Biomedical subjects
Publications and source records attributed to R D Smith.
We report a case of a profundus tendon sheath fibroma which initially presented as triggering on wrist movements but rapidly caused a flexor tenosynovitis limiting both finger and wrist movements. Surgical excision was curative.
Resistance to antimicrobial drugs is increasing worldwide. This resistance is, at least in part, associated with high antimicrobial usage. Despite increasing awareness, economists (and policy analysts more generally) have paid little attention to the problem. In this paper antimicrobial resistance is conceptualised as a negative externality associated with the consumption of antimicrobials and is set within the broader context of the costs and benefits associated with antimicrobial usage. It is difficult to determine the overall impact of attempting to reduce resistance, given the extremely limited ability to model the epidemiology of resistant and sensitive micro-organisms. It is assumed for the purposes of the paper, however, that dealing with resistance by reducting antimicrobial usage would lead to a positive societal benefit. Three policy options traditionally associated with environmental economics (regulation, permits and charges) are examined in relation to their potential ability to impact upon the problem of resistance. The primary care sector of the U.K.'s National Health Service provides the context for this examination. Simple application of these policies to health care is likely to be problematic, with difficulties resulting particularly from the potential reduction in clinical freedom to prescribe when appropriate, and from the desire for equity in health care provision. The paper tentatively concludes that permits could offer the best policy response to antimicrobial resistance, with the caveat that empirical research is needed to develop the most practical and efficient system. This research must be conducted alongside the required epidemiological research.
Electrospray ionization (ESI) Fourier transform ion cyclotron resonance (FTICR) mass spectrometry has been used to characterize heterotetrameric corynebacterial sarcosine oxidase. By using a conventional quadrupole mass spectrometer, no spectra for the intact complex could be obtained (i.e., electrospraying protein at neutral pH), but spectra showing the four protein subunits were obtained when electrospraying from acidic solution. Initial low resolution ESI-FTICR mass spectra of the intact heterotetramer revealed a typical narrow charge state distribution in the range 6000 < m/z < 9000, consistent with retention of a compact structure in the gas phase, and gave a mass measurement about 1000 u higher than predicted. Efficient in-trap clean up, based upon low energy collisionally induced dissociation of adducts, allowed significant improvement in mass measurement accuracy. The present results represent the largest heteromultimeric protein complex successfully analyzed using FTICR mass spectrometry, and clearly illustrate the importance of sample clean up methods for large molecule characterization.
Initial results obtained using a new electrospray ionization (ESI) Fourier transform ion cyclotron resonance (FTICR) mass spectrometer operated at a magnetic field 11.5 tesla are presented. The new instrument utilized an electrostatic ion guide between the ESI source and FTICR trap that provided up to 5% overall transmission efficiency for light ions and up to 30% efficiency for heavier biomolecules. The higher magnetic field in combination with an enlarged FTICR ion trap made it possible to substantially improve resolving power and operate in a more robust fashion for large biopolymers compared to lower field instruments. Mass resolution up to 10(6) has been achieved for intermediate size biopolymers such as bovine ubiquitin (8.6 kDa) and bovine cytochrome c (12.4 kDa) without the use of frequency drift correction methods. A mass resolution of 370,000 has been demonstrated for isotopically resolved molecular ions of bovine serum albumin (66.5 kDa). Comparative measurements were made with the same spectrometer using a lower field 3.5-tesla magnet allowing the performance gains to be more readily quantified. Further improvements in pumping capacity of the vacuum system and efficiency of ion transmission from the source are expected to lead to further substantial sensitivity gains.
Type one serine/threonine protein phosphatases (PP1s) have been implicated in various processes of plant growth and development. In all plant species studied, PP1s are encoded by multigene families. Previous studies in our laboratory identified five Arabidopsis thaliana PP1 genes (TOPP1, TOPP2, TOPP3, TOPP4 and TOPP5). In the present study, we report the isolation of three additional PP1 genes (TOPP6, TOPP7 and TOPP8). Southern blot analyses indicate that these three newly isolated genes are single-copy genes in A. thaliana genome. All the three genes are expressed in roots, rosettes and flowers, although their expression levels appear to be lower than those of the five previously identified TOPP genes. Six of the eight TOPP genes were mapped to different positions on four of five A. thaliana chromosomes. Sequence comparison revealed that TOPP genes belong to different subgroups of plant PP1 genes, suggesting that they may encode proteins with distinct functions.
