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Biomedical subjects

L R Brown

Publications and source records attributed to L R Brown.

At least 19 recordsLinked to original sources
PubMed2001

Radiographic measurements of cardiac size as predictors of outcome in patients with dilated cardiomyopathy.

BACKGROUND: Cardiac dilatation is a predictor of poor outcome in patients with dilated cardiomyopathy. Whereas cardiac chamber dimensions or volumes can be assessed by various noninvasive and invasive techniques, simple chest radiography also may provide a valuable assessment of cardiac size. METHODS AND RESULTS: To determine the relative power of radiographic heart measurements for predicting outcome in dilated cardiomyopathy, we retrospectively studied 88 adult patients with chest radiographs obtained within 35 days of echocardiography. Standard radiographic variables were measured for each patient, and the cardiothoracic (CT) ratio, frontal cardiac area, and volume were calculated. During a mean 4.1-year follow-up, 62 of the 88 (71%) patients died. CT ratio was the best predictor of mortality among the radiographic cardiac measurements. By multivariate analysis, a model including echocardiographic ejection fraction, New York Heart Association (NYHA) functional class, and history of heart failure was highly predictive of survival. When added to this model, CT ratio also was independently associated with mortality, but not radiographic cardiac area or volume. When radiographic variables were each added to CT ratio, they did not add incremental predictive value to the model that included CT ratio alone. Echocardiographic measurement of left ventricular (LV) size, especially when indexed for body size, was independently predictive of outcome, but it did not supersede the predictive power of CT ratio. CONCLUSION: The simply derived radiographic CT ratio is a useful predictor of outcome in patients with dilated cardiomyopathy and compares favorably with other clinical and selected echocardiographic variables.

Adolescent↗
PubMed2001

How water scarcity will shape the new century.

Water resources are increasingly being overexploited, such that current food production, which relies heavily on irrigation schemes, is unsustainable. Many steps, including improved irrigation techniques, more water-efficient crops and animal protein production, etc., will be needed to raise water productivity across the board. Water must in future be recognised as a scarce resource and not taken for granted.

Agriculture↗
PubMed2000

Dominant sensitization variants of human O(6)-methylguanine-DNA-methyltransferase obtained by a mutational screen of surface residues.

A scanning mutagenesis experiment was performed on human O(6)-methylguanine methyltransferase (hMGMT), directed largely at non-conserved surface residues that have not previously been studied. Variants typically contained two or more substitutions. Two of the 16 variants characterized in detail are inactive for methyltransfer, but increase the cytotoxicity and mutagenic effects of methylating agents. This phenotype is reminiscent of a variant (C145A) that has a mutation in the methyl-accepting cysteine. C145A is inactive, but reportedly binds methylated DNA and confers sensitivity to methylating agents. The sensitization phenotype of the two new variants is more striking in strains that are wild-type for DNA repair than in strains that are deficient for repair, suggesting that these proteins inhibit functional DNA repair proteins by competitively binding to methylated DNA. Both variants have multiple substitutions in the last helix of the protein. These results suggest that the C-terminal helix is necessary for methyltransfer activity, but not for methylguanine-specific binding.

Amino Acid Sequence↗
PubMed2000

The NMR solution structure of the ion channel peptaibol chrysospermin C bound to dodecylphosphocholine micelles.

Chrysospermin C is a 19-residue peptaibol capable of forming transmembrane ion channels in phospholipid bilayers. The conformation of chrysospermin C bound to dodecylphosphocholine micelles has been solved using heteronuclear NMR spectroscopy. Selective 15N-labeling and 13C-labeling of specific alpha-aminoisobutyric acid residues was used to obtain complete stereospecific assignments for all eight alpha-aminoisobutyric acid residues. Structures were calculated using 339 distance constraints and 40 angle constraints obtained from NMR data. The NMR structures superimpose with mean global rmsd values to the mean structure of 0. 27 A (backbone heavy atoms) and 0.42 A (all heavy atoms). Chrysospermin C bound to decylphosphocholine micelles displays two well-defined helices at the N-terminus (residues Phe1-Aib9) and C-terminus (Aib13-Trp-ol19). A slight bend preceding Pro14, i.e. encompassing residues 10-12, results in an angle of approximately 38 degrees between the mean axes of the two helical regions. The bend structure observed for chrysospermin C is compatible with the sequences of all 18 long peptaibols and may represent a common 'active' conformation. The structure of chrysospermin C shows clear hydrophobic and hydrophilic surfaces which would be appropriate for the formation of oligomeric ion channels.

