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R D Smith

Publications and source records attributed to R D Smith.

At least 55 records · Page 3Linked to original sources
PubMed2001

Controlled ion fragmentation in a 2-D quadrupole ion trap for external ion accumulation in ESI FTICR mass spectrometry.

Undesired fragmentation of electrospray generated ions in an rf multipole traps can be problematic in many applications. Of special interest here is ion dissociation in a 2-D quadrupole ion trap external to a Fourier transform ion cyclotron resonance mass spectrometer (FTICR MS) used in proteomic studies. In this work, we identified the experimental parameters that determine the efficiency of ion fragmentation. We have found that under the pressure conditions used in this study there is a specific combination of the radial and axial potential well depths that determines the fragmentation threshold. This combination of rf and dc fields appears to be universal for ions of different mass-to-charge ratios, molecular weights, and charge states. Such universality allows the fragmentation efficiency of the trapped ions in the course of capillary liquid chromatography (LC) separation studied to be controlled and can increase the useful duty cycle and dynamic range of a FTICR mass spectrometer equipped with an external rf only 2-D quadrupole ion trap.

Algorithms↗
PubMed2001

Dietary monounsaturated fatty acids and haemostasis.

Diets high in monounsaturated fatty acids (MUFA) are increasingly being recommended as a highly-effective cholesterol-lowering strategy in populations at risk of CHD. However, the need for a re-appraisal of the benefits of diets rich in MUFA became apparent as a result of recent studies showing that meals high in olive oil cause greater postprandial activation of blood coagulation factor VII than meals rich in saturated fatty acids. The present review evaluates the evidence for the effects of MUFA-rich diets on fasting and postprandial measurements of haemostasis, and describes data from a recently-completed long-term controlled dietary intervention study. The data show that a background diet high in MUFA has no adverse effect on fasting haemostatic variables and decreases the postprandial activation of factor VII in response to a standard fat-containing meal. Since the same study also showed a significant reduction in the ex vivo activation of platelets in subjects on the high-MUFA diet, the overall findings suggest that there is no reason for concern regarding adverse haemostatic consequences of high-MUFA diets.

Cardiovascular Diseases↗
PubMed2001

Expression of the developmental and oncogenic PAX2 gene in human prostate cancer.

PURPOSE: In the human prostate cancer cell lines LNCaP, DU145 and PC3, 27 primary prostate cancers, 10 benign prostatic hyperplasia specimens and 5 normal prostates we investigated the expression pattern of PAX2, a member of the PAX family of developmental control genes. PAX2 is expressed at high levels in developing undifferentiated cells of the urogenital system and is repressed upon terminal differentiation with no expression in normal adult cells. It is also been shown to be a proto-oncogene in mice and is expressed in human renal cell carcinoma. MATERIALS AND METHODS: PAX2 expression was assessed at the RNA level by reverse transcriptase-polymerase chain reaction and Southern blot analysis using specific sets of nucleotides. The expression pattern of PAX2 was reconfirmed at the protein level by immunofluorescence in the cell lines, and by Western blot analysis in primary human prostate cancers and benign prostatic tissue. RESULTS: Using reverse transcription-polymerase chain reaction combined with Southern hybridization PAX2 expression was detected in 52% of primary cancers and all 3 cell lines. PAX2 expression in these samples was confirmed at a protein level using immunoblotting and immunofluorescence. PAX2 messenger RNA was not detected in any benign or normal prostatic samples. Immunoblotting of protein from benign prostatic hyperplasia samples confirmed the lack of expression of PAX2 protein. CONCLUSIONS: The expression of PAX2 in prostate cancer compared to nonmalignant prostates is statistically significant (Fisher's exact test p = 0.0004). These results suggest a possible role for PAX2 in prostate cancer. Although previous studies have suggested a role for PAX2 for supporting proliferation in undifferentiated cells, no correlation of PAX2 expression with Gleason score was found in prostate cancer.

Blotting, Western↗
PubMed2001

Identification of intrinsic order and disorder in the DNA repair protein XPA.

