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At least 289 records · Page 16

Over-Expression, Purification and Crystallization of Human Dihydrolipoamide Dehydrogenase

Dehydrolipoamide dehydrogenase (E3; dihydrolipoan-tide:NAD+ oxidoreductase, EC 1.8.1.4) is a common catalytic component found in pyruvate dehydrogenase complex, alpha-ketoglutarate dehydrogenase complex, and branched-chain cc-keto acid dehydrogenase complex. E3 is also a component (referred to as L protein) of the glycine cleavage system in bacterial metabolism (2). Active E3 forms a homodimer with four distinctive subdomain structures (FAD binding, NAD+ binding, central and interface domains) with non-covalently but tightly bound FAD in the holoenzyme. Deduced amino acids from cloned full-length human E3 gene showed a total of 509 amino acids with a leader sequence (N-terminal 35 amino acids) that is excised (mature form) during transportation of expressed E3 into mitochondria membrane. So far, three-dimensional structure of human E3 has not been reported. Our effort to achieve the elucidation of the X-ray crystal structure of human E3 will be presented. Recombinant pPROEX-1 expression vector (from GIBCO BRL Life Technologies) having the human E3 gene without leader sequence was constructed by Polymerase Chain Reaction (PCR) and subsequent ligation, and cloned in E.coli XL1-Blue by transformation. Since pPROEX-1 vector has an internal His-tag (six histidine peptide) located at the upstream region of a multicloning site, one-step affinity purification of E3 using nickelnitriloacetic acid (Ni-NTA) agarose resin, which has a strong affinity to His-tag, was feasible. Also a seven-amino-acid spacer peptide and a recombinant tobacco etch virus protease recognition site (seven amino acids peptide) found between His-tag and first amino acid of expressed E3 facilitated the cleavage of His-tag from E3 after the affinity purification. By IPTG induction, ca. 15 mg of human E3 (mature form) was obtained from 1L LB culture with overnight incubation at 25C. Over 98% of purity of E3 from one-step Ni-NTA agarose affinity purification was confirmed by SDS-PAGE analysis. For crystallization, E3 samples were prepared with and without His-tag. To minimize the aggregation of E3, apo- and holo- forms of E3s were tested, as well as a mutated E3. Dynamic light scattering measurements revealed that the E3 preparations without His-tag and substrate are highly monodispersive with regard to homodimers. Consequent crystallization trials of this E3 preparation led to single crystals of E3 grown by the vapor diffusion method. Crystals were obtained within a few days from solution containing poly (ethylene glycol) monomethyl ether 5000 as a precipitant. Autoindexing and integration of the X-ray diffraction data showed that E3 crystals belong to an orthorhombic system with unit cell parameters a-- 123. 1, b= 165.3 and c=214.3A. Further optimization of protein preparation and crystallization experiments for the structural determination will be discussed.

