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At least 613 records · Page 34
All sourcesNASA NTRSDOE OSTI
NASA NTRS2000

Rhodoferax antarcticus sp. nov., a moderately psychrophilic purple nonsulfur bacterium isolated from an Antarctic microbial mat

A new species of purple nonsulfur bacteria isolated from an Antarctic microbial mat is described. The organism, designated strain ANT.BR, was mildly psychrophilic, growing optimally at 15-18 degrees C with a growth temperature range of 0-25 degrees C. Cells of strain ANT.BR were highly motile curved rods and spirals, contained bacteriochlorophyll a, and showed a multicomponent in vivo absorption spectrum. A specific phylogenetic relationship was observed between strain ANT.BR and the purple bacterium Rhodoferax fermentans FR2T, and the two organisms shared several physiological and other phenotypic properties, with the notable exception of growth temperature optimum. Tests of genomic DNA hybridization, however, showed Rfx. fermentans FR2T and strain ANT.BR to be genetically distinct bacteria. Because of its unique set of properties, especially its requirement for low growth temperatures, we propose to recognize strain ANT.BR as a new species of the genus Rhodoferax, Rhodoferax antarcticus, named for its known habitat, the Antarctic.

NASA Discipline Exobiology↗
NASA NTRS2000

Quantitative assessment of Hox complex expression in the indirect development of the polychaete annelid Chaetopterus sp

A prediction from the set-aside theory of bilaterian origins is that pattern formation processes such as those controlled by the Hox cluster genes are required specifically for adult body plan formation. This prediction can be tested in animals that use maximal indirect development, in which the embryonic formation of the larva and the postembryonic formation of the adult body plan are temporally and spatially distinct. To this end, we quantitatively measured the amount of transcripts for five Hox genes in embryos of a lophotrochozoan, the polychaete annelid Chaetopterus sp. The polychaete Hox complex is shown not to be expressed during embryogenesis, but transcripts of all measured Hox complex genes are detected at significant levels during the initial stages of adult body plan formation. Temporal colinearity in the sequence of their activation is observed, so that activation follows the 3'-5' arrangement of the genes. Moreover, Hox gene expression is spatially localized to the region of teloblastic set-aside cells of the later-stage embryos. This study shows that an indirectly developing lophotrochozoan shares with an indirectly developing deuterostome, the sea urchin, a common mode of Hox complex utilization: construction of the larva, whether a trochophore or dipleurula, does not involve Hox cluster expression, but in both forms the complex is expressed in the set-aside cells from which the adult body plan derives.

Non-NASA Center↗
NASA NTRS2000

Role of hypoxia-inducible factor-1 in transcriptional activation of ceruloplasmin by iron deficiency

A role of the copper protein ceruloplasmin (Cp) in iron metabolism is suggested by its ferroxidase activity and by the tissue iron overload in hereditary Cp deficiency patients. In addition, plasma Cp increases markedly in several conditions of anemia, e.g. iron deficiency, hemorrhage, renal failure, sickle cell disease, pregnancy, and inflammation. However, little is known about the cellular and molecular mechanism(s) involved. We have reported that iron chelators increase Cp mRNA expression and protein synthesis in human hepatocarcinoma HepG2 cells. Furthermore, we have shown that the increase in Cp mRNA is due to increased rate of transcription. We here report the results of new studies designed to elucidate the molecular mechanism underlying transcriptional activation of Cp by iron deficiency. The 5'-flanking region of the Cp gene was cloned from a human genomic library. A 4774-base pair segment of the Cp promoter/enhancer driving a luciferase reporter was transfected into HepG2 or Hep3B cells. Iron deficiency or hypoxia increased luciferase activity by 5-10-fold compared with untreated cells. Examination of the sequence showed three pairs of consensus hypoxia-responsive elements (HREs). Deletion and mutation analysis showed that a single HRE was necessary and sufficient for gene activation. The involvement of hypoxia-inducible factor-1 (HIF-1) was shown by gel-shift and supershift experiments that showed HIF-1alpha and HIF-1beta binding to a radiolabeled oligonucleotide containing the Cp promoter HRE. Furthermore, iron deficiency (and hypoxia) did not activate Cp gene expression in Hepa c4 hepatoma cells deficient in HIF-1beta, as shown functionally by the inactivity of a transfected Cp promoter-luciferase construct and by the failure of HIF-1 to bind the Cp HRE in nuclear extracts from these cells. These results are consistent with in vivo findings that iron deficiency increases plasma Cp and provides a molecular mechanism that may help to understand these observations.