The DNA-repair protein XPA is required to recognize a wide variety of bulky lesions during nucleotide excision repair. Independent NMR solution structures of a human XPA fragment comprising approximately 40% of the full-length protein, the minimal DNA-binding domain, revealed that one-third of this molecule was disordered. To better characterize structural features of full-length XPA, we performed time-resolved trypsin proteolysis on active recombinant Xenopus XPA (xXPA). The resulting proteolytic fragments were analyzed by electrospray ionization interface coupled to a Fourier transform ion cyclotron resonance mass spectrometry and SDS-PAGE. The molecular weight of the full-length xXPA determined by mass spectrometry (30922.02 daltons) was consistent with that calculated from the sequence (30922.45 daltons). Moreover, the mass spectrometric data allowed the assignment of multiple xXPA fragments not resolvable by SDS-PAGE. The neural network program Predictor of Natural Disordered Regions (PONDR) applied to xXPA predicted extended disordered N- and C-terminal regions with an ordered internal core. This prediction agreed with our partial proteolysis results, thereby indicating that disorder in XPA shares sequence features with other well-characterized intrinsically unstructured proteins. Trypsin cleavages at 30 of the possible 48 sites were detected and no cleavage was observed in an internal region (Q85-I179) despite 14 possible cut sites. For the full-length xXPA, there was strong agreement among PONDR, partial proteolysis data, and the NMR structure for the corresponding XPA fragment.

Amino Acid Motifs↗
PubMed2001

Aberrant mobility phenomena of the DNA repair protein XPA.

The DNA repair protein XPA recognizes a wide variety of bulky lesions and interacts with several other proteins during nucleotide excision repair. We recently identified regions of intrinsic order and disorder in full length Xenopus XPA (xXPA) protein using an experimental approach that combined time-resolved trypsin proteolysis and electrospray ionization interface coupled to a Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry (MS). MS data were consistent with the interpretation that xXPA contains no post-translational modifications. Here we characterize the discrepancy between the calculated molecular weight (31 kDa) for xXPA and its apparent molecular weight on SDS-PAGE (multiple bands from approximately 40-45 kDa) and gel filtration chromatography ( approximately 92 kDa), as well as the consequences of DNA binding on its anomalous mobility. Iodoacetamide treatment of xXPA prior to SDS-PAGE yielded a single 42-kDa band, showing that covalent modification of Cys did not correct aberrant mobility. Determination of sulfhydryl content in xXPA with Ellman's reagent revealed that all nine Cys in active protein are reduced. Unexpectedly, structural constraints induced by intramolecular glutaraldehyde crosslinks in xXPA produced a approximately 32-kDa monomer in closer agreement with its calculated molecular weight. To investigate whether binding to DNA alters xXPA's anomalous migration, we used gel filtration chromatography. For the first time, we purified stable complexes of xXPA and DNA +/- cisplatin +/- mismatches. xXPA showed at least 10-fold higher affinity for cisplatin DNA +/- mismatches compared to undamaged DNA +/- mismatches. In all cases, DNA binding did not correct xXPA's anomalous migration. To test predictions that a Glu-rich region (EEEEAEE) and/or disordered N- and C-terminal domains were responsible for xXPA's aberrant mobility, the molecular weights of partial proteolytic fragments from approximately 5 to 25 kDa separated by reverse-phase HPLC and precisely determined by ESI-FTICR MS were correlated with their migration on SDS-PAGE. Every partial tryptic fragment analyzed within this size range exhibited 10%-50% larger molecular weights than expected. Thus, both the disordered domains and the Glu-rich region in xXPA are primarily responsible for the aberrant mobility phenomena.

Amino Acid Sequence↗
PubMed2001

The physiological basis of uterine contractility: a short review.

In this review we discuss our current understanding of the cellular basis of uterine contractility, highlighting those areas requiring further study. It is clear that the basic processes of excitation-contraction coupling lie within the myometrial cell, and that these may be modified by agonists. Pacemaker activity, however, remains a mystery. The contribution of extracellular calcium entry to contraction is shown to be vital, whilst the role of the sarcoplasmic reticulum remains controversial. Much current experimental focus is on pathways controlling and regulating contraction, and we discuss sensitisation mechanisms and question their role in intact uterine preparations. Experimental Physiology (2001) 86.2, 239-246.

Animals↗
PubMed2001

Lone star tick-infecting borreliae are most closely related to the agent of bovine borreliosis.

Although Borrelia theileri, the agent of bovine borreliosis, was described at the turn of the century (in 1903), its relationship with borreliae causing Lyme disease or relapsing fever remains undescribed. We tested the previously published hypothesis that spirochetes infecting Lone Star ticks (Amblyomma americanum) may comprise B. theileri by analyzing the 16S ribosomal DNAs (rDNAs) and flagellin genes of these spirochetes. B. theileri, the Amblyomma agent, and B. miyamotoi formed a natural group or clade distinct from but most closely related to that of the relapsing fever spirochetes. B. theileri and the Amblyomma agent were 97 and 98% similar at the nucleotide level within the analyzed portions of the 16S rDNA and the flagellin gene respectively, suggesting a recent divergence. The agent of bovine borreliosis might be explored as a surrogate antigen for the as-yet-uncultivatable Amblyomma agent in studies designed to explore the etiology of a Lyme disease-like infection associated with Lone Star ticks.

