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Biomedical subjects

R B Thompson

Publications and source records attributed to R B Thompson.

At least 37 records · Page 2Linked to original sources
PubMed2000

Zinc biosensing with multiphoton excitation using carbonic anhydrase and improved fluorophores.

Previously, we had shown that the zinc-dependent binding of certain fluorescent aryl sulfonamide inhibitors could be used with apocarbonic anhydrase II to transduce the level of free zinc as a change in the fluorescence of the inhibitor. While inhibitors such as dansylamide, ABD-M, and ABD-N made possible quantitation of free zinc in the picomolar range with high selectivity, they have only modest absorbance which limits their utility. We describe here the synthesis and properties of two new probes, Dapoxyl sulfonamide and BTCS, and their use in zinc biosensing. Dapoxyl sulfonamide exhibits a dramatic increase and blue shift in its emission upon binding to holocarbonic anhydrase II, as well as a 20-fold increase in lifetime: it is thus well suited for quantitating free Zn(II) down to picomolar ranges. The anisotropy of BTCS increases fivefold upon binding to the holoprotein, making this probe well suited for anisotropy-based determination of zinc. BTCS and ABD-N are efficiently excited with two photon excitation using 1.5 ps pulses from a titanium sapphire laser, and exhibit the increased zinc-dependent anisotropy response anticipated on the basis of photoselection.

Anisotropy↗
PubMed1999

Selectivity and sensitivity of fluorescence lifetime-based metal ion biosensing using a carbonic anhydrase transducer.

A key performance criterion for metal ion determinations in complex media like serum, cytoplasm of the cell, and sea water is selectivity: the ability to determine the analyte(s) of interest, in the presence of relatively high concentrations of interferents. Cu(II), Zn(II), Cd(II), Co(II), and Ni(II) may be determined by changes they induce in the fluorescence lifetime and intensity of site-specifically labeled fluorescent variants of apocarbonic anhydrase II. Free metal ion concentrations in the picomolar range (for Cu(II) and Zn(II)) and the nanomolar range (for Cd(II), Co(II), and Ni(II)) were determined, based on the affinity of the apoenzyme for these ions. Mg(II) at 50 mM and Ca(II) at 10 mM produced no effect. By the use of different fluorescent labels, transducers were made which responded well to Cu(II), Co(II), and Ni(II), but not to Zn(II) and Cd(II), and vice versa.

Biosensing Techniques↗
PubMed1999

Sources of variability in the response of coupled spins to the PRESS sequence and their potential impact on metabolite quantification.

Using a numerical method of solving the equation of motion of the density matrix, an evaluation is presented of the sources of the marked variability in the response to the point resolved spectroscopy (PRESS) pulse sequence of coupled proton spin systems. The consequences of an inappropriate 180 degrees pulse design and of the limitations on radiofrequency power are demonstrated for a weakly coupled example, lactate. The dominating role of strong coupling, which is present in most brain metabolites, is demonstrated for glutamate, in which 160 terms in the density operator were tracked to monitor the gross changes in lineshape and signal intensity as a function of the two echo times. The predictions of the numerical solutions were confirmed by experiments on phantoms of aqueous metabolite solutions.

Brain↗
PubMed1999

Residual dipolar coupling of the Cr/PCr methyl resonance in resting human medial gastrocnemius muscle.

This paper presents a detailed analysis of the TE dependence of the creatine methyl proton signal at 3.02 ppm, resulting from a symmetric PRESS sequence applied to the resting human gastrocnemius muscle. The analysis shows that a two-component decay of the central peak of the dipolar-coupled-methyl triplet should be interpreted as the superposition of a rapid ( approximately 34 msec) dipolar dephasing and a less rapid ( approximately 162 msec) transverse relaxation. These data do not support a two-pool hypothesis for TE dependence of this signal. Magn Reson Med 42:421-424, 1999.

Algorithms↗
PubMed1999

Metastasis correlates with production of vascular endothelial growth factor in a murine model of human Wilms' tumor.