Hong, Y. S.↗

Modeling the Activity of Single Genes

The central dogma of molecular biology states that information is stored in DNA, transcribed to messenger RNA (mRNA) and then translated into proteins. This picture is significantly augmentated when we consider the action of certain proteins in regulating transcription. These transcription factors provide a feedback pathway by which genes can regulate one another's expression as mRNA and then as protein. To review: DNA, RNA and proteins have different functions. DNA is the molecular storehouse of genetic information. When cells divide, the DNA is replicated, so that each daughter cell maintains the same genetic information as the mother cell. RNA acts as a go-between from DNA to proteins. Only a single copy of DNA is present, but multiple copies of the same piece of RNA may be present, allowing cells to make huge amounts of protein. In eukaryotes (organisms with a nucleus), DNA is found in the nucleus only. RNA is copied in the nucleus then translocates(moves) outside the nucleus, where it is transcribed into proteins. Along the way, the RNA may be spliced, i.e., may have pieces cut out. RNA then attaches to ribosomes and is translated to proteins. Proteins are the machinery of the cell other than DNA and RNA, all the complex molecules of the cell are proteins. Proteins are specialized machines, each of which fulfills its own task, which may be transporting oxygen, catalyzing reactions, or responding to extracellular signals, just to name a few. One of the more interesting functions a protein may have is binding directly or indirectly to DNA to perform transcriptional regulation, thus forming a closed feedback loop of gene regulation. The structure of DNA and the central dogma were understood in the 50s; in the early 80s it became possible to make arbitrary modifications to DNA and use cellular machinery to transcribe and translate the resulting genes; more recently, genomes (i.e., the complete DNA sequence) of many organisms have been sequenced. This large-scale sequencing began with simple organisms, viruses and bacteria, progressed to eukaryotes such as yeast, and more recently (1998) progressed to a multi-cellular animal, the nematode Caenorhabditis elegans. Sequencers have now moved on to the fruit fly Drosophila melanogaster, whose sequence is slated for completion by the end of 1999. The human genome project is expected to determine the complete sequence of all 3 billion bases of human DNA within the next five years. In the wake of genome-scale sequencing, further instrumentation is being developed to assay gene expression and function on a comparably large scale. Much of the work in computational biology focuses on computational tools used in sequencing, finding genes that are related to a particular gene, finding which parts of the DNA code for proteins and which do not, understanding what proteins will be formed from a given length of DNA, predicting how the proteins will fold from a one-dimensional structure into a three dimensional structure, and so on. Much less computational work has been done regarding the function of proteins. One reason for this is that different proteins function very differently, and so work on protein function is very specific to certain classes of proteins. There are, for example, proteins such enzymes that catalyze various intracellular reactions, receptors that respond to extracellular signals and ion channels that regulate the flow of charged particles into and out of the cell. In this chapter, we will consider a particular class of proteins called transcription factors(TFs), which are responsible for regulating when a certain gene is expressed in a certain cell, which cells it is express in, and how much is expressed. Understanding these processes will involve developing a deeper understanding of transcription, translation, and the cellular processes that control those processes. All of these elements fall under the aegis of gene regulation or more narrowly transcriptional regulation. Some of the key questions in gene regulation are: What genes are expressed in a certain cell at a certain time? How does gene expression differ from cell to cell in a multicellular organism? Which proteins act as transcription factors, i.e., are important in regulating gene expression? From questions like these, we hope to understand which genes are important for various macroscopic processes. Nearly all of the cells of a multicellular organism contain the same DNA. Yet this same genetic information yields a large number of different cell types. The fundamental difference between a neuron and a liver cell, for example, is which genes are expressed. Thus understanding gene regulation is an important step in understanding development. Furthermore, understanding the usual genes that are expressed in cells may give important clues about various diseases. Some diseases, such as sickle cell anemia and cystic fibrosis, are caused by defects in single, non-regulatory genes; others, such as certain cancers, are caused when the cellular control circuitry malfunctions - an understanding of these diseases will involve pathways of multiple interacting gene products. There are numerous challenges in the area of understanding and modeling gene regulation. First and foremost, biologists would like to develop a deeper understanding of the processes involved, including which genes and families of genes are important, how they interact, etc. From a computation point of view, there has been embarrassingly little work done. In this chapter there are many areas in which we can phrase meaningful, non-trivial computational questions, but questions that have not been addressed. Some of these are purely computational (what is a good algorithm for dealing with a model of type X) and others are more mathematical (given a system with certain characteristics, what sort of model can one use? How does one find biochemical parameters from system-level behavior using as few experiments as possible?). In addition to biological and algorithmic problems, there is also the ever-present issue of theoretical biology - what general principles can be derived from these systems, what can one do with models other than just simulate time-courses, what can be deduced about a class of systems without knowing all the details? The fundamental challenge to computationalists and theorists is to add value to the biology - to use models, modeling techniques and algorithms to understand the biology in new ways.

