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R D Smith

Publications and source records attributed to R D Smith.

At least 127 records · Page 7Linked to original sources
PubMed1998

Characterization of microdialysis acidification for capillary isoelectric focusing-microelectrospray ionization mass spectrometry.

A microdialysis junction, based on a microdialysis membrane connecting a separation capillary and a short, sharply tapered microelectrospray emitter capillary, is demonstrated for on-line combination of capillary isoelectric focusing (CIEF) with electrospray ionization mass spectrometry (ESI-MS). The microdialysis junction provides the necessary electrical connection across the dialysis membrane for defining the electric fields needed for the CIEF separation and the electrospray process. Additionally, postseparation acidification of focused protein zones eluted from the CIEF capillary is achieved using the microdialysis junction while separation efficiency and resolution is maintained. A microelectrospray emitter produces a stable electrospray of protein analytes without the need for a makeup liquid flow and eliminates any subsequent sample dilution and reduction in MS sensitivity. The microdialysis junction is advantageous over the coaxial liquid sheath interface as evidenced by the simplicity in operation procedures, the enhancement in detection sensitivity, and the linear correlation between protein migration time and isoelectric point in CIEF-ESI-MS.

Buffers↗
PubMed1998

An ion funnel interface for improved ion focusing and sensitivity using electrospray ionization mass spectrometry.

To improve upon the already impressive sensitivity achievable with electrospray ionization sources, a novel electrohydrodynamic ion funnel interface has been developed and implemented with a triple-quadrupole mass spectrometer. The ion funnel interface effectively consists of a series of ring electrodes of increasingly small internal diameters to which rf and dc electric potentials are coapplied. In the 1-10-Torr pressure range, the electric fields cause the collisionally damped ions to be more effectively focused and transmitted as a collimated ion beam. This paper describes the ion funnel design and presents an evaluation of its performance using a triple-quadrupole mass spectrometer. Ion transmission and m/z discriminating parameters (resulting in both effective low- and high-m/z cutoffs) are presented based upon both ion current measurements and mass spectra. Electrospray ionization mass spectra of selected protein solutions demonstrated well over 1 order of magnitude increase in signal relative to that of the instrument operated in its standard (inlet capillary-skimmer) configuration under similar conditions. The present results suggest that it will be feasible to realize close to 100% ion transmission efficiency for analytically relevant ions through the electrospray ionization interface and into the mass analyzer.

Animals↗
PubMed1998

A microfabricated dialysis device for sample cleanup in electrospray ionization mass spectrometry.

A laser microfabricated device was constructed for rapid microdialysis cleanup of biological samples for analysis by electrospray ionization mass spectrometry (ESI-MS) in both off-line and on-line modes. A microdialysis membrane was sandwiched between two chips having micromachined serpentine channels. The total volume of the sample serpentine channel used for the microdialysis was 1 microL. Efficient desalting was demonstrated for both DNA and protein samples using ESI with an ion trap mass spectrometer after microdialysis against a counter flow of ESI-compatible buffer. Signal-to-noise ratios were also greatly enhanced compared to direct infusion of the original nondialyzed samples. Importantly, the microfabricated device allowed use of sample flow rates 1 order of magnitude smaller than previous designs based on a microdialysis fiber, allowing reduced sample utilization and improved sensitivity with ESI-MS. The effectiveness of the cleanup was attributed to the size difference between the sample channel and the buffer channel and the fact that the sample is continuously refreshed by the buffer counterflow. The results indicate substantial potential for construction of highly compact and rugged devices enabling field applications of ESI-MS.

DNA↗
PubMed1998

Probing RegA/RNA interactions using electrospray ionization-fourier transform ion cyclotron resonance-mass spectrometry.