BACKGROUND/PURPOSE: The growth and spread of solid tumors are critically dependent on the induction of angiogenesis. We hypothesized that vascular endothelial growth factor (VEGF) would be detected in Wilms' tumors, and that both growth and metastasis would parallel VEGF levels in a murine model. METHODS: Primary tumors were established in the right kidneys of nude mice (n = 21). Mice were killed at 3, 4.5, or 6 weeks. Tumor-bearing and control kidneys were subjected to enzyme-linked immunosorbent assay (ELISA) for VEGF. Representative sections were assessed by histology and immunohistochemistry. Lungs were examined for metastases. Clinical specimens of Wilms' tumor (n = 12) also were assayed for VEGF. RESULTS: The authors detected VEGF by ELISA with increasing frequency, and in increasing quantity, as experimental Wilms' tumors were grown over time. Immunohistochemistry demonstrated accumulation of VEGF in areas of viable tumor. Lung metastases occurred in 8 of 10 animals with VEGF-positive tumors, but in only 3 of 11 animals with VEGF-negative tumors, an association that was statistically significant. VEGF was found in 10 of 12 clinical Wilms' tumor specimens tested. CONCLUSIONS: VEGF is present in both clinical and experimental Wilms' tumors. In a murine model, absolute VEGF levels increase as primary tumors grow, and VEGF production is significantly associated with tumor metastasis.

Animals↗
PubMed1999

Gender differences in preschoolers' help-eliciting communication.

Gender differences in help-eliciting communication and the relationship of such utterances with ability were explored. A sample of 71 preschoolers (38 boys, 33 girls; mean age 4 years 3 months) were videotaped as they solved a difficult puzzle. Spontaneous talk was analyzed for orientation (to whom or to what an utterance referred) and for the frequency of utterances coded as help eliciting. Significant main effects for gender were observed, with more frequent help-eliciting utterances (HEUs) made by the girls than by the boys, particularly HEUs about themselves (self-disclosing). Although the girls' HEUs were not predictive of ability on the puzzle, the boys' were. No gender differences in puzzle-solving ability were observed. Findings are discussed with regard to problem solving and social/linguistic development.

Child↗
PubMed1999

Thirteen-week subchronic rat inhalation toxicity study with a recovery phase of trivalent chromium compounds, chromic oxide, and basic chromium sulfate.

The toxicity of trivalent chromium compounds; chromic oxide and basic chromium sulfate, was investigated in rats in a 13-week nose-only inhalation study that included a 13-week recovery period. Nose-only exposures to insoluble chromic oxide dust at 4.4, 15, or 44 mg/m3 or soluble basic chromium sulfate dust at 17, 54, or 168 mg/m3 (trivalent chromium equivalent concentrations of 3, 10, and 30 mg/m3) were carried out for 6 h/day, 5 days/week. No compound-related mortality occurred. General toxic effects, only observed with high-exposure levels of basic chromium sulfate, included sporadic signs of labored breathing and depressed body weights. No apparent compound-related effects were noted for sperm motility or morphology, for any concentration of either test material. Bronchoalveolar lavage fluid evaluations showed test material in mononuclear cells with chromic oxide and increased neutrophils, protein, lactic dehydrogenase and cellular debris with basic chromium sulfate. The principle effects for both materials were primarily to the respiratory tract. Chromic oxide caused pathological changes in the bronchial and mediastinal lymphatic tissue and lungs, consisting of the presence of pigment-laden macrophages, lymphoid and septal hyperplasia, and interstitial inflammation similar to that observed with other inert dusts. Basic chromium sulfate produced more severe and widespread effects in the nasal cavity, larynx, lungs, and mediastinal lymph node. Effects were characterized by accumulation of foreign material, infiltration of alveolar macrophages, septal cell hyperplasia, and granulomatous and chronic inflammation. Pigment was still present in chromic oxide and, to a lesser extent, in basic chromium sulfate-treated animals after the 13-week recovery period, with partial recovery of the pathological lesions. A NOAEL was not established for either test material, but 4.4 mg/m3 was thought to be near the NOAEL level for subchronic exposure to chromic oxide. The results of this study indicate significant differences in toxicity to the respiratory tract between trivalent chromium compounds chromic oxide and basic chromium sulfate. These are likely related to differences in acidity and water solubility, rather than chromium concentration per se. This conclusion is substantiated by the lack of effect on other internal organs.