Amino Acid Sequence↗
PubMed2000

Use of sublimation to prepare solid microbial media with water-insoluble substrates.

A method was developed to deposit a visible layer of water-insoluble compounds via sublimation onto the surface of solid media. The compound is sublimed from a heated aluminum dish containing the compound onto the surface of an inverted, ice-cooled, inoculated agar petri dish. The method results in the deposition of a thin, even layer on the agar surface without the use of solvent. After incubation, clearing zones around colonies indicate the presence of compound-degrading microorganisms.

Anthracenes↗
PubMed1999

The NMR structure of the 5S rRNA E-domain-protein L25 complex shows preformed and induced recognition.

The structure of the complex between ribosomal protein L25 and a 37 nucleotide RNA molecule, which contains the E-loop and helix IV regions of the E-domain of Escherichia coli 5S rRNA, has been determined to an overall r.m.s. displacement of 1.08 A (backbone heavy atoms) by heteronuclear NMR spectroscopy (Protein Databank code 1d6k). The interacting molecular surfaces are bipartite for both the RNA and the protein. One side of the six-stranded beta-barrel of L25 recognizes the minor groove of the E-loop with very little change in the conformations of either the protein or the RNA and with the RNA-protein interactions occurring mainly along one strand of the E-loop duplex. This minor groove recognition module includes two parallel beta-strands of L25, a hitherto unknown RNA binding topology. Binding of the RNA also induces conversion of a flexible loop to an alpha-helix in L25, the N-terminal tip of which interacts with the widened major groove at the E-loop/helix IV junction of the RNA. The structure of the complex reveals that the E-domain RNA serves as a preformed docking partner, while the L25 protein has one preformed and one induced recognition module.

Amino Acid Sequence↗
PubMed1999

Direct identification of NH...N hydrogen bonds in non-canonical base pairs of RNA by NMR spectroscopy.

It is shown that the recently developed quantitative J(NN)HNN-COSY experiment can be used for the direct identification of hydrogen bonds in non-canonical base pairs in RNA. Scalar(2h)J(NN)couplings across NH.N hydrogen bonds are observed in imino hydrogen bonded GA base pairs of the hpGA RNA molecule, which contains a tandem GA mismatch, and in the reverse Hoogsteen AU base pairs of the E-loop of Escherichia coli 5S rRNA. These scalar couplings correlate the imino donor(15)N nucleus of guanine or uridine with the acceptor N1 or N7 nucleus of adenine. The values of the corresponding(2h)J(NN)coupling constants are similar in size to those observed in Watson-Crick base pairs. The reverse Hoogsteen base pairs could be directly detected for the E-loop of E.coli 5S rRNA both in the free form and in a complex with the ribosomal protein L25. This supports the notion that the E-loop is a pre-folded RNA recognition site that is not subject to significant induced conformational changes. Since Watson-Crick GC and AU base pairs are also readily detected the HNN-COSY experiment provides a useful and sensitive tool for the rapid identification of RNA secondary structure elements.

Base Pair Mismatch↗
PubMed1999

Triple-resonance experiments for correlation of H5 and exchangeable pyrimidine base hydrogens in (13)C,(15)N-labeled RNA.

Triple-resonance two-dimensional H5(C5C4N)H experiments are described that provide through-bond H5 to imino/amino connectivities in uridines and cytidines in (13)C, (15)N-labeled RNAs. The experiments employ selective INEPT steps for transferring magnetization from the H5 hydrogens through the intervening C5, C4, and N3/N4 nuclei to the imino/amino hydrogens. The improved sensitivity of these experiments for assignments in a large 43-nucleotide RNA is demonstrated.

Carbon Isotopes↗
PubMed1998

The NMR structure of Escherichia coli ribosomal protein L25 shows homology to general stress proteins and glutaminyl-tRNA synthetases.