The interactions of bacteriophage T4 regA protein, a unique translational regulator, with RNAs of various size and sequence were studied using electrospray ionization-Fourier transform ion cyclotron resonance-mass spectrometry. Using very gentle interface conditions, regA/RNA complexes with a 1:1 binding stoichiometry were observed for all four target RNAs studied, consistent with solution binding studies. Competitive binding of target RNAs and their degradation products with regA demonstrated that the loss of a single nucleotide resulted in a dramatic change in binding affinity in some cases. Competitive binding of regA with four target RNAs revealed similar relative binding affinity order to that suggested by previous in vitro repression experiments. The use of sustained off-resonance irradiation for collisionally induced dissociation of a regA/RNA complex suggested the potential for directly obtaining information regarding the regA binding domain.

Bacterial Proteins↗
PubMed1998

Functional analysis of the mouse Scn8a sodium channel.

The mouse Scn8a sodium channel and its ortholog Na6 in the rat are abundantly expressed in the CNS. Mutations in mouse Scn8a result in neurological disorders, including paralysis, ataxia, and dystonia. In addition, Scn8a has been observed to mediate unique persistent and resurgent currents in cerebellar Purkinje cells (Raman et al., 1997). To examine the functional characteristics of this channel, we constructed a full-length cDNA clone encoding the mouse Scn8a sodium channel and expressed it in Xenopus oocytes. The electrophysiological properties of the Scn8a channels were compared with those of the Rat1 and Rat2 sodium channels. Scn8a channels were sensitive to tetrodotoxin at a level comparable to that of Rat1 or Rat2. Scn8a channels inactivated more rapidly and showed differences in their voltage-dependent properties compared with Rat1 and Rat2 when only the alpha subunits were expressed. Coexpression of the beta1 and beta2 subunits modulated the properties of Scn8a channels, but to a lesser extent than for the Rat1 or Rat2 channels. Therefore, all three channels showed similar voltage dependence and inactivation kinetics in the presence of the beta subunits. Scn8a channels coexpressed with the beta subunits exhibited a persistent current that became larger with increasing depolarization, which was not observed for either Rat1 or Rat2 channels. The unique persistent current observed for Scn8a channels is consistent with the hypothesis that this channel is responsible for distinct sodium conductances underlying repetitive firing of action potentials in Purkinje neurons.

Amino Acid Sequence↗
PubMed1998

Capillary isoelectric focusing-electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry for protein characterization.

On-line combination of capillary isoelectric focusing (CIEF) with electrospray ionization Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry is demonstrated for high-resolution analysis of model proteins, human hemoglobin variants, and Escherichia coli proteins. The acquisition of high-resolution mass spectra of hemoglobin beta chains allows direct identification of hemoglobin variants A and C, differing in molecular mass by 1 Da. Direct mass determination of cellular proteins separated in the CIEF capillary is achieved using their isotopic envelopes obtained from ESI-FTICR. The factors which dictate overall performance of CIEF-ESI-FTICR, including duty cycle, mass resolution, scan rate, and sensitivity, are discussed in the context of protein variants and cell lysates analyzed in this study.

Bacterial Proteins↗
PubMed1998

On-line dual microdialysis with ESI-MS for direct analysis of complex biological samples and microorganism lysates.

A novel dual-microdialysis approach has been developed for fast and efficient fractionation and cleanup for ESI-MS and ESI MS/MS analyses of biological samples. A modified dynamic microdialyzer utilizing two mirror-image serpentine channels, which sandwich a regenerated cellulose membrane of selected molecular weight cutoff, serves as the first stage for the removal of high-molecular-weight components and cellular residue. The second stage employs a hollow microdialysis capillary to remove low-molecular-weight species (e.g., salts) which can degrade or preclude analysis ESI-MS. A protein mixture consisting of 30 microM bovine serum albumin (BSA), 4.0 microM cytochrome c, 2.3 microM ubiquitin, and 9.4 microM bradykinin in 0.5 M NaCl was used to evaluate the performance of this system. Essentially complete removal of both BSA and NaCl was achieved, resulting in high-quality mass spectra containing only the lower molecular weight proteins. After passing through the on-line dual-microdialysis system, a crude bacteria cell lysate yielded clean ESI-mass spectra in approximately 20 min. MS/MS of selected ions demonstrated abundant fragment ions and provided a second-dimension "fingerprint" of the complex cellular fraction. Preliminary application of this technique for direct characterization of microorganism lysates is presented.