Mjolsness, Eric↗

Microbial Impact on Success of Human Exploration Missions

The purpose of this study is to identify microbiological risks associated with space exploration and identify potential countermeasures available. Identification of microbial risks associated with space habitation requires knowledge of the sources and expected types of microbial agents. Crew data along with environmental data from water, surfaces, air, and free condensate are utilized in risk examination. Data from terrestrial models are also used. Microbial risks to crew health include bacteria, fungi, protozoa, and viruses. Adverse effects of microbes include: infections, allergic reactions, toxin production, release of volatiles, food spoilage, plant disease, material degradation, and environmental contamination. Risk is difficult to assess because of unknown potential changes in microbes (e.g., mutation) and the human host (e.g., immune changes). Prevention of adverse microbial impacts is preferred over remediation. Preventative measures include engineering measures (e.g., air filtration), crew microbial screening, acceptability standards, and active verification by onboard monitoring. Microbiological agents are important risks to human health and performance during space flight and risks increase with mission duration. Acceptable risk level must be defined. Prevention must be given high priority. Careful screening of crewmembers and payloads is an important element of any risk mitigation plan. Improved quantitation of microbiological risks is a high priority.

Pierson, Duane L.↗

Fluid Physics and Macromolecular Crystal Growth in Microgravity

The first protein crystallization experiment in microgravity was launched in April, 1981 and used Germany's Technologische Experimente unter Schwerelosigkeit (TEXUS 3) sounding rocket. The protein P-galactosidase (molecular weight 465Kda) was chosen as the sample with a liquid-liquid diffusion growth method. A sliding device brought the protein, buffer and salt solution into contact when microgravity was reached. The sounding rocket gave six minutes of microgravity time with a cine camera and schlieren optics used to monitor the experiment, a single growth cell. In microgravity a strictly laminar diffusion process was observed in contrast to the turbulent convection seen on the ground. Several single crystals, approx 100micron in length, were formed in the flight which were of inferior but of comparable visual quality to those grown on the ground over several days. A second experiment using the same protocol but with solutions cooled to -8C (kept liquid with glycerol antifreeze) again showed laminar diffusion. The science of macromolecular structural crystallography involves crystallization of the macromolecule followed by use of the crystal for X-ray diffraction experiments to determine the three dimensional structure of the macromolecule. Neutron protein crystallography is employed for elucidation of H/D exchange and for improved definition of the bound solvent (D20). The structural information enables an understanding of how the molecule functions with important potential for rational drug design, improved efficiency of industrial enzymes and agricultural chemical development. The removal of turbulent convection and sedimentation in microgravity, and the assumption that higher quality crystals will be produced, has given rise to the growing number of crystallization experiments now flown. Many experiments can be flown in a small volume with simple, largely automated, equipment - an ideal combination for a microgravity experiment. The term "protein crystal growth" is often historically used to describe these microgravity experiments. This is somewhat inaccurate as the field involves the study of many varied biological molecules including viruses, proteins, DNA, RNA and complexes of those structures. For this reason we use the term macromolecular crystal growth. In this chapter we review a series of diagnostic microgravity crystal growth experiments carried out principally using the European Space Agency (ESA) Advanced Protein Crystallization Facility (APCF). We also review related research, both experimental and theoretical, on the aspects of microgravity fluid physics that affect microgravity protein crystal growth. Our experiments have revealed some surprises that were not initially expected. We discuss them here in the context of practical lessons learnt and how to maximize the limited microgravity opportunities available.

Helliwell, John R.↗

Biological Sterilization of Returned Mars Samples

Martian rock and soil, collected by robotic spacecraft, will be returned to terrestrial laboratories early in the next century. Current plans call for the samples to be immediately placed into biological containment and tested for signs of present or past life and biological hazards. It is recommended that "Controlled distribution of unsterilized materials from Mars should occur only if rigorous analyses determine that the materials do not constitute a biological hazard. If any portion of the sample is removed from containment prior to completion of these analyses it should first be sterilized." While sterilization of Mars samples may not be required, an acceptable method must be available before the samples are returned to Earth. The sterilization method should be capable of destroying a wide range of organisms with minimal effects on the geologic samples. A variety of biological sterilization techniques and materials are currently in use, including dry heat, high pressure steam, gases, plasmas and ionizing radiation. Gamma radiation is routinely used to inactivate viruses and destroy bacteria in medical research. Many commercial sterilizers use Co-60 , which emits gamma photons of 1.17 and 1.33 MeV. Absorbed doses of approximately 1 Mrad (10(exp 8) ergs/g) destroy most bacteria. This study investigates the effects of lethal doses of Co-60 gamma radiation on materials similar to those anticipated to be returned from Mars. The goals are to determine the gamma dose required to kill microorganisms in rock and soil samples and to determine the effects of gamma sterilization on the samples' isotopic, chemical and physical properties. Additional information is contained in the original extended abstract.