The structure of the Escherichia coli ribosomal protein L25 has been determined to an r.m.s. displacement of backbone heavy atoms of 0.62 +/- 0.14 A by multi-dimensional heteronuclear NMR spectroscopy on protein samples uniformly labeled with 15N or 15N/13C. L25 shows a new topology for RNA-binding proteins consisting of a six-stranded beta-barrel and two alpha-helices. A putative RNA-binding surface for L25 has been obtained by comparison of backbone 15N chemical shifts for L25 with and without a bound cognate RNA containing the eubacterial E-loop that is the site for binding of L25 to 5S ribosomal RNA. Sequence comparisons with related proteins, including the general stress protein, CTC, show that the residues involved in RNA binding are highly conserved, thereby providing further confirmation of the binding surface. Tertiary structure comparisons indicate that the six-stranded beta-barrels of L25 and of the tRNA anticodon-binding domain of glutaminyl-tRNA synthetase are similar.

Amino Acid Sequence↗
PubMed1998

Nuclear magnetic resonance solution structure of the plasminogen-activator protein staphylokinase.

Staphylokinase, a 15.5 kDa protein from Staphylococcus aureus, is a plasminogen activator which is currently undergoing clinical trials for the therapy of myocardial infarction and peripheral thrombosis. The three-dimensional (3D) NMR solution structure has been determined by multidimensional heteronuclear NMR spectroscopy on uniformly 15N- and 15N,13C-labeled samples of staphylokinase. Structural constraints were obtained from 82 3JHNH alpha as well as 22 3JNH beta scalar coupling constants and 2345 NOE cross-peaks, derived from 15N-edited and 13C-edited 3D NOE spectra. NOE cross-peak assignments were confirmed by analysis of ¿15N,13C¿-edited and ¿13C,13C¿-edited 4D NOE spectra. The structure is presented as a family of 20 conformers which show an average rmsd of 1.02 +/- 0.15 A from the mean structure for the backbone atoms. The tertiary structure of staphylokinase shows a well-defined global structure consisting of a central 13-residue alpha-helix flanked by a two-stranded beta-sheet, both of which are located above a five-stranded beta-sheet. Two of the connecting loops exhibit a higher conformational heterogeneity. Overall, staphylokinase shows a strong asymmetry of hydrophilic and hydrophobic surfaces. The N-terminal sequence, including Lys10 which is the site of the initial proteolytic cleavage during activation of plasminogen, folds back onto the protein core, thereby shielding amino acids with functional importance in the plasminogen activation process. From a comparison of the structure with mutational studies, a binding region for plasminogen is proposed.

Crystallography, X-Ray↗
PubMed1998

Advanced multiple beam equalization radiography: receiver operating characteristic comparison with screen-film chest radiography.

OBJECTIVE: To test the hypothesis that the advanced multiple beam equalization radiography (AMBER) imaging system is superior to conventional chest radiography in the demonstration of diffuse infiltrative lung disease, emphysema, pulmonary nodules, calcification within nodules, and mediastinal or hilar masses and lymphadenopathy. MATERIAL AND METHODS: The study involved 115 patients, each of whom underwent chest computed tomography (CT), AMBER, posteroanterior chest radiography, and conventional posteroanterior stereoscopic chest radiography (two films). All radiographs were obtained with the InSight Thoracic Imaging System. Four chest radiologists independently analyzed the 115 AMBER studies, 115 unpaired single conventional radiographs (a single film from a stereoscopic pair), and 115 stereoscopic conventional radiographs (2 films) for the presence of diffuse infiltrative lung disease, emphysema, pulmonary nodules, calcification within nodules, and mediastinal or hilar masses and lymphadenopathy. For each abnormality detected, the radiologists described their level of confidence based on a scale of 1 to 5. The 115 CT examinations were interpreted by consensus among 3 of the chest radiologists. The CT results were considered the standard. Receiver operating characteristic (ROC) techniques were used for statistical analysis. RESULTS: No statistically significant differences were found with ROC techniques between the AMBER system and single or stereoscopic conventional screen-film radiography for the abnormalities studied. CONCLUSION: We noted no clinically significant difference between AMBER and either single or stereoscopic conventional screen-film radiography in this prospective study of 115 patients in which CT (performed within 1 week of both radiographic examinations) was the standard.

Humans↗
PubMed1998

A solution NMR study of the selectively 13C, 15N-labeled peptaibol chrysospermin C in methanol.