NASA Discipline Cardiopulmonary↗
NASA NTRS2000

Intramolecular activation of a Ca(2+)-dependent protein kinase is disrupted by insertions in the tether that connects the calmodulin-like domain to the kinase

Ca(2+)-dependent protein kinases (CDPK) have a calmodulin-like domain (CaM-LD) tethered to the C-terminal end of the kinase. Activation is proposed to involve intramolecular binding of the CaM-LD to a junction sequence that connects the CaM-LD to the kinase domain. Consistent with this model, a truncated CDPK (DeltaNC) in which the CaM-LD has been deleted can be activated in a bimolecular interaction with an isolated CaM-LD or calmodulin, similar to the activation of a calmodulin-dependent protein kinase (CaMK) by calmodulin. Here we provide genetic evidence that this bimolecular activation requires a nine-residue binding segment from F436 to I444 (numbers correspond to CPK-1 accession number L14771). Two mutations at either end of this core segment (F436/A and VI444/AA) severely disrupted bimolecular activation, whereas flanking mutations had only minor effects. Intramolecular activation of a full-length kinase was also disrupted by a VI444/AA mutation, but surprisingly not by a F436/A mutation (at the N-terminal end of the binding site). Interestingly, intramolecular but not bimolecular activation was disrupted by insertion mutations placed immediately downstream of I444. To show that mutant enzymes were not misfolded, latent kinase activity was stimulated through binding of an antijunction antibody. Results here support a model of intramolecular activation in which the tether (A445 to G455) that connects the CaM-LD to the kinase provides an important structural constraint and is not just a simple flexible connection.

NASA Discipline Plant Biology↗
NASA NTRS2000

Constructing high complexity synthetic libraries of long ORFs using in vitro selection

We present a method that can significantly increase the complexity of protein libraries used for in vitro or in vivo protein selection experiments. Protein libraries are often encoded by chemically synthesized DNA, in which part of the open reading frame is randomized. There are, however, major obstacles associated with the chemical synthesis of long open reading frames, especially those containing random segments. Insertions and deletions that occur during chemical synthesis cause frameshifts, and stop codons in the random region will cause premature termination. These problems can together greatly reduce the number of full-length synthetic genes in the library. We describe a strategy in which smaller segments of the synthetic open reading frame are selected in vitro using mRNA display for the absence of frameshifts and stop codons. These smaller segments are then ligated together to form combinatorial libraries of long uninterrupted open reading frames. This process can increase the number of full-length open reading frames in libraries by up to two orders of magnitude, resulting in protein libraries with complexities of greater than 10(13). We have used this methodology to generate three types of displayed protein library: a completely random sequence library, a library of concatemerized oligopeptide cassettes with a propensity for forming amphipathic alpha-helical or beta-strand structures, and a library based on one of the most common enzymatic scaffolds, the alpha/beta (TIM) barrel. Copyright 2000 Academic Press.

Non-NASA Center↗
NASA NTRS2000

Orthostatic intolerance and tachycardia associated with norepinephrine-transporter deficiency

