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At least 865 records · Page 48

Crystallization Physics in Biomacromolecular Systems

The crystals are built of molecules of protein, nucleic acid and their complexes, like viruses, approx. 5x10(exp 3)+ 3x10(exp 6) Da in weight and 2 + 20 nm in effective diameter. This size strongly exceeds action range of molecular forces and makes a big difference with inorganic crystals. Intermolecular contacts form patches on the biomacromolecular surface. Each patch may occupy only a small percent of the whole surface and vary from polymorph to polymorph of the same protein. Thus, under different conditions (pH, solution chemistry, temperature, any area on the macromolecular surface may form a contact. The crystal Young moduli, E approx. equals 0.1 + 0.5 GPa are more than 10 times lower than that of inorganics and the biomolecules themselves. Water within biocrystals (30-70%) is unable to flow unless typical deformation time is longer than approx. 10(exp -5)s. This explains the discrepancy between light scattering and static measurements of E. Nucleation and Growth requires typically concentrations exceeding the equilibrium ones up to 100 times - because of the new size scale results in 10 - 10(exp 3) times lower kinetic coefficients than that needed for inorganic solution growth. All phenomena observed in the latter occur with protein crystallization and are even better studied by AFM. Crystals are typically facetted. Among unexpected findings of general significance are - net molecular exchange flux at kinks is much lower than that expected from supersaturation, steps with low (< approx. 10(exp -2)) kink density at steps follow Gibbs-Thomson law only at very low supersaturations, step segment growth rate may be independent of step energy. Crystal perfection is a must of biocrystallization to achieve the major goal to find 3-D atomic structure of biomacromolecules by x-ray diffraction. Poor diffraction resolution (> 3Angstrom) makes crystallization a bottleneck for structural biology. All defects typical of small molecule crystals are found in biocrystals, but the defects responsible for poor resolution are not identified. Conformational changes are one of them. Biocrystallization in microgravity reportedly results in 20% cases of better crystals. The mechanism of how lack of convection can do this is still not clear. Lower supersaturation, self-purification &om preferentially trapped homologous impurities and step bunching are viable hypotheses.

Chernov, A. A.↗

Investigating the Impact of UV Radiation on High-Altitude Shallow Lake Habitats, Life Diversity, and Life Survival Strategies: Clues for Mars' Past Habitability Potential?

We present data and results from an ongoing project of astrobiological high-altitude expeditions investigating the highest and least explored perennial lakes on Earth in the Bolivian and Chilean Andes, including several volcanic crater lakes nearing and beyond 6,000 m in elevation. In the next five years, they will provide the first integrated long-term astrobiological characterization and monitoring of lacustrine environments and their biology for such altitude. These extreme lakes are natural laboratories. They provide the field data missing beyond 4,000 m to complete our understanding of terrestrial lakes and biota. Research on the effects of UV has been performed in lower altitude lakes and models of UV flux over time are being developed. Lakes showing a high content of dissolved organic material (DOM) shield organisms from UV. DOM acts as a natural sunscreen as it influences the water transparency, therefore is a determinant of photic zone depth. In sparsely vegetated alpine areas, lakes are clearer and offer less protection from UV to organisms living in the water. Transparent water and high UV irradiance may maximize the penetration and effect of UV radiation. Shallow-water communities in these lakes are particularly sensitive to UV radiation. The periphyton can live on various susbtrates. While on rocks, it includes immobile species that cannot seek low UV refuges unlike sediment-dwelling periphyton or alpine phytoflagellates which undergo vertical migration. Inhibition of algal photosynthesis by UV radiation has been documented in laboratory and showed that phytoplankton production is reduced by formation of nucleic acid lesions or production of peroxides and free oxygen radicals. of peroxides and free oxygen radicals. Our project is providing the field data that is missing from natural laboratories beyond 4,000 m and will complement the vision of the effects of UV on life and its adaptation modes (or lack thereof).

