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At least 199 records · Page 11

Tissue Culture in Microgravity

Attempts to simulate normal tissue micro-environments in vitro have been thwarted by the complexity and plasticity of the extracellular matrix, which is important in regulating cytoskeletal and nuclear matrix proteins. Gravity is one of the problems, tending to separate components that should be kept together. For space shuttle experiments, NASA engineers devised a double-walled rotating bioreactor, which is proving to be a useful tissue culture device on earth as well as in space.

Pellis, Neal R.↗

Plasma membrane disruption: repair, prevention, adaptation

Many metazoan cells inhabit mechanically stressful environments and, consequently, their plasma membranes are frequently disrupted. Survival requires that the cell rapidly repair or reseal the disruption. Rapid resealing is an active and complex structural modification that employs endomembrane as its primary building block, and cytoskeletal and membrane fusion proteins as its catalysts. Endomembrane is delivered to the damaged plasma membrane through exocytosis, a ubiquitous Ca2+-triggered response to disruption. Tissue and cell level architecture prevent disruptions from occurring, either by shielding cells from damaging levels of force, or, when this is not possible, by promoting safe force transmission through the plasma membrane via protein-based cables and linkages. Prevention of disruption also can be a dynamic cell or tissue level adaptation triggered when a damaging level of mechanical stress is imposed. Disease results from failure of either the preventive or resealing mechanisms.

NASA Discipline Cell Biology↗

Models of the Protocellular Structures, Functions and Evolution

In the absence of extinct or extant record of protocells, the most direct way to test our understanding of the origin of cellular life is to construct laboratory models that capture important features of protocellular systems. Such efforts are currently underway in a collaborative project between NASA-Ames, Harvard medical School and University of California. They are accompanied by computational studies aimed at explaining self-organization of simple molecules into ordered structures. The centerpiece of this project is a method for the in vitro evolution of protein enzymes toward arbitrary catalytic targets. A similar approach has already been developed for nucleic acids: First, a very large population of candidate molecules is generated using a random synthetic approach. Next, the small numbers of molecules that can accomplish the desired task are selected. These molecules are next vastly multiplied using the polymerase chain reaction. A mutagenic approach, in which the sequences of selected molecules are randomly altered, can yield further improvements in performance or alterations of specificities. Unfortunately, the catalytic potential of nucleic acids is rather limited. Proteins are more catalytically capable but cannot be directly amplified. In the new technique, this problem is circumvented by covalently linking each protein of the initial, diverse, pool to the RNA sequence that codes for it. Then, selection is performed on the proteins, but the nucleic acids are replicated. To date, we have obtained "a proof of concept" by evolving simple, novel proteins capable of selectively binding adenosine tri-phosphate (ATP). Our next goal is to create an enzyme that can phosphorylate amino acids and another to catalyze the formation of peptide bonds in the absence of nucleic acid templates. This latter reaction does not take place in contemporary cells. once developed, these enzymes will be encapsulated in liposomes so that they will function in a simulated cellular environment. To provide a continuous energy supply, usually needed to activate the substrates, an energy transduction complex which generates ATP from adenosine diphosphate, inorganic phosphate and light will be used. This system, consisting of two modern proteins, ATP synthase and bacteriorhodopsin, has already been built and shown to work efficiently. By coupling chemical synthesis to such a system, it will be possible to drive chemical reactions by light if only the substrates for these reactions are supplied.

Pohorille, Andrew↗

Combinatorial Multidomain Mesoporous Chips and a Method for Fractionation, Stabilization, and Storage of Biomolecules

A new fractionation device shows desirable features for exploratory screening and biomarker discovery. The constituent MSCs may be tailored for desired pore sizes and surface properties and for the sequestration and enrichment of extremely low abundant protein and peptides in desired ranges of the mass/charge spectrum. The MSCs are effective in yielding reproducible extracts from complex biological samples as small as 10 microliter in a time as short as 30 minutes. They are inexpensive to manufacture, and allow for scaled up production to attain the simultaneous processing of a large number of samples. The MSCs are multiplexed, label-free diagnostic tools with the potential of biological recognition moiety modification for enhanced specificity. The MSCs may store, protect and stabilize biological fluids, enabling the simplified and cost-effective collection and transportation of clinical samples. The MSC-based device may serve as a diagnostic tool to complement histopathology, imaging, and other conventional clinical techniques. The MSCs mediated identification of disease-specific protein signatures may help in the selection of personalized therapeutic combinations, in the real-time assessment of therapeutic efficacy and toxicity, and in the rational modulation of therapy based on the changes in the protein networks associated with the prognosis and the drug resistance of the disease.

