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PubMed2002

The out of phase 1 mutant defines a role for PHYB in circadian phase control in Arabidopsis.

Arabidopsis displays circadian rhythms in stomatal aperture, stomatal conductance, and CO(2) assimilation, each of which peaks around the middle of the day. The rhythmic opening and closing of stomata confers a rhythm in sensitivity and resistance, respectively, to the toxic gas sulfur dioxide. Using this physiological assay as a basis for a mutant screen, we isolated mutants with defects in circadian timing. Here, we characterize one mutant, out of phase 1 (oop1), with the circadian phenotype of altered phase. That is, the timing of the peak (acrophase) of multiple circadian rhythms (leaf movement, CO(2) assimilation, and LIGHT-HARVESTING CHLOROPHYLL a/b-BINDING PROTEIN transcription) is early with respect to wild type, although all circadian rhythms retain normal period length. This is the first such mutant to be characterized in Arabidopsis. oop1 also displays a strong photoperception defect in red light characteristic of phytochrome B (phyB) mutants. The oop1 mutation is a nonsense mutation of PHYB that results in a truncated protein of 904 amino acids. The defect in circadian phasing is seen in seedlings entrained by a light-dark cycle but not in seedlings entrained by a temperature cycle. Thus, PHYB contributes light information critical for proper determination of circadian phase.

Adaptation, Physiological↗
PubMed2003

Accurate triplet phase determination in non-perfect crystals--a general phasing procedure.

A completely different approach to the problem of physically measuring the invariant triplet phases by three-beam X-ray diffraction is proposed. Instead of simulating the three-beam diffraction process to reproduce the experimental intensity profiles, the proposed approach makes use of a general parametric equation for fitting the profiles and extracting the triplet phase values. The inherent flexibility of the parametric equation allows its applicability to be extended to non-perfect crystals. Exploitation of the natural linear polarization of synchrotron radiation is essential for eliminating systematic errors and to provide accurate triplet phase values. Phasing procedures are suggested and demonstrative examples from simulated data are given.

Journal Article↗
PubMed2006

Structure model for the tau(mu) phase in Al-Cr-Si alloys deduced from the lambda phase by the strong-reflections approach.

There are very obvious common features in the electron diffraction patterns of the lambda and tau(mu) phases in the Al-Cr-Si system. The positions of the strong reflections and their intensity distributions are similar for the two structures. The relation of the reciprocal lattices of the lambda and tau(mu) phases is studied. By applying the strong-reflections approach, the structure factors of tau(mu) are deduced from the corresponding structure factors of the known lambda phase. Rules for selecting reflections for the strong-reflections approach are described. Similar to that of lambda, the structure of tau(mu) contains six layers stacked along the c axis in each unit cell. There are 752 atoms in each unit cell, 53 of them are unique. The corresponding composition of the tau(mu) model is Al(3.82 - x)CrSi(x). Simulated electron diffraction patterns from the structure model are in good agreement with the experimental ones. The arrangement of interpenetrated icosahedral clusters in the tau(mu) phase is discussed.

Journal Article↗
PubMed2004

Radiation-damage-induced phasing with anomalous scattering: substructure solution and phasing.

Substructure-solution and phasing procedures using a combination of anomalous scattering and radiation-damage-induced isomorphous differences have been investigated. The tyrosine residues in thaumatin were iodinated with N-iodosuccinimide in the crystalline form as well as prior to crystallization. Several data sets were collected from both forms and used for substructure solution and phasing using various protocols, employing anomalous, isomorphous or both these signals. It was shown that combination of the anomalous and isomorphous signals in the form of the RIPAS (radiation-damage-induced phasing with anomalous scattering) strategy is beneficial for both locating the substructure and subsequent phasing.

Crystallography, X-Ray↗
PubMed1994

On the application of phase relationships to complex structures. XXXIV. VFOM - a new figure of merit for protein phase sets at moderate resolution.