Escherichia coli↗
PubMed1998

Heterogeneity in Bacillus cereus PCR products detected by ESI-FTICR mass spectrometry.

PCR amplification of a segment of the 16/23S rDNA interspace region (ISR) from Bacillus cereus 6464 produced a mixture of products. An 89-bp product was predicted on the basis of the reported sequence. The ESI-FTICR analysis revealed three double-stranded products, differing in size by a single nucleotide corresponding to two homoduplexes of 89 and 88 base pairs and a heteroduplex of 89 and 88 nucleotide strands. These were produced from a single preparation of genomic DNA and a single primer pair. ESI-FTICR analysis of the single strands identified a deletion of a T in the coding strand and a corresponding loss of an A in the noncoding strand of this product. The ESI-FTICR analysis indicated the presence of an unreported sequence variation between rRNA operons in this organism. This report illustrates that PCR products amplified from templates differing by a single nucleotide can be resolved and identified using ESI-FTICR at the 89-bp level. Furthermore, the ESI-FTICR mass measurements provided the identity of the deletion, which is indicative of interoperon variability.

Bacillus cereus↗
PubMed1998

Characterization of a novel covalent monoadduct on the RNA overhang of an RNA-DNA hybrid induced by antitumor antibiotic neocarzinostatin.

Neocarzinostatin chromophore (NCS-chrom) has been found previously to induce thiol-dependent abasic site formation and cleavage on the RNA strand of RNA-DNA hybrids. When a four-base nucleotide was deleted from the 5' end of the DNA strand, however, an unexpected product with slow mobility, potentially a drug adduct or interstrand cross-linked material, was observed instead. End-labeling of the RNA and DNA strands showed that only the RNA strand is present in the product. The product, isolated from a denaturing polyacrylamide gel, has a fluorescence spectrum similar to that of activated NCS-chrom, and thiols with different charges give rise to products with various mobilities on the gel, indicating that the product consists of a drug adduct with NCS-chrom and thiol attached to the RNA strand of the hybrid. Mass spectroscopic (MS) analysis shows that it is a monoadduct. Chemical sequencing of 5'- and 3'-end-labeled monoadduct and MS/MS data show that drug is attached only to the U9 of the RNA in the hybrid 5'-r(CACAGAAUU9CG)/5'-d(TTCTGTG). Unlike most other DNA adducts and cross-linked products found previously, this adduct can be formed readily in the presence of atmospheric oxygen. 2'-O-Methyl and 2'-H substitutions and nucleotide replacements on the 4-base RNA overhang affect monoadduct formation to different extents, reflecting the existence of a tight three-dimensional binding pocket for the activated drug. Stability of drug adduct to heat and aniline-HOAc treatment suggest that the drug is covalently bound to the ribose of U9. Since no deuterium isotope selection effect was observed for C-1' of U9, it is possible, although not required, that the drug abstracts H from another hydrogen source on the sugar of the targeted ribonucleotide.

Aerobiosis↗
PubMed1998

Functional analysis of the rat I sodium channel in xenopus oocytes.

