Molecular evolution and genetic engineering of protein domains involving aspartate transcarbamoylase.
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Amino Acid Sequence↗
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Results for “Molecular engineering”
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Metabolic engineering--integrating methodologies of molecular breeding and bioprocess systems engineering.
Metabolic engineering is an integrating methodology of analysis and synthesis for the improvement of flux distribution of metabolic pathways in complicated bioprocesses, which are highly multi-hierarchical systems to extend from macroscopic to microscopic levels. Recent progress in metabolic engineering methodologies to improve metabolic pathways in microorganisms was reviewed with many studies in this paper. Metabolic flux distribution was analyzed under different environmental conditions, using a metabolic reaction model. The physiological states of microorganisms were understood by interpreting metabolic flux analysis (MFA). This analysis was also used for development of process operation and control strategy. Cell capability to form a targeted product was analyzed with a metabolic reaction model and linear programming (LP). The use of a 13C-enriched carbon source and nuclear magnetic resonance (NMR) and gas chromatography-mass spectrometry (GCMS) analyses of intracellular and extracellular metabolites enabled determination of a metabolic flux distribution more accurately than the flux distribution determined only by the metabolic reaction model, which involves not only metabolite balances but also energy and redox balances. The comparison of metabolic flux distributions between before and after genetic modification of cells yielded information on the mechanism of regulation of metabolic flux in microorganisms. Finally, integration of bioinformatics and metabolic engineering is discussed, and cyclic modification of the complex bionetwork and process development were emphasized.
Journal Article↗
Re-engineering butyrylcholinesterase as a cocaine hydrolase.
To address the problem of acute cocaine overdose, we undertook molecular engineering of butyrylcholinesterase (BChE) as a cocaine hydrolase so that modest doses could be used to accelerate metabolic clearance of this drug. Molecular modeling of BChE complexed with cocaine suggested that the inefficient hydrolysis (k(cat) = 4 min(-1)) involves a rotation toward the catalytic triad, hindered by Tyr332. To eliminate rotational hindrance and retain substrate affinity, we introduced two amino acid substitutions (Ala328Trp/Tyr332Ala). The resulting mutant BChE reduced cocaine burden in tissues, accelerated plasma clearance by 20-fold, and prevented cocaine-induced hyperactivity in mice. The enzyme's kinetic properties (k(cat) = 154 min(-1), K(M) = 18 microM) satisfy criteria suggested previously for treating cocaine overdose (k(cat) >120 min(-1), K(M) < 30 microM). This success demonstrates that computationally guided mutagenesis can generate functionally novel enzymes with clinical potential.
Alanine↗
Molecular beacons.
This opinion covers the field of molecular beacons (MBs), in which nucleic acids are molecularly engineered to have unique functions for the investigation of biomolecules. Molecular beacons have been used in a variety of formats, and this review discusses four: first, in vitro RNA and DNA monitoring; second, biosensors and biochips based on MBs; third, real-time monitoring of genes and gene expression in living systems; and finally, the next generation of molecular beacons that will be highly useful for studies with proteins, molecular beacon aptamers. These unique applications have shown that MBs holds great potential in genomics and proteomics where real-time molecular recognition with high sensitivity and excellent specificity is critical.
Biosensing Techniques↗
Bioinspired laser-operated molecular locomotive.
Biomotors kinesin and dynein show us that robust track-walking is possible down to molecular scale. Here I design a laser-powered molecular locomotive that is able to do that on an easily constructed track. The core of the machine is its work cycle that periodically converts optical energy into mechanical work, which is further rectified into processive, directional motion. Thus the molecular locomotive is essentially beyond the famous design of molecular shuttles. Under automated laser operation, the locomotive can move a few mum per second comparable to its biological counterparts. However, this artificial motor is capable of conveniently switchable, dual directional motion in contrast to common unidirectionality of biomotors. The locomotive is also different from the big category of Brownian motors in the sense that move of the locomotive is not a result of biasing pre-existing fluctuations, rather it is directly and decisively driven by optomechanical strokes of the work cycle, generating a pulling force ten times greater than those of biomotors. Being a novel type of molecular motor as well as a powerful molecular engine, this machine will potentially enable automatic, forceful delivery of molecular building blocks with nanometer accuracy. Well within reach of established techniques, its implementation will be a significant advance in nanoscience and nanotechnology.
Biomimetic Materials↗
Advances in cell therapy for renal failure.