Administration, Inhalation↗
PubMed1998

Determination of picomolar concentrations of metal ions using fluorescence anisotropy: biosensing with a "reagentless" enzyme transducer.

Because of their high affinity and selectivity, metalloproteins can be used as transducers in novel sensors, i.e., biosensors, for the determination of trace levels of metal ions in solution. Here, we exploit carbonic anhydrase to determine picomolar to nanomolar concentrations of free transition metal ions by fluorescence anisotropy (polarization) in a reagentless format. Carbonic anhydrase variants engineered with a cysteine replacing a residue chosen near the active site (F131C and H64C) were covalently labeled with derivatives of benzoxadiazole sulfonamide. These labeled variants exhibited changes in anisotropy up to 0.07 upon binding free Cu(II), Co(II), and Zn(II) with apparent Kd's close to the values observed with wild-type apocarbonic anhydrase. The covalent attachment of the label has significant advantages over noncovalent labels we have described previously. Furthermore, the metal ion-dependent anisotropy changes were predictable using simple theory. The results demonstrate that free transition metal ions can be determined at trace levels in aqueous solution using inexpensive instruments.

Anisotropy↗
PubMed1998

A new multiple quantum filter design procedure for use on strongly coupled spin systems found in vivo: its application to glutamate.

A numerical procedure is outlined that is appropriate for the design of multiple quantum filter sequences targeted for the strongly coupled, multiple spin systems that occur in metabolites present in brain. The procedure uses numerical methods of solution of the density matrix equations, first, to establish the most appropriate resonance to target with the filter; second, to provide contour plots of a performance index of the filter in terms of critical sequence parameters; and third, to produce the response signals of the target and the background metabolites to the optimized filter. The procedure is exemplified for the AMNPQ spin system of the amino acid glutamate at a field strength of 3 T. The 2.3 ppm peak of the PQ multiplet of glutamate was identified as the target resonance, and the performance of the filter so derived was evaluated experimentally on phantom solutions and in human brain. These experiments clearly demonstrate that a linewidth of <or=4 Hz is required for full resolution of glutamate from glutamine at 3 T using this double quantum filter. Nevertheless, even at a linewidth of approximately 7 Hz in vivo, the 2.3 ppm peak of glutamate dominates the filter response and thereby removes a significant cause of uncertainty in measuring changes in glutamate by eliminating most of the background observed in unedited spectra obtained using PRESS or STEAM.

Brain↗
PubMed1998

Differential onset of median nerve block: randomized, double-blind comparison of mepivacaine and bupivacaine in healthy volunteers.

We have compared the delay in onset of 1% mepivacaine and 0.33% bupivacaine in different nerve fibre types in 10 volunteers undergoing median nerve blocks, in a randomized, double-blind, crossover study. Hot, cold, pinprick and light touch sensations, compound motor action potentials (CMAP), sensory nerve action potentials (SNAP) and skin temperature were recorded at 2-min intervals. Hot, cold, pinprick, light touch sensations, SNAP and CMAP were significantly inhibited, and skin temperature was significantly increased after administration of both agents. The first noticeable reduction in cold sensation was detected later after bupivacaine compared with mepivacaine, but after a delay similar to that of other nerve functions. Bupivacaine and mepivacaine inhibited SNAP and CMAP with a similar time delay to steady-state. Bupivacaine produced steady-state inhibition of hot and cold sensations significantly later than mepivacaine; nevertheless, the sequence that sensory modalities failed, with few exceptions, and the extent of anaesthesia at 40 min were similar for both agents. Our technique provides a novel, multi-modal method of comparing local anaesthetics and related agents over time.

