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At least 307 records · Page 17

Three stages during the evolution of the genetic code

A diversification of the genetic code based on the number of codons available for the proteinous amino acids is established. Three groups of amino acids during evolution of the code are distinguished. On the basis of their chemical complexity and a small codon number those amino acids emerging later in a translation process are derived. Both criteria indicate that His, Phe, Tyr, Cys and either Lys or Asn were introduced in the second stage, whereas the number of codons alone gives evidence that Trp and Met were introduced in the third stage. The amino acids of stage one use purines rich codons, thus purines have been retained in their third codon position. All the amino acids introduced in the second stage, in contrast, use pyrimidines in this codon position. A low abundance of pyrimidines during early translation is derived. This assumption is supported by experiments on non enzymatic replication and interactions of DNA hairpin loops with a complementary strand. A back extrapolation concludes a high purine content of the first nucleic acids which gradually decreased during their evolution. Amino acids independently available form prebiotic synthesis were thus correlated to purine rich codons. Conclusions on prebiotic replication are discussed also in the light of recent codon usage data.

Baumann, U.↗

Monoclonal Antibody Testing for Cancer Metastasis

Malignant cells are characterized by the ability to invade surrounding normal tissues. Tumor invasion is abetted by proteolytic enzymes that have been correlated with recurrent disease and metastasis. These enzymes are involved in a cascade of proteolytic interactions with other enzymes and inhibitors which allow cancer cells to dissolve surrounding extracellular matrix, thereby enabling the cells to rapidly invade adjacent tissues and migrate to metastatic sites distant from the primary tumor. Among these proteases are the plasminogen activators (PA), collagenase IV, faminase, and in some cases cathepsin D, which together mediate key steps in the invasion process of metastasis. Cells which have the selective advantage for invasion and metastasis are those capable of regulating their proteolytic activity and proliferation. Cells in the process of invasion would be probably down-regulated for proliferation, but subsequent to attachment and adhesion at a distant site, would then be in a proliferative mode, up-regulating DNA replication. Urokinase (uPA) can be present in the tissues in several molecular forms. The inactive proenzyme is a single chain protein (scuPA) that is cleaved at Lys. 158 to form the double chain, high molecular weight active form (HMW-uPA) of 54 kD. A low molecular weight form (LMW-uPA) can also be produced by cleavage of the HMW-U PA at Lys. 135 - Lys. 136 giving a 35 kD active enzyme. Recently, it has been shown that the HMW active form of urokinase, bound to the tumor cell membrane, is responsible for the local lysis of the extracellular matrix, hence the tissue invasion mechanism for metastasis (Andreasen et al, 19861. Receptor- (membrane) bound uPA is twice as efficient (catalytically) as free fluid-phase uPA. Tho unbound uPA and the LMW form is not responsible for most of the local dissolution of extracellular matrix in the immediate vicinity of the metastatic tumor cell. High levels of urokinase (greater than 3.49 ng/mg of total protein) extracted from breast tumor tissues have recently been shown, together with plasminogen activator inhibitor 1 (PAI-1), to be a good prognostic indicator for high risk of recurrence and shorter patient survival times. In this project, we have attempted to develop immunocytochemical methodologies for the clinical assessment of the expression of urokinase plasminogen activator, which has been implicated to be important for initial steps in tumor invasion, and to relate it to cell proliferation and DNA replication at the single-cell level.

Source record↗

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↗

Nonlinear Response of Layer Growth Dynamics in the Mixed Kinetics-Bulk-Transport Regime

In situ high-resolution interferometry on horizontal facets of the protein lysozyme reveal that the local growth rate R, vicinal slope p, and tangential (step) velocity v fluctuate by up to 80% of their average values. The time scale of these fluctuations, which occur under steady bulk transport conditions through the formation and decay of step bunches (macrosteps), is of the order of 10 min. The fluctuation amplitude of R increases with growth rate (supersaturation) and crystal size, while the amplitude of the v and p fluctuations changes relatively little. Based on a stability analysis for equidistant step trains in the mixed transport-interface-kinetics regime, we argue that the fluctuations originate from the coupling of bulk transport with nonlinear interface kinetics. Furthermore, step bunches moving across the interface in the direction of or opposite to the buoyancy-driven convective flow increase or decrease in height, respectively. This is in agreement with analytical treatments of the interaction of moving steps with solution flow. Major excursions in growth rate are associated with the formation of lattice defects (striations). We show that, in general, the system-dependent kinetic Peclet number, Pe(sub k) , i.e., the relative weight of bulk transport and interface kinetics in the control of the growth process, governs the step bunching dynamics. Since Pe(sub k) can be modified by either forced solution flow or suppression of buoyancy-driven convection under reduced gravity, this model provides a rationale for the choice of specific transport conditions to minimize the formation of compositional inhomogeneities under steady bulk nutrient crystallization conditions.