Animals↗
PubMed2001

Self-assembly and binding of a sorting nexin to sorting endosomes.

The fate of endocytosed membrane proteins and luminal contents is determined by a materials processing system in sorting endosomes. Endosomal retention is a mechanism that traps specific proteins within this compartment, and thereby prevents their recycling. We report that a sorting nexin SNX1, a candidate endosomal retention protein, self-assembles in vitro and in vivo, and has this property in common with its yeast homologue Vps5p. A comparison of SNX1 expressed in bacterial and in mammalian systems and analyzed by size-exclusion chromatography indicates that in cytosol SNX1 tetramers are part of a larger complex with additional proteins. An endosomal retention function would require that SNX1 bind to endosomal membranes, yet the complexes that we analyzed were largely soluble and little SNX1 was found in pellet fractions. Using green fluorescent protein fusions, endocytic compartment markers and fluorescence recovery after photobleaching, we found that there is an equilibrium between free cytoplasmic and early/sorting endosome-bound pools of green fluorescent protein-SNX1. Fluorescence resonance energy transfer indicated that spectral variants of green fluorescent protein-SNX1 were oligomeric in vivo. In cell extracts, these green fluorescent protein-SNX1 oligomers corresponded to tetrameric and larger complexes of green fluorescent protein-SNX1. Using video microscopy, we observed small vesicle docking and tubule budding from large green fluorescent protein-SNX1 coated endosomes, which are features consistent with their role as sorting endosomes. http://www.biologists.com/JCS/movies/jcs2058.html

Amino Acid Sequence↗
PubMed2001

Exclusive amplification of cDNA template (EXACT) RT-PCR to avoid amplifying contaminating genomic pseudogenes.

Genomic DNA contamination within RNA samples has important implications for RT-PCR, particularly if there is a pseudogene related to the gene under investigation, because amplification from pseudogenes and reverse-transcribed cDNA can be very difficult to distinguish. Methods to remove DNA contamination cannot guarantee the absolute absence of DNA from the sample without a loss of RNA quantity or quality, which can be crucial for small amounts of RNA or for the investigation of transcripts with a low level of expression. Here, we describe a general technique for RT-PCR that applies a sequence to the 5' tail of reverse-transcribed cDNA that is not present in genomic DNA and uses this for annealing the reverse PCR primer to exclude genomic DNA amplification in unmodified RNA samples.

Base Sequence↗
PubMed2001

Blockade of rat alpha3beta4 nicotinic receptor function by methadone, its metabolites, and structural analogs.

The opioid agonist properties of (+/-)-methadone are ascribed almost entirely to the (-)-methadone enantiomer. To extend our knowledge of the pharmacological actions of methadone at ligand-gated ion channels, we investigated the effects of the two enantiomers of methadone and its metabolites R-(+)-2-ethyl-1,5-dimethyl-3,3-diphenylpyrrolinium perchlorate (EDDP) and R-(+)-2-ethyl-5-methyl-3,3-diphenyl-1-pyrroline hydrochloride (EMDP), as well as structural analogs of methadone, including (-)-alpha-acetylmethadol hydrochloride (LAAM) and (+)-alpha-propoxyphene, on rat alpha3beta4 neuronal nicotinic acetylcholine receptors (nAChRs) stably expressed in a human embryonic kidney 293 cell line, designated KXalpha3beta4R2. (+/-)-methadone inhibited nicotine-stimulated 86Rb+ efflux from the cells in a concentration-dependent manner with an IC50 value of 1.9 +/- 0.2 microM, indicating that it is a potent nAChR antagonist. The (-)- and (+)-enantiomers of methadone have similar inhibitory potencies on nicotine-stimulated 86Rb+ efflux, with IC50 values of approximately 2 microM. EDDP, the major metabolite of methadone, is even more potent, with an IC50 value of approximately 0.5 microM, making it one of the most potent nicotinic receptor blockers reported. In the presence of (+/-)-methadone, EDDP, or LAAM, the maximum nicotine-stimulated 86Rb+ efflux was markedly decreased, but the EC50 value for nicotine stimulation was altered only slightly, if at all, indicating that these compounds block alpha3beta4 nicotinic receptor function by a noncompetitive mechanism. Consistent with a noncompetitive mechanism, (+/-)-methadone, its metabolites, and structural analogs have very low affinity for nicotinic receptor agonist binding sites in membrane homogenates from KXalpha3beta4R2 cells. We conclude that both enantiomers of methadone and its metabolites as well as LAAM and (+)-alpha-propoxyphene are potent noncompetitive antagonists of alpha3beta4 nAChRs.