Adult↗
PubMed1998

Use of green fluorescent protein to assess urease gene expression by uropathogenic Proteus mirabilis during experimental ascending urinary tract infection.

Proteus mirabilis, a cause of complicated urinary tract infection, expresses urease when exposed to urea. While it is recognized that the positive transcriptional activator UreR induces gene expression, the levels of expression of the enzyme during experimental infection are not known. To investigate in vivo expression of P. mirabilis urease, the gene encoding green fluorescent protein (GFP) was used to construct reporter fusions. Translational fusions of urease accessory gene ureD, which is preceded by a urea-inducible promoter, were made with gfp (modified to express S65T/V68L/S72A [B. P. Cormack et al. Gene 173:33-38, 1996]). Constructs were confirmed by sequencing of the fusion junctions. UreD-GFP fusion protein was induced by urea in both Escherichia coli DH5alpha and P. mirabilis HI4320. By using Western blotting with antiserum raised against GFP, expression level was shown to correlate with urea concentration (tested from 0 to 500 mM), with highest induction at 200 to 500 mM urea. Fluorescent E. coli and P. mirabilis bacteria were observed by fluorescence microscopy following urea induction, and the fluorescence intensity of GFP in cell lysates was measured by spectrophotofluorimetry. P. mirabilis HI4320 carrying the UreD-GFP fusion plasmid was transurethrally inoculated into the bladders of CBA mice. One week postchallenge, fluorescent bacteria were detected in thin sections of both bladder and kidney samples; the fluorescence intensity of bacteria in bladder tissue was higher than that in the kidney. Kidneys were primarily infected with single-cell-form fluorescent bacteria, while aggregated bacterial clusters were observed in the bladder. Elongated swarmer cells were only rarely observed. These observations demonstrate that urease is expressed in vivo and that using GFP as a reporter protein is a viable approach to investigate in vivo expression of P. mirabilis virulence genes in experimental urinary tract infection.

Animals↗
PubMed1998

The role of the N-acetylaspartate multiplet in the quantification of brain metabolites.

N-Acetylaspartate, whose methyl singlet is the primary magnetic resonance marker of neuronal dysfunction, also gives rise to a sequence-dependent, strongly coupled multiplet that overlaps the resonances of several other metabolites. Results are presented in this paper of a full numerical calculation of the response of the strongly coupled aspartate multiplet of N-acetylaspartate to a PRESS pulse sequence employing practical slice-selective pulses. These calculations, confirmed by experiments on phantoms, demonstrate the ability to predict the dependence of the response of strongly coupled spins on pulse design, as well as on interpulse evolutions, thereby facilitating a more rigorous comparison of the use of spectral fitting routines employed to extract metabolite concentrations on different instruments.

Aspartic Acid↗
PubMed1997

Metabolite-specific NMR spectroscopy in vivo.

An outline is presented of metabolite-specific in vivo NMR spectroscopy (particularly in brain). It reviews from a physical spectroscopist's perspective, the need for and the methods of observation of, individual metabolite resonances.

Animals↗
PubMed1997

Nonuniformity intrinsic flood artifact caused by point-source syringe needle.

UNLABELLED: The purpose of this study was to investigate the cause of a nonuniformity artifact seen on an intrinsic flood. METHODS: Intrinsic floods were acquired on three different cameras using a point source in a 1-cc syringe, with and without a needle attached. Floods were obtained with the syringe placed perpendicular and parallel to the camera face. RESULTS: A nonuniformity artifact was evident if a syringe with a needle was placed perpendicular to the crystal face. CONCLUSION: If a syringe is used as a point source during the acquisition of an intrinsic quality control flood, the needle should not be placed perpendicular to the camera face.

Artifacts↗
PubMed1996

Topoisomerase II.etoposide interactions direct the formation of drug-induced enzyme-DNA cleavage complexes.

