The synthesis of beta-galactosidase by constitutive and other regulatory mutants of Escherichia coli in chemostat culture.

The synthesis of beta-galactosidase by an E. coli constitutive mutant was examined in a chemostat using glucose-, glycerol-, succinate- or N-limited growth media. Except for glucose-grown bacteria, the steady-state intracellular level of beta-galactosidase was maximal at dilution rates between 0-2 and 0-3 h-1. At higher dilution rates enzyme synthesis was reduced by catabolite repression, which could be relieved by the addition of cyclic AMP. With a catabolite-resistant mutant (UV5c), no decrease in enzyme level at high dilution rates were observed. All mutants examined were constitutive and gave decreased enzyme levels at low dilution rates, with the exception of lac-/F'lac UV5c mutants where the enzyme levels rose at low dilution rates. Hyper-producing mutants were isolated but were unstable. A constitutive mutant growing on glycerol-limited media was considered the most suitable for large-scale production of beta-galactosidase in a chemostat.     

Combined structure- and ligand-based pharmacophore modeling and molecular dynamics simulation studies to identify selective inhibitors of MMP-8

Matrix metalloproteinase-8 (MMP-8) is the key mediator in initiating type I collagen degradation and is associated with rheumatoid arthritis. In the present study, a pharmacophore hypothesis was developed based on selective non zinc binding inhibitors of MMP-8. The pharmacophore hypothesis was refined manually and validated by observing structures and the interactions of MMP-8 inhibitors. The refined pharmacophore model was able to discriminate the non-zinc binding inhibitors of MMP-8 with respect to other inhibitors. Hence this study proposes a combined structure- and ligand-based pharmacophore model that is suitable for retrieving the novel inhibitors of MMP-8. The pharmacophore hypothesis AADRH was used as query for retrieving potential compounds from the Zinc database and hits were selected based on the catalytic selective amino acid residues of Arg 222, and Tyr 227. We identified six compounds as potent inhibitors and their selectivity profile were checked against different subtypes of MMPs using the cross-docking method. Molecular dynamics results indicated that ZINC 00673680 forms a stable interaction with the key amino acid residues and avoids the zinc atom with a distance of 5.49?Å. Our computational study might be useful for further development of selective MMP-8 inhibitors.     

Coordination of escape and spatial navigation circuits orchestrates versatile flight from threats

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The theoretical aspects of biochemical fuel cells

Biological systems can be used in three types of fuel cell: depolarization (or concentration) cell, product cell, and redox cell. The possibilities and theoretical limitations of each type of cell have been considered in terms of the metabolic activities of microorganisms and the coupling of these to electrochemical systems. The use of cell extracts and enzymes, particularly in an insoluble form, has been discussed.     

Immobilized enzyme reaction stability: Attrition of the support material

One of the main reasons for immobilizing an enzyme is to enable its reuse, or continuous use, in a reactor. Consequently immobilized enzyme stability is an important factor in enzyme reactor design. The performance of the reactor will decrease if during operation the support material disintegrates into smaller particles that pass out of the reactor system. When β-galactosidase is immobilized by covalent attachment to AE-cellulose, the smaller particles have a higher activity. After subjection of the immobilized enxyme to a shear stress the average particle size decreases and the total enzymic activity increases. A loss of small particles from the reactor, although constituting a small weight percent loss of support, will result in a disproportionately large loss in activity. The relevance of these observations to reactor performance is discussed.     

Proton-transport activity,sidedness, and morphometry of tonoplast and plasma-membrane vesicles purified by free-flow electrophoresis from roots of Lepidium sativum L. and hypocotyls of Cucurbita pepo L.

Large-scale preparations of highly purified tonoplast and plasma-membrane vesicles were obtained from roots (garden cress, Lepidium sativum L.) and shoots (etiolated zucchini hypocotyl, Cucurbita pepo L.) of representative dicotyledonous seedlings. When tonoplast-enriched fractions of cress roots were prepared by centrifugation and then subjected to free-flow electrophoresis a highly purified tonoplast fraction was obtained. This fraction from cress roots was characterized by morphometry of filipin-treated freeze-fractured preparations and by enzymology to be about 90% homogeneous. Using latency of nitrate-inhibited ATPase and H⁺-pumping as criteria we found that the majority of the tonoplast vesicles from both sources were oriented right(cytoplasmic)-side-out. Plasma-membrane vesicles were first purified by two-phase partitioning and then subjected to free-flow electrophoresis for further purification. From cress roots, the fraction of highest purity contained 89% plasma-membrane vesicles as judged by morphometry of filipin-treated, freeze-fractured preparations and by enzymology. From both sources, the major plasma-membrane subfraction in the upper phase after two-phase partitioning was shown to have the least electrophoretic mobility in free-flow electrophoresis and to be oriented right(extracytoplasmic)-side-out a slightly more mobile plasma-membrane subfraction was oriented inside-out and originated after freezing thawing from outside-out plasma-membrane vesicles.Part of the doctoral thesis (D5) of B. vom DorpWe thank the Bundesministerium für Forschung und Technologie for financial support.