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At least 91 records · Page 5

Sequence data - Magnitude and implications of some ambiguities.

A stochastic model is applied to the divergence of the horse-pig lineage from a common ansestor in terms of the alpha and beta chains of hemoglobin and fibrinopeptides. The results are compared with those based on the minimum mutation distance model of Fitch (1972). Buckwheat and cauliflower cytochrome c sequences are analyzed to demonstrate their ambiguities. A comparative analysis of evolutionary rates for various proteins of horses and pigs shows that errors of considerable magnitude are introduced by Glx and Asx ambiguities into evolutionary conclusions drawn from sequences of incompletely analyzed proteins.

Holmquist, R.↗

Narcosis studies and oxygen poisoning of mice

The research for a mechanism by which narcotic gases alter metabolism is reported. Possible sites of action by narcotic and anesthetic gases in isolated electron transport particles were explored. Using the relative activities of the NADH-oxygen, NADH-ferricyanide, succinate-cytochrome C and succinate-NAD oxidoreductase systems as parameters, the relative potency of volatile anesthetics were tested. Testing the relative ability of human subjects to contract and repay an oxygen debt while in the narcotic versus alert state, it was found that narcosis induced by 33% nitrous oxide increased the size of the oxygen debt contracted and the amount of oxygen required to repay it during recovery. Mice acclimatized to sea level (760 mm Hg), 5000 feet (632 mm Hg) or 15,000 feet 437 mm Hg) for from one to eight weeks were found to be more susceptible to convulsion and death as a function of altitude acclimatization when tested in hyperoxic environments. There were no reasonable explanations for the connection between hypoxia and oxygen poisoning but several practical implications for persons living at altitude are discussed.

Source record↗

Comments on papers by Reichert and Wong

Papers of Reichert and Wong (1971) and Reichert (1972) are referred to as erroneous in stating that in their work with cytochrome c sequences they demonstrated that vertebrates have achieved a higher organized genetic message with a higher fidelity in the DNA-to-protein information processing channel. Arguments are given to contest that statement.

Gatlin, L. L.↗

Inferences from protein and nucleic acid sequences - Early molecular evolution, divergence of kingdoms and rates of change

Description of new sensitive, objective methods for establishing the probable common ancestry of very distantly related sequences and the quantitative evolutionary change which has taken place. These methods are applied to four families of proteins and nucleic acids and evolutionary trees will be derived where possible. Of the three families containing duplications of genetic material, two are nucleic acids: transfer RNA and 5S ribosomal RNA. Both of these structures are functional in the synthesis of coded proteins, and prototypes must have been present in the cell at the inception of the fundamental coding process that all living things share. There are many types of tRNA which recognize the various nucleotide triplets and the 20 amino acids. These types are thought to have arisen as a result of many gene duplications. Relationships among these types are discussed. The 5S ribosomal RNA, presently functional in both eukaryotes and prokaryotes, is very likely descended from an early form incorporating almost a complete duplication of genetic material. The amount of evolution in the various lines can again be compared. The other two families containing duplications are proteins; ferredoxin and cytochrome c.

Dayhoff, M. O.↗

Research and development of a luminol-carbon monoxide flow system

Adaption of the luminol-carbon monoxide injection system to a flowing type system is reported. Analysis of actual wastewater samples was carried out and revealed that bacteria can be associated with particles greater than 10 microns in size in samples such as mixed liquor. Research into the luminol reactive oxidation state indicates that oxidized iron porphyrins, cytochrome-c in particular, produce more luminol chemiluminescence than the reduced form. Correlation exists between the extent of porphyrin oxidation and relative chemiluminescence. In addition, the porphyrin nucleus is apparently destroyed under the current chemiluminescent reaction conditions.

Thomas, R. R.↗

A measure of the denseness of a phylogenetic network

An objective measure of phylogenetic denseness is developed to examine various phylogenetic criteria: alpha- and beta-hemoglobin, myoglobin, cytochrome c, and the parvalbumin family. Attention is given to the number of nucleotide replacements separating homologous sequences, and to the topology of the network (in other words, to the qualitative nature of the network as defined by how closely the studied species are related). Applications include quantitative comparisons of species origin, relation, and rates of evolution.