Cabrol, A.↗

Plasma Enhanced Growth of Carbon Nanotubes For Ultrasensitive Biosensors

The multitude of considerations facing nanostructure growth and integration lends itself to combinatorial optimization approaches. Rapid optimization becomes even more important with wafer-scale growth and integration processes. Here we discuss methodology for developing plasma enhanced CVD growth techniques for achieving individual, vertically aligned carbon nanostructures that show excellent properties as ultrasensitive electrodes for nucleic acid detection. We utilize high throughput strategies for optimizing the upstream and downstream processing and integration of carbon nanotube electrodes as functional elements in various device types. An overview of ultrasensitive carbon nanotube based sensor arrays for electrochemical bio-sensing applications and the high throughput methodology utilized to combine novel electrode technology with conventional MEMS processing will be presented.

Cassell, Alan M.↗

Ultrasensitive Label-free Electronic Chip for DNA Analysis Using Carbon Nanotube Nanoelectrode Arrays

There is a strong need for faster, cheaper, and simpler methods for nucleic acid analysis in today s clinical tests. Nanotechnologies can potentially provide solutions to these requirements by integrating nanomaterials with biofunctionalities. Dramatic improvement in the sensitivity and multiplexing can be achieved through the high-degree miniaturization. Here, we present our study in the development of an ultrasensitive label-free electronic chip for DNA/RNA analysis based on carbon nanotube nanoelectrode arrays. A reliable nanoelectrode array based on vertically aligned multi-walled carbon nanotubes (MWNTs) embedded in a SiO2 matrix is fabricated using a bottom-up approach. Characteristic nanoelectrode behavior is observed with a low-density MWNT nanoelectrode array in measuring both the bulk and surface immobilized redox species. The open-end of MWNTs are found to present similar properties as graphite edge-plane electrodes, with a wide potential window, flexible chemical functionalities, and good biocompatibility. A BRCA1 related oligonucleotide probe with 18 bases is covalently functionalized at the open ends of the MWNTs and specifically hybridized with an oligonucleotide target as well as a PCR amplicon. The guanine bases in the target molecules are employed as the signal moieties for the electrochemical measurements. Ru(bpy)3(2+) mediator is used to further amplify the guanine oxidation signal. This technique has been employed for direct electrochemical detection of label-free PCR amplicon through specific hybridization with the BRCAl probe. The detection limit is estimated to be less than approximately 1000 DNA molecules, approaching the limit of the sensitivity by laser-based fluorescence techniques in DNA microarray. This system provides a general electronic platform for rapid molecular diagnostics in applications requiring ultrahigh sensitivity, high-degree of miniaturization, simple sample preparation, and low- cost operation.

Li, Jun↗

Plasma Enhanced Growth of Carbon Nanotubes For Ultrasensitive Biosensors

The multitude of considerations facing nanostructure growth and integration lends itself to combinatorial optimization approaches. Rapid optimization becomes even more important with wafer-scale growth and integration processes. Here we discuss methodology for developing plasma enhanced CVD growth techniques for achieving individual, vertically aligned carbon nanostructures that show excellent properties as ultrasensitive electrodes for nucleic acid detection. We utilize high throughput strategies for optimizing the upstream and downstream processing and integration of carbon nanotube electrodes as functional elements in various device types. An overview of ultrasensitive carbon nanotube based sensor arrays for electrochemical biosensing applications and the high throughput methodology utilized to combine novel electrode technology with conventional MEMS processing will be presented.

Cassell, Alan M.↗

The action of a dietary retinoid on gene expression and cancer induction in electron-irradiated rat skin

Current models of radiation carcinogenesis generally assume that the DNA is damaged in a variety of ways by the radiation and that subsequent cell divisions contribute to the conversion of the damage to heritable mutations. Cancer may seem complex and intractable, but its complexity provides multiple opportunities for preventive interventions. Mitotic inhibitors are among the strongest cancer preventive agents, not only slowing the growth rate of preneoplasias but also increasing the fidelity of DNA repair processes. Ionizing radiation, including electrons, is a strong inducer of cancer in rat skin, and dietary retinoids have shown potent cancer preventive activity in the same system. A non-toxic dietary dose of retinyl acetate altered gene expression levels 24 hours after electron irradiation of rat skin. Of the 8740 genes on an Affymetrix rat expression array, the radiation significantly (5 fold or higher) altered 188, while the retinoid altered 231, including 16 radiation-altered genes that were reversely altered. While radiation strongly affected the expression of stress response, immune/inflammation and nucleic acid metabolism genes, the retinoid most strongly affected proliferation-related genes, including some significant reversals, such as, keratin 14, retinol binding protein, and calcium binding proteins. These results point to reversal of proliferation-relevant genes as a likely basis for the anti-radiogenic effects of dietary retinyl acetate.