Binding, Competitive↗
PubMed2001

Directly observed treatment for multidrug-resistant tuberculosis: an economic evaluation in the United States of America and South Africa.

OBJECTIVE: To estimate the cost-effectiveness of directly observed treatment compared to conventional therapy in reducing the spread of multidrug-resistant tuberculosis, for an industrialised country (represented by the United States of America) and a developing country (South Africa). METHODS: Monte Carlo analysis using published data on probability, cost and health impact. RESULTS: In both countries, directly observed treatment is the dominant strategy, yielding cost savings and improved health outcomes. Cost savings for directly observed treatment relative to conventional therapy become more significant as more expensive second-line drugs are used in treatments. CONCLUSIONS: The cost-effectiveness of directly observed treatment relative to conventional therapy is demonstrated for both the USA and South Africa. Cost savings are more pronounced (especially for South Africa) as the likelihood of multidrug-resistant tuberculosis increases and more expensive second-line therapies are used. Given that health care resources are more severely constrained in developing countries, the data contained in this study are useful in guiding the design of policies for the effective management of multidrug-resistant tuberculosis in settings with limited resources.

Antitubercular Agents↗
PubMed2000

Quantitative analysis of globin gene induction in single human erythroleukemic cells.

The mechanisms involved in the normal developmental regulation of globin gene expression, and the response to pharmacological agents that elevate fetal hemoglobin, may be expected to involve either changes in each cell or a selection process affecting subsets of differentiating erythroid cells. To study these mechanisms we have developed assays to measure mRNA levels in single erythroid cells. The assay involved the use of globin-specific probes, with no detectable cross-reactivity, in real-time, fluorescence-based quantitative PCR (Q-PCR). We had previously used this Q-PCR method to measure globin mRNA levels in cultures of primary erythroid cells demonstrating that drugs like hydroxyurea, 5-azacytidine and butyric acid each yielded increases in gamma/( gamma + ss) mRNA ratios, with differential effects on ss-globin levels. We have now extended this approach to measure globin mRNA levels in single K562 cells, a human erythroleukemic cell line, with and without 30 microM hemin treatment. Hemin exposure increases total hemoglobin levels by approximately 9-fold and total alpha-, epsilon- and gamma-globin mRNA levels by 1.5-2.3-fold. Single cell analyses showed initial wide distributions of each of the three individual globin mRNA levels with most cells having detectable but very low levels of each globin transcript. Hemin induction shifted the distributions to higher levels, with a tendency to residual left skewing as some cells remained with very low expression levels despite the effect of hemin in increasing expression in most of these low expressing cells. Thus transcriptional heterogeneity remains a crucial variable, even in this extensively used model of human erythroid biology, and clearly influences strongly the response to inducing agents. These methods may enable us to define better possible molecular and/or cellular models of globin gene modulation.

Cell Count↗
PubMed2000

Separation and detection of intact noncovalent protein complexes from mixtures by on-line capillary isoelectric focusing-mass spectrometry.

Separation and mass spectrometric analysis of intact noncovalent protein-protein complexes from mixtures is described. Protein complexes were separated using isoelectric focusing in a capillary under native conditions. During the mobilization, molecular masses of the intact complexes were measured on-line (as they emerged from the capillary) using Fourier transform ion cyclotron resonance (FTICR) mass spectrometry. An FTICR "in-trap" ion cleanup procedure was necessary for some complexes to reduce high levels of adduction and to obtain accurate molecular mass measurements. Optimization of the conditions for analysis of different intact complexes is discussed. We have shown that either the intact noncovalent complexes or their constituent protein subunits can be detected by variation of sheath liquid (i.e., NH4OAc vs HOAc) added at the electrospray-mass spectrometer interface. Thus, two successive experiments permit a fast and efficient characterization of intact complex stoichiometry, the individual complex subunits and the possible presence of metal or other adducted species.

Animals↗
PubMed2000

Nociceptin attenuates opioid and gamma-aminobutyric acid(B) receptor-mediated analgesia in the mouse tail-flick assay.

