Biomedical subjects

D Cowburn

Publications and source records attributed to D Cowburn.

At least 55 records · Page 3Linked to original sources

PubMed1995

Solution structure of pleckstrin homology domain of dynamin by heteronuclear NMR spectroscopy.

The pleckstrin homology (PH) domain is a recognition motif thought to be involved in signal-transduction pathways controlled by a variety of cytoplasmic proteins. Assignments of nearly all 1H, 13C, and 15N resonances of the PH domain from dynamin have been obtained from homonuclear and heteronuclear NMR experiments. The secondary structure has been elucidated from the pattern of nuclear Overhauser enhancements, from 13C chemical shift deviations, and from observation of slowly exchanging amide hydrogens. The secondary structure contains one alpha-helix and eight beta-strands, seven of which are arranged in two contiguous, antiparallel beta-sheets. The structure is monomeric, in contrast to the well-defined intimate dimerization of the crystal structure of this molecule. Residues possibly involved in ligand binding are in apparently flexible loops. Steady-state 15N(1H) nuclear Overhauser effect measurements indicate unequivocally the boundaries of this PH domain, and the structured portion of the domain appears to be more extended to the C terminus than previously suggested for other PH domains.

Amino Acid Sequence↗

PubMed1994

Radiation dose-dependent changes in tumor metabolism measured by 31P nuclear magnetic resonance spectroscopy.

The effects of radiation dose upon a hypoxic murine mammary carcinoma were followed using 31P nuclear magnetic resonance spectroscopy. Animals were studied before and over the course of 9 days after tumors were irradiated with a single dose of 0, 4, 8, or 17 Gy. The current data is compared to our previous studies of the effects of 32 or 65 Gy on the same tumor model. The energy status of the tumors, as reflected in nucleotide triphosphate:Pi and phosphocreatine:Pi ratios, improved after receiving a dose of 8 to 65 Gy and decreased after receiving 0 or 4 Gy doses. The energy status of the 8- to 65-Gy dose cohorts reached a maximum between 1 and 4 days after irradiation. Additionally, the change in the hypoxic cell fraction 48 h after a 17-Gy dose was determined; it was calculated from changes in the doses required to control 50% of the tumors post radiation for clamped (hypoxic) and unclamped (normoxic) tumors in parallel animal cohorts. A significant decrease compared to preirradiation values was observed in the hypoxic cell fraction following 17 Gy irradiation. This decrease was temporally coincident with increases in tumor energy status measured using nuclear magnetic resonance and was similar to our previously reported results of the change in hypoxic fraction 48 h after a 32-Gy dose. Changes in the relative ratio of phosphomonoesters showed a strong dose dependence after irradiation. The downfield component of the phosphomonoester peak, which consists largely of phosphoethanolamine, increased relative to the upfield component, phosphocholine. This dose-dependent ratio reached a maximum approximately 7 days post radiation. Changes in the levels of membrane phospholipid precursors may be related to alterations in cell proliferation or may be a result of radiation-induced membrane damage.

Animals↗

PubMed1994

Interferon activation of the transcription factor Stat91 involves dimerization through SH2-phosphotyrosyl peptide interactions.

Stat91 (a 91 kd protein that acts as a signal transducer and activator of transcription) is inactive in the cytoplasm of untreated cells but is activated by phosphorylation on tyrosine in response to a number of polypeptide ligands, including interferon alpha (IFN-alpha) and IFN-gamma. We report here that the inactive Stat91 in the cytoplasm of untreated cells is a monomer and that upon IFN-gamma-induced phosphorylation it forms a stable homodimer. Only the dimer is capable of binding to a specific DNA sequence directing transcription. Through dissociation and reassociation assays, we show that dimerization of Stat91 is mediated through SH2-phosphotyrosyl peptide interactions. Dimerization involving SH2 recognition of specific phosphotyrosyl peptides may well provide a prototype for interactions among family members of STAT proteins to form different transcription complexes.

Amino Acid Sequence↗

PubMed1994

Lectin domains in the toxin of Bordetella pertussis: selectin mimicry linked to microbial pathogenesis.

The pathogenesis of many infectious diseases is critically determined by prokaryotic lectins which enable differential recognition and activation of targeted eukaryotic cells. Some bacterial adhesins mimic and co-opt eukaryotic cell-cell adhesion motifs. This is illustrated by the toxin of Bordetella pertussis. Pertussis toxin mediates intoxication of eukaryotic cells by elevation of cAMP and it serves as an adhesin binding the bacteria to ciliated cells and respiratory macrophages. These activities are mediated by the lectin-like properties of the binding oligomer of the toxin. A comparison of pertussis toxin and the selectins involved in leukocyte trafficking indicates that these prokaryotic and eukaryotic C-type lectins share some element of primary sequence similarity, three dimensional structure, and biological activities. Such mimicry suggests a link between eukaryotic cell-cell adhesion motifs and microbial pathogenesis.