Cell therapy is one of the most exciting fields in translational medicine. It stands at the intersection of a variety of rapidly developing scientific disciplines: stem cell biology, immunology, tissue engineering, molecular biology, biomaterials, transplantation biology, regenerative medicine and clinical research. Cell-based therapy may develop into a new therapeutic platform to treat a vast array of clinical disorders. Blood transfusions and bone marrow transplantation are prime examples of the successful application of cell-based therapeutics; but recent advances in cellular and molecular biology have expanded the potential applications of this approach. Although recombinant genetic engineering to produce a variety of therapeutics, such as human erythropoietin and insulin has proven successful, these treatments are unable to completely correct or reverse disease states, because most common disease processes are not due to the deficiency of a single protein but develop due to alterations in the complex interactions of a variety of cell components. In these complex situations, cell-based therapy may be a more successful strategy by providing a dynamic, interactive and individualized therapeutic approach that responds to the pathophysiological condition of the patient. In this regard, cells may provide innovative methods for drug delivery of biologics, immunotherapy, and tissue regenerative or replacement engineering [Nature 392 (1998) 518-524, Nat Biotechnol 20 (2002) 339-343]. The translation of this discipline to medicinal practice has tremendous potential, but in many applications technological issues need to be overcome. Since many cell-based indications are already being evaluated in the clinic, the field appears to be on the threshold of a number of successes. This review will focus on our group's use of human stem/progenitor cells in the treatment of acute and chronic renal failure as extensions to current successful renal substitution processes of hemodialysis and hemofiltration.
Acute Kidney Injury↗
Stem cells: the next therapeutic frontier.
Cell therapy is one of the most exciting fields in translational medicine. It stands at the intersection of a variety of rapidly developing scientific disciplines: stem cell biology, immunology, tissue engineering, molecular biology, biomaterials, transplantation biology, regenerative medicine, and clinical research. Cell-based therapy may develop into a new therapeutic platform to treat a vast array of clinical disorders. Blood transfusions and bone marrow transplantation are prime examples of the successful application of cell-based therapeutics; but recent advances in cellular and molecular biology have expanded the potential applications of this approach. Although recombinant genetic engineering to produce a variety of therapeutics such as human erythropoietin and insulin has proven successful, these treatments are unable to completely correct or reverse disease states, because most common disease processes are not due to the deficiency of a single protein but develop due to alterations in the complex interactions of a variety of cell components. In these complex situations, cell-based therapy may be a more successful strategy by providing a dynamic, interactive, and individualized therapeutic approach that responds to the pathophysiological condition of the patient. In this regard, cells may provide innovative methods for drug delivery of biologics, immunotherapy, and tissue regenerative or replacement engineering (1,2). The translation of this discipline to medical practice has tremendous potential, but in many applications technological issues need to be overcome. Since many cell-based indications are already being evaluated in the clinic, the field appears to be on the threshold of a number of successes. This review will focus on our group's use of human stem/progenitor cells in the treatment of acute and chronic renal failure as extensions to the current successful renal substitution processes of hemodialysis and hemofiltration.
Animals↗
Recent biotechnological developments in the use of peroxidases.
Peroxidases are ubiquitous oxidoreductases that use hydrogen peroxide or alkyl peroxides as oxidants. Advances have recently been made in using them to prepare, under mild and controlled conditions, chiral organic molecules that are valuable for the chemoenzymatic synthesis of a wide range of useful compounds. Horseradish peroxidase can be converted into a peroxygenative enzyme by molecular engineering. Chloroperoxidase, the most versatile peroxidase, behaves like a 'true' monooxygenase in sulfoxidations with molecular oxygen and an external reductant, with substantial increases in enantioselectivity and enzyme stability.
Alcohols↗
A dedication in memoriam of Dr. Richard Skalak.
Richard Skalak (1923-1997) played a leadership role in the formative decades of the discipline of biomedical engineering through his technical contributions in biomechanics, his educational influence on students, and his service to many developing societies and journals. But always, the distinguishing marks of his involvement with any activity or person were his generosity, respect and tolerance for others, integrity, and curiosity. These very qualities are what first brought him as a traditional engineering trained in engineering mechanics into the young field of biomedical engineering in the 1960s, and they are what led him to new approaches to cellular and molecular engineering, tissue engineering, and orthopedic biomechanics. His technical papers and lectures on blood cell mechanics, pulmonary circulation, dental implants, and tissue growth were models of clarity and often pointed the way to new areas of exploration, while his personal writings offer advice on life, academic organizations, and the pursuit of significant work. He would be deeply appreciative that this first volume of the Annual Review of Biomedical Engineering is dedicated to his memory.