Voltage-gated sodium channels in the mammalian CNS initiate and propagate action potentials when excitatory inputs achieve threshold membrane depolarization. There are multiple sodium channel isoforms expressed in rat brain (types I, II, III, 6, and NaG). We have constructed a full-length cDNA clone encoding type I and compared the electrophysiological properties of type I (Rat1) and II (Rat2) channels in the absence and presence of the two accessory subunits beta1 and beta2. Injection into Xenopus oocytes of RNA encoding Rat1 resulted in functional sodium currents that were blocked by tetrodotoxin, with Kapp = 9.6 nM. Rat1 sodium channels had a slower time course of fast inactivation than Rat2. Coexpression of beta1 accelerated inactivation of both Rat1 and Rat2, resulting in comparable inactivation kinetics. Rat1 recovered from fast inactivation more rapidly than Rat2, regardless of whether beta1 or beta2 was present. The voltage dependence of activation was similar for Rat1 and Rat2 without the beta subunits, but it was more positive for Rat1 when beta1 and beta2 were coexpressed. The voltage dependence of inactivation was more positive for Rat1 than for Rat2, and coexpression with beta1 and beta2 accentuated that difference. Finally, sodium current amplitudes were reduced by 7-9% for both Rat1 and Rat2 channels when protein kinase A phosphorylation was induced. It has been suggested previously that Rat1 and Rat6 channels mediate transient and maintained sodium conductances, respectively, in Purkinje cells, and the electrophysiological properties of Rat1 currents are consistent with a role for this channel in mediating the rapidly inactivating, transient current.

Animals↗
PubMed1998

In-trap cleanup of proteins from electrospray ionization using soft sustained off-resonance irradiation with fourier transform ion cyclotron resonance mass spectrometry.

Electrospray ionization Fourier transform ion cyclotron resonance (FTICR) mass spectrometry is capable of producing high mass resolving power and improved mass accuracy for large proteins and noncovalent complexes when coupled with collisionally induced dissociation (CID) of noncovalent adducts and consequent minimization of ion charge density in the ICR trap during measurements. This work demonstrates the application of in-trap cleanup to several biologically relevant systems, including carbonic anhydrase, 4-oxalocrotonate tautomerase (4OT) analogue, and SecB, a chaperone from Escherichia coli. In-trap cleanup yields improved mass measurements for these systems and is expected to further enable measurements for even more complex systems where adduction levels have precluded study of intact complexes.

Bacterial Proteins↗
PubMed1998

The observation of chaperone-ligand noncovalent complexes with electrospray ionization mass spectrometry.

Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) was applied for the study of noncovalent chaperone SecB-ligand complexes produced in solution and examined in the gas phase with the aid of electrospray ionization (ESI). Since chaperone proteins are believed to recognize and bind only with ligands with nonnative tertiary structure, this work required careful unfolding of the ligand and subsequent reaction with the intact chaperone (the noncovalent tetrameric protein, SecB). A high denaturant concentration was employed to produce nonnative structures of the OppA, and microdialysis of the resulting solutions containing the chaperone-ligand complexes was carried out to rapidly remove the denaturant prior to analysis. Multistage mass spectrometry was essential to the successful study of these complexes since the initial mass spectra indicated extensive adduction that precluded mass measurements, even after microdialysis. However, low energy collisional activation of the ions in the FTICR trap proved useful for adduct removal, and careful control of excitation level preserved the intact complexes of interest, revealing a 1:1 SecB:OppA stoichiometry. To our knowledge, these results present the first direct observation of chaperone-ligand noncovalent complexes and the highest molecular weight heterogeneous noncovalent complex observed to date by mass spectrometry. Furthermore, these results highlight the capabilities of FTICR for the study of such complex systems, and the development of a greater understanding of chaperone interactions in protein export.

Bacterial Proteins↗
PubMed1998

The interaction between the chaperone SecB and its ligands: evidence for multiple subsites for binding.

The chaperone protein SecB is dedicated to the facilitation of export of proteins from the cytoplasm to the periplasm and outer membrane of Escherichia coli. It functions to bind and deliver precursors of exported proteins to the membrane-associated translocation apparatus before the precursors fold into their native stable structures. The binding to SecB is characterized by a high selectivity for ligands having nonnative structure but a low specificity for consensus in sequence among the ligands. A model previously presented (Randall LL, Hardy SJS, 1995, Trends Biochem Sci 20:65-69) to rationalize the ability of SecB to distinguish between the native and nonnative states of a polypeptide proposes that the SecB tetramer contains two types of subsites for ligand binding: one kind that would interact with extended flexible stretches of polypeptides and the other with hydrophobic regions. Here we have used titration calorimetry, analytical ultracentrifugation, and electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry to obtain evidence that such distinguishable subsites exist.

