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At least 1,603 records · Page 89Linked to original sources
PubMed1992

Human immunodeficiency virus gp120 binding C'C" ridge of CD4 domain 1 is also involved in interaction with class II major histocompatibility complex molecules.

Using site-directed mutagenesis informed by high-resolution CD4 structural data, we have investigated the role of residues of the C'C'' ridge region of human CD4 on class II major histocompatibility complex (MHC) binding. This C'C'' ridge is homologous to the CDR2 loop of an immunoglobulin variable domain and is known to contain the binding site for human immunodeficiency virus (HIV) coat glycoprotein gp120. Here we report that this region is also involved in interaction with class II MHC. Exposed positively charged residues Lys-35, Lys-46, and Arg-59 and the exposed hydrophobic residue Phe-43 contribute significantly to class II MHC binding. Moreover, mutations in the buried residues Trp-62 and Ser-49, which support the top and bottom of the C'C'' ridge, respectively, disrupt class II MHC interaction. The HIV binding region appears to involve a restricted area of the larger class II MHC binding site on CD4. Strategies of drug design aimed at interrupting CD4-HIV interaction will need to consider the extensive overlap between class II MHC and HIV gp120 binding surfaces in this region of CD4.

Animals↗
PubMed1993

Delineation of an extended surface contact area on human CD4 involved in class II major histocompatibility complex binding.

We describe a detailed mapping of the class II major histocompatibility complex (MHC) binding site using site-directed mutagenesis in conjunction with high-resolution CD4 structural data. Residues on all lateral surfaces of domain 1 and the neighboring portions of domain 2 participate in contacting class II MHC. Thus, in addition to the C'C" ridge that forms the human immunodeficiency virus type 1 gp120 binding site, apparent MHC contacts extend over the BED face of domain 1 and across the interdomain groove onto the FG loop of domain 2. Several models of the CD4/class II MHC interaction accounting for the extent of the CD4 surface involved are discussed, including the possibility that CD4 may contact more than one class II MHC molecule using different surfaces.

Amino Acid Sequence↗
PubMed1993

Crystal structure of Escherichia coli L-asparaginase, an enzyme used in cancer therapy.

The crystal structure of Escherichia coli asparaginase II (EC 3.5.1.1), a drug (Elspar) used for the treatment of acute lymphoblastic leukemia, has been determined at 2.3 A resolution by using data from a single heavy atom derivative in combination with molecular replacement. The atomic model was refined to an R factor of 0.143. This enzyme, active as a homotetramer with 222 symmetry, belongs to the class of alpha/beta proteins. Each subunit has two domains with unique topological features. On the basis of present structural evidence consistent with previous biochemical studies, we propose locations for the active sites between the N- and C-terminal domains belonging to different subunits and postulate a catalytic role for Thr-89.

Amino Acid Sequence↗
PubMed1994

The structure of a complex of hexameric insulin and 4'-hydroxyacetanilide.

X-ray crystallographic studies have been carried out on human insulin crystals grown in the presence of 4'-hydroxyacetanilide (Tylenol) and show that this nontoxic phenolic derivative can induce the T-->R transition, producing a T3R3 hexamer. Two different crystals, grown under different conditions, are rhombohedral, space group R3, with cell constants a = 81.11, c = 37.97 and a = 80.88, c = 37.60 A. The T3R3 hexamer is symmetric, resulting from the presence of a crystallographic threefold axis, and the asymmetric unit consists of a TR dimer. Data to a resolution of 1.9 A were measured on a crystal from each of the two crystallizations and the structures have been refined to residuals of 0.168 and 0.173. The guest molecule is bound by the R-state monomer through the formation of two hydrogen bonds from the hydroxy group of Tylenol to the carbonyl oxygen and the nitrogen of A6 Cys and A11 Cys, respectively. Due to steric constraints of the phenolic binding site, the acetamide group of Tylenol is rotated approximately 50 degrees out of the plane of the phenyl group and the methyl group is cis; no hydrogen bonds exist between the acetamide group and the hexamer. Although the zinc ion, which is bound to the R-state trimer, has tetrahedral coordination in both structures, the T-state zinc is observed to have octahedral coordination in one structure but tetrahedral coordination in the other. The side chain of A10 Ile in the R-state monomer adopts a high-energy conformation as a result of close contact to a residue in an adjacent dimer and may explain in part the differences between therapeutic preparations of beef insulin, for which A10 is a Val residue, and human insulin.