Non-NASA Center↗

A new version of the RDP (Ribosomal Database Project)

The Ribosomal Database Project (RDP-II), previously described by Maidak et al. [ Nucleic Acids Res. (1997), 25, 109-111], is now hosted by the Center for Microbial Ecology at Michigan State University. RDP-II is a curated database that offers ribosomal RNA (rRNA) nucleotide sequence data in aligned and unaligned forms, analysis services, and associated computer programs. During the past two years, data alignments have been updated and now include >9700 small subunit rRNA sequences. The recent development of an ObjectStore database will provide more rapid updating of data, better data accuracy and increased user access. RDP-II includes phylogenetically ordered alignments of rRNA sequences, derived phylogenetic trees, rRNA secondary structure diagrams, and various software programs for handling, analyzing and displaying alignments and trees. The data are available via anonymous ftp (ftp.cme.msu. edu) and WWW (http://www.cme.msu.edu/RDP). The WWW server provides ribosomal probe checking, approximate phylogenetic placement of user-submitted sequences, screening for possible chimeric rRNA sequences, automated alignment, and a suggested placement of an unknown sequence on an existing phylogenetic tree. Additional utilities also exist at RDP-II, including distance matrix, T-RFLP, and a Java-based viewer of the phylogenetic trees that can be used to create subtrees.

Non-NASA Center↗

The first living systems: a bioenergetic perspective

The first systems of molecules having the properties of the living state presumably self-assembled from a mixture of organic compounds available on the prebiotic Earth. To carry out the polymer synthesis characteristic of all forms of life, such systems would require one or more sources of energy to activate monomers to be incorporated into polymers. Possible sources of energy for this process include heat, light energy, chemical energy, and ionic potentials across membranes. These energy sources are explored here, with a particular focus on mechanisms by which self-assembled molecular aggregates could capture the energy and use it to form chemical bonds in polymers. Based on available evidence, a reasonable conjecture is that membranous vesicles were present on the prebiotic Earth and that systems of replicating and catalytic macromolecules could become encapsulated in the vesicles. In the laboratory, this can be modeled by encapsulated polymerases prepared as liposomes. By an appropriate choice of lipids, the permeability properties of the liposomes can be adjusted so that ionic substrates permeate at a sufficient rate to provide a source of monomers for the enzymes, with the result that nucleic acids accumulate in the vesicles. Despite this progress, there is still no clear mechanism by which the free energy of light, ion gradients, or redox potential can be coupled to polymer bond formation in a protocellular structure.

Review↗

Random-breakage mapping method applied to human DNA sequences

The random-breakage mapping method [Game et al. (1990) Nucleic Acids Res., 18, 4453-4461] was applied to DNA sequences in human fibroblasts. The methodology involves NotI restriction endonuclease digestion of DNA from irradiated calls, followed by pulsed-field gel electrophoresis, Southern blotting and hybridization with DNA probes recognizing the single copy sequences of interest. The Southern blots show a band for the unbroken restriction fragments and a smear below this band due to radiation induced random breaks. This smear pattern contains two discontinuities in intensity at positions that correspond to the distance of the hybridization site to each end of the restriction fragment. By analyzing the positions of those discontinuities we confirmed the previously mapped position of the probe DXS1327 within a NotI fragment on the X chromosome, thus demonstrating the validity of the technique. We were also able to position the probes D21S1 and D21S15 with respect to the ends of their corresponding NotI fragments on chromosome 21. A third chromosome 21 probe, D21S11, has previously been reported to be close to D21S1, although an uncertainty about a second possible location existed. Since both probes D21S1 and D21S11 hybridized to a single NotI fragment and yielded a similar smear pattern, this uncertainty is removed by the random-breakage mapping method.