Vekilov, Peter G.↗

DNA Recombinase Proteins, their Function and Structure in the Active Form, a Computational Study

Homologous recombination is a crucial sequence of reactions in all cells for the repair of double strand DNA (dsDNA) breaks. While it was traditionally considered as a means for generating genetic diversity, it is now known to be essential for restart of collapsed replication forks that have met a lesion on the DNA template (Cox et al., 2000). The central stage of this process requires the presence of the DNA recombinase protein, RecA in bacteria, RadA in archaea, or Rad51 in eukaryotes, which leads to an ATP-mediated DNA strand-exchange process. Despite many years of intense study, some aspects of the biochemical mechanism, and structure of the active form of recombinase proteins are not well understood. Our theoretical study is an attempt to shed light on the main structural and mechanistic issues encountered on the RecA of the e-coli, the RecA of the extremely radio resistant Deinococcus Radiodurans (promoting an inverse DNA strand-exchange repair), and the homolog human Rad51. The conformational changes are analyzed for the naked enzymes, and when they are linked to ATP and ADP. The average structures are determined over 2ns time scale of Langevian dynamics using a collision frequency of 1.0 ps(sup -1). The systems are inserted in an octahedron periodic box with a 10 Angstrom buffer of water molecules explicitly described by the TIP3P model. The corresponding binding free energies are calculated in an implicit solvent using the Poisson-Boltzmann solvent accessible surface area, MM-PBSA model. The role of the ATP is not only in stabilizing the interaction RecA-DNA, but its hydrolysis is required to allow the DNA strand-exchange to proceed. Furthermore, we extended our study, using the hybrid QM/MM method, on the mechanism of this chemical process. All the calculations were performed using the commercial code Amber 9.

Carra, Claudio↗

Conservation of Endo16 expression in sea urchins despite evolutionary divergence in both cis and trans-acting components of transcriptional regulation

Evolutionary changes in transcriptional regulation undoubtedly play an important role in creating morphological diversity. However, there is little information about the evolutionary dynamics of cis-regulatory sequences. This study examines the functional consequence of evolutionary changes in the Endo16 promoter of sea urchins. The Endo16 gene encodes a large extracellular protein that is expressed in the endoderm and may play a role in cell adhesion. Its promoter has been characterized in exceptional detail in the purple sea urchin, Strongylocentrotus purpuratus. We have characterized the structure and function of the Endo16 promoter from a second sea urchin species, Lytechinus variegatus. The Endo16 promoter sequences have evolved in a strongly mosaic manner since these species diverged approximately 35 million years ago: the most proximal region (module A) is conserved, but the remaining modules (B-G) are unalignable. Despite extensive divergence in promoter sequences, the pattern of Endo16 transcription is largely conserved during embryonic and larval development. Transient expression assays demonstrate that 2.2 kb of upstream sequence in either species is sufficient to drive GFP reporter expression that correctly mimics this pattern of Endo16 transcription. Reciprocal cross-species transient expression assays imply that changes have also evolved in the set of transcription factors that interact with the Endo16 promoter. Taken together, these results suggest that stabilizing selection on the transcriptional output may have operated to maintain a similar pattern of Endo16 expression in S. purpuratus and L. variegatus, despite dramatic divergence in promoter sequence and mechanisms of transcriptional regulation.