Nociceptin (NC) and the opioid receptor like-1 receptors are widely distributed in areas of the neuraxis that are part of the descending modulatory pain system. We used the tail-flick assay in mice to assess the interaction between NC and other analgesic compounds acting on different areas of the descending pathway. Given by intracerebroventricular injection, NC induced hyperalgesia at 10 nmol (39% of reduction vs. control group). The same dose of NC reversed analgesia induced by distinct classes of analgesia-producing compounds such as morphine, dynorphin A or baclofen. NC caused a reduction of their antinociceptive effects: 61, 41 and 27%, respectively. Thus, NC at the supraspinal level appears to interact with both opioid and gamma-aminobutyric acid(B) systems producing anti-analgesic effects probably through the descending pathway for pain control.

Analgesics, Opioid↗
PubMed2000

Capillary isoelectric focusing of yeast cells.

In the present work, capillary isoelectric focusing (CIEF) methods were developed for the separation and identification of yeast cells. Yeast cells (approximately 4-microm diameter) cultured to various phases of growth were shown to be reproducibly resolved by CIEF using 100-microm-i.d. fused-silica capillaries coated with hydroxypropyl methylcellulose. Separation efficiencies corresponding to peak capacities of >4000 were obtained. The suitable cell concentration range for obtaining repeatable elution in CIEF separations was found to be quite low (<3 cells/microL). CIEF experiments showed that yeast cell populations at early log, mid log, and stationary growth phases differ in isoelectric point, with values ranging from 5.2 to 6.4. The broader application of CIEF are projected for microorganism identification and separation based upon growth conditions.

Isoelectric Focusing↗
PubMed2000

MS for identification of single nucleotide polymorphisms and MS/MS for discrimination of isomeric PCR products.

ESI (electrospray ionization) MS and tandem mass spectrometry (MS/MS) were used for the analysis of single nucleotide polymorphisms (SNPs) and more complex genetic variations. Double-stranded (ds) PCR products were studied. PCR products of the proline [5'-x(G17)-x(C38)x-3'] and arginine variants [(5'-x(Gl7)-x(G38)x-3'] of the p53 gene are distinguished by an SNP (cytosine or guanine) and were discriminated using both quadrupole and quadrupole ion trap MS analysis. A 69 bp arginine mutant PCR product [5'-x(C17)-x(G38)x-3'] with a negating switch has the same mass as the proline variant but was readily distinguishable on ion trap MS/MS analysis; fragments containing the mutation site, but not the polymorphism, were identified. The 69 bp PCR products were restriction-enzyme-digested, to create 43 bp fragments. ESI quadrupole ion trap MS/MS analysis of the 43 bp product-ion spectra readily demonstrated both polymorphism and negating switch sites. MS and MS/MS are powerful and complementary techniques for analysis of DNA. MS can readily distinguish SNPs but MS/MS is required to differentiate isomeric PCR products (same nucleotide composition but different sequence).

Mass Spectrometry↗
PubMed2000

Utility of accurate mass tags for proteome-wide protein identification.

An enabling capability for proteomics would be the ability to study protein expression on a global scale. While several different separation and analysis options are being investigated to advance the practice of proteomics, mass spectrometry (MS) is rapidly becoming the core instrumental technology used to characterize the large number of proteins that constitute a proteome. To be most effective, proteomic measurements must be high-throughput, ideally allowing thousands of proteins to be identified on a time scale of hours. Most strategies of identification by MS rely on the analysis of enzymatically produced peptides originating from an isolated protein followed by either peptide mapping or tandem MS (MS/MS) to obtain sequence information for a single peptide. In the case of peptide mapping, several peptide masses are needed to unambiguously identify a protein with the typically achieved mass measurement accuracies (MMA). The ability to identify proteins based on the mass of a single peptide (i.e., an accurate mass tag; AMT) is proposed and is largely dependent on the MMA that can be achieved. To determine the MMA necessary to enable the use of AMTs for proteome-wide protein identification, we analyzed the predicted proteins and their tryptic fragments from Saccharomyces cerevisiae and Caenorhabditis elegans. The results show that low ppm (i.e., approximately 1 ppm) level measurements have practical utility for analysis of small proteomes. Additionally, up to 85% of the peptides predicted from these organisms can function as AMTs at sub-ppm MMA levels attainable using Fourier transform ion cyclotron resonance MS. Additional information, such as sequence constraints, should enable even more complex proteomes to be studied at more modest mass measurement accuracies. Once AMTs are established, subsequent high-throughput measurements of proteomes (e.g., after perturbations) will be greatly facilitated.

Cyclotrons↗