NASA Discipline Radiation Health↗

Continuous in vitro evolution of bacteriophage RNA polymerase promoters

Rapid in vitro evolution of bacteriophage T7, T3, and SP6 RNA polymerase promoters was achieved by a method that allows continuous enrichment of DNAs that contain functional promoter elements. This method exploits the ability of a special class of nucleic acid molecules to replicate continuously in the presence of both a reverse transcriptase and a DNA-dependent RNA polymerase. Replication involves the synthesis of both RNA and cDNA intermediates. The cDNA strand contains an embedded promoter sequence, which becomes converted to a functional double-stranded promoter element, leading to the production of RNA transcripts. Synthetic cDNAs, including those that contain randomized promoter sequences, can be used to initiate the amplification cycle. However, only those cDNAs that contain functional promoter sequences are able to produce RNA transcripts. Furthermore, each RNA transcript encodes the RNA polymerase promoter sequence that was responsible for initiation of its own transcription. Thus, the population of amplifying molecules quickly becomes enriched for those templates that encode functional promoters. Optimal promoter sequences for phage T7, T3, and SP6 RNA polymerase were identified after a 2-h amplification reaction, initiated in each case with a pool of synthetic cDNAs encoding greater than 10(10) promoter sequence variants.

Non-NASA Center↗

Nucleobases in Space: Laboratory Studies of Polycyclic Aromatic Nitrogen Heterocycles

Polycyclic Aromatic Nitrogen Heterocycles (PANHs) are heterocyclic aromatics Le., PAHs with carbon atoms replaced by a nitrogen atom. These molecules have been detected in meteorite extracts, and in general these nitrogen heterocycles are of astrobiological interest since this class of molecules include nucleobases, basic components of our nucleic acids. These compounds are predicted to be present in the interstellar medium and in Titan tholin, but have received relatively little attention. We will present spectra and reactions of PANHs, frozen in solid H2O at 12 K, conditions germane to astronomical observations. In contrast to simple PAHs, that do not interact strongly with solid H2O, the nitrogen atoms in PANHs are potentially capable of hydrogen bonding with H20 changing their spectra, complicating their remote detection on the surfaces of icy bodies. Moreover, we have studied the photo-chemistry of these interesting compounds under astrophysical conditions and will use our lab studies to assess a potential interstellar heritage of these compounds in carbonaceous chondrites.

Elsila, Jamie↗

Nanoscale Bio-engineering Solutions for Space Exploration: The Nanopore Sequencer

Characterization of biological systems at the molecular level and extraction of essential information for nano-engineering design to guide the nano-fabrication of solid-state sensors and molecular identification devices is a computational challenge. The alpha hemolysin protein ion channel is used as a model system for structural analysis of nucleic acids like DNA. Applied voltage draws a DNA strand and surrounding ionic solution through the biological nanopore. The subunits in the DNA strand block ion flow by differing amounts. Atomistic scale simulations are employed using NASA supercomputers to study DNA translocation, with the aim to enhance single DNA subunit identification. Compared to protein channels, solid-state nanopores offer a better temporal control of the translocation of DNA and the possibility to easily tune its chemistry to increase the signal resolution. Potential applications for NASA missions, besides real-time genome sequencing include astronaut health, life detection and decoding of various genomes.

Stolc, Viktor↗

Use of a Novel Rover-mounted Fluorescence Imager and Fluorescent Probes to Detect Biological Material in the Atacama Desert in Daylight

We have developed an imaging system, the Fluorescence Imager (FI), for detecting fluorescence signals from sparse microorganisms and biofilms during autonomous rover exploration. The fluorescence signals arise both from naturally occurring chromophores, such as chlorophyll of cyanobacteria and lichens, and from fluorescent probes applied to soil and rocks. Daylight imaging has been accomplished by a novel use of a high-powered flashlamp synchronized to a CCD camera. The fluorescent probes are cell permanent stains that have extremely low intrinsic fluorescence (quantum yields less than 0.01) and a large fluorescence enhancement (quantum yields greater than 0.4) when bound to the target. Each probe specifically targets either carbohydrates, proteins, nucleic acids or membrane lipids, the four classes of macromolecules found in terrestrial life. The intent of the probes is to interrogate the environment for surface and endolithic life forms.