Amino Acid Sequence↗

PubMed1994

Helical encounter.

Explore the source record for details and available documents.

Amino Acid Sequence↗

PubMed1993

Binding of a high affinity phosphotyrosyl peptide to the Src SH2 domain: crystal structures of the complexed and peptide-free forms.

The crystal structure of the Src SH2 domain complexed with a high affinity 11-residue phosphopeptide has been determined at 2.7 A resolution by X-ray diffraction. The peptide binds in an extended conformation and makes primary interactions with the SH2 domain at six central residues: PQ(pY)EEI. The phosphotyrosine and the isoleucine are tightly bound by two well-defined pockets on the protein surface, resulting in a complex that resembles a two-pronged plug engaging a two-holed socket. The glutamate residues are in solvent-exposed environments in the vicinity of basic side chains of the SH2 domain, and the two N-terminal residues cap the phosphotyrosine-binding site. The crystal structure of Src SH2 in the absence of peptide has been determined at 2.5 A resolution, and comparison with the structure of the high affinity complex reveals only localized and relatively small changes.

Amino Acid Sequence↗

PubMed1992

Secondary structure of Src homology 2 domain of c-Abl by heteronuclear NMR spectroscopy in solution.

The Src homology 2 (SH2) domain is a recognition motif thought to mediate the association of the cytoplasmic proteins involved in signal transduction by binding to phosphotyrosyl-containing sequences in proteins. Assignments of nearly all 1H and 15N resonances of the SH2 domain from the c-Abl protein-tyrosine kinase have been obtained from homonuclear and heteronuclear NMR experiments. The secondary structure has been elucidated from the pattern of nuclear Overhauser effects, from vicinal coupling constants, and from observation of slowly exchanging amino hydrogens. The secondary structure contains two alpha-helices and eight beta-strands, six of which are arranged in two contiguous, antiparallel beta-sheets. Residues believed to be involved in phosphotyrosyl ligand binding are on a face of one beta-sheet. The alignment of homologous sequences on the basis of secondary structure suggests a conserved global fold in a family of SH2 domains.

Amino Acid Sequence↗

PubMed1992

Quantitative changes in tumor metabolism, partial pressure of oxygen, and radiobiological oxygenation status postradiation.

Hypoxia is considered to be a major cause of tumor radioresistance. Reoxygenation of previously hypoxic areas after a priming dose of radiation is associated with an increase in tumor radiosensitivity. In a study of a hypoxic mammary carcinoma, 31P nuclear magnetic resonance spectra showed statistically significant increases in metabolite ratios (phosphocreatine/Pi and nucleotide triphosphate/Pi) after 65 and 32 Gy. The maximum changes in metabolite ratios after 32 Gy occurred at 48 h, although significant changes were detected at 24 h. A corresponding increase in the mean tumor pO2 (polarographic microelectrode measurements) and a decrease in hypoxic cell fraction [changes in paired (clamped versus unclamped) tumor control dose for 50% of tumors] were also shown to occur 48 h after a priming dose of 32 Gy. A significant increase in the mean tumor pO2, phosphocreatine/Pi, and nucleotide triphosphate/Pi, compared to initial values, was noted at 24, 48, and 96 h post 65-Gy radiation. An increase in the downfield component of the phosphomonoester peak relative to the upfield component (phosphoethanolamine), is also noted after doses of 65 and 32 Gy. These are likely to be due to cell kill and/or decreased cell proliferation. In this tumor model, 31P nuclear magnetic resonance spectroscopic changes postradiation are temporally coincident with and may be indicative of tumor reoxygenation as measured by the tumor control dose for 50% of tumors and oxygen-sensitive microelectrodes.

Animals↗

PubMed1992

Three-dimensional solution structure of the src homology 2 domain of c-abl.

SH2 regions are protein motifs capable of binding target protein sequences that contain a phosphotyrosine. The solution structure of the abl SH2 product, a protein of 109 residues and 12.1 kd, has been determined by multidimensional nuclear magnetic resonance spectroscopy. It is a compact spherical domain with a pair of three-stranded antiparallel beta sheets and a C-terminal alpha helix enclosing the hydrophobic core. Three arginines project from a short N-terminal alpha helix and one beta sheet into the putative phosphotyrosine-binding site, which lies on a face distal from the termini. Comparison with other SH2 sequences supports a common global fold and mode of phosphotyrosine binding for this family.