In recent years it has been shown that direct methods are capable of solving the structures of small proteins. Mukherjee & Woolfson [Acta Cryst. (1993), D49, 9-12] have shown that useful phase sets can be produced even at 3 A resolution but that the standard figures of merit could not distinguish the better phase sets from others. They found modified forms of the standard figures of merit that could pick out better phase sets for 2 A resolution or higher. Gilmore, Henderson & Bricogne [Acta Cryst. (1991), A47, 842-846] have shown that evaluation of the log-likelihood gain, coming from entropy-maximization procedures, is also very successful in picking out good protein phases sets. A new figure of merit is described, based on the expected charactistics of an electron-density map for a protein, and comparisons are made with the other figures of merit mentioned above.

Journal Article↗
PubMed1996

Direct-space methods in phase extension and phase refinement. IV. The double-histogram method.

In the conventional histogram-matching technique for phase extension and refinement for proteins a simple one-to-one transformation is made in the protein region to modify calculated density so that it will have some target histogram in addition to solvent flattening. This work describes an investigation where the density modification takes into account not only the current calculated density at a grid point but also some characteristic of the environment of the grid point within some distance R. This characteristic can be one of the local maximum density, the local minimum density or the local variance of density. The grid points are divided into ten groups, each containing the same number of grid points, for ten different ranges of value of the local characteristic. The ten groups are modified to give different histograms, each corresponding to that obtained under the same circumstances from a structure similar to the one under investigation. This process is referred to as the double-histogram matching method. Other processes which have been investigated are the weighting of structure factors when calculating maps with estimated phases and also the use of a factor to dampen the change of density and so control the refinement process. Two protein structures were used in numerical trials, RNApl [Bezborodova, Ermekbaeva, Shlyapnikov, Polyakov & Bezborodov (1988). Biokhimiya, 53, 965-973] and 2-Zn insulin [Baker, Blundell, Cutfield, Cutfield, Dodson, Dodson, Hodgkin, Hubbard, lsaacs, Reynolds, Sakabe, Sakabe & Vijayan (1988). Philos. Trans. R. Soc. London Ser. B, 319, 456--469]. Comparison of the proposed procedures with the normal histogram-matching technique without structure-factor weighting or damping gives mean phase errors reduced by up to 10 degrees with map correlation coefficients improved by as much as 0.14. Compared to the normal histogram used with weighting of structure factors and damping, the improvement due to the use of the double-histogram method is usually of order 4 degrees in mean phase error and an increase of 0.06-0.08 in the map correlation coefficient. It is concluded that the most reliable results are found with the local-maximum condition and with R in the range 0.5-0.6 A.

Journal Article↗
PubMed1996

A minimalist's approach to the phase problem - phasing selenomethionyl protein structures using Cu Kalpha data.

The feasibility of phasing protein structures through the use of the isomorphous and anomalous signal of selenomethionyl (Se-Met) derivative and diffraction data collected with a standard laboratory Cu Kalpha X-ray source has been investigated. Interpretable electron-density maps were obtained for the core domain of avian sarcoma virus integrase, a typical medium-sized protein having four Met residues in a sequence of 156 amino acids. The r.m.s. difference between 3.1 A experimental phases obtained from Se-Met Cu Kalpha data and the final phases calculated from the refined model is 55 degrees. A procedure combining single isomorphous replacement/single anomalous scattering phasing and solvent flattening for data based on a single Se-Met derivative and Cu Kalpha radiation has been tested on this and another protein. The results are encouraging enough to indicate that such procedures might be recommended when a synchrotron source is not readily available.

Journal Article↗
PubMed1996

Direct-space methods in phase extension and phase refinement. V. The histogram moments method.