NASA Discipline Evolutionary Biology↗

Long-term effects of acute irradiation and isolation on crew age-matched mice

The future of space missions beyond low-Earth orbit presents exciting opportunities but may come with formidable health challenges. Astronauts face a unique combination of space stressors capable of exerting effects on the central nervous system (CNS) with possible sex-dependent differences. The interaction of these combined spaceflight stressors may induce oxidative stress, thereby altering brain integrity and behavioral performance. Understanding these changes is critical for safeguarding astronaut health and enabling successful exploration. By understanding the physiological responses to the combined effects of ionizing radiation (IR) and social isolation in the brain through immunohistochemistry (IHC), proteomics and cytokine expression analyses, we aim to identify CNS pathways ripe for countermeasure development to mitigate adverse brain changes with future deep space exploration. Here, we study the combined effects of simulated 5-ion galactic cosmic radiation (GCRsim) at acute dosage (5, 15, and 50 cGy) and social isolation on crew age-matched male and female mice at 124 days post-irradiation. We conducted analyses of hippocampal cytokine biomarkers and proteomic profiling on hippocampal lysates. Our findings reveal sex-specific regulation of hippocampal cytokines and proteomic profiles. Ongoing immunohistochemistry findings on the hippocampus will provide qualitative information regarding key protein expression and cellular changes such as microglial activation, blood brain barrier integrity, and neuroinflammation. With future plans for long-duration space missions, such as Mars exploration, understanding CNS changes over extended periods in mice can provide insights into potential long-term health impacts on astronauts. This project will provide us with valuable insights into sex-differences in neurobiological processes and disease mechanisms impacting men and women in space.

space radiation↗

Interaction of Vascular Smooth Muscle Cells Under Low Shear Stress

The blood vessel wall consists of three cellular layers, an outer adventitial, a middle medial and an inner intimal layer. When the blood vessel forms in the embryo it begins as a tube composed of a single cell type called endothelial cells. Over time, other cells are recruited from the surrounding tissue to form additional layers on the outer surface of the endothelial tube. The cells that are recruited are called mesenchymal cells. Mesenchymal cells are responsible for the production of connective tissue that holds the blood vessel together and for developing into vascular smooth muscle cells that are responsible for regulating the diameter of the vessel (1) and therefore, blood flow. In a fully developed blood vessel, the endothelial cells make- up the majority of cells in the intimal layer while the mesenchymal cells make-up the majority of cells in the medial and adventitial layers. Within the medial layer of a mature vessel, cells are organized into multiple circular layers of alternating bands of connective tissue and cells. The cell layer is composed of a mixture of mesenchymal cells that have not developed into smooth muscle cells and fully developed smooth muscle cells (2). The assembly and organization of complex tissues is directed in part by a signaling system composed of proteins on the cell surface called adhesion molecules. Adhesion molecules enable cells to recognize each other as well as the composition of the connective tissue in which they reside (3). It was hypothesized that the different cell types that compose the vascular wall possess different adhesion molecules that enable them to recognize each other and through this recognition system, form the complex layered organization of the vascular wall. In other words, the layered organization is an intrinsic property of the cells. If this hypothesis is correct then the different cells that make up the vessel wall, when mixed together, should organize themselves into a layered structure resembling an intact blood vessel. Experiments described below were designed to test this hypothesis.

Seidel, Charles L.↗

Positive selection moments identify potential functional residues in human olfactory receptors

Correlated mutation analysis and molecular models of olfactory receptors have provided evidence that residues in the transmembrane domains form a binding pocket for odor ligands. As an independent test of these results, we have calculated positive selection moments for the alpha-helical sixth transmembrane domain (TM6) of human olfactory receptors. The moments can be used to identify residues that have been preferentially affected by positive selection and are thus likely to interact with odor ligands. The results suggest that residue 622, which is commonly a serine or threonine, could form critical H-bonds. In some receptors a dual-serine subsite, formed by residues 622 and 625, could bind hydroxyl determinants on odor ligands. The potential importance of these residues is further supported by site-directed mutagenesis in the beta-adrenergic receptor. The findings should be of practical value for future physiological studies, binding assays, and site-directed mutagenesis.

Non-NASA Center↗

Microscopy Methods for Life Detection on Ocean Worlds

On Earth, light microscopy is commonly used in microbiology to identify organisms and observe their interactions with the environment; this makes it an attractive technique for in situ life detection methods on ocean worlds. As a standalone technique, brightfield microscopy, while able to provide important contextual information, has limited usefulness as a life detection technique because it is often challenging to differentiate between abiotic and biotic particles based solely on their size and shape, which may introduce risks of false positive or false negative interpretations. However, these risks can be reduced by combining brightfield microscopy with fluorescence microscopy to provide a method that correlate sample chemistry with sample morphology. In this work, we have used the Luminescence Imager for Exploration (LIfE), a brightfield and epifluorescence microscope with an integrated sample processing system (matured under the Concepts for Ocean worlds Life Detection Technology and Instrument Concepts of Europa Exploration programs) to develop methods that increase the fidelity of in situ microscopy life detection measurements through two main approaches. First, native fluorescence is excited in molecules that contain aromatic moieties such as proteins (using deep UV excitation), and energy carrying molecules and endogenous chromophores (using visible-light excitation), to correlate the location of these species with cell-like structural features (brightfield imaging). Second, fluorescent stains are used to selectively image cells and cell fragments by targeting proteins, lipids, and nucleic acids. We discuss the results of tests, obtained using ocean world analog samples, that have examined trades associated with implementing these methods autonomously in planetary missions, including the intrinsic properties of candidate fluorescence dyes and long-term storage and radiation stability.