Holmquist, R.↗

Gravity as a biochemical determinant

Hypogravity effects on the biochemistry of living organisms are surveyed, and the differences found under orbital, clinostat, and flotation conditions are examined. Changes can be transient - such as altered serum hormone and electrolyte levels in man - and disappear shortly after return to earth, or changes can be more persistent or permanent, as seems to be the case for changes in the skeletal system mineral and protein constituents for humans who have been in space. While plant ethylene and peroxidase both increase under orbital, clinostat, or flotation conditions, 3-PGA-dehydrogenase increases under orbital but not clinostat conditions and cytochrome C reductase and malic dehydrogenase are affected by clinostat but not actual free fall conditions. Reasons for these discrepancies are considered.

Siegel, S. M.↗

The REH theory of protein and nucleic acid divergence - A retrospective update

The random evolutionary hits (REH) theory of evolutionary divergence, originally proposed in 1972, is restated with attention to certain aspects of the theory that have caused confusion. The theory assumes that natural selection and stochastic processes interact and that natural selection restricts those codon sites which may fix mutations. The predicted total number of fixed nucleotide replacements agrees with data for cytochrome c, a-hemoglobin, beta-hemoglobin, and myoglobin. The restatement analyzes the magnitude of possible sources of errors and simplifies calculational methodology by supplying polynomial expressions to replace tables and graphs.

Holmquist, R.↗

Theoretical foundations for quantitative paleogenetics. III - The molecular divergence of nucleic acids and proteins for the case of genetic events of unequal probability

Theoretical equations are derived for molecular divergence with respect to gene and protein structure in the presence of genetic events with unequal probabilities: amino acid and base compositions, the frequencies of nucleotide replacements, the usage of degenerate codons, the distribution of fixed base replacements within codons and the distribution of fixed base replacements among codons. Results are presented in the form of tables relating the probabilities of given numbers of codon base changes with respect to the original codon for the alpha hemoglobin, beta hemoglobin, myoglobin, cytochrome c and parvalbumin group gene families. Application of the calculations to the rabbit alpha and beta hemoglobin mRNAs and proteins indicates that the genes are separated by about 425 fixed based replacements distributed over 114 codon sites, which is a factor of two greater than previous estimates. The theoretical results also suggest that many more base replacements are required to effect a given gene or protein structural change than previously believed.

Holmquist, R.↗

The spatial distribution of fixed mutations within genes coding for proteins

An examination has been conducted of the extensive amino acid sequence data now available for five protein families - the alpha crystallin A chain, myoglobin, alpha and beta hemoglobin, and the cytochromes c - with the goal of estimating the true spatial distribution of base substitutions within genes that code for proteins. In every case the commonly used Poisson density failed to even approximate the experimental pattern of base substitution. For the 87 species of beta hemoglobin examined, for example, the probability that the observed results were from a Poisson process was the minuscule 10 to the -44th. Analogous results were obtained for the other functional families. All the data were reasonably, but not perfectly, described by the negative binomial density. In particular, most of the data were described by one of the very simple limiting forms of this density, the geometric density. The implications of this for evolutionary inference are discussed. It is evident that most estimates of total base substitutions between genes are badly in need of revision.

Holmquist, R.↗

The rRNA evolution and procaryotic phylogeny

Studies of ribosomal RNA primary structure allow reconstruction of phylogenetic trees for prokaryotic organisms. Such studies reveal major dichotomy among the bacteria that separates them into eubacteria and archaebacteria. Both groupings are further segmented into several major divisions. The results obtained from 5S rRNA sequences are essentially the same as those obtained with the 16S rRNA data. In the case of Gram negative bacteria the ribosomal RNA sequencing results can also be directly compared with hybridization studies and cytochrome c sequencing studies. There is again excellent agreement among the several methods. It seems likely then that the overall picture of microbial phylogeny that is emerging from the RNA sequence studies is a good approximation of the true history of these organisms. The RNA data allow examination of the evolutionary process in a semi-quantitative way. The secondary structures of these RNAs are largely established. As a result it is possible to recognize examples of local structural evolution. Evolutionary pathways accounting for these events can be proposed and their probability can be assessed.