BACKGROUND: Orthostatic intolerance is a syndrome characterized by lightheadedness, fatigue, altered mentation, and syncope and associated with postural tachycardia and plasma norepinephrine concentrations that are disproportionately high in relation to sympathetic outflow. We tested the hypothesis that impaired functioning of the norepinephrine transporter contributes to the pathophysiologic mechanism of orthostatic intolerance. METHODS: In a patient with orthostatic intolerance and her relatives, we measured postural blood pressure, heart rate, plasma catecholamines, and systemic norepinephrine spillover and clearance, and we sequenced the norepinephrine-transporter gene and evaluated its function. RESULTS: The patient had a high mean plasma norepinephrine concentration while standing, as compared with the mean (+/-SD) concentration in normal subjects (923 vs. 439+/-129 pg per milliliter [5.46 vs. 2.59+/-0.76 nmol per liter]), reduced systemic norepinephrine clearance (1.56 vs. 2.42+/-0.71 liters per minute), impairment in the increase in the plasma norepinephrine concentration after the administration of tyramine (12 vs. 56+/-63 pg per milliliter [0.07 vs. 0.33+/-0.37 pmol per liter]), and a disproportionate increase in the concentration of plasma norepinephrine relative to that of dihydroxyphenylglycol. Analysis of the norepinephrine-transporter gene revealed that the proband was heterozygous for a mutation in exon 9 (encoding a change from guanine to cytosine at position 237) that resulted in more than a 98 percent loss of function as compared with that of the wild-type gene. Impairment of synaptic norepinephrine clearance may result in a syndrome characterized by excessive sympathetic activation in response to physiologic stimuli. The mutant allele in the proband's family segregated with the postural heart rate and abnormal plasma catecholamine homeostasis. CONCLUSIONS: Genetic or acquired deficits in norepinephrine inactivation may underlie hyperadrenergic states that lead to orthostatic intolerance.

Non-NASA Center↗
NASA NTRS2000

New genes and new biological roles for expansins

Expansins are extracellular proteins that loosen plant cell walls in novel ways. They are thought to function in cell enlargement, pollen tube invasion of the stigma (in grasses), wall disassembly during fruit ripening, abscission and other cell separation events. Expansins are encoded by two multigene families and each gene is often expressed in highly specific locations and cell types. Structural analysis indicates that one expansin region resembles the catalytic domain of family-45 endoglucanases but glucanase activity has not been detected. The genome projects have revealed numerous expansin-related sequences but their putative wall-loosening functions remain to be assessed.

NASA Program Fundamental Space Biology↗
NASA NTRS1999

A molecular description of the evolution of resistance

BACKGROUND: In vitro evolution has been used to obtain nucleic acid molecules with interesting functional properties. The evolution process usually is carried out in a stepwise manner, involving successive rounds of selection, amplification and mutation. Recently, a continuous in vitro evolution system was devised for RNAs that catalyze the ligation of oligonucleotide substrates, allowing the evolution of catalytic function to be studied in real time. RESULTS: Continuous in vitro evolution of an RNA ligase ribozyme was carried out in the presence of a DNA enzyme that was capable of cleaving, and thereby inactivating, the ribozyme. The DNA concentration was increased steadily over 33.5 hours of evolution, reaching a final concentration that would have been sufficient to inactivate the starting population in one second. The evolved population of ribozymes developed resistance to the DNA enzyme, reducing their vulnerability to cleavage by 2000-fold but retaining their own catalytic function. Based on sequencing and kinetic analysis of the ribozymes, two mechanisms are proposed for this resistance. One involves three nucleotide substitutions, together with two compensatory mutations, that alter the site at which the DNA enzyme binds the ribozyme. The other involves enhancement of the ribozyme's ability to bind its own substrate in a way that protects it from cleavage by the DNA enzyme. CONCLUSIONS: The ability to direct the evolution of an enzyme's biochemical properties in response to the behavior of another macromolecule provides insight into the evolution of resistance and may be useful in developing enzymes with novel or enhanced function.

Non-NASA Center↗
NASA NTRS1999

Non-unity molecular heritability demonstrated by continuous evolution in vitro

INTRODUCTION: When catalytic RNA is evolved in vitro, the molecule's chemical reactivity is usually the desired selection target. Sometimes the phenotype of a particular RNA molecule cannot be unambiguously determined from its genotype, however. This can occur if a nucleotide sequence can adopt multiple folded states, an example of non-unity heritability (i.e. one genotype gives rise to more than one phenotype). In these cases, more rounds of selection are required to achieve a phenotypic shift. We tested the influence of non-unity heritability at the molecular level by selecting for variants of a ligase ribozyme via continuous evolution. RESULTS: During 20 bursts of continuous evolution of a 152-nucleotide ligase ribozyme in which the Mg2+ concentration was periodically lowered, a nine-error variant of the starting 'wild-type' molecule became dominant in the last eight bursts. This variant appears to be more active than the wild type. Kinetic analyses of the mutant suggest that it may not possess a higher first-order catalytic rate constant, however. Examination of the multiple RNA conformations present under the continuous evolution conditions suggests that the mutant is superior to the wild type because it is less likely to misfold into inactive conformers. CONCLUSIONS: The evolution of genotypes that are more likely to exhibit a particular phenotype is an epiphenomenon usually ascribed only to complex living systems. We show that this can occur at the molecular level, demonstrating that in vitro systems may have more life-like characteristics than previously thought, and providing additional support for an RNA world.