Allen, C. C.↗

Genetic Optimization of a Tensegrity Structure

Marshall Space Flight Center (MSFC) is charged with developing advanced technologies for space telescopes. The next generation of space optics will be very large and lightweight. Tensegrity structures are built of compressive members (bars), and tensile members (strings). For most materials, the tensile strength of a longitudinal member is larger than its buckling strength; therefore a large stiffness to mass ratio can be achieved by increasing the use of tensile members. Tensegrities are the epitome of lightweight structures, since they take advantage of the larger tensile strength of materials. The compressive members of tensegrity structures are disjoint allowing compact storage of the structure. The structure has the potential to eliminate the requirement for assembly by man in space; it can be deployed by adjustments in its cable tension. A tensegrity structure can be more reliably modeled since none of the individual members experience bending moments. (Members that experience deformation in more than one dimension are much harder to model.) Structures that can be more precisely modeled can be more precisely controlled. Furthermore, an astoundingly wide variety of natural systems, including carbon atoms, water molecules, proteins, viruses, cells, tissues and even human and other living creatures are tensegrity structures. Through the process of evolution, nature continually improves the design of living creatures for the environment they live in. Since tensegrities are nature's structure of choice, it is conceivable that they have other benefits we are unaware of. A. Keane and S. Brown designed a satellite boom truss system with an enhanced vibration performance. They started with a standard truss system, then used a genetic algorithm to alter the design, while optimizing the vibration performance. An improvement of over 20,000% in frequency-averaged energy levels was obtained using this approach. In this report an introduction to tensegrity structures is given, along with a description of how to generate the nodal coordinates and connectivity of a multiple stage cylindrical tensegrity structure. A description of how finite elements can be used to develop a stiffness and mass matrix so that the modes of vibration can be determined from the eigenvalue problem is shown. A brief description of a micro genetic algorithm is then presented.

Jaime R. Taylor↗

Water Purification

The Vision Catalyst Purifier employs the basic technology developed by NASA to purify water aboard the Apollo spacecraft. However, it also uses an "erosion" technique. The purifier kills bacteria, viruses, and algae by "catalytic corrosion." A cartridge contains a silver-impregnated alumina bed with a large surface area. The catalyst bed converts oxygen in a pool of water to its most oxidative state, killing over 99 percent of the bacteria within five seconds. The cartridge also releases into the pool low levels of ionic silver and copper through a controlled process of erosion. Because the water becomes electrochemically active, no electricity is required.

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Airing Out Anthrax

The AiroCide TiO2 is an air-purifier that kills 93.3 percent of airborne pathogens that pass through it, including Bacillus anthraci, more commonly known as anthrax. It is essentially a spinoff of KES Science & Technology, Inc.'s Bio-KES system, a highly effective device used by the produce industry for ethylene gas removal to aid in preserving the freshness of fruits, vegetables, and flowers. The TiO2-based ethylene removal technology that is incorporated into the company's AiroCide TiO2 and Bio-KES products was first integrated into a pair of plant-growth chambers known as ASTROCULTURE(TM) and ADVANCED ASTROCULTURE(TM). Both chambers have housed commercial plant growth experiments in space on either the Space Shuttle or the International Space Station. The AiroCide TiO2 also has a proven record of destroying 98 percent of other airborne pathogens, such as microscopic dust mites, molds, and fungi. Moreover, the device is a verified killer of Influenza A (flu), E. coli, Staphylococcus aureas, Streptococcus pyogenes, and Mycoplasma pneumoniae, among many other harmful viruses.

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Water Purification

Silver ionization water purification technology was originally developed for Apollo spacecraft. It was later used to cleanse swimming pools and has now been applied to industrial cooling towers and process coolers. Sensible Technologies, Inc. has added two other technologies to the system, which occupies only six square feet. It is manufactured in three capacities, and larger models are custom built on request. The system eliminates scale, corrosion, algae, bacteria and debris, and because of the NASA technology, viruses and waterborne bacteria are also destroyed. Applications include a General Motors cooling tower, amusement parks, ice manufacture and a closed-loop process cooling system.