Fox, G. E.↗

Synthesis and degradation of nitrate reductase during the cell cycle of Chlorella sorokiniana

Studies on the diurnal variations of nitrate reductase (NR) activity during the life cycle of synchronized Chlorella sorokiniana cells grown with a 7:5 light-dark cycle showed that the NADH:NR activity, as well as the NR partial activities NADH:cytochrome c reductase and reduced methyl viologen:NR, closely paralleled the appearance and disappearance of NR protein as shown by sodium dodecyl sulfate gel electrophoresis and immunoblots. Results of pulse-labeling experiments with [35S]methionine further confirmed that diurnal variations of the enzyme activities can be entirely accounted for by the concomitant synthesis and degradation of the NR protein.

Non-NASA Center↗

Mechanisms of cell signaling by nitric oxide and peroxynitrite: from mitochondria to MAP kinases

Many of the biological and pathological effects of nitric oxide (NO) are mediated through cell signaling pathways that are initiated by NO reacting with metalloproteins. More recently, it has been recognized that the reaction of NO with free radicals such as superoxide and the lipid peroxyl radical also has the potential to modulate redox signaling. Although it is clear that NO can exert both cytotoxic and cytoprotective actions, the focus of this overview are those reactions that could lead to protection of the cell against oxidative stress in the vasculature. This will include the induction of antioxidant defenses such as glutathione, activation of mitogen-activated protein kinases in response to blood flow, and modulation of mitochondrial function and its impact on apoptosis. Models are presented that show the increased synthesis of glutathione in response to shear stress and inhibition of cytochrome c release from mitochondria. It appears that in the vasculature NO-dependent signaling pathways are of three types: (i) those involving NO itself, leading to modulation of mitochondrial respiration and soluble guanylate cyclase; (ii) those that involve S-nitrosation, including inhibition of caspases; and (iii) autocrine signaling that involves the intracellular formation of peroxynitrite and the activation of the mitogen-activated protein kinases. Taken together, NO plays a major role in the modulation of redox cell signaling through a number of distinct pathways in a cellular setting.

Non-NASA Center↗

BIGEL analysis of gene expression in HL60 cells exposed to X rays or 60 Hz magnetic fields

We screened a panel of 1,920 randomly selected cDNAs to discover genes that are differentially expressed in HL60 cells exposed to 60 Hz magnetic fields (2 mT) or X rays (5 Gy) compared to unexposed cells. Identification of these clones was accomplished using our two-gel cDNA library screening method (BIGEL). Eighteen cDNAs differentially expressed in X-irradiated compared to control HL60 cells were recovered from a panel of 1,920 clones. Differential expression in experimental compared to control cells was confirmed independently by Northern blotting of paired total RNA samples hybridized to each of the 18 clone-specific cDNA probes. DNA sequencing revealed that 15 of the 18 cDNA clones produced matches with the database for genes related to cell growth, protein synthesis, energy metabolism, oxidative stress or apoptosis (including MYC, neuroleukin, copper zinc-dependent superoxide dismutase, TC4 RAS-like protein, peptide elongation factor 1alpha, BNIP3, GATA3, NF45, cytochrome c oxidase II and triosephosphate isomerase mRNAs). In contrast, BIGEL analysis of the same 1,920 cDNAs revealed no differences greater than 1.5-fold in expression levels in magnetic-field compared to sham-exposed cells. Magnetic-field-exposed and control samples were analyzed further for the presence of mRNA encoding X-ray-responsive genes by hybridization of the 18 specific cDNA probes to RNA from exposed and control HL60 cells. Our results suggest that differential gene expression is induced in approximately 1% of a random pool of cDNAs by ionizing radiation but not by 60 Hz magnetic fields under the present experimental conditions.

Non-NASA Center↗

Regulation and Maturation of the Shewanella oneidensis Sulfite Reductase SirA

Shewanella oneidensis, a metal reducer and facultative anaerobe, expresses a large number of c-type cytochromes, many of which function as anaerobic reductases. All of these proteins contain the typical heme-binding motif CXXCH and require the Ccm proteins for maturation. Two c-type cytochrome reductases also possess atypical heme-binding sites, the NrfA nitrite reductase (CXXCK) and the SirA sulfte reductase (CX12NKGCH). S. oneidensis MR-1 encodes two cytochrome c synthetases (CcmF and SirE) and two apocytochrome c chaperones (CcmI and SirG). SirE located in the sir gene cluster is required for the maturation of SirA, but not NrfA. Here we show that maturation of SirA requires the combined function of the two apocytochrome c chaperones CcmI and SirG. Loss of either protein resulted in decreased sulfte reductase. Furthermore, SirA was not detected in a mutant that lacked both chaperones, perhaps due to misfolding or instability. These results suggest that CcmI interacts with SirEFG during SirA maturation, and with CcmF during maturation of NrfA. Additionally, we show that CRP regulates expression of sirA via the newly identifed transcriptional regulatory protein, SirR.