Ferrari, Mauro↗

Preliminary Design of a Downstream Processing System for Protein Production in Space

Biomanufacturing is a promising technology to convert in situ resources into essential products including enzymes, therapeutics, biopolymers and other chemicals required to support deep-space missions that may not be easily supplied or produced by alternative means. In addition to the biomass production operations, vital down-stream steps including biomass harvesting/concentration, cell lysis, protein capture and purification are needed to produce an application-ready product. Commercially available terrestrial processes commonly require complex, heavy equipment and highly trained operators, which are not practical in deep space environments. In this work, we aim to identify approaches required to produce an intracellular, His-tagged recombinant enzyme using E. coli at 1 L production scales within the constraints of a deep-space mission as a model use-case scenario. Based on extensive literature review and commercially available products, we identified candidate technologies and products that could be integrated for deep space biomanufacturing. Different preliminary designs were then compared in terms of total system impacts on up-mass, processing time, and consumables required. Our analysis indicated that a biomass concentrator would significantly reduce the processing time and consumables required for the overall system without a large increase in the total mass. We also identified viable technologies for other steps such as cell lysis and protein purification. Predictions from our trade study will be validated in the laboratory by testing the most promising products with the results used to optimize the design. This research will help transfer technology that is well developed on Earth to a space-ready format to produce biological products from a wide variety of microorganisms that can support deep-space missions.

Biomanufacturing↗

Bio-Organic Nanotechnology: Using Proteins and Synthetic Polymers for Nanoscale Devices

While the ability of proteins to self-assemble makes them powerful tools in nanotechnology, in biological systems protein-based structures ultimately depend on the context in which they form. We combine the self-assembling properties of synthetic diblock copolymers and proteins to construct intricately ordered, three-dimensional polymer protein structures with the ultimate goal of forming nano-scale devices. This hybrid approach takes advantage of the capabilities of organic polymer chemistry to build ordered structures and the capabilities of genetic engineering to create proteins that are selective for inorganic or organic substrates. Here, microphase-separated block copolymers coupled with genetically engineered heat shock proteins are used to produce nano-scale patterning that maximizes the potential for both increased structural complexity and integrity.

Molnar, Linda K.↗

The Importance of Sample Return in Establishing Chemical Evidence for Life on Mars or Other Solar System Bodies

The search for evidence of life on Mars and elsewhere will continue to be one of the primary goals of NASA s robotic exploration program over the next decade. NASA and ESA are currently planning a series of robotic missions to Mars with the goal of understanding its climate, resources, and potential for harboring past or present life. One key goal will be the search for chemical biomarkers including complex organic compounds important in life on Earth. These include amino acids, the monomer building blocks of proteins and enzymes, nucleobases and sugars which form the backbone of DNA and RNA, and lipids, the structural components of cell membranes. Many of these organic compounds can also be formed abiotically as demonstrated by their prevalence in carbonaceous meteorites [1], though, their molecular characteristics may distinguish a biological source [2]. It is possible that in situ instruments may reveal such characteristics, however, return of the right sample (i.e. one with biosignatures or having a high probability of biosignatures) to Earth would allow for more intensive laboratory studies using a broad array of powerful instrumentation for bulk characterization, molecular detection, isotopic and enantiomeric compositions, and spatially resolved chemistry that may be required for confirmation of extant or extinct Martian life. Here we will discuss the current analytical capabilities and strategies for the detection of organics on the Mars Science Laboratory (MSL) using the Sample Analysis at Mars (SAM) instrument suite and how sample return missions from Mars and other targets of astrobiological interest will help advance our understanding of chemical biosignatures in the solar system.

Glavin, D. P.↗

A microarray immunoassay for simultaneous detection of proteins and bacteria

We report the development and characterization of an antibody microarray biosensor for the rapid detection of both protein and bacterial analytes under flow conditions. Using a noncontact microarray printer, biotinylated capture antibodies were immobilized at discrete locations on the surface of an avidin-coated glass microscope slide. Preservation of capture antibody function during the deposition process was accomplished with the use of a low-salt buffer containing sucrose and bovine serum albumin. The slide was fitted with a six-channel flow module that conducted analyte-containing solutions over the array of capture antibody microspots. Detection of bound analyte was subsequently achieved using fluorescent tracer antibodies. The pattern of fluorescent complexes was interrogated using a scanning confocal microscope equipped with a 635-nm laser. This microarray system was employed to detect protein and bacterial analytes both individually and in samples containing mixtures of analytes. Assays were completed in 15 min, and detection of cholera toxin, staphylococcal enterotoxin B, ricin, and Bacillus globigii was demonstrated at levels as low as 8 ng/mL, 4 ng/mL, 10 ng/mL, and 6.2 x 10(4) cfu/mL, respectively. The assays presented here are very fast, as compared to previously published methods for measuring antibody-antigen interactions using microarrays (minutes versus hours).