Any distribution is completely defined by its moments. It is shown that a process of phase refinement can be carried out, based on Fourier transforms, which modifies the moments of electron density, separately in the protein and solvent regions, towards target values. Tests have been carried out on two moderate-sized proteins with 800-900 atoms in the asymmetric unit, one containing heavy atoms and the other not. It has been found that refinement using the third moment about zero in the protein region is most effective and that refinement with higher moments, or in the solvent region, adds nothing useful. Two kinds of weights are necessary in the method. One is for giving a weighted mixture of new phase indications with original phase estimates from, say, multiple isomorphous replacement. The other weights are applied to the Fourier coefficients of density maps to give the best possible signal:noise ratio. These weights have been explored empirically and the best ones found are described. It is concluded that since the moments method, which changes phases in reciprocal space, is independent of other histogram-matching procedures, which change density in real space, it has something to offer in a refinement package containing several procedures.

Journal Article↗
PubMed2004

A discrepancy between liquid phase and gel phase assays for evaluation of total complement activity and some possible explanations.

This study was conducted to investigate the frequency and origin of discrepant assay results between two haemolytic assays which both measure activity of the classical pathway of complement (CH50) by haemolysis of sheep red blood cells (SRBCs). One is conducted in gel phase using undiluted sera and the other in liquid phase with sera in 1/100 dilution. The majority of discrepant readings are observed as low or absent haemolysis in the gel phase, with values within or above the normal range in the liquid phase. The incidence of discrepant assay readings was evaluated in 300 samples. Furthermore, 28 samples showing the most discrepant readings were investigated further for disturbing factors. Factors evaluated in the test sera were mannose-binding lectin, C-reactive protein (CRP), immune complexes, antibodies to SRBCs, rheumatoid factor and immunoglobulin A (IgA) and IgG anti-C1q antibodies. The results showed that discrepant readings are present in 10% of the 300 samples and false low gel assay readings account for 6.3%. The majority (68%) of the discrepant samples contained a heat-stable-inhibiting factor, and the main mediators found were elevated levels of IgA anti-C1q antibodies and antibodies to SRBCs. This could indicate a clinically relevant factor in the test sera but can also result from the difference in assay design.

Antigen-Antibody Complex↗
PubMed1979

Tone-segregation by phase: on the phase sensitivity of the single ear.

A monaural complex tone is synthesized from 12 harmonically related pure tones, played in phase. In each of 12 segments, one of the tones (the target) is played out of phase so that the sequence of targets is increasing or decreasing in frequency. If the target is at least 30 degrees out of phase, the targets are perceptually segregated. This tone-segregation by phase raises doubts concerning several current theories of pitch perception. The phenomenon is conjectured to be caused by the ear's nonlinear compressive transfer characteristic or by a temporal analysis of the stimulus.

Acoustic Stimulation↗
PubMed2005

Arsenite-induced aryl hydrocarbon receptor nuclear translocation results in additive induction of phase I genes and synergistic induction of phase II genes.

Complex mixtures of carcinogenic metalloids, such as arsenic, and polycyclic aromatic hydrocarbons or halogenated aromatic hydrocarbons are common environmental contaminants. The biological consequences of exposure to these mixtures are unpredictable and, although the health effects of individual chemicals may be known, the toxicity of environmental mixtures is largely unexplored. Arsenic, not a potent mutagen by itself, is co-mutagenic with many DNA-damaging agents. Mixtures of arsenite plus benzo[a]pyrene (B[a]P) augment B[a]P mutagenicity, suggesting that arsenite might uncouple expression of phase I and II genes responsible for detoxification. We have studied the effects of arsenite exposure on the activation of the aryl hydrocarbon receptor (AHR) and its subsequent role in gene transactivation. Treatment of mouse Hepa-1 cells with arsenite induces AHR nuclear translocation and binding to the Cyp1a1 gene promoter with the same efficiency as tetrachlorodibenzo-p-dioxin (TCDD), the most potent ligand of the AHR; however, TCDD and B[a]P are an order of magnitude more potent than arsenite in up-regulating Cyp1a1 transcription. Global profiling analyses of cells treated with arsenite plus B[a]P indicate that several phase I and II detoxification genes are in some cases additively and in others synergistically deregulated by the mixtures. Real-time reverse transcription-polymerase chain reaction analyses of mouse embryonic fibroblasts showed that the mixtures had an additive effect on the mRNA levels of Cyp1b1, a prototypical phase I detoxification gene, and an AHR-dependent synergistic effect on the corresponding levels of Nqo1, a prototypical phase II gene. We conclude that exposure to arsenite/B[a]P mixtures causes regulatory changes in the expression of detoxification genes that ultimately affect the metabolic activation and disposition of toxicants.