Animals↗
Open computing grid for molecular science and engineering.
Grid is an emerging infrastructure for distributed computing that provides secure and scalable mechanisms for discovering and accessing remote software and data resources. Applications built on this infrastructure have great potential for addressing and solving large scale chemical, pharmaceutical, and material science problems. The article describes the concept behind grid computing and will present the OpenMolGRID system that is an open computing grid for molecular science and engineering. This system provides grid enabled components, such as a data warehouse for chemical data, software for building QSPR/QSAR models, and molecular engineering tools for generating compounds with predefined chemical properties or biological activities. The article also provides an overview about the availability of chemical applications in the grid.
Journal Article↗
Molecular and cellular characterization of mesenchymal progenitors for skeletal biomedical devices.
Mesenchymal cells are successfully used to create cell-loaded devices in tissue engineering. Molecular properties of the cells and interaction with polymer scaffolds regulate the development of desired tissues. The present study compared the molecular markers in mesenchymal pleuripotent C3H10T1/2 and osteogenic MBA-15 cells. The cells express transcription factors (TF) of chondro-ostegenic pathway (cbfa-1 and c-fos) and MyoD - TF of muscle differentiation pathway, but not myogenin. Analyzed cells expressed receptors for glucocorticoids, growth hormone, prolactin, and PTH, which indicates their potential responsiveness to systemic signals. Analysis of mRNA encoding for receptors of TGFbeta, TNF, and various interleukins revealed differential expression of IL-2r and TGFbeta-1r receptors, which were expressed by MBA-15 but not by C3H10T1/2 cells. Expression of functional genes indicates differences in the stages of cell differentiation: ALK was present in MBA-15 only, while both cell types expressed collagen-I. Furthermore, we evaluated the expression of adhesion proteins that mediate cell-polymer interactions by flow cytometry analysis. Cell adhesion molecules (CAMs) analyzed were integrinalpha-M (CD11b), selectin-E (CD62E), and PECAM-1 (CD31), which have shown differential expression on cells cultured on plastic, poly(L-lactic acid) (PLLA) or poly(DL-lactide-glycolide acid) (PDLGA) polymer films. Detailed molecular characterization of mesenchymal cells will enable optimization of culture conditions for successful creation of implantable cell-loaded constructs.
Animals↗
Protein engineering and the development of generic biosensors.
Biosensors exploit the remarkable specificity of biomolecular recognition to provide analytical tools that can measure the presence of a single molecular species in a complex mixture. A new strategy is emerging in the development of biosensor technologies: molecular-engineering techniques are being used to adapt the properties of proteins to simple, generic detector instrumentation, rather than adapting instruments to the unique requirements of a natural molecule.
Binding Sites↗
Self-assembled hexa-peri-hexabenzocoronene graphitic nanotube.
An amphiphilic hexa-peri-hexabenzocoronene self-assembles to form a pi-electronic, discrete nanotubular object. The object is characterized by an aspect ratio greater than 1000 and has a uniform, 14-nanometer-wide, open-ended hollow space, which is an order of magnitude larger than those of carbon nanotubes. The wall is 3 nanometers thick and consists of helical arrays of the pi-stacked graphene molecule, whose exterior and interior surfaces are covered by hydrophilic triethylene glycol chains. The graphitic nanotube is redox active, and a single piece of the nanotube across 180-nanometer-gap electrodes shows, upon oxidation, an electrical resistance of 2.5 megohms at 285 kelvin [corrected]. This family of molecularly engineered graphite with a one-dimensional tubular shape and a chemically accessible surface constitutes an important step toward molecular electronics.
Journal Article↗
Droplets as microreactors for high-throughput biology.
Inspired by the principles of biological evolution, biologists--and others--have in recent decades harnessed the power of "natural" selection to sift through huge libraries of genes and find those with desirable properties. At the same time, the demand for high-throughput biochemical and genetic assays and screens has driven the development of increasingly miniaturised assay systems. An exciting synergy is now emerging between these two fields, whereby the tools of ultrahigh-throughput screening promise to open up new directions in molecular engineering.
Bacteria↗
Self-assembled nanoparticle probes for recognition and detection of biomolecules.