Acetaminophen↗
PubMed2001

In planta sequential hydroxylation and glycosylation of a fungal phytotoxin: Avoiding cell death and overcoming the fungal invader.

To facilitate plant colonization, some pathogenic fungi produce phytotoxic metabolites that damage tissues; plants may be resistant to a particular pathogen if they produce an enzyme(s) that catalyzes detoxification of this metabolite(s). Alternaria blackspot is one of the most damaging and significant fungal diseases of brassica crops, with no source of resistance known within the Brassica species. Destruxin B is the major phytotoxin produced by the blackspot-causing fungus, Alternaria brassicae (Berkley) Saccardo. We have established that a blackspot-resistant species (Sinapis alba) metabolized (14)C-labeled destruxin B to a less toxic product substantially faster than any of the susceptible species. The first metabolite, hydroxydestruxin B ((14)C-labeled), was further biotransformed to the beta-d-glucosyl derivative at a slower rate. The structures of hydroxydestruxin B and beta-d-glucosyl hydroxydestruxin B were deduced from their spectroscopic data [NMR, high resolution (HR)-MS, Fourier transform infrared (FTIR)] and confirmed by total chemical synthesis. Although these hydroxylation and glucosylation reactions occurred in both resistant (S. alba) and susceptible (Brassica napus, Brassica juncea, and Brassica rapa) species, hydroxylation was the rate limiting step in the susceptible species, whereas glucosylation was the rate limiting step in the resistant species. Remarkably, it was observed that the hydroxydestruxin B induced the biosynthesis of phytoalexins in blackspot-resistant species but not in susceptible species. This appears to be a unique example of phytotoxin detoxification and simultaneous phytoalexin elicitation by the detoxification product. Our studies suggest that S. alba can overcome the fungal invader through detoxification of destruxin B coupled with production of phytoalexins.

Alternaria↗
PubMed2001

First crystal structure of Rubisco from a green alga, Chlamydomonas reinhardtii.

The crystal structure of Rubisco (ribulose 1,5-bisphosphate carboxylase/oxygenase) from the unicellular green alga Chlamydomonas reinhardtii has been determined to 1.4 A resolution. Overall, the structure shows high similarity to the previously determined structures of L8S8 Rubisco enzymes. The largest difference is found in the loop between beta strands A and B of the small subunit (betaA-betaB loop), which is longer by six amino acid residues than the corresponding region in Rubisco from Spinacia. Mutations of residues in the betaA-betaB loop have been shown to affect holoenzyme stability and catalytic properties. The information contained in the Chlamydomonas structure enables a more reliable analysis of the effect of these mutations. No electron density was observed for the last 13 residues of the small subunit, which are assumed to be disordered in the crystal. Because of the high resolution of the data, some posttranslational modifications are unambiguously apparent in the structure. These include cysteine and N-terminal methylations and proline 4-hydroxylations.

Animals↗
PubMed2003

Crystal structure of the Yersinia protein-tyrosine phosphatase YopH complexed with a specific small molecule inhibitor.

The pathogenic bacteria Yersinia are causative agents in human diseases ranging from gastrointestinal syndromes to bubonic plague. There is increasing risk of misuse of infectious agents, such as Yersinia pestis, as weapons of terror as well as instruments of warfare for mass destruction. Because the phosphatase activity of the Yersinia protein tyrosine phosphatase, YopH, is essential for virulence in the Yersinia pathogen, potent and selective YopH inhibitors are expected to serve as novel anti-plague agents. We have identified a specific YopH small molecule inhibitor, p-nitrocatechol sulfate (pNCS), which exhibits a Ki value of 25 microM for YopH and displays a 13-60-fold selectivity in favor of YopH against a panel of mammalian PTPs. To facilitate the understanding of the underlying molecular basis for tight binding and specificity, we have determined the crystal structure of YopH in complex with pNCS at a 2.0-A resolution. The structural data are corroborated by results from kinetic analyses of the interactions of YopH and its site-directed mutants with pNCS. The results show that while the interactions of the sulfuryl moiety and the phenyl ring with the YopH active site contribute to pNCS binding affinity, additional interactions of the hydroxyl and nitro groups in pNCS with Asp-356, Gln-357, Arg-404, and Gln-446 are responsible for the increased potency and selectivity. In particular, we note that residues Arg-404, Glu-290, Asp-356, and a bound water (WAT185) participate in a unique H-bonding network with the hydroxyl group ortho to the sulfuryl moiety, which may be exploited to design more potent and specific YopH inhibitors.