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Microbiology System

Technology originating in a NASA-sponsored study of the measurement of microbial growth in zero gravity led to the development of Biomerieux Vitek, Inc.'s VITEK system. VITEK provides a physician with accurate diagnostic information and identifies the most effective medication. Test cards are employed to identify organisms and determine susceptibility to antibiotics. A photo-optical scanner scans the card and monitors changes in the growth of cells contained within the card. There are two configurations - VITEK and VITEK JR as well as VIDAS, a companion system that detects bacteria, viruses, etc. from patient specimens. The company was originally created by McDonnell Douglas, the NASA contractor.

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A Universal Antidote

Last fall two of the Big Three U.S. automakers concluded arrangements with Alcide Corporation for distribution of Alcide's patented Ren New Air Conditioning Disinfectant. Special properties of Alcide formulation enable it to destroy mold and fungus as well as bacteria and viruses with minimal harm to humans, animals and/or plants. This allows use of the product to disinfect and deodorize auto air conditioners without removing them and without any lingering toxicity. Disinfectant/deodorizer is one of a wide range of Alcide formulations engineered for a variety of purposes, spanning automotive, medical, agricultural, pharmaceutical and consumer markets. Alcide is not strictly speaking a spinoff from aerospace technology, but the products and company that makes them are beneficiaries of assistance provided by NERAC, Inc.

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Research Help

The Electrosynthesis Co. Inc.'s Electrocinerator System integrates a highly effective air scrubber with an electrochemical cell to provide an apparatus capable of destroying virtually all toxic chemicals and airborne bacteria. Project (funded by DOD) is prospective means of decontaminating airborne chemicals and biological warfare agents. Also has broad civil use applicable to hospitals for destruction of airborne viruses and bacteria, and industrial use for elimination of toxic solvent vapors and malodorous emissions.

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Irradiated HMEC from A-T Heterozygous Breast Tissue

Women who are heterozygous for ataxia-telangiectasia (A-T) carry a single defective ATM gene in chromosome 11 q22-23, and have been statistically determined with high significance within a defined database to be approximately 5-fold more susceptible for developing breast cancer than their noma1 counterpart. Breast cancer susceptibility of these A-T heterozygotes has been hypothesized to include consequence of response to damage caused by low levels of ionizing radiation. Prophylactic mastectomy specimens were donated by a 41 year-old obligate A-T heterozygote who was located prior to her elective surgery through an existing pedigree. Harvest of that breast tissue provided an isolate of long-term growth human mammary epithelial cells (HMEC), designated WH612/3. An isolate of presumed normal long-term growth HMEC, designated 48R, was obtained from Dr. Martha Stampfer (Lawrence Berkeley Laboratory, University of California), and the A-T heterozygous HMEC were transformed with E6 and E7 oncogenes of human papilloma virus Type-16 in the laboratory of Dr. Ray White (Hunt- Cancer Institute, University of Utah) for use in this study. The objective of this study is to study the expression of end points that may bear on cancer outcome following irradiation of HMEC. Specific end points are cell survival, cell cycle, p53 expression, and apoptosis. Survival curves, immunostaining, and flow cytometery are used to examine these end points. Radiation-induced cell killing shows less shoulder development in the survival curve for WH61U3 compared to 48R HMEC, suggesting less repair of damage in the former HMEC. Additional information is included in the original extended abstract.

Richmond, Robert↗

Multipurpose Compound

Specially formulated derivatives of an unusual basic compound known as Alcide may be the answer to effective treatment and prevention of the disease bovine mastitis, a bacterial inflammation of a cow's mammary gland that results in loss of milk production and in extreme cases, death. Manufactured by Alcide Corporation the Alcide compound has killed all tested bacteria, virus and fungi, shortly after contact, with minimal toxic effects on humans or animals. Alcide Corporation credits the existence of the mastitis treatment/prevention products to assistance provided the company by NERAC, Inc.