Pavel E. Z. Klier↗

Microscopy Methods for Life Detection on Ocean Worlds

On Earth, light microscopy is commonly used in microbiology to identify organisms and observe their interactions with the environment; this makes it an attractive technique for in situ life detection methods on ocean worlds. As a standalone technique, brightfield microscopy, while able to provide important contextual information, has limited usefulness as a life detection technique because it is often challenging to differentiate between abiotic and biotic particles based solely on their size and shape, which may introduce risks of false positive or false negative interpretations. However, these risks can be reduced by combining brightfield microscopy with fluorescence microscopy to provide a method that correlate sample chemistry with sample morphology. In this work, we have used the Luminescence Imager for Exploration (LIfE), a brightfield and epifluorescence microscope with an integrated sample processing system (matured under the Concepts for Ocean worlds Life Detection Technology and Instrument Concepts of Europa Exploration programs) to develop methods that increase the fidelity of in situ microscopy life detection measurements through two main approaches. First, native fluorescence is excited in molecules that contain aromatic moieties such as proteins (using deep UV excitation), and energy carrying molecules and endogenous chromophores (using visible-light excitation), to correlate the location of these species with cell-like structural features (brightfield imaging). Second, fluorescent stains are used to selectively image cells and cell fragments by targeting proteins, lipids, and nucleic acids. We discuss the results of tests, obtained using ocean world analog samples, that have examined trades associated with implementing these methods autonomously in planetary missions, including the intrinsic properties of candidate fluorescence dyes and long-term storage and radiation stability.

Pavel E Z Klier↗

Glycogen synthase kinase 3 phosphorylates kinesin light chains and negatively regulates kinesin-based motility

Membrane-bounded organelles (MBOs) are delivered to different domains in neurons by fast axonal transport. The importance of kinesin for fast antero grade transport is well established, but mechanisms for regulating kinesin-based motility are largely unknown. In this report, we provide biochemical and in vivo evidence that kinesin light chains (KLCs) interact with and are in vivo substrates for glycogen synthase kinase 3 (GSK3). Active GSK3 inhibited anterograde, but not retrograde, transport in squid axoplasm and reduced the amount of kinesin bound to MBOs. Kinesin microtubule binding and microtubule-stimulated ATPase activities were unaffected by GSK3 phosphorylation of KLCs. Active GSK3 was also localized preferentially to regions known to be sites of membrane delivery. These data suggest that GSK3 can regulate fast anterograde axonal transport and targeting of cargos to specific subcellular domains in neurons.

NASA Discipline Neuroscience↗

Conservation of Fold and Topology of Functional Elements in Thiamin Pyrophosphate Enzymes

Thiamin pyrophosphate (TPP)-dependent enzymes are a highly divergent family of proteins binding both TPP and metal ions. They perform decarboxylation-hydroxyaldehydes. Prior -ketoacids and of a common - (O=)C-C(OH)- fragment of to knowledge of three-dimensional structures of these enzmes, the GDGY25-30NN sequence was used to identify these enzymes. Subsequently, a number of structural studies on those enzymes revealed multi-subunit organization and the features of the two duplicate cofactor binding sites. Analyzing the structures of 44 structurally known enzymes, we found that the common structure of these enzymes is reduced to 180-220 amino acid long fragments of two PP and two PYR domains that form the [PP:PYR]2 binding center of two cofactor molecules. The structures of PP and PYR are arranged in a similar fold-sheet with triplets of helices on both sides.Dconsisting of a six-stranded Residues surrounding the cofactors are not strictly conserved, but they provide the same interatomic contacts required for the catalytic functions that these enzymes perform while maintaining interactive structural integrity. These structural and functional amino acids are topological counterparts located in the same positions of the conserved fold of sets of PP and PYR domains. Additional parallels include short fragments of sequences that link these amino acids to the fold and function. This report on the structural commonalities amongst TPP dependent enzymes is thought to contribute new approaches to annotation that may assist in advancing the functional proteomics of TPP dependent enzymes, and trace their complexity within evolutionary context.