Amino Acid Sequence↗
PubMed2004

Three-dimensional structure of P-glycoprotein: the transmembrane regions adopt an asymmetric configuration in the nucleotide-bound state.

Multidrug resistance of cancer cells and pathogens is a serious clinical problem. A major factor contributing to drug resistance in cancer is the over-expression of P-glycoprotein, a plasma membrane ATP-binding cassette (ABC) drug efflux pump. Three-dimensional structural data with a resolution limit of approximately 8 A have been obtained from two-dimensional crystals of P-glycoprotein trapped in the nucleotide-bound state. Each of the two transmembrane domains of P-glycoprotein consists of six long alpha-helical segments. Five of the alpha-helices from each transmembrane domain are related by a pseudo-2-fold symmetry, whereas the sixth breaks the symmetry. The two alpha-helices positioned closest to the (pseudo-) symmetry axis at the center of the molecule appear to be kinked. A large loop of density at the extracellular surface of the transporter is likely to correspond to the glycosylated first extracellular loop, whereas two globular densities at the cytoplasmic side correspond to the hydrophilic, nucleotide-binding domains. This is the first three-dimensional structure for an intact eukaryotic ABC transporter. Comparison with the structures of two prokaryotic ABC transporters suggests significant differences in the packing of the transmembrane alpha-helices within this protein family.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2004

Stable carbon isotopic composition of tree rings from a pine tree from Augustów Wilderness, Poland, as a temperature and local environment conditions indicator.

Tree rings can be used as archives of climatic and environmental data with annual resolution. Tree rings widths, maximum late wood density and other parameters as stable composition in tree rings can be used for the reconstruction of past climatic and environmental changes. Stable carbon isotope ratios in tree rings may provide valuable information on past climatic conditions. 13C/12C ratios of plant organic matter can reflect corresponding 13C/12C ratio of atmospheric CO2 during formation of the rings. Investigations of isotopic carbon composition in tree rings from in the ecologically clean the Augustów Wilderness region in the north-eastern part of Poland (22 degrees 58'E, 53 degrees 51'N) (nowadays a sanctuary) were undertaken. Series of delta13C in alpha-cellulose and in wholewood were acquired. Those measurements constituted a part of more complex investigations of carbon isotope composition in tree rings including the measurements of radiocarbon concentration and tree ring widths. This article presents preliminary results. It is argued that contrary to the tree ring widths and delta13C in wholewood that do not reveal significant correlation with temperature, the variation of delta13C in the latewood alpha-cellulose is correlated with combined July and August temperatures.

Carbon Isotopes↗
PubMedDate not supplied

Replication of human tracheobronchial hollow airway models using a selective laser sintering rapid prototyping technique.

Exposures to toxic or pathogenic aerosols are known to produce adverse health effects. The nature and severity of these effects often are governed in large part by the location and amount of aerosol deposition within the respiratory tract. Morphologically detailed replica hollow lung airway casts are widely used in aerosol deposition research; however, techniques are not currently available that allow replicate deposition studies in identical morphologically detailed casts produced from a common reference anatomy. This project developed a technique for the precision manufacture of morphologically detailed human tracheobronchial airway models based on high-resolution anatomical imaging data. Detailed physical models were produced using the selective laser sintering (SLS) rapid prototyping process. Input to the SLS process was a three-dimensional computer model developed by boundary-based two-dimension to three-dimension conversion of anatomical images from the original National Institutes of Health/National Library of Medicine Visible Human male data set. The SLS process produced identical replicate models that corresponded exactly to the anatomical section images, within the limits of the measurement. At least five airway generations were achievable, corresponding to airways less than 2 mm in diameter. It is anticipated that rapid prototyping manufacture of respiratory tract structures based on reference anatomies such as the Visible Male and Visible Female may provide "gold standard" models for inhaled aerosol deposition studies. Adaptations of the models to represent various disease states may be readily achieved, thereby promoting exploration of pharmaceutical research on targeted drug delivery via inhaled aerosols.

Aerosols↗
PubMed1977

Gap junction structures. II. Analysis of the x-ray diffraction data.