Amino Acid Sequence↗

PubMed1992

Crystal structure of the phosphotyrosine recognition domain SH2 of v-src complexed with tyrosine-phosphorylated peptides.

Three-dimensional structures of complexes of the SH2 domain of the v-src oncogene product with two phosphotyrosyl peptides have been determined by X-ray crystallography at resolutions of 1.5 and 2.0 A, respectively. A central antiparallel beta-sheet in the structure is flanked by two alpha-helices, with peptide binding mediated by the sheet, intervening loops and one of the helices. The specific recognition of phosphotyrosine involves amino-aromatic interactions between lysine and arginine side chains and the ring system in addition to hydrogen-bonding interactions with the phosphate.

Amino Acid Sequence↗

PubMed1992

The corrected nucleotide sequences of the TaqI restriction and modification enzymes reveal a thirteen-codon overlap.

The nucleotide sequence of the genes encoding methyltransferase TaqI (M.TaqI) and restriction endonuclease TaqI (R.TaqI) with the recognition sequence, TCGA, were analyzed in clones isolated from independent libraries. The genes, originally reported as 363 and 236 codons long [Slatko et al., Nucleic Acids Res. 15 (1987) 9781-9796] were redetermined as 421 and 263 codons long, respectively. The C terminus of the taqIM gene overlaps the N terminus of the taqIR gene by 13 codons, as observed with the isoschizomeric TthHB8I restriction-modification system [Barany et al., Gene 112 (1992) 13-20]. Removal of the overlapping codons did not interfere with in vivo M.TaqI activity. We postulate the overlap plays a role in regulating taqIR expression.

Amino Acid Sequence↗

PubMed1991

Heteronuclear two-dimensional 15N- and 13C-NMR studies of DNA oligomers and their netropsin complexes using indirect proton detection.

Heteronuclear multispin coherence proton-detected two-dimensional nmr spectroscopic experiments were used to obtain information on protonated carbons and nitrogens of the self-complementary d(G-G-T-A-T-A-C-C) and d(G-G-A-A-T-T-C-C) duplexes, with and without the drug netropsin dissolved in aqueous solution. Many correlations of protons coupled to 13C nuclei on the base and sugar rings of the octanucleotides were detected, allowing the carbon resonances to be assigned based on previous homonuclear proton two-dimensional nmr studies. Imino nitrogen assignments can also be made using the proton assignments from previous one-dimensional nuclear overhauser effect experiments. Imino nitrogen shifts may be useful indicators of changes in local hydrogen-bonding interactions to base-pair edges.

Base Sequence↗

PubMed1991

In vivo monitoring of changes in 5-fluorouracil metabolism induced by methotrexate measured by 19F NMR spectroscopy.

We have used in vivo 19F NMR spectroscopy to study the metabolism of 5-fluorouracil (FUra) in tumors with and without pretreatment with methotrexate (MTX). Using the CD8F1 murine mammary tumor as an in vivo model, we observed signals from FUra, alpha-fluoro-beta-alanine (F beta ALA), alpha-fluoro-beta-ureidopropionic acid (FUPA), and 5-fluorouracil-nucleotides (FUN) after intravenous or intraperitoneal injection of 150 mg/kg FUra. Formation of FUN was increased about 1.7-fold in CD8F1 tumor with methotrexate pretreatment as determined by acid extraction and HPLC analysis. A comparison of in vivo NMR spectra from FUra and sequential MTX + FUra-treated tumors showed a significantly higher ratio of the FUN signal to the initial total 19F signal in the MTX + FUra-treated tumors (p less than 0.001) for animals receiving FUra either intravenously or intraperitoneally. In addition, tumors treated with MTX + FUra had significantly longer time durations during which FUN was detected, independent of the mode of administration. These experiments indicate that in vivo 19F NMR spectroscopy can be used to noninvasively monitor alterations of 5-fluorouracil metabolism that occur with administration of modulating agents such as methotrexate. Further studies, in both murine tumor models and patients, are indicated to determine if these results can be correlated with tumor response.

Animals↗

PubMed1990

Changes in radiation sensitization induced by Fluosol-DA as measured by 31P nuclear magnetic resonance spectroscopy.