The conformation of the 19-residue peptaibol chrysospermin C in methanol has been investigated by NMR spectroscopy using selective 15N and 13C labeling of the alpha-aminoisobutyric acid (Aib) residues. Complete 1H and 13C sequential assignments, including stereospecific assignments for the heavily overlapped resonances from the two Cbeta methyl groups of the eight Aib residues, are reported for a peptaibol for the first time. An Aib residue followed by a Pro is an exception to previous suggestions regarding stereospecific assignment of the two Cbeta methyl groups of Aib residues. Local nuclear Overhauser effects and 3J(HNC') and 3J(HNCbeta) scalar couplings indicate that the phi angles of the Aib residues are restricted sterically to local conformations consistent with right-handed helices. Despite these constraints on the eight Aib residues, the NMR data for chrysospermin C in methanol are generally most consistent with an ensemble of transient conformations, including backbone conformations inconsistent with helical structures. Initial NMR measurements for chrysospermin C bound to micelles suggest structural and dynamic differences relative to alamethicin bound to micelles which may be related to differences in gating voltages for formation of ion channels.

Amino Acid Sequence↗
PubMed1998

Detection of subtle abnormalities on chest radiographs after irreversible compression.

PURPOSE: To assess the effect of wavelet-based compression of posteroanterior chest radiographs on detection of small uncalcified pulmonary nodules and fibrosis. MATERIALS AND METHODS: Computed tomography (CT) of the chest was used to identify 20 patients with normal posteroanterior chest radiographs, 20 with a solitary uncalcified pulmonary nodule 1-2 cm in diameter, and 20 with fibrotic disease. A double-blind protocol for readings of original images and images compressed at 40:1 and 80:1 was analyzed by using the nonparametric receiver operating characteristic to measure differences in diagnostic accuracy and their statistical significance. RESULTS: There was no substantial difference in the overall diagnostic accuracy (measured by the area under the curve index) for both nodules and fibrosis between images compressed at 40:1 and 80:1 and uncompressed images. Readers tended to perform better on images compressed at 40:1 compared with uncompressed images. The "high-sensitivity" portion of the 80:1 compression curve for nodules was below that for the uncompressed curve, although this was not statistically significant. CONCLUSION: Lossy compression of chest radiographs at 40:1 can be used without decreased diagnostic accuracy for detection of pulmonary nodules and fibrosis. There is no statistically significant difference in diagnostic accuracy at 80:1 compression, but detection ability is decreased.

Algorithms↗
PubMed1997

Structure of an RNA hairpin loop with a 5'-CGUUUCG-3' loop motif by heteronuclear NMR spectroscopy and distance geometry.

Structural features of a 19-nucleotide RNA hairpin loop (5'-GGCGUACGUUUCGUACGCC-3'), a loop motif which occurs in eukaryotic 18S rRNA, have been derived using multidimensional heteronuclear NMR spectroscopy in combination with local conformational analysis and torsion angle distance geometry followed by restrained energy minimization. A method to obtain both the 3JC4'P3' and 3JC4'P5' coupling constants from a set of spin-echo difference constant time HSQC spectra is introduced, and it is shown how these couplings can be assigned to the backbone angles beta and epsilon. A total of 280 distance constraints as well as 132 homo- and heteronuclear three-bond scalar coupling constants were derived from the NMR data. The structure which has been determined is a pentaloop rather than a triloop with no base pairing between G8 and C12. G8 is pointed to the minor groove where it forms a base triplet with C7-G13 that is further stabilized by hydrogen bonding to the 2'-hydroxyl group of C7. C12 is directed to the major groove where its conformation is stabilized by hydrogen bonding between O2 and HO2'. The NMR data suggest two possible, interconverting conformations with stacking of bases U10-G8 or U11-C7. Overall, the loop provides a variety of interaction sites for RNA or protein interactions.

Carbon Isotopes↗
PubMed1997

An NMR study of the interaction of 15N-labelled bradykinin with an antibody mimic of the bradykinin B2 receptor.

