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Biomedical subjects

R B Thompson

Publications and source records attributed to R B Thompson.

At least 19 recordsLinked to original sources
PubMed2000

Improving polymeric microemulsions with block copolymer polydispersity.

Recent experiments have demonstrated that block copolymers are capable of stabilizing immiscible homopolymer blends producing bicontinuous microemulsion. The stability of these polymeric alloys requires the copolymer to form flexible, nonattractive monolayers along the homopolymer interfaces. We predict that copolymer polydispersity can substantially and simultaneously improve the monolayers in both of these respects. Furthermore, polydispersity should provide similar improvements in systems, such as colloidal suspensions and polymer/clay composites, that utilize polymer brushes to suppress attractive interactions.

Journal Article↗
PubMed2000

Fluorescence microscopy of stimulated Zn(II) release from organotypic cultures of mammalian hippocampus using a carbonic anhydrase-based biosensor system.

We demonstrate here that electrical stimulation of organotypic cultures of rat hippocampus results in the prompt release of significant amounts of Zn(II) by a fluorescence microscopic method. The fluorescence imaging of free Zn(II) is achieved using a highly selective biosensing indicator system consisting of human apo-carbonic anhydrase II (apoCAII) and a fluorescent aryl sulfonamide inhibitor of the enzyme, ABD-N. The apoenzyme and ABD-N in the absence of Zn(II) exhibit weak, reddish fluorescence typical of the ABD-N alone; when Zn(II) is added it binds to the apoenzyme (K(D) = 4 pM), which strongly promotes binding of ABD-N to the holoenzyme (K(D) = 0.9 microM). Binding of ABD-N to the holoenzyme results in a 9-fold increase in apparent quantum yield, significant blue shifts in excitation and emission, an increase in average fluorescence lifetime, a 4-fold increase in the ratio of intensities at 560 and 680 nm, and a large increase in anisotropy. Prior to stimulation, cultures immersed in phosphate-buffered saline with glucose and apoCAII with ABD-N emitted negligible fluorescence, but within 20 s after electrical stimulation a diffuse cloud of greenish fluorescence emerged and subsequently covered most of the culture, indicating release of zinc into the extracellular medium.

Animals↗
PubMed2000

Green fluorescent protein as a probe of rotational mobility within bacteriophage T4.

Green fluorescent protein (GFP) was targeted into bacteriophage T4 heads and proheads as a probe of the internal environment. Targeting was accomplished with internal protein III (IPIII) fusion proteins or capsid targeting sequence (CTS)-tagged proteins, where CTS is the 10-amino acid residue CTS of IPIII. Recombinant phage T4[CTS/IPIII/GFP], T4[CTS/IPIII(T)GFP], and T4[CTS/GFP] packaged GFP fusion proteins and processed them at cleavage sites designated /. Steady-state and time-resolved fluorescence measurements suggest that packaged GFP is concentrated to a high density, that fusion protein IPIII(T)GFP occurs in a tightly clustered arrangement, and that the internal milieu of the phage head reduces rotational mobility of GFP. Phage, but not proheads, packaged with fusion protein IPIII(T)GFP gave an unexpectedly lower anisotropy than phage and proheads packaged with GFP, which suggests IPIII(T)GFP is bound to DNA in a manner that causes close associations between GFP molecules resulting in homotransfer between fluorophores within packaged phage. Targeting of reporter proteins into active virions is a promising approach for determining the structure of the condensed DNA, and properties of encapsidated viral enzymes.

Bacteriophage T4↗
PubMed2000

Importance of zinc in the central nervous system: the zinc-containing neuron.

Zinc is essential to the structure and function of myriad proteins, including regulatory, structural and enzymatic. It is estimated that up to 1% of the human genome codes for zinc finger proteins. In the central nervous system, zinc has an additional role as a neurosecretory product or cofactor. In this role, zinc is highly concentrated in the synaptic vesicles of a specific contingent of neurons, called "zinc-containing" neurons. Zinc-containing neurons are a subset of glutamatergic neurons. The zinc in the vesicles probably exceeds 1 mmol/L in concentration and is only weakly coordinated with any endogenous ligand. Zinc-containing neurons are found almost exclusively in the forebrain, where in mammals they have evolved into a complex and elaborate associational network that interconnects most of the cerebral cortices and limbic structures. Indeed, one of the intriguing aspects of these neurons is that they compose somewhat of a chemospecific "private line" of the mammalian cerebral cortex. The present review outlines (1) the methods used to discover, define and describe zinc-containing neurons; (2) the neuroarchitecture and synaptology of zinc-containing neural circuits; (3) the physiology of regulated vesicular zinc release; (4) the "life cycle" and molecular biology of vesicular zinc; (5) the importance of synaptically released zinc in the normal and pathological processes of the cerebral cortex; and (6) the role of specific and nonspecific stressors in the release of zinc.

Alzheimer Disease↗
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↗