Numerous agents have been studied in attempts to sensitize radioresistant hypoxic tumor cells. We have investigated the effect of Fluosol-DA plus carbogen (95% oxygen and 5% CO2) on the sensitivity of a radioresistant mammary carcinoma in C3H/He mice and also on tumor metabolism by 31P nuclear magnetic resonance spectroscopy. Statistically significant increases in phosphocreatine/Pi were noted for small- (150-350 mm3) and medium- (351-650 mm3) sized tumors treated with Fluosol-DA plus carbogen. Small tumors were shown to undergo significant radiosensitization in the presence of Fluosol-DA plus carbogen and medium-sized tumors showed a lesser degree of radiosensitization. Large tumors (greater than 900 mm3) showed no effect. Fluosol-DA or carbogen alone had no effects on animals with any tumor volume, as monitored by significant changes in radiosensitivity or nuclear magnetic resonance parameters. An approximately linear relationship was found between the decrease in the values for radiation dose which yields 50% tumor control and the increase in phosphocreatine/Pi, with a correlation of r = -0.93. 31P nuclear magnetic resonance spectroscopy may be useful for monitoring changes in radiosensitivity induced by agents which alter tumor oxygenation and subsequent metabolic status.

Animals↗

PubMed1990

The binding of ATP and AMP to Escherichia coli adenylate kinase investigated by 1H and 15N NMR spectroscopy.

[N6 15N]ATP and [N6 15N]AMP, complexed with E.coli adenylate kinase (AKe), were observed with 15N isotope-filtered NMR pulse sequences and 1H[15N] heterocorrelated experiments to determine differences between binding sites based on chemical shifts and competition by substrate analogs. The chemical shifts of the N6 amino proton and nitrogen signals changed significantly after mixing with adenylate kinase. Differences in chemical shifts between the bound ATP and AMP signals are slight. The response of these shifts to further addition of other substrates or Mg2+ supports the view that the unchelated nucleotides can bind to both the sites, whereas the metal complexed species are restricted to the MgATP/MgADP binding site.

Adenosine Monophosphate↗

PubMed1990

Relationship of changes in pH and energy status to hypoxic cell fraction and hyperthermia sensitivity.

The relative concentrations of nucleotide triphosphates, creatine phosphate, inorganic phosphate, and pH have been evaluated as a function of tumor volume in a murine fibrosarcoma (FSaII) by 31P NMR spectroscopy. As the tumor volume increased from 60-1250 mm3, the ratio of phosphocreatine to inorganic phosphate systemically decreased. This decrease paralleled a decrease in the ratio of nucleotide triphosphate to inorganic phosphate in the same tumor volume range. The tumor pH as measured by 31P NMR decreased slightly with tumor growth. A pH of 7.17 +/- 0.07 (n = 17) was found for tumors between 60 and 150 mm3, whereas for tumors greater than 900 mm3, a pH of 7.05 +/- .03 (n = 6) was noted. Intermediate size tumors (151-900) had a pH of 7.12 +/- 0.09 (n = 18). The change in tumor energy status with tumor volume inversely paralleled the change in tumor radiobiologic hypoxic cell fraction and suggested a causal relationship between tumor nutrient status and energy status. Tumor thermal sensitivity also increased with tumor volume, suggesting a relationship between pH, energy status, and thermal sensitivity, as has been demonstrated under in vitro conditions. Each NMR parameter was found to correlate significantly with tumor volume independent of the other NMR parameters.

Adenosine Triphosphate↗

PubMed1989

Observation of arginyl-deoxyoligonucleotide interactions in Taq I endonuclease by detection of specific 1H NMR signals from 140kD [N eta 1, N eta 2, 15N Arg]Taq I/oligomer complexes.

Proton and nitrogen signals of the guanidinium amines in [N eta 1, N eta 2 15N Arg]Taq I endonuclease were observed using isotope filtered experiments and proton detected 1H[15N] heterocorrelated two dimensional NMR spectroscopy. These rapidly exchanging protons could be detected in the free enzyme only at pH 4.5; at pH 8.5, no signals were measured after extensive signal averaging. Addition of deoxyribonucleotide oligomers resulted in the appearance of two groups of signals at about 6.8 and 7.5 ppm. Since these signals are independent of the presence of cognate sequence or Mg2+, it is assumed they represent nonspecific arginyl-DNA interactions. This labeling/NMR approach provides a new method for investigating the role of arginine in protein-DNA interactions.

Arginine↗

PubMed1988

13C NMR of the bases of three DNA oligonucleotide duplexes: assignment methods and structural features.

Natural abundance 13C NMR spectra of three DNA oligomers have been obtained. Most of the base resonances are well resolved from one another. A combination of two independent methods was used in making assignments: a one-dimensional spectral comparison method and a two-dimensional proton-detected 1H-13C correlated experiment for the protonated carbons. There are large shielding changes (between 1.62 and -1.40 ppm) upon thermal dissociation of the duplex. The shapes of the chemical shift vs temperature curves are largely independent of sequence. The base carbon resonance frequencies are sensitive to hydrogen bonding, base stacking, sugar conformation, and changes in the glycosyl torsion angle.

Base Composition↗