Non-NASA Center↗
NASA NTRS1999

Mutations in a new Arabidopsis cyclophilin disrupt its interaction with protein phosphatase 2A

The heterotrimeric protein phosphatase 2A (PP2A) is a component of multiple signaling pathways in eukaryotes. Disruption of PP2A activity in Arabidopsis is known to alter auxin transport and growth response pathways. We demonstrated that the regulatory subunit A of an Arabidopsis PP2A interacts with a novel cyclophilin, ROC7. The gene for this cyclophilin encodes a protein that contains a unique 30-amino acid extension at the N-terminus, which distinguishes the gene product from all previously identified Arabidopsis cyclophilins. Altered forms of ROC7 cyclophilin with mutations in the conserved DENFKL domain did not bind to PP2A. Unlike protein phosphatase 2B, PP2A activity in Arabidopsis extracts was not affected by the presence of the cyclophilin-binding molecule cyclosporin. The ROC7 transcript was expressed to high levels in all tissues tested. Expression of an ROC7 antisense transcript gave rise to increased root growth. These results indicate that cyclophilin may have a role in regulating PP2A activity, by a mechanism that differs from that employed for cyclophilin regulation of PP2B.

NASA Discipline Plant Biology↗
NASA NTRS1999

A complex ligase ribozyme evolved in vitro from a group I ribozyme domain

Like most proteins, complex RNA molecules often are modular objects made up of distinct structural and functional domains. The component domains of a protein can associate in alternative combinations to form molecules with different functions. These observations raise the possibility that complex RNAs also can be assembled from preexisting structural and functional domains. To test this hypothesis, an in vitro evolution procedure was used to isolate a previously undescribed class of complex ligase ribozymes, starting from a pool of 10(16) different RNA molecules that contained a constant region derived from a large structural domain that occurs within self-splicing group I ribozymes. Attached to this constant region were three hypervariable regions, totaling 85 nucleotides, that gave rise to the catalytic motif within the evolved catalysts. The ligase ribozymes catalyze formation of a 3',5'-phosphodiester linkage between adjacent template-bound oligonucleotides, one bearing a 3' hydroxyl and the other a 5' triphosphate. Ligation occurs in the context of a Watson-Crick duplex, with a catalytic rate of 0.26 min(-1) under optimal conditions. The constant region is essential for catalytic activity and appears to retain the tertiary structure of the group I ribozyme. This work demonstrates that complex RNA molecules, like their protein counterparts, can share common structural domains while exhibiting distinct catalytic functions.

NASA Discipline Exobiology↗
NASA NTRS1999

A role for cysteine 3635 of RYR1 in redox modulation and calmodulin binding

Oxidation of the skeletal muscle Ca(2+) release channel (RYR1) increases its activity, produces intersubunit disulfide bonds, and blocks its interaction with calmodulin. Conversely, bound calmodulin protects RYR1 from the effects of oxidants (Zhang, J.-Z., Wu, Y., Williams, B. Y., Rodney, G., Mandel, F., Strasburg, G. M., and Hamilton, S. L. (1999) Am. J. Physiol. 276, Cell Physiol. C46-C53). In addition, calmodulin protects RYR1 from trypsin cleavage at amino acids 3630 and 3637 (Moore, C. P., Rodney, G., Zhang, J.-Z., Santacruz-Toloza, L., Strasburg, G. M., and Hamilton, S. L. (1999) Biochemistry 38, 8532-8537). The sequence between these two tryptic sites is AVVACFR. Alkylation of RYR1 with N-ethylmaleimide (NEM) blocks both (35)S-apocalmodulin binding and oxidation-induced intersubunit cross-linking. In the current work, we demonstrate that both cysteines needed for the oxidation-induced intersubunit cross-link are protected from alkylation with N-ethylmaleimide by bound calmodulin. We also show, using N-terminal amino acid sequencing together with analysis of the distribution of [(3)H]NEM labeling with each sequencing cycle, that cysteine 3635 of RYR1 is rapidly labeled by NEM and that this labeling is blocked by bound calmodulin. We propose that cysteine 3635 is located at an intersubunit contact site that is close to or within a calmodulin binding site. These findings suggest that calmodulin and oxidation modulate RYR1 activity by regulating intersubunit interactions in a mutually exclusive manner and that these interactions involve cysteine 3635.