Weinstein, S.↗

Nanoscale Bioengineering Solutions for Space Exploration the Nanopore Sequencer

Characterization of biological systems at the molecular level and extraction of essential information for nano-engineering design to guide the nano-fabrication of solid-state sensors and molecular identification devices is a computational challenge. The alpha hemolysin protein ion channel is used as a model system for structural analysis of nucleic acids like DNA. Applied voltage draws a DNA strand and surrounding ionic solution through the biological nanopore. The subunits in the DNA strand block ion flow by differing amounts. Atomistic scale simulations are employed using NASA supercomputers to study DNA translocation. with the aim to enhance single DNA subunit identification. Compared to protein channels, solid-state nanopores offer a better temporal control of the translocation of DNA and the possibility to easily tune its chemistry to increase the signal resolution. Potential applications for NASA missions, besides real-time genome sequencing include astronaut health, life detection and decoding of various genomes. http://phenomrph.arc.nasa.gov/index.php

Ioana, Cozmuta↗

Simulation experiments of the effect of space environment on bacteriophage and DNA thin films

The main goal of PUR experiment (phage and uracil response) is to examine and quantify the effect of specific space conditions on nucleic acid models. To achieve this an improved method was elaborated for the preparation of DNA and bacteriophage thin films. The homogeneity of the films was controlled by UV spectroscopy and microscopy. To provide experimental evidence for the hypothesis that interplanetary transfer of the genetic material is possible, phage T7 and isolated T7 DNA thin films have been exposed to selected space conditions: intense UVC radiation (lambda=254 nm) and high vacuum (10(-4) Pa). The effects of DNA hydration, conformation and packing on UV radiation damage were examined. Characteristic changes in the absorption spectrum, in the electrophoretic pattern of DNA and the decrease of the amount of PCR products have been detected indicating the photodamage of isolated and intraphage DNA. c2004 COSPAR. Published by Elsevier Ltd. All rights reserved.

Bacteriophage T7/genetics/radiation effects↗

Nanophase Iron Oxides as an Ultraviolet Sunscreen for Ancient Photosynthetic Microbes: A Possible Link Between Early Organisms, Banded-Iron Formations, and the Oxygenation of the Atmosphere

We propose that nanophase iron oxide-bearing materials provided important niches for ancient photosynthetic microbes on the early Earth that ultimately led to the oxygenation of the Earth s atmosphere and the formation of iron oxide deposits. Atmospheric oxygen and ozone attenuate UV radiation on the Earth today providing substantial protection for photosynthetic organisms. With ultraviolet radiation fluxes likely to have been even higher on the early Earth than today, accessing solar radiation was particularly risky for early organisms. Yet, we know that photosynthesis arose then and played a critical role in subsequent evolution. Of primary importance was protection at approx.250-290 nm, where peak nucleic acid (approx.260 nm) and protein (approx.280 nm) absorptions occur. Nanophase ferric oxide/oxyhydroxide minerals absorb, and thus block, the lethal UV radiation, while transmitting light through much of the visible and near-infrared regions of interest to photosynthesis (400 to 1100 nm). Further, they were available in early environments, and are synthesized by many organisms. Based on ferric oxide/oxyhydroxide spectral properties, likely geologic processes, and the results of experiments with the photosynthetic organisms, Euglena sp. and Chlumydomonus reinhardtii, we propose a scenario where photosynthesis, and ultimately the oxygenation of the atmosphere, depended on the protection of early microbes by nanophase ferric oxides/oxyhydroxides. The results of this study are also applicable to other potentially habitable iron-bearing planetary bodies because of the evolutionary pressure to utilize solar radiation when available as an energy source.

Bishop, Janice L.↗

Nucleozymes

Nucleozymes containing ribonucleotides and deoxyribonucleotides or nucleic acid analogues are described herein. The nucleozymes have catalytic activity and are significantly more resistant to degradation than their all-RNA ribozyme counterparts. Also described are methods for preparing the nucleozymes along with methods of using nucleozymes, e.g., as therapeutic agents.

Usman, Nassim↗