Models for the spatial distribution of protein, lipid and water in gap junction structures have been constructed from the results of the analysis of X-ray diffraction data described here and the electron microscope and chemical data presented in the preceding paper (Caspar, D. L. D., D. A. Goodenough, L. Makowski, and W.C. Phillips. 1977. 74:605-628). The continuous intensity distribution on the meridian of the X-ray diffraction pattern was measured, and corrected for the effects of the partially ordered stacking and partial orientation of the junctions in the X-ray specimens. The electron density distribution in the direction perpendicular to the plane of the junction was calculated from the meridional intensity data. Determination of the interference function for the stacking of the junctions improved the accuracy of the electron density profile. The pair-correlation function, which provides information about the packing of junctions in the specimen, was calculated from the interference function. The intensities of the hexagonal lattice reflections on the equator of the X-ray pattern were used in coordination with the electron microscope data to calculate to the two-dimensional electron density projection onto the plane of the membrane. Differences in the structure of the connexons as seen in the meridional profile and equatorial projections were shown to be correlated to changes in lattice constant. The parts of the junction structure which are variable have been distinguished from the invariant parts by comparison of the X-ray data from different specimens. The combination of these results with electron microscope and chemical data provides low resolution three- dimensional representations of the structures of gap junctions.

Animals↗
PubMed1999

The Tangled Web: Gene Genealogies and the Origin of Eukaryotes.

Accessing data from the genomes of organisms (individual genes) and analyzing these data using sophisticated alignment and phylogenetic methods led to the expectation that we would be able to paint a clear picture of the evolution of eukaryotes. Previous analyses based on morphology and ultrastructure failed to pinpoint both the sister taxon to eukaryotes and the branching order of eukaryotic lineages. However, the expectation that molecular data would provide resolution has not been met since a growing number of gene genealogies present conflicting hypotheses for the origin and diversification of eukaryotes. Instead of reconstructing a simple bifurcating tree of life, these gene genealogies have generated a complex picture of eukaryotic genomes whereby ancient lateral transfers (of individual genes or perhaps even entire genomes) has tangled the evolutionary history of eukaryotes. Resolution of these conflicting genealogies comes in recognizing that eukaryotes are chimeric, containing genetic information from multiple ancestral lineages.

Journal Article↗
PubMed2004

Validation of the GATE Monte Carlo simulation platform for modelling a CsI(Tl) scintillation camera dedicated to small-animal imaging.

Monte Carlo simulations are increasingly used in scintigraphic imaging to model imaging systems and to develop and assess tomographic reconstruction algorithms and correction methods for improved image quantitation. GATE (GEANT4 application for tomographic emission) is a new Monte Carlo simulation platform based on GEANT4 dedicated to nuclear imaging applications. This paper describes the GATE simulation of a prototype of scintillation camera dedicated to small-animal imaging and consisting of a CsI(Tl) crystal array coupled to a position-sensitive photomultiplier tube. The relevance of GATE to model the camera prototype was assessed by comparing simulated 99mTc point spread functions, energy spectra, sensitivities, scatter fractions and image of a capillary phantom with the corresponding experimental measurements. Results showed an excellent agreement between simulated and experimental data: experimental spatial resolutions were predicted with an error less than 100 microns. The difference between experimental and simulated system sensitivities for different source-to-collimator distances was within 2%. Simulated and experimental scatter fractions in a [98-182 keV] energy window differed by less than 2% for sources located in water. Simulated and experimental energy spectra agreed very well between 40 and 180 keV. These results demonstrate the ability and flexibility of GATE for simulating original detector designs. The main weakness of GATE concerns the long computation time it requires: this issue is currently under investigation by the GEANT4 and the GATE collaborations.

Animals↗
PubMed2006

Measuring nanometer scale gradients in spindle microtubule dynamics using model convolution microscopy.

A computational model for the budding yeast mitotic spindle predicts a spatial gradient in tubulin turnover that is produced by kinetochore-attached microtubule (kMT) plus-end polymerization and depolymerization dynamics. However, kMTs in yeast are often much shorter than the resolution limit of the light microscope, making visualization of this gradient difficult. To overcome this limitation, we combined digital imaging of fluorescence redistribution after photobleaching (FRAP) with model convolution methods to compare computer simulations at nanometer scale resolution to microscopic data. We measured a gradient in microtubule dynamics in yeast spindles at approximately 65-nm spatial intervals. Tubulin turnover is greatest near kinetochores and lowest near the spindle poles. A beta-tubulin mutant with decreased plus-end dynamics preserves the spatial gradient in tubulin turnover at a slower time scale, increases average kinetochore microtubule length approximately 14%, and decreases tension at kinetochores. The beta-tubulin mutant cells have an increased frequency of chromosome loss, suggesting that the accuracy of chromosome segregation is linked to robust kMT plus-end dynamics.

