme.

Binding Sites↗
PubMed1998

Design and synthesis of fluorogenic trypsin peptide substrates based on resonance energy transfer.

An assay based on new internally quenched fluorogenic peptide substrates with the general structure 4-(4-dimethylaminophenylazo)benzoic acid (DABCYL)-Gly-Pro-Ala-Xaa-Leu-Ala-Ile-Gly-5-(2-aminoethylamino++ +)naphtha lene-1-sulfonic acid (EDANS), where Xaa = Arg, Lys, has been developed to measure proteolytic activity of trypsin and similar proteases. The kinetic parameters for the tryptic hydrolysis of DABCYL-Gly-Pro-Ala-Arg-Leu-Ala-Ile-Gly-EDANS are Km = 34 microM, kcat = 40 s-1, and kcat/Km = 1.17 x 10(6) M-1 s-1. The substrates offer two advantages over common substrates. First they are very sensitive. Applications to chemically modified trypsin and engineered variants show the ability to detect traces of proteolytic activity. In addition, these substrates are adapted to the S'-specificity of the investigated protease. These features and the prospect of miniaturization makes the assay suitable for applications to high-throughput screening.

Amino Acid Sequence↗
PubMed1995

Bispecific antibodies retarget murine T cell cytotoxicity against syngeneic breast cancer in vitro and in vivo.

Bispecific antibodies with specificity for CD3 and a tumor antigen can redirect cytolytic T cells to kill tumor targets, regardless of their natural specificity. To assess the clinical potential of bispecific antibodies for treatment of human cancers we have, in the present study, adapted a totally synergeic mouse model to the targeting of mouse T cells against mouse tumors in immunocompetent mice. We show that gp52 of the mouse mammary tumor virus (MTV) can serve as a tumor-specific antigen for redirected cellular cytotoxicity. Chemically crosslinked and genetically engineered bispecific antibodies with specificities for gp52 and murine CD3 epsilon-chain induced activated mouse T cells to specifically lyse mouse mammary tumor cells from cultured lines and primary tumors from C3H-MTV+ mice. Retargeted T cells also blocked the growth of mammary tumors in vitro as well as their growth in syngeneic mice. These findings identify murine MTV-induced mammary adenocarcinomas as a solid-tumor, animal model for retargeting T cells with bispecific antibodies against syngeneic breast cancer.

Adenocarcinoma↗
PubMed2006

Parkinson's disease: a rethink of rodent models.

Parkinson's disease (PD) is a multifactorial disease with a complex etiology that results from genetic risk factors, environmental exposures and most likely a combination of both. Rodent models of parkinsonism aim to reproduce key pathogenic features of the syndrome including movement disorder induced by the progressive loss of dopaminergic neurons in the substantia nigra, accompanied by the formation of alpha-synuclein containing Lewy body inclusions. Despite the creation of many excellent models, both chemically induced and genetically engineered, there is none that accurately demonstrates these features. Recent pathological staging studies in man have also emphasized the significant non-CNS component of PD that has yet to be tackled. Herein, we summarize rodent models of PD and what they offer to the field, and suggest future challenges and opportunities.

Animals↗
PubMed2005

Enhanced radical scavenging activity of genetically modified Arabidopsis seeds.

The proanthocyanidin (PA) content was increased in seeds of pap1-D mutant of Arabidopsis thaliana, in which the expression of endogenous PAP1 gene encoding a Myb-like transcription factor was induced by activation-tagging with enhancer sequences derived from cauliflower mosaic virus 35S promoter. In contrast, the PA contents decreased in seeds of transgenic plants transformed with a PAP1 cDNA or with a PAP1 chimeric repressor, although the amount of soluble anthocyanins increased in seeds of transgenic plants over-expressing PAP1 cDNA. The enhanced radical scavenging activity was observed only in the seed extracts of pap1-D mutant, indicating that PAs are primarily responsible for radical scavenging activity in seeds. The present study suggests the feasibility of engineering a transcription factor of flavonoid biosynthesis for health beneficial plant seeds.

Arabidopsis↗
PubMed2006

The surface functionalization of 45S5 Bioglass-based glass-ceramic scaffolds and its impact on bioactivity.

The first and foremost function of a tissue engineering scaffold is its role as a substrate for cell attachment, and their subsequent growth and proliferation. However, cells do not attach directly to the culture substrate; rather they bind to proteins that are adsorbed to the scaffold's surface. Like standard tissue culture plates, tissue engineering scaffolds can be chemically treated to couple proteins without losing the conformational functionality; a process called surface functionalization. In this work, novel highly porous 45S5 Bioglass-based scaffolds have been functionalized applying 3-AminoPropyl-TriethoxySilane (APTS) and glutaraldehyde (GA) without the use of organic solvents. The efficiency and stability of the surface modification was assessed by X-ray photoemission spectroscopy (XPS). The bioactivity of the functionalized scaffolds was investigated using simulated body fluid (SBF) and characterized by scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS) and X-ray diffraction (XRD). It was found that the aqueous heat-treatment applied at 80 degrees C for 4 hrs during the surface functionalization procedure accelerated the structural transition of the crystalline Na2Ca2Si3O9 phase, present in the original scaffold structure as a result of the sintering process used for fabrication, to an amorphous phase during SBF immersion. The surface functionalized scaffolds exhibited an accelerated crystalline hydroxyapatite layer formation upon immersion in SBF caused by ion leaching and the increased surface roughness induced during the heat treatment step. The possible mechanisms behind this phenomenon are discussed.