Non-NASA Center↗

Strategies for Distinguishing Abiotic Chemistry from Martian Biochemistry in Samples Returned from Mars

A key goal in the search for evidence of extinct or extant life on Mars will be the identification of chemical biosignatures including complex organic molecules common to all life on Earth. These include amino acids, the monomer building blocks of proteins and enzymes, and nucleobases, which serve as the structural basis of information storage in DNA and RNA. However, many of these organic compounds can also be formed abiotically as demonstrated by their prevalence in carbonaceous meteorites [1]. Therefore, an important challenge in the search for evidence of life on Mars will be distinguishing between abiotic chemistry of either meteoritic or martian origin from any chemical biosignatures from an extinct or extant martian biota. Although current robotic missions to Mars, including the 2011 Mars Science Laboratory (MSL) and the planned 2018 ExoMars rovers, will have the analytical capability needed to identify these key classes of organic molecules if present [2,3], return of a diverse suite of martian samples to Earth would allow for much more intensive laboratory studies using a broad array of extraction protocols and state-of-theart analytical techniques for bulk and spatially resolved characterization, molecular detection, and isotopic and enantiomeric compositions that may be required for unambiguous confirmation of martian life. Here we will describe current state-of-the-art laboratory analytical techniques that have been used to characterize the abundance and distribution of amino acids and nucleobases in meteorites, Apollo samples, and comet- exposed materials returned by the Stardust mission with an emphasis on their molecular characteristics that can be used to distinguish abiotic chemistry from biochemistry as we know it. The study of organic compounds in carbonaceous meteorites is highly relevant to Mars sample return analysis, since exogenous organic matter should have accumulated in the martian regolith over the last several billion years and the analytical techniques previously developed for the study of extraterrestrial materials can be applied to martian samples.

Glavin, D. P.↗

Prochloron research

The purpose was to prepare Prochloron photosynthetic membranes for the isolation of the two major chlorophyll-proteins, the P700-chlorophyll a-protein and the light-harvesting chlorophyll a/b-protein, using SDS-polyacrylamide gel electrophoresis. The prepared proteins (purified) were examined for their cross-reactivity to polyclonal antibodies prepared from higher plant proteins. In addition, material was prepared for electron microscopy, and isolation of the DNA for determination of its general complexity (COT analysis) and similarity to barley chloroplast DNA and Anabaena DNA by using restriction-endonuclease analysis. Kleinschmidt spreads of the DNA were in the electron microscope to identify and measure the extent and size of the circlar DNA.

Lewin, R. A.↗

Prochloron expedition

The purpose was to prepare Prochloron phototsynthetic membranes for the isolation of the two major chlorophyll-proteins, the p700-chlorophyll a protein and the light harvesting chlorophyll a/b-protein, using SDS-polyacrylamide get electrophoreses. The prepared proteins (purified) were examined for their cross-reactivity to polyclonal antibodies prepared from higher plant proteins. In addition, material was prepared for electron microscopy, and isolation of the DNA for determination of its general complexity (COT analysis) and similarity to barley chloroplast DNA and Anabaena DNA by using restriction-endonuclease analysis Kleinschmidt spreads of the DNA were in the electron microscope to identify and measure the extent and size of the circular DNA.

Alberte, R. S.↗

Possible prebiotic significance of polyamines in the condensation, protection, encapsulation, and biological properties of DNA

Some properties of DNA condensed with spermidine have been compared with the properties of DNA condensed with Co3+(NH3)6 to determine whether condensation of DNA with these trivalent cations protects DNA against the action of DNase I and increases transcription and encapsulation of DNA into liposomes. It was shown that DNA condensed with Co3+(NH3)6 was resistant to the action of the endonuclease DNase I such as DNA condensed with spermidine was. However, DNA condensed with Co3+(NH3)6 was significantly less active in transcription with the E. coli RNA polymerase than DNA-spermidine condensed forms. In addition, it was demonstrated that both compacted forms of DNA were more efficiently encapsulated into neutral liposomes; however, negatively, charged liposomes were scarcely formed in the presence of DNA condensed with Co3+(NH3)6. These experiments and the well documented properties of polyamines increasing the resistance to radiations and hydrolysis of nucleic acids, as well as their biological activities, such as replication, transcription, and translation, together with the low concentration of Co3+ in the environment, lead us to propose spermidine as a plausible prebiotic DNA condensing agent rather than Co3+ and the basic proteins proposed by other authors. Then, we consider the possible role and relevance of the polyamine-nucleic acids complexes in the evolution of life.