Active Transport, Cell Nucleus↗
PubMed2004

Genetic evidence that Legionella pneumophila RpoS modulates expression of the transmission phenotype in both the exponential phase and the stationary phase.

The opportunistic pathogen Legionella pneumophila alternates between two states: replication within phagocytes and transmission between host amoebae or macrophages. In broth cultures that model this life cycle, during the replication period, CsrA inhibits expression of transmission traits. When nutrients become limiting, the alarmone (p)ppGpp accumulates and the sigma factors RpoS and FliA and the positive activators LetA/S and LetE promote differentiation to the transmissible form. Here we show that when cells enter the postexponential growth phase, RpoS increases expression of the transmission genes fliA, flaA, and mip, factors L. pneumophila needs to establish a new replication niche. In contrast, in exponential (E)-phase cells whose (p)ppGpp levels are low, rpoS inhibits expression of transmission traits, on the basis of three separate observations. First, rpoS RNA levels peak in the E phase, suggestive of a role for RpoS during replication. Second, in multiple copies, rpoS decreases the amounts of csrA, letE, fliA, and flaA transcripts and inhibits the transmission traits of motility, infectivity, and cytotoxicity. Third, rpoS blocks expression of cytotoxicity and motility by E-phase bacteria that have been induced to express the LetA activator ectopically. The data are discussed in the context of a model in which the alarmone (p)ppGpp enables RpoS to outcompete other sigma factors for binding to RNA polymerase to promote transcription of transmission genes, while LetA/S acts in parallel to relieve CsrA posttranscriptional repression of the transmission regulon. By coupling transcriptional and posttranscriptional control pathways, intracellular L. pneumophila could respond to stress by rapidly differentiating to a transmissible form.

Animals↗
PubMed1993

Complex transcriptional control of the sigma s-dependent stationary-phase-induced and osmotically regulated osmY (csi-5) gene suggests novel roles for Lrp, cyclic AMP (cAMP) receptor protein-cAMP complex, and integration host factor in the stationary-phase response of Escherichia coli.

osmY (csi-5) is a representative of a large group of sigma s-dependent genes in Escherichia coli that exhibit both stationary-phase induction and osmotic regulation. A chromosomal transcriptional lacZ fusion (csi-5::lacZ) was used to study the regulation of osmY. We show here that in addition to sigma s, the global regulators Lrp, cyclic AMP (cAMP) receptor protein-cAMP complex (cAMP-CRP), and integration host factor (IHF) are involved in the control of osmY. All three regulators negatively modulate the expression of osmY, and they act independently from sigma s. Stationary-phase induction of osmY in minimal medium can be explained by stimulation by sigma s combined with a relief of Lrp repression. Stationary-phase induction of osmY in rich medium is mediated by the combined action of sigma s, Lrp, cAMP-CRP, and IHF, with the latter three proteins acting as transition state regulators. The transcriptional start site of osmY was determined and revealed an mRNA with an unusual long nontranslated leader of 244 nucleotides. The regulatory region is characterized by a sigma 70-like -10 promoter region and contains potential binding sites for Lrp, CRP, and IHF. Whereas sigma s, Lrp, CRP, and IHF are clearly involved in stationary-phase induction, none of these regulators is essential for osmotic regulation of osmY.

Amino Acid Sequence↗
PubMed1993

Phase variation in Xenorhabdus luminescens: cloning and sequencing of the lipase gene and analysis of its expression in primary and secondary phases of the bacterium.