Biocompatible Materials↗
PubMed1994

Structure of RecA-DNA complex and mechanism of DNA strand exchange reaction in homologous recombination.

The importance of filament formation of RecA for the DNA strand exchange reaction, both in vivo and in vitro, is established. RecA forms a very long and relatively stiff filament by binding around DNA with high cooperativity. The monomer units are assembled in the filament in a head-to-tail arrangement in a helical manner, similar to the organization of RecA molecules found in the crystal of pure RecA or including ADP. This filament of RecA, containing a DNA molecule in its interior, can bind another DNA molecule and yet a third one in the presence of cofactor (ATP or its analogs). Each filament may have three DNA binding sites, each able to bind one DNA strand of either ss or ds DNA. According to linear dichroism and fluorescence spectroscopies, the DNA molecules in the RecA filament are well organized with a well defined but modified structure. This organization and modification of DNA by RecA probably has the purpose of facilitating Watson-Crick base-pair recognition and strand exchange reaction. RecA is thus actively involved in the reaction. The phosphoribose backbone of DNA follows the RecA helix and the DNA is stretched 50% and unwound. The nucleobases are destacked but still firmly oriented almost perpendicular to the axis for the first DNA and are immobile even in the case of ssDNA. Even in the second DNA the motion of DNA bases is very restricted although their orientation appears to be less perpendicular. All DNA strands in the complex show some sequence dependent interaction with each other. It could be a non-conventional base-base pairing, that could provide a mechanism of search for homologous DNA. In order to understand how RecA organizes DNA, a 3-D model building of the RecA-DNA complex is in progress undertaken based on the crystal structure of RecA and results obtained using chemical interference and protein engineering techniques. A characterization of structure of the second DNA and the mode of DNA-DNA interaction may further clarify the reaction mechanism of strand exchange.

Amino Acid Sequence↗
PubMed1993

Controlling environmental nitrogen through microbial metabolism.

The major sources of environmental nitrogen are the result of process intensification by human activities such as the chemical synthesis of nitrogenous fertilizers, internal-combustion engines, intensive farming and the use of xenobiotic chemicals in industrial processes. Recent advances in the biochemistry, genetics and ecology of nitrifying and denitrifying bacteria can now be exploited in the control of environmental pollution from these sources. However, frequently, biotreatment must be combined with physical and chemical processes to achieve satisfactory remediation.

Biodegradation, Environmental↗
PubMed1992

Synthetic approaches to vaccines for infectious and autoimmune diseases.

The development is outlined of some synthetic vaccines against infectious diseases, in particular cholera, shigella and influenza. In the last case, use of the synthetic adjuvant MDP in combination with a haemagglutinin peptide has led to a synthetic vaccine with built-in adjuvanticity. The production of vaccines both by chemical synthesis and genetic engineering is described. The successful use of the synthetic amino acid copolymer COP-1 as an immunomodulatory vaccine to suppress the onset of allergic encephalomyelitis in experimental animals has led to clinical trials with patients suffering from exacerbating remitting multiple sclerosis. T-cell vaccination is an alternative approach to immunization against autoimmune diseases.

Adjuvants, Immunologic↗
PubMed1990

Refinement of long-term toxicity and carcinogenesis studies.

The chance that alternatives will completely replace animals for toxicology research in the foreseeable future is nil. Continual refinement of animal toxicity and carcinogenesis studies, however, can be an effective means of reducing the numbers of animals used and conserving time and resources without compromising scientific quality. We must continue to strive to find species and strains that can metabolize chemicals similar to humans, are small enough to be housed in large numbers, and have low prevalence of spontaneous lesions with sufficient life span to express the toxic and carcinogenic potential of chemicals. Adequate care of animals with control of variables such as light, temperature, diet, bedding, diseases, and genetic characters of laboratory animals will decrease the variability. Humane considerations and euthanasia of animals with large masses and other conditions interfering with eating and drinking, major injuries and ulcers related to husbandry and treatment, and diseases indicating pain and suffering will help not only to alleviate further pain and distress but also to facilitate collection of tissues without secondary complications for detection of chemical treatment-related lesions. Limiting the duration of studies to decrease the variability due to age-associated changes will also refine long-term studies. Other considerations for refinement of carcinogenesis studies include selection of the most sensitive sex of one or more species for evaluation of selected chemicals in a class where toxic and carcinogenic potential of other representative chemicals are known. Genetically engineered animal models with known oncogenes may reduce the duration and increase the sensitivity of carcinogenesis studies with a reduction in the use of animals.

Animal Husbandry↗
PubMed1992

Behavior of large nucleic acids in reversed-phase high-performance liquid chromatography.

Large nucleic acids can be separated by reversed-phase high-performance liquid chromatography. Analysis shows that the retention time depends not only on the chain length but also on the base composition and the secondary structure of the molecule. A model is proposed to interpret their behavior. This model, called "multiple-point interaction theory" is based on the observation that macromolecules are flexible and very large compared to the hydrophobic phase (octadecylsilane) of the column. It explains the behavior of large nucleic acids in terms of an equilibrium of the macromolecule between the two phases through a multiple-point attachment to the chromatographic matrix, the parameters of the equilibrium being both the hydrophobicity of the base and the number of attachment points. This model fits the experimental data and can be applied to all types of flexible macromolecules, especially proteins and nucleic acids, when they are chromatographed on reversed-phase columns. The model is used to explain the separation of nucleic acids of importance in molecular biology.

Chromatography, High Pressure Liquid↗