59 BASIC BIOLOGICAL SCIENCES↗

Maturation of Rhodobacter capsulatus Multicopper Oxidase CutO Depends on the CopA Copper Efflux Pathway and Requires the cutF Product (Final Report)

The goal is to achieve a comprehensive understanding of the nature, mechanism of function and biogenesis of c-type cytochromes and their oligomeric complexes. These cytochrome are ubiquitous heme proteins that are required for cellular bioenergetics, including photosynthesis (Ps) and respiration (Res), which are central to chemical energy (ATP) production by oxidative- and photophosphorylation. We contribute to the molecular characterization of individual and membrane-integral cytochromes as parts of Ps and Res electron transport (ET) pathways, using the facultative phototrophic bacteria of as excellent models for multidisciplinary investigations. Supported by this grant we discovered novel membrane-bound cytochromes and cyts c complexes that are important for Ps- and Res-ET. We worked out their biogenesis including maturation (covalent heme addition) and assembly of mature subunits into complexes. These involve fundamental process found virtually in all organisms and are fundamental processes with major significance to the “Photosynthetic Systems” program of DOE-BES, because the cytochromes are integral components of natural energy production systems. Elucidation of basic principles, and the knowledge gained by their study greatly impact on our ability to design, engineer, and create new natural, bio-hybrid, bio-inspired and artificial systems to better capture light, transduce and store its energy with improved efficacy and are of broad significance.

14 SOLAR ENERGY↗

Evolutionary connections of biological kingdoms based on protein and nucleic acid sequence evidence

Prokaryotic and eukaryotic evolutionary trees are developed from protein and nucleic-acid sequences by the methods of numerical taxonomy. Trees are presented for bacterial ferredoxins, 5S ribosomal RNA, c-type cytochromes , cytochromes c2 and c', and 5.8S ribosomal RNA; the implications for early evolution are discussed; and a composite tree showing the branching of the anaerobes, aerobes, archaebacteria, and eukaryotes is shown. Single lines are found for all oxygen-evolving photosynthetic forms and for the salt-loving and high-temperature forms of archaebacteria. It is argued that the eukaryote mitochondria, chloroplasts, and cytoplasmic host material are descended from free-living prokaryotes that formed symbiotic associations, with more than one symbiotic event involved in the evolution of each organelle.

Dayhoff, M. O.↗

Mimicking Natural Photosynthesis: Designing Ultrafast Photosensitized Electron Transfer into Multiheme Cytochrome Protein Nanowires

Efficient nanomaterials for artificial photosynthesis require fast and robust unidirectional electron transfer (ET) from photosensitizers through charge-separation and accumulation units to redox-active catalytic sites. We explored the ultrafast time-scale limits of photo-induced charge transfer between a Ru(II)tris(bipyridine) derivative photosensitizer and PpcA, a 3-heme c-type cytochrome serving as a nanoscale biological wire. Four covalent attachment sites (K28C, K29C, K52C, and G53C) were engineered in PpcA enabling site-specific covalent labeling with expected donor-acceptor (DA) distances of 4–8 Å. X-ray scattering results demonstrated that mutations and chemical labeling did not disrupt the structure of the proteins. Time-resolved spectroscopy revealed three orders of magnitude difference in charge transfer rates for the systems with otherwise similar DA distances and the same number of covalent bonds separating donors and acceptors. All-atom molecular dynamics simulations provided additional insight into the structure-function requirements for ultrafast charge transfer and the requirement of van der Waals contact between aromatic atoms of photosensitizers and hemes in order to observe sub-nanosecond ET. This work demonstrates opportunities to utilize multi-heme c-cytochromes as frameworks for designing ultrafast light-driven ET into charge-accumulating biohybrid model systems, and ultimately for mimicking the photosynthetic paradigm of efficiently coupling ultrafast, light-driven electron transfer chemistry to multi-step catalysis within small, experimentally versatile photosynthetic biohybrid assemblies.

36 MATERIALS SCIENCE↗