NASA Discipline Exobiology↗

Coprecipitation of thermal lysine-rich proteinoids with polyribonucleotides

An experimental study was conducted to determine whether the precipitation of thermal proteinoids with homopolynucleotides can serve as a tool for studying the specificities between proteins and polynucleotides. Attention is given to exploring the best means of quantitation of the precipitate and the effect of varying the lysine content and the amount of Mg(2+) on the results. The formation of microparticles was monitored both by turbidity and by the mass of precipitate formed. Only under certain conditions was the turbidity a reliable indication of the amount of precipitate. Increasing concentration of Mg(2+) tended to displace proteinoid from the complex with polynucleotide. The results indicate that the interaction of thermal proteinoids with polynucleotides appears to be a suitable tool for studying specificities of interactions between proteins and nucleic acids.

Lacey, J. C., Jr.↗

Protein structure, electron transfer and evolution of prokaryotic photosynthetic reaction centers

Photosynthetic reaction centers from a variety of organisms have been isolated and characterized. The groups of prokaryotic photosynthetic organisms include the purple bacteria, the filamentous green bacteria, the green sulfur bacteria and the heliobacteria as anoxygenic representatives as well as the cyanobacteria and prochlorophytes as oxygenic representatives. This review focuses on structural and functional comparisons of the various groups of photosynthetic reaction centers and considers possible evolutionary scenarios to explain the diversity of existing photosynthetic organisms.

NASA Discipline Exobiology↗

XCTK1: A Xenopus C-terminal Kinesin-like Protein

XCTK1 is 97kDa kinesin-like protein homologous to FKIF2 and KIFC3. XCTK1 is present at picomolar levels in eggs, embryos and cultured cells in a soluble high-molecular weight complex that is not associated with membranes. XCKT1 localizes to centrosomes in Xenopus A6 cells. Anti-XCTK1 antibodies also localize to spindle poles when injected into A6 cells or when added to extracts during in vitro spindle assembly reactions. XCTK1 is associated with the center of taxol-induced microtubule asters in extracts. Therefore its localization to poles is dependent on microtubule minus-ends and not on centrosomes per se. Overexpression of XCTK1 leads to centrosome destruction in cultured cells. XCTK1 was tagged at either the N- or C-terminus and transfected into Xenopus A6 cells At low expression levels, XCTK1 associated with centrosomes. At higher levels, the protein localized to insoluble cytoplasmic structures. Gamma-tubulin staining was dramatically decreased from centrosomes or altogether absent. The centrosomal SPJ antigen colocalized with XCTK1-containing structures. Upon nocodozole treatment, microtubules failed to regrow from the centrosomes indicating that overexpression of XCTK1 severely compromises centrosomal function. Current studies are aimed at determining whether XCTK1 interacts directly with centrosomal proteins and to determine the effects of XCTK1 depletion on oocyte maturation and embryogenesis.

Winfree, Seth↗

The Origins of Transmembrane Ion Channels

Even though membrane proteins that mediate transport of ions and small molecules across cell walls are among the largest and least understood biopolymers in contemporary cells, it is still possible to shed light on their origins and early evolution. The central observation is that transmembrane portions of most ion channels are simply bundles of -helices. By combining results of experimental and computer simulation studies on synthetic models and natural channels, mostly of non-genomic origin, we show that the emergence of -helical channels was protobiologically plausible, and did not require highly specific amino acid sequences. Despite their simple structure, such channels could possess properties that, at the first sight, appear to require markedly larger complexity. Specifically, we explain how the antiamoebin channels, which are made of identical helices, 16 amino acids in length, achieve efficiency comparable to that of highly evolved channels. We further show that antiamoebin channels are extremely flexible, compared to modern, genetically coded channels. On the basis of our results, we propose that channels evolved further towards high structural complexity because they needed to acquire stable rigid structures and mechanisms for precise regulation rather than improve efficiency. In general, even though architectures of membrane proteins are not nearly as diverse as those of water-soluble proteins, they are sufficiently flexible to adapt readily to the functional demands arising during evolution.

Pohorille, Andrew↗

Structures of the Nitrogenase Complex Prepared Under Catalytic Turnover Conditions

The enzyme nitrogenase couples adenosine triphosphate (ATP) hydrolysis to the multielectron reduction of atmospheric dinitrogen into ammonia. Despite extensive research, the mechanistic details of ATP-dependent energy transduction and dinitrogen reduction by nitrogenase are not well understood, requiring new strategies to monitor its structural dynamics during catalytic action. Here, we report cryo–electron microscopy structures of the nitrogenase complex prepared under enzymatic turnover conditions. We observe that asymmetry governs all aspects of the nitrogenase mechanism, including ATP hydrolysis, protein-protein interactions, and catalysis. Conformational changes near the catalytic iron-molybdenum cofactor are correlated with the nucleotide-hydrolysis state of the enzyme.

Hannah L. Rutledge↗