Non-NASA Center↗
NASA NTRS1999

Peptide-formation on cysteine-containing peptide scaffolds

Monomeric cysteine residues attached to cysteine-containing peptides by disulfide bonds can be activated by carbonyldiimidazole. If two monomeric cysteine residues, attached to a 'scaffold' peptide Gly-Cys-Glyn-Cys-Glu10, (n = 0, 1, 2, 3) are activated, they react to form the dipeptide Cys-Cys. in 25-65% yield. Similarly, the activation of a cysteine residue attached to the 'scaffold' peptide Gly-Cys-Gly-Glu10 in the presence of Arg5 leads to the formation of Cys-Arg5 in 50% yield. The significance of these results for prebiotic chemistry is discussed.

NASA Discipline Exobiology↗
NASA NTRS1999

Ionizing radiation-induced mutagenesis: radiation studies in Neurospora predictive for results in mammalian cells

Ionizing radiation was the first mutagen discovered and was used to develop the first mutagenicity assay. In the ensuing 70+ years, ionizing radiation became a fundamental tool in understanding mutagenesis and is still a subject of intensive research. Frederick de Serres et al. developed and used the Neurospora crassa ad-3 system initially to explore the mutagenic effects of ionizing radiation. Using this system, de Serres et al. demonstrated the dependence of the frequency and spectra of mutations induced by ionizing radiation on the dose, dose rate, radiation quality, repair capabilities of the cells, and the target gene employed. This work in Neurospora predicted the subsequent observations of the mutagenic effects of ionizing radiation in mammalian cells. Modeled originally on the mouse specific-locus system developed by William L. Russell, the N. crassa ad-3 system developed by de Serres has itself served as a model for interpreting the results in subsequent systems in mammalian cells. This review describes the primary findings on the nature of ionizing radiation-induced mutagenesis in the N. crassa ad-3 system and the parallel observations made years later in mammalian cells.

NASA Discipline Radiation Health↗
NASA NTRS1999

Cloning the promoter for transforming growth factor-beta type III receptor. Basal and conditional expression in fetal rat osteoblasts

Transforming growth factor-beta binds to three high affinity cell surface molecules that directly or indirectly regulate its biological effects. The type III receptor (TRIII) is a proteoglycan that lacks significant intracellular signaling or enzymatic motifs but may facilitate transforming growth factor-beta binding to other receptors, stabilize multimeric receptor complexes, or segregate growth factor from activating receptors. Because various agents or events that regulate osteoblast function rapidly modulate TRIII expression, we cloned the 5' region of the rat TRIII gene to assess possible control elements. DNA fragments from this region directed high reporter gene expression in osteoblasts. Sequencing showed no consensus TATA or CCAAT boxes, whereas several nuclear factors binding sequences within the 3' region of the promoter co-mapped with multiple transcription initiation sites, DNase I footprints, gel mobility shift analysis, or loss of activity by deletion or mutation. An upstream enhancer was evident 5' proximal to nucleotide -979, and a silencer region occurred between nucleotides -2014 and -2194. Glucocorticoid sensitivity mapped between nucleotides -687 and -253, whereas bone morphogenetic protein 2 sensitivity co-mapped within the silencer region. Thus, the TRIII promoter contains cooperative basal elements and dispersed growth factor- and hormone-sensitive regulatory regions that can control TRIII expression by osteoblasts.