Aprotinin↗
PubMed1998

The effects of changing intracellular pH on calcium and potassium currents in smooth muscle cells from the guinea-pig ureter.

Guinea-pig ureteric smooth muscle is unusual in that intracellular acidification increases and alkalinization decreases force production. To help elucidate the mechanism underlying these effects on force we have investigated the effects of changing intracellular pH on both calcium and potassium currents in single cells isolated from the guinea-pig ureter to determine their possible role in force development. Depolarization to +40 mV resulted in a fast transient outward current which was inhibited by 4-aminopyridine but not tetraethylammonium. Intracellular alkalinization (20 mM trimethylamine) increased this current to 179 +/- 24% of the control and resulted in the development of a slowly activating large outward current which was inhibited by tetraethylammonium and washout. Acidification (40 mM sodium butyrate) decreased the fast transient outward current to 58 +/- 3% of the control and did not produce a slowly activating current. When potassium was replaced by caesium in the pipette solution, depolarization to 0 mV resulted in an inward calcium current which was abolished by nifedipine. Intracellular alkalinization increased this current to 126 +/- 11% of the control whereas acidification had the opposite effect, decreasing it to 55 +/- 10%. Furthermore, current-clamp experiments showed that intracellular alkalinization inhibited the amplitude of the action potential, therefore decreasing excitability of the cell. From our results, we suggest that the predominant effects of intracellular pH on force production in the guinea-pig ureter are mediated via the modulation of outward potassium currents (thereby reducing excitability of the tissue) rather than the effects on the inward calcium current.

Animals↗
PubMed1998

Controlling antimicrobial resistance: a proposed transferable permit market.

Resistance to antimicrobials results in those antimicrobials becoming ineffective in treating common bacterial infections. To prevent the spread of such resistance the use of antimicrobials requires control. The authors have previously argued that use of regulation or charges to control resistance would not be efficient. Regulation will not account for different marginal costs of reducing antimicrobial prescription amongst GPs, and charges, although based on sound economic concepts, are based on an unrealistic amount of required information. It was therefore argued that a system of tradable permits, by combining the targets of regulation and the market flexibility of charges, would achieve control more efficiently than simple regulation or charges. In this paper the authors progress this proposed policy by considering various important issues which arise when designing such a tradable permit system for antimicrobials. The paper does not provide an exhaustive plan enabling a blueprint for such a market to be designed, but a proposal which may be used as platform for further specific development of such an initiative to deal with resistance.

Anti-Bacterial Agents↗
PubMed1998

Primary care reform: a three country comparison of 'budget holding'.

Governments in most developed nations have been looking to organisational and financial reform of health systems over the last decade. Although the structure and problems of the health care sector in each country may differ, with countries correspondingly adopting different reform agendas, there has been some element of commonality in reforms: that of (managed) competition. Of particular importance in such reforms has been the strengthening of primary care. General practitioners and primary care physicians, as 'gatekeepers' to the health system, are increasingly being called upon to be accountable; not only for their patients' health but also for the wider resource implications of any treatments prescribed. In some countries this role has been formalised through establishing 'budget holding' for general practitioners and primary care physicians, for example, through general practice 'fund holding' in the UK, Health Maintenance Organisations in the USA, and Independent Practice Associations in New Zealand. This paper examines: (i) what such budget holding seeks to achieve; (ii) the effectiveness of the budget holding experience to date in achieving these objectives; and (iii) factors which appear to determine the success of budget holding in achieving its objectives.

Budgets↗