The phenomenon of phase variation in the insect-pathogenic bacterium Xenorhabdus luminescens was investigated. Differential activity of the lipase enzyme (EC 3.1.1.3) was observed between the two phases of the bacteria. The enzyme was found to be secreted into the culture medium, and about five to six times greater specific activity was secreted by the primary phase than by the secondary form. The lipase gene (lip-1) was cloned and sequenced. The data imply that there is only a single Tween 80-utilizing lipase gene in X. luminescens K122. The sequence revealed a translation product of 645 amino acids, from which a hydrophobic leader sequence of 24 amino acids is removed during processing. The structure of the gene was shown to be the same in the primary and secondary forms of X. luminescens. In addition, transcription was found to start at the same position, 169 bp upstream of the translation initiation codon, in the two forms of the bacteria. Equal amounts of lipase RNA accumulated in the two forms, and at least as much lipase protein was secreted by the secondary form as by the primary. This suggests that the difference in specific activity between the enzymes secreted by the two phases probably arises from a posttranslational type of regulation.

Amino Acid Sequence↗
PubMed1962

Isocitrate lyase and malate synthase in Pseudomonas indigofera. II. Enzyme changes during the phase of adjustment and the early exponential phase.

Howes, William V. (Washington State University, Pullman) and Bruce A. McFadden. Isocitrate lyase and malate synthase in Pseudomonas indigofera. II. Enzyme changes during the phase of adjustment and the early exponential phase. J. Bacteriol.84:1222-1227. 1962.-The differential rates of synthesis (DRS) of malate synthase and isocitrate lyase were investigated at 30 C during the phase of adjustment (lag) and the early exponential phase in Pseudomonas indigofera. Cells grown on ethanol-acetate-yeast extract or succinate-yeast extract were added to a medium containing ethanol or succinate, and the DRS of each enzyme was followed. A reproducible decline in both enzymatic activities occurred after inoculation when a lag in growth was observed. To define the DRS occurring after this decline, a new parameter, epsilon, was defined and calculated for linear segments of a DRS plot. Certain variations of epsilon during DRS suggested that a balance between induction (or derepression) and repression controls, in part at least, malate synthase and isocitrate lyase levels. Effects of chloramphenicol upon enzyme synthesis after the period of expected decline were studied. No degradation or synthesis of malate synthase occurred in the presence of chloramphenicol, although, after degradation, slight synthesis of isocitrate lyase occurred. Acetate, butyrate, ethanol, glyoxylate, and isocitrate did not stimulate production of either enzyme in nongrowing cultures of high cell density. In fact, some degradation of each enzyme occurred.

Acetates↗
PubMed2002

The mental health of UK Gulf war veterans: phase 2 of a two phase cohort study.

OBJECTIVES: To examine the prevalence of psychiatric disorders in veterans of the Gulf war with or without unexplained physical disability (a proxy measure of ill health) and in similarly disabled veterans who had not been deployed to the Gulf war (non-Gulf veterans). DESIGN: Two phase cohort study. SETTING: Current and ex-service UK military personnel. PARTICIPANTS: Phase 1 consisted of three randomly selected samples of Gulf veterans, veterans of the 1992-7 Bosnia peacekeeping mission, and UK military personnel not deployed to the Gulf war (Era veterans) who had completed a postal health questionnaire. Phase 2 consisted of randomly selected subsamples from phase 1 of Gulf veterans who reported physical disability (n=111) or who did not report disability (n=98) and of Bosnia (n=54) and Era (n=79) veterans who reported physical disability. MAIN OUTCOME MEASURE: Psychiatric disorders assessed by the schedule for clinical assessment in neuropsychiatry and classified by the Diagnostic and Statistical Manual of Mental Disorders, fourth edition. RESULTS: Only 24% (n=27) of the disabled Gulf veterans had a formal psychiatric disorder (depression, anxiety, or alcohol related disorder). The prevalence of psychiatric disorders in non-disabled Gulf veterans was 12%. Disability and psychiatric disorders were weakly associated in the Gulf group when confounding was adjusted for (adjusted odds ratio 2.4, 99% confidence interval 0.8 to 7.2, P=0.04). The prevalence of psychiatric disorders was similar in disabled non-Gulf veterans and disabled Gulf veterans ( 19% v 24%; 1.3, 0.5 to 3.4). All groups had rates for post-traumatic stress disorder of between 1% and 3%. CONCLUSIONS: Most disabled Gulf veterans do not have a formal psychiatric disorder. Post-traumatic stress disorder is not higher in Gulf veterans than in other veterans. Psychiatric disorders do not fully explain self reported ill health in Gulf veterans; alternative explanations for persistent ill health in Gulf veterans are needed.