Topoisomerase II is the target for several highly active anticancer drugs that induce cell death by enhancing enzyme-mediated DNA scission. Although these agents dramatically increase levels of nucleic acid cleavage in a site-specific fashion, little is understood regarding the mechanism by which they alter the DNA site selectivity of topoisomerase II. Therefore, a series of kinetic and binding experiments were carried out to determine the mechanistic basis by which the anticancer drug, etoposide, enhances cleavage complex formation at 22 specific nucleic acid sequences. In general, maximal levels of DNA scission (i.e. Cmax) varied over a considerably larger range than did the apparent affinity of etoposide (i.e. Km) for these sites, and there was no correlation between these two kinetic parameters. Furthermore, enzyme.drug binding and order of addition experiments indicated that etoposide and topoisomerase II form a kinetically competent complex in the absence of DNA. These findings suggest that etoposide. topoisomerase II (rather than etoposide.DNA) interactions mediate cleavage complex formation. Finally, rates of religation at specific sites correlated inversely with Cmax values, indicating that maximal levels of etoposide-induced scission reflect the ability of the drug to inhibit religation at specific sequences rather than the affinity of the drug for site-specific enzyme-DNA complexes.

Animals↗
PubMed1995

Increased drug affinity as the mechanistic basis for drug hypersensitivity of a mutant type II topoisomerase.

Altered sensitivity of topoisomerase II to anticancer drugs profoundly affects the response of eukaryotic cells to these agents. Therefore, several approaches were employed to elucidate the mechanism of drug hypersensitivity of the mutant yeast type II topoisomerase, top2H1012Y. This mutant, which is approximately 5-fold hypersensitive to ellipticine, formed DNA cleavage complexes more rapidly than the wild-type yeast enzyme in the presence of the drug. Conversely, no change in the rate of DNA religation was observed. There was, however, a correlation between increased cleavage rates and enhanced drug binding affinity. The apparent dissociation constant for ellipticine in the mutant topoisomerase II.drug.DNA ternary complex was approximately 5-fold lower than in the wild-type ternary complex. Furthermore, the apparent KD value for the mutant binary (topoisomerase II.drug) complex was approximately 2-fold lower than the corresponding wild-type complex, indicating that drug hypersensitivity is intrinsic to the enzyme. These findings strongly suggest that the enhanced ellipticine binding affinity for topoisomerase II is the mechanistic basis for drug hypersensitivity of top2H1012Y.

Amino Acid Sequence↗
PubMed1995

Topoisomerase II binds to ellipticine in the absence or presence of DNA. Characterization of enzyme-drug interactions by fluorescence spectroscopy.

Although a number of drugs currently in use for the treatment of human cancers act by stimulating topoisomerase II-mediated DNA breakage, little is known regarding interactions between these agents and the enzyme. To further define the mechanism of drug action, interactions between ellipticine (an intercalative drug with clinical relevance) and yeast topoisomerase II were characterized. By utilizing a yeast genetic system, topoisomerase II was identified as the primary cellular target of the drug. Furthermore, ellipticine did not inhibit enzyme-mediated DNA religation, suggesting that it stimulates DNA breakage by enhancing the forward rate of cleavage. Finally, ellipticine binding to DNA, topoisomerase II, and the enzyme-DNA complex was assessed by steady-state and frequency domain fluorescence spectroscopy. As determined by changes in fluorescence intensity and emission maximum wavelength, and by lifetime analysis, only the protonated species of ellipticine bound to a double-stranded 40-mer oligonucleotide containing a topoisomerase II cleavage site (KD approximately 65 nM). In contrast, predominantly deprotonated ellipticine bound to the enzyme.DNA complex (KD approximately 1.5 microM) or to the enzyme in the absence of nucleic acids (KD approximately 160 nM). These findings suggest that ellipticine interacts directly with topoisomerase II and that the enzyme dictates the ionic state of the drug in the ternary complex. A model is presented in which the topoisomerase II.ellipticine.DNA complex is formed via initial drug binding to either the enzyme or DNA.

Base Sequence↗