An isotope-edited NMR study of the peptide hormone bradykinin (RPPGFSPFR) bound to the Fab fragment of a monoclonal antibody against bradykinin (MBK3) is reported. MBK3 was previously shown to provide a binding site model of the B2 bradykinin receptor [Haasemann, M., Buschko, J., Faussner, A., Roscher, A. A., Hoebeke, J., Burch, R. M. & Muller-Esterl, W. (1991) Anti-idiotypic antibodies bearing the internal image of a bradykinin epitope, J. Immunol. 147, 3882-3892]. Bradykinin was obtained in a uniformly 15N-labelled form using recombinant expression of a fusion protein consisting of the glutathione-binding domain of glutathione S-transferase fused to residues 354-375 of the high-molecular-mass kininogen from which bradykinin was released by proteolytic digestion with its natural protease plasma kallikrein. Bradykinin forms a complex with the Fab fragment of MBK3 which exchanges slowly on the NMR time scale. The 15N and 1H resonances of the tightly bound residues of bradykinin show appreciable changes in chemical shift with respect to the free form, while the 15N and 1H linewidths indicate that the hydrodynamic behaviour of bound bradykinin is dominated by the high-molecular-mass Fab fragment. The NMR data indicate that essentially the entire nonapeptide is involved in binding. The kinetics of the ligand-exchange process, together with resonance assignments obtained via exchange spectroscopy. indicate that bradykinin binds to MBK3 only in the all-trans conformation at all three Xaa-Pro amide bonds. NH-NH NOE connectivities suggest that bradykinin is bound in an extended conformation. The spectroscopic data obtained from this study are compared to recently proposed computational models of the conformation of bradykinin bound to the B2 receptor.

Amino Acid Sequence↗
PubMed1997

Construction and overexpression of a synthetic gene for human DNA methylguanine methyltransferase: renaturation and rapid purification of the protein.

A synthetic gene was constructed that encodes human DNA methylguanine methyltransferase (hMGMT). The synthetic gene was designed with a number of unique restriction sites to facilitate cassette mutagenesis and to reflect the preferences found among genes in Escherichia coli. Both the full-length gene and a gene for a functional variant (hMGMT delta C) that lacks the C-terminal 28 codons were constructed, and the genes were overexpressed using a T7 RNA polymerase promoter. The proteins are made in the form of insoluble aggregates but the truncated form of the protein (hMGMT delta C) has been successfully denatured, renatured, and purified to near homogeneity by ion exchange. Methyltransferase activity assays of hMGMT delta C demonstrate that the reconstituted protein has substantial DNA repair activity, though somewhat less than full-length hMGMT that had been expressed and purified in a soluble form. Mass spectrometry of a mixture of proteolytic fragments confirmed the protein sequence and indicated no detectable oxidation of the active site cysteine. The protein was determined to be monomeric by gel filtration chromatography, and circular dichroism spectra for renatured hMGMT delta C and fully soluble hMGMT are consistent with the renatured protein preparation being fully folded. Refolded hMGMT delta C had a curious propensity to form large aggregates in a time-dependent manner when injected into a dynamic light scattering instrument; this aggregation behavior was not observed for hMGMT purified in a soluble form. Differences in susceptibility to aggregation may account for differences in methyltransfer activity. Yields of purified protein were approximately 5 mg/liter of culture.

Amino Acid Sequence↗
PubMed1997

Evaluation of irreversible compression of digitized posterior-anterior chest radiographs.

The purpose of this article is to assess lossy image compression of digitized chest radiographs using radiologist assessment of anatomic structures and numerical measurements of image accuracy. Forty posterior-anterior (PA) chest radiographs were digitized and compressed using an irreversible wavelet technique at 10, 20, 40, and 80:1. These were presented in a blinded fashion with an uncompressed image for A-B comparison of 11 anatomic structures as well as overall quality assessments. Mean error, root-mean square (RMS) error, maximum pixel error, and number of pixels within 1% of original value were also computed for compression ratios from ratios from 5:1 to 80:1. We found that at low compression (10:1) there was a slight preference for compressed images. There was no significant difference at 20:1 and 40:1. There was a slight preference on some structures for the original compared with 80:1 compressed images. Numerical measures showed high image faithfulness, both in terms of number of pixels that were within 1% of their original value, and by the average error for all pixels. Our findings suggest that lossy compression at 40:1 or more can be used without perceptible loss in the representation of anatomic structures. On this finding, we will do a receiver-operator characteristic (ROC) analysis of nodule detection in lossy compressed images using 40:1 compression.

Artifacts↗