Cohort Studies↗
PubMed2001

Adrenal masses: quantification of fat content with double-echo chemical shift in-phase and opposed-phase FLASH MR images for differentiation of adrenal adenomas.

PURPOSE: To quantify fat content in adrenal lesions with double-echo chemical shift magnetic resonance (MR) imaging in a phantom study and to differentiate adrenal adenomas from other adrenal masses by assessing fat content in a clinical study. MATERIALS AND METHODS: The study consisted of two parts: a phantom study and a clinical study. To explore the effect of the T1 value on in- and opposed-phase MR images of fat-containing tissues, phantom models with various proportions of fat and gadopentetate dimeglumine concentrations were implemented. Signal intensity (SI) indexes ([SI in-phase - SI opposed-phase]/SI in-phase) were calculated with double-echo fast low-angle shot (FLASH) MR imaging. In the clinical study, 23 patients with 28 adrenal masses (16 adrenal adenomas, nine adrenal metastases, and three pheochromocytomas) underwent double-echo FLASH MR imaging, and SI indexes were calculated. RESULTS: SI index reached a maximum of 0.87 at 53% fat fraction for gadopentetate dimeglumine concentration at 0.5 mmol/L as the simulated T1 of the adrenal mass. The SI indexes of the adrenal adenomas, adrenal metastases, and pheochromocytomas, respectively, were 0.36, -0.15, and -0.07, and estimated fat fraction from the phantom study was 26.5%, 0%, and 0%. All adrenal adenomas contained fat on double-echo FLASH images. There was no overlap in SI index between adenomas and other tumors. CONCLUSION: Preliminary experience indicates that quantitative measurement of the fat fraction of adrenal masses is possible with the double-echo chemical shift FLASH technique and allows for differentiating adrenal adenomas from other adrenal masses.

Adenoma↗
PubMed2005

Development of a transactivator in hepatoma cells that allows expression of phase I, phase II, and chemical defense genes.

Precise control of the level of protein expression in cells can yield quantitative and temporal information on the role of a given gene in normal cellular physiology and on exposure to chemicals and drugs. This is particularly relevant to liver cells, in which the expression of many proteins, such as phase I and phase II drug-metabolizing enzymes, vary widely between species, among individual humans, and on exposure to xenobiotics. The most widely used gene regulatory system has been the tet-on/off approach. Although a second-generation tet-on transactivator was recently described, it has not been widely investigated for its potential as a tool for regulating genes in cells and particularly in cells previously recalcitrant to the first-generation tet-on approach, such as hepatocyte-derived cells. Here we demonstrate the development of two human (HepG2 and HuH7) and one mouse (Hepa1c1c7) hepatoma-derived cell lines incorporating a second-generation doxycycline-inducible gene expression system and the application of the human lines to control the expression of different transgenes. The two human cell lines were tested for transient or stable inducibility of five transgenes relevant to liver biology, namely phase I (cytochrome P-450 2E1; CYP2E1) and phase II (glutathione S-transferase P1; GSTP1) drug metabolism, and three transcription factors that respond to chemical stress [nuclear factor erythroid 2 p45-related factors (NRF)1 and 2 and NFKB1 subunit of NF-kappaB]. High levels of functional expression were obtained in a time- and dose-dependent manner. Importantly, doxycycline did not cause obvious changes in the cellular proteome. In conclusion, we have generated hepatocyte-derived cell lines in which expression of genes is fully controllable.

Amino Acid Sequence↗