Non-NASA Center↗
NASA NTRS1999

Rapid and systemic accumulation of chloroplast mRNA-binding protein transcripts after flame stimulus in tomato

It has been shown that tomato (Lycopersicon esculentum) plants respond to flame wounding and electrical stimulation by a rapid (15 min) and systemic up-regulation of proteinase inhibitor (pin) genes. To find other genes having a similar expression pattern, we used subtractive cDNA screening between flamed and control plants to select clones up-regulated by flame wounding. We report the characterization of one of them, a chloroplast mRNA-binding protein encoded by a single gene and expressed preferentially in the leaves. Systemic gene expression in response to flaming in the youngest terminal leaf exhibited three distinct phases: a rapid and transient increase (5-15 min) in transcript accumulation, a decline to basal levels (15-45 min), and then a second, more prolonged increase (60-90 min). In contrast, after a mechanical wound the rapid, transient increase (5 min) was followed by a rapid decline to basal levels but no later, prolonged accumulation. In the petiole, the initial flame-wound-evoked transient increase (15 min) was followed by a continuous decline for 3 h. The nature of the wound signal(s) causing such rapid changes in transcript abundance is discussed in relation to electrical signaling, which has recently been implicated in plant responses to wounding.

NASA Discipline Plant Biology↗
NASA NTRS1999

Developmental regulation of the gene for chimeric calcium/calmodulin-dependent protein kinase in anthers

Chimeric Ca(2+)/calmodulin-dependent protein kinase (CCaMK) was cloned from developing anthers of lily (Lilium longiflorum Thumb. cv. Nellie White) and tobacco (Nicotiana tabacum L. cv. Xanthi). Previous biochemical characterization and structure/function studies had revealed that CCaMK has dual modes of regulation by Ca(2+) and Ca(2+)/calmodulin. The unique structural features of CCaMK include a catalytic domain, a calmodulin-binding domain, and a neural visinin-like Ca(2+)-binding domain. The existence of these three features in a single polypeptide distinguishes it from other kinases. Western analysis revealed that CCaMK is expressed in a stage-specific manner in developing anthers. Expression of CCaMK was first detected in pollen mother cells and continued to increase, reaching a peak around the tetrad stage of meiosis. Following microsporogenesis, CCaMK expression rapidly decreased and at later stages of microspore development, no expression was detected. A tobacco genomic clone of CCaMK was isolated and transgenic tobacco plants were produced carrying the CCaMK promoter fused to the beta-glucuronidase reporter gene. Both CCaMK mRNA and protein were detected in the pollen sac and their localizations were restricted to the pollen mother cells and tapetal cells. Consistent results showing a stage-specific expression pattern were obtained by beta-glucuronidase analysis, in-situ hybridization and immunolocalization. The stage- and tissue-specific appearance of CCaMK in anthers suggests that it could play a role in sensing transient changes in free Ca(2+) concentration in target cells, thereby controlling developmental events in the anther.

NASA Discipline Plant Biology↗
NASA NTRS1999

Haloanaerobium kushneri sp. nov., an obligately halophilic, anaerobic bacterium from an oil brine

Three strains, designated VS-751T, VS-511 and VS-732, of a strictly anaerobic, moderately halophilic, Gram-negative, rod-shaped bacterium were isolated from a highly saline (15-20%) brine from an oil reservoir in central Oklahoma, USA. The optimal concentration of NaCl for growth of these three strains was 2 M (12%), and the strains also grew in the presence of an additional 1 M MgCl2. The strains were mesophilic and grew at a pH range of 6-8. Carbohydrates used by all three strains included glucose, fructose, arabinose, galactose, maltose, mannose, cellobiose, sucrose and inulin. Glucose fermentation products included ethanol, acetate, H2 and CO2, with formate produced by two of the three strains. Differences were noted among strains in the optimal temperature and pH for growth, the maximum and minimum NaCl concentration that supported growth, substrate utilization and cellular fatty acid composition. Despite the phenotypic differences among the three strains, analysis of the 16S rRNA gene sequences and DNA-DNA hybridizations showed that these three strains were members of the same genospecies which belonged to the genus Haloanaerobium. The phenotypic and genotypic characteristics of strains VS-751T, VS-511 and VS-732 are different from those of previously described species of Haloanaerobium. It is proposed that strain VS-751T (ATCC 700103T) be established as the type strain of a new species, Haloanaerobium kushneri.

NASA Discipline Exobiology↗