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Membrane-Based Functions in the Origin of Cellular Life

How simple membrane peptides performed such essential proto-cellular functions as transport of ions and organic matter across membranes separating the interior of the cell from the environment, capture and utilization of energy, and transduction of environmental signals, is a key question in protobiological evolution. On the basis of detailed, molecular-level computer simulations we investigate how these peptides insert into membranes, self-assemble into higher-order structures and acquire functions. We have studied the insertion of an a-helical peptide containing leucine (L) and serine (S) of the form (LSLLLSL)S into a model membrane. The transmembrane state is metastable, and approximately 15 kcal/mol is required to insert the peptide into the membrane. Investigations of dimers formed by (LSLLLSL)S and glycophorin A demonstrate how the favorable free energy of helix association can offset the unfavorable free energy of insertion, leading to self- assembly of peptide helices in the membrane. An example of a self-assembled structure is the tetrameric transmembrane pore of the influenza virus M2 protein, which is an efficient and selective voltage-gated proton channel. Our simulations explain the gating mechanism and provide guidelines how to reengineering the channel to act as a simple proton pump. In general, emergence of integral membrane proteins appears to be quite feasible and may be easier to envision than the emergence of water-soluble proteins.

Wilson, Michael A.↗

Human Peripheral Blood Mononuclear Cells Cultured in Normal and Hyperglycemic Media in Simulated Microgravity Using NASA Bioreactors

We sought answers to several questions this summer at NASA Johnson Space Center. Initial studies involved the in vitro culture of human peripheral blood mononuclear in cells in different conditioned culture media. Several human cancer clones were similarly studied to determine responses to aberrant glycosylation by the argon laser. The cells were grown at unit gravity in flasks and in simulated microgravity using NASA bioreactors. The cells in each instance were analyzed by flow cytometry. Cell cycle analysis was acquired by staining nuclear DNA with propidium iodide. Responses to the laser stimulation was measured by observing autofluorescence emitted in the green and red spectra after stimulation. Extent of glycosylation correlated with the intensity of the laser stimulated auto-fluorescence. Our particular study was to detect and monitor aberrant glycosylation and its role in etiopathogenesis. Comparisons were made between cells known to be neoplastic and normal cell controls using the same Laser Induced Autofluorescence technique. Studies were begun after extensive literature searches on using the antigen presenting potential of dendritic cells to induce proliferation of antigen specific cytotoxic T-cells. The Sendai virus served as the antigen. Our goal is to generate sufficient numbers of such cells in the simulated microgravity environment for use in autologous transplants of virally infected individuals including those positive for hepatitis and HIV.

Lawless, DeSales↗

The Occurrence of Type S1A Serine Proteases in Sponge and Jellyfish

Although serine proteases are found in all kinds of cellular organisms and many viruses, the classic "chymotrypsin family" (Group S1A by th e 1998 Barrett nomenclature) has an unusual phylogenetic distribution , being especially common in animals, entirely absent from plants and protists, and rare among fungi. The distribution in Bacteria is larg ely restricted to the genus Streptomyces, although a few isolated occ urrences in other bacteria have been reported. The family may be enti rely absent from Archaea. Although more than a thousand sequences have been reported for enzymes of this type from animals, none of them ha ve been from early diverging phyla like Porifera or Cnidaria, We now report the existence of Group SlA serine proteases in a sponge (phylu m Porifera) and a jellyfish (phylum Cnidaria), making it safe to conc lude that all animal groups possess these enzymes.

Rojas, Ana↗

The Grand Assault

Plasmodium falciparum, the protozoan parasite responsible for most human malaria, is among the most studied pathogens of all time, probably only exceeded by the human immunodeficiency virus HTV and the bacterium Mycobacterium tuberculosis. The extent of human suffering and the devastating costs of malaria have long been recognized by world bodies, and numerous initiatives have been taken over the years in an effort to defeat this insidious microbe. Beginning in 1996, an international consortium of scientists from more than a dozen institutions set about to determine the sequence of the organism's 23-megabase genome. Their massive effort-which ended up going well beyond simple sequencing is reported in this special issue of Nature. The avowed goal of the project was to search for chinks in the parasite's armor so that new and effective drugs and vaccines might be developed.

Doolittle, Russell F.↗