Chromosome Segregation↗
PubMed2025

Doblin: inferring dominant clonal lineages from high-resolution DNA barcoding time series.

MOTIVATION: The lineage dynamics and history of cells in a population reflect the interplay of evolutionary forces they experience, including mutation, drift, and selection. When the population is polyclonal, lineage dynamics also manifest the extent of clonal competition among co-existing mutational variants. If the population exists in a community of other species, the lineage dynamics could also reflect the population's ecological interaction with the rest of the community. Recent advances in high-resolution lineage tracking via DNA barcoding, coupled with next-generation sequencing of bacteria, yeast, and mammalian cells, allow for precise quantification of clonal dynamics in these organisms. RESULTS: In this work, we introduce Doblin, an R suite for identifying dominant barcode lineages based on high-resolution lineage tracking data. We first benchmarked Doblin's accuracy using lineage data from evolutionary simulations, showing that it recovers the clones' identity and relative fitness in the simulation. Next, we applied Doblin to analyze clonal dynamics in laboratory evolutions of Escherichia coli populations undergoing antibiotic treatment and in colonization experiments of the gut microbial community. Doblin's versatility allows it to be applied to lineage time-series data across different experimental setups. AVAILABILITY AND IMPLEMENTATION: Doblin is available on CRAN (https://CRAN.R-project.org/package=doblin) and Github (https://github.com/dagagf/doblin).

DNA Barcoding, Taxonomic↗
PubMed2001

Real and illusory contour processing in area V1 of the primate: a cortical balancing act.

It is known that neurons in area V2 (the second visual area) can signal the orientation of illusory contours in the primate. Whether area V1 (primary visual cortex) can signal illusory contour orientation is more controversial. While some electrophysiology studies have ruled out illusory signaling in V1, other reports suggest that V1 shows some illusory-specific response. Here, using optical imaging and single unit electrophysiology, we report that primate V1 does show an orientation-specific response to the 'abutting line grating' illusory contour. However, this response does not signal an illusory contour in the conventional sense. Rather, we find that illusory contour stimulation leads to an activation map that, after appropriate subtraction of real line signal, is inversely related to the real orientation map. The illusory contour orientation is thus negatively signaled or de-emphasized in V1. This 'activation reversal' is robust, is not due merely to presence of line ends, is not dependent on inducer orientation, and is not due to precise position of line end stimulation of V1 cells. These data suggest a resolution for previous apparently contradictory experimental findings. We propose that the de-emphasis of illusory contour orientation in V1 may be an important signal of contour identity and may, together with illusory signal from V2, provide a unique signature for illusory contour representation.

Animals↗
PubMed2005

Microgenomics: gene expression analysis at the tissue-specific and single-cell levels.

Plant organs are composed of many different cell types and the analysis of 'bulk' material results in the average of all information in these cells. Therefore, this does not reflect any individuality of the tissues present in plants. This review briefly summarizes different sampling methods which provide tissue- and cell-specific samples, respectively. In addition, gene expression analysis tools that allow the analysis of transcripts in minute samples are discussed in detail. The combination of both approaches results in high resolution gene expression data, which increases understanding of plant physiology in such diverse areas as primary and secondary metabolism, plant defence or stress response.

Base Sequence↗
PubMed2006

Elongation complexes of Thermus thermophilus RNA polymerase that possess distinct translocation conformations.

We have characterized elongation complexes (ECs) of RNA polymerase from the extremely thermophilic bacterium, Thermus thermophilus. We found that complexes assembled on nucleic acid scaffolds are transcriptionally competent at high temperature (50-80 degrees C) and, depending upon the organization of the scaffold, possess distinct translocation conformations. ECs assembled on scaffolds with a 9 bp RNA:DNA hybrid are highly stable, resistant to pyrophosphorolysis, and are in the posttranslocated state. ECs with an RNA:DNA hybrid longer or shorter than 9 bp appear to be in a pretranslocated state, as evidenced by their sensitivity to pyrophosphorolysis, GreA-induced cleavage, and exonuclease footprinting. Both pretranslocated (8 bp RNA:DNA hybrid) and posttranslocated (9 bp RNA:DNA hybrid) complexes were crystallized in distinct crystal forms, supporting the homogeneity of the conformational states in these complexes. Crystals of a posttranslocated complex were used to collect diffraction data at atomic resolution.

Crystallization↗