Dominiak, P.↗

The role of pyridoxine as a countermeasure for in-flight loss of lean body mass

Ground based and in flight research has shown that humans, under conditions of microgravity, sustain a loss of lean body tissue (protein) and changes in several biological processes including, reductions in red blood cell mass, and neurotransmitters. The maintenance of muscle mass, the major component of lean body mass, is required to meet the needs of space station EVAs. Central to the biosynthesis of amino acids, the building blocks of protein, is pyridoxine (vitamin B-6). Muscle mass integrity requires the availability of vitamin B-6 for protein metabolism and neurotransmitter synthesis. Furthermore, the formation of red blood cells require pyridoxine as a cofactor in the biosynthesis of hemoglobin, a protein that carries oxygen to tissues. In its active form, pyridoxal-5'-phosphate (PLP), vitamin B-6 serves as a link between amino acid and carbohydrate metabolism through intermediates of glycolysis and the tricarboxylic acid cycle. In addition to its role in energy metabolism, PLP is involved in the biosynthesis of hemoglobin and neurotransmitter which are necessary for neurological functions. Alterations in pyridoxine metabolism may affect countermeasures designed to overcome some of these biochemical changes. The focus of this research is to determine the effects of microgravity on the metabolic utilization of vitamin B-6, integrating nutrition as an integral component of the countermeasure (exercise) to maintain lean body mass and muscle strength. The objectives are: 1) to determine whether microgravity effects the metabolic utilization of pyridoxine and 2) to quantitate changes in B-6 vitamer distribution in tissue and excreta relative to loss of lean body tissue. The rationale for this study encompasses the unique challenge to control biochemical mechanisms effected during space travel and the significance of pyridoxine to maintain and counter muscle integrity for EVA activities. This experiment will begin to elucidate the importance of biochemical interactions between micronutrients and the homeostasis condition of biological processes in the space environment. To address this research topic a simulated microgravity model has been developed. The experiment uses radioisotopically labelled pyridoxine administered as an oral dose to rats which are maintained by tail suspension to simulate a microgravity environment. At the termination of the study, liver, muscle, blood and urine are collected and analyzed by reverse phase high pressure liquid chromatography to determine the quantity and distribution of the B-6 vitamers in tissue and excreta relative to lean body tissue loss. Earlier studies, published by this investigator, have shown that differences in vitamer distribution among samples from experimental versus control subjects indicate changes in metabolic utilization and storage of vitamin B-6.

Gilbert, Joyce A.↗

Mutational analysis of photosystem I polypeptides in the cyanobacterium Synechocystis sp. PCC 6803. Targeted inactivation of psaI reveals the function of psaI in the structural organization of psaL

We cloned, characterized, and inactivated the psaI gene encoding a 4-kDa hydrophobic subunit of photosystem I from the cyanobacterium Synechocystis sp. PCC 6803. The psaI gene is located 90 base pairs downstream from psaL, and is transcribed on 0.94- and 0.32-kilobase transcripts. To identify the function of PsaI, we generated a cyanobacterial strain in which psaI has been interrupted by a gene for chloramphenicol resistance. The wild-type and the mutant cells showed comparable rates of photoautotrophic growth at 25 degrees C. However, the mutant cells grew slower and contained less chlorophyll than the wild-type cells, when grown at 40 degrees C. The PsaI-less membranes from cells grown at either temperature showed a small decrease in NADP+ photoreduction rate when compared to the wild-type membranes. Inactivation of psaI led to an 80% decrease in the PsaL level in the photosynthetic membranes and to a complete loss of PsaL in the purified photosystem I preparations, but had little effect on the accumulation of other photosystem I subunits. Upon solubilization with nonionic detergents, photosystem I trimers could be obtained from the wild-type, but not from the PsaI-less membranes. The PsaI-less photosystem I monomers did not contain detectable levels of PsaL. Therefore, a structural interaction between PsaL and PsaI may stabilize the association of PsaL with the photosystem I core. PsaL in the wild-type and PsaI-less membranes showed equal resistance to removal by chaotropic agents. However, PsaL in the PsaI-less strain exhibited an increased susceptibility to proteolysis. From these data, we conclude that PsaI has a crucial role in aiding normal structural organization of PsaL within the photosystem I complex and the absence of PsaI alters PsaL organization, leading to a small, but physiologically significant, defect in photosystem I function.