Injury sustained through falling is a significant risk for the elderly and a significant burden on the health service. Although many risk factors have been detected and interventions proposed, there remains limited evidence concerning the cost-effectiveness of fall prevention. This study addressed the cost-effectiveness of a home assessment and modification program hypothesised to reduce risk of falling for the independent elderly. Due to a lack of direct clinical trial evidence concerning such an intervention, a decision analytic model was developed to simulate the potential costs and outcomes of the intervention. The model was developed using available published literature concerning injury in the elderly, focusing on Australian data where possible. Cost-effectiveness was estimated as the cost per fall prevented and cost per injury prevented. Over a one-year period, the incremental cost of introducing the intervention was $172 per person, resulting in an incremental cost per fall prevented of $1,721 and cost per injury prevented of $17,208. Over a 10-year period, the intervention resulted in a cost saving of $92 per person (i.e. dominance, with cost savings in addition to reduced falls and injuries). This analysis indicates that there is potential for considerable benefit to be gained from this intervention, in terms of less morbidity, fewer hospitalisations and, possibly, improved quality of life. However, these results are based on a model constructed from various data sources and assumptions so, although results are indicative, further research is required to provide firm data before definitive policy conclusions and recommendations may be made.
The agonist-induced phosphorylation sites of the rat AT1a angiotensin receptor were analyzed using epitope-tagged mutant receptors expressed in Cos-7 cells. Angiotensin II-stimulated receptor phosphorylation was unaffected by truncation of the cytoplasmic tail of the receptor at Ser342 (Delta342) but was abolished by truncation at Ser325 (Delta325). Truncation at Ser335 (Delta335), or double-point mutations of Ser335 and Thr336 to alanine (ST-AA), reduced receptor phosphorylation by approximately 50%, indicating that in addition to Ser335 and/or Thr336, amino acids within the Ser326-Thr332 segment are also phosphorylated. Agonist-induced phosphorylation of the ST-AA and Delta335 receptors was partially inhibited by staurosporine, suggesting that the single protein kinase C consensus site in the Ser326-Thr332 segment (Ser331) is phosphorylated. The impairment of receptor phosphorylation was broadly correlated with the attenuation of agonist-induced internalization rates (Delta325 < Delta335 < ST-AA < Delta342 < wild-type) and with the increasing rank order of magnitude of inositol phosphate production normalized to an equal number of receptors (Delta325 > Delta335 > ST-AA = Delta342 > wild-type). These results demonstrate that agonist-induced phosphorylation of the AT1a receptor is confined to an 11-amino-acid serine/threonine-rich segment of its carboxyl-terminal cytoplasmic tail and implicate this region in the mechanisms of receptor internalization and desensitization.
A renal biopsy from a 36-year-old man with AIDS showed a severe tubulointerstitial nephritis with intranuclear inclusions in epithelial cells. Electron microscopy revealed the characteristic findings of a polyomavirus (PyV) infection, and immunofluorescence indicated the presence of BK virus (BKV) antigen. Inoculation of rhesus monkey kidney cell cultures both with urine and with buffy coat blood cells resulted in a cytopathic response which was subsequently confirmed to be due to BKV. Further characterization of the viral DNA from the kidney by PCR amplification and Southern blot analysis with PyV and strain-specific primers and probes indicated that the virus was closely related to the BK(Dun) strain but different in its apparent sequence arrangement. Subsequent cycle sequencing showed a dinucleotide mutation of TG-->AA which substitutes hydrophilic Gln for hydrophobic Leu in a sequence homologous to an origin DNA-binding domain of simian virus 40 T antigen. It is suggested that the mutation and a coding region rearrangement of this strain of BKV designated BKV(Cin) has the potential to alter viral DNA replication and enhance pathogenicity.
Angiotensin II (Ang II) stimulates growth and mitogenesis in bovine adrenal glomerulosa cells, but little is known about the signaling pathways that mediate these responses. An analysis of the growth-promoting pathways in cultured bovine adrenal glomerulosa cells revealed that Ang II, acting via the AT1 receptor, caused rapid but transient activation of mitogen-activated protein kinase (MAPK), with an ED50 of 10-50 pM. Although neither Ca2+ influx nor Ca2+ release from intracellular stores was sufficient to activate MAPK, Ca2+ appeared to play a permissive role in this response. A major component of Ang II-induced MAPK activation was insensitive to pertussis toxin (PTX), although a minor PTX-sensitive component could not be excluded. Ang II also induced the rapid activation of ras and raf-1 kinase with time-courses that correlated with that of MAPK. Activation of protein kinase C (PKC) by phorbol 12-myristate 13-acetate was sufficient to activate both MAPK and raf-1 kinase. However, whereas PKC depletion had no effect on Ang II-induced raf-1 kinase activation, it attenuated Ang II-induced MAPK activation. Ang II also stimulated a mobility shift of raf-1, reflecting hyperphosphorylation of the kinase. However, unlike its activation, raf-1 hyperphosphorylation was dependent on PKC and its time-course correlated not with activation, but rather with deactivation of the kinase. Taken together, these findings indicate that Ang II stimulates multiple pathways to MAPK activation via PKC and ras/raf-1 kinase in bovine adrenal glomerulosa