Colloidal gold nanocrystals have been used to develop a new class of nanobiosensors that is able to recognize and detect specific DNA sequences and single-base mutations in a homogeneous format. At the core of this biosensor is a 2.5-nm gold nanoparticle that functions as both a nano-scaffold and a nano-quencher (efficient energy acceptor). Attached to this core are oligonucleotide molecules labeled with a thiol group at one end and a fluorophore at the other. This hybrid bio/inorganic construct is found to spontaneously assemble into a constrained arch-like conformation on the particle surface. Binding of target molecules results in a conformational change, which restores the fluorescence of the quenched fluorophore. Unlike conventional molecular beacons with a stem-and-loop structure, the nanoparticle probes do not require a stem, and their background fluorescence increases little with temperature. In comparison with the organic quencher Dabcyl (4,4'-dimethylaminophenyl azo benzoic acid), metal nanoparticles have unique structural and optical properties for new applications in biosensing and molecular engineering.
Base Sequence↗
New insights into the immunoultrastructural organization of cutaneous basement membrane zone molecules.
The epidermal basement membrane zone (BMZ) is composed of various molecules, each of which plays an important role in dermo-epidermal adhesion. Genetic abnormality of certain BMZ molecules leads to an inherited group of skin diseases collectively referred to as epidermolysis bullosa, whose hallmark is skin fragility of varying degrees. Furthermore, development of autoantibodies to certain BMZ molecules leads to the onset of a number of acquired autoimmune blistering diseases in which dermo epidermal separation occurs, including bullous pemphigoid and epidermolysis bullosa acquisita. The ultrastructural location of each BMZ molecule has been studied using a range of immunoelectron microscopy (immuno-EM) techniques. Recent technical advances in immuno-EM and in molecular engineering for production of epitope-specific antibodies have enabled a more correct and precise elucidation of the native ultrastructural molecular organization of the respective molecules and their relationship to each other. These recent studies have also revealed several misinterpretations in the previously established model of the immunoultrastructural organization of BMZ molecules. In response to these findings, this review focuses on three major BMZ-related molecules, type VII collagen, BPAG2 and laminin 5, for which recent immuno-EM studies have produced a revision in the accepted dogma on their ultrastructural distribution at the BMZ.
Basement Membrane↗
Supramolecular chemistry and technology
Supramolecular chemistry deals with the association of several chemical species, in an organized way and according to well defined purposes. Based on a molecular engineering approach, supramolecular structures can be designed from pre-formed building blocks, providing a promising route from chemistry to molecular nanotechnology. New supramolecular systems have been assembled in our laboratory with the use of bridging unities such as tetrapyridylporphyrins, porphyrazines and polypyrazines, connecting transition metal complexes and clusters. These systems display a very exciting electrochemical and catalytic behavior, and interact with DNA, generating 1O2 and leading to efficient oxidative clivage for photodynamic terapy applications. Molecular interfaces have been developed, exhibiting photocurrent response in the presence of visible-UV light, and rectifying properties in the presence of electroactive species. Successful applications of the supramolecular species in chemical and bio-sensors have been developed.
Journal Article↗
A review of molecular recognition technologies for detection of biological threat agents.
The present review summarizes the state of the art in molecular recognition of biowarfare agents and other pathogens and emphasizes the advantages of using particular types of reagents for a given target (e.g. detection of bacteria using antibodies versus nucleic acid probes). It is difficult to draw firm conclusions as to type of biorecognition molecule to use for a given analyte. However, the detection method and reagents are generally target-driven and the user must decide on what level (genetic versus phenotypic) the detection should be performed. In general, nucleic acid-based detection is more specific and sensitive than immunological-based detection, while the latter is faster and more robust. This review also points out the challenges faced by military and civilian defense components in the rapid and accurate detection and identification of harmful agents in the field. Although new and improved sensors will continue to be developed, the more crucial need in any biosensor may be the molecular recognition component (e.g. antibody, aptamer, enzyme, nucleic acid, receptor, etc.). Improvements in the affinity, specificity and mass production of the molecular recognition components may ultimately dictate the success or failure of detection technologies in both a technical and commercial sense. Achieving the ultimate goal of giving the individual soldier on the battlefield or civilian responders to an urban biological attack or epidemic, a miniature, sensitive and accurate biosensor may depend as much on molecular biology and molecular engineering as on hardware engineering. Fortunately, as this review illustrates, a great deal of scientific attention has and is currently being given to the area of molecular recognition components. Highly sensitive and specific detection of pathogenic bacteria and viruses has increased with the proliferation of nucleic acid and immuno-based detection technologies. If recent scientific progress is a fair indicator, the future promises remarkable new developments in molecular recognition elements for use in biosensors with a vast array of applications.
Biological Warfare↗