NASA Discipline Cell Biology↗

Complex high affinity interactions occur between MHCI and superantigens

Staphylococcal enterotoxins A and C1 (SEA or SEC1) bound to major histocompatibility-I (MHCI) molecules with high affinity (binding constants ranging from 1.1 microM to 79 nM). SEA and SEC1 directly bound MHCI molecules that had been captured by monoclonal antibodies specific for H-2Kk, H-2Dk, or both. In addition, MHCI-specific antibodies inhibited the binding of SEC1 to LM929 cells and SEA competitively inhibited SEC1 binding; indicating that the superantigens bound to MHCI on the cell surface. The affinity and number of superantigen binding sites differed depending on whether MHCI was expressed in the membrane of LM929 cells or whether it was captured. These data support the hypothesis that MHCI molecules can serve as superantigen receptors.

NASA Discipline Cell Biology↗

Fluid Physics and Macromolecular Crystal Growth in Microgravity

The molecular structure of biological macromolecules is important in understanding how these molecules work and has direct application to rational drug design for new medicines and for the improvement and development of industrial enzymes. In order to obtain the molecular structure, large, well formed, single macromolecule crystals are required. The growth of macromolecule crystals is a difficult task and is often hampered on the ground by fluid flows that result from the interaction of gravity with the crystal growth process. One such effect is the bulk movement of the crystal through the fluid due to sedimentation. A second is buoyancy driven convection close to the crystal surface. On the ground the crystallization process itself induces both of these flows. Buoyancy driven convection results from density differences between the bulk solution and fluid close to the crystal surface which has been depleted of macromolecules due to crystal growth. Schlieren photograph of a growing lysozyme crystal illustrating a 'growth plume' resulting from buoyancy driven convection. Both sedimentation and buoyancy driven convection have a negative effect on crystal growth and microgravity is seen as a way to both greatly reduce sedimentation and provide greater stability for 'depletion zones' around growing crystals. Some current crystal growth hardware however such as those based on a vapor diffusion techniques, may also be introducing unwanted Marangoni convection which becomes more pronounced in microgravity. Negative effects of g-jitter on crystal growth have also been observed. To study the magnitude of fluid flows around growing crystals we have attached a number of different fluorescent probes to lysozyme molecules. At low concentrations, less than 40% of the total protein, the probes do not appear to effect the crystal growth process. By using these probes we expect to determine not only the effect of induced flows due to crystal growth hardware design but also hope to optimize crystallization hardware so that destructive flows are minimized both on the ground and in microgravity.

Pusey, M.↗

Modeled Microgravity-Induced Protein Kinase C Isoform Expression in Human Lymphocytes

In long-term space travel, the crew is exposed to microgravity and radiation that invoke potential hazards to the immune system. T cell activation is a critical step in the immune response. Receptor-mediated signaling is inhibited both in microgravity and modeled microgravity (MMG) as reflected in diminished DNA synthess in peripheral blood lymphocytes and their locomotion through gelled type 1 collagen. Direct activation of Protein Kinase C (PKC) bypassing cell surface events using the phorbol ester PMA rescues MMG-inhibited lymphocyte activation and locomotion, whereas calcium ionophore ionomycin had no rescue effect. Thus calcium-independent PKC isoforms may be affected in MMG-induced locomotion inhibition and rescue. Both calcium-dependent isoforms and calcium-independent PKC isoforms were investigated to assess their expression in lymphocytes in 19 and MMG-culture. Human lymphocytes were cultured and harvested at 24, 48, 72 and 96 hours and serial samples assessed for locomotion using type I collagen and expression of PKC isoforms. Expression of PKC-alpha, -delta and -epsilon was assessed by RT-PCR, flow cytometry and immunoblotting. Results indicated that PKC isoforms delta and epsilon were down-regulated by more than 50% at the transcriptional and translational levels in MMG-cultured lymphocytes compared with 19 controls. Events upstream of PKC such as phosphorylation of Phospholipase C(gamma) (PLC-gamma) in MMG, revealed accumulation of inactive enzyme. Depressed Ca++ -independent PKC isoforms may be a consequence of an upstream lesion in the signal transduction pathway. The differential response among calcium-dependent and calcium-independent isoforms may actually result from MMG intrusion events earlier than, but after ligand-receptor interaction. Keywords: Signal transduction, locomotion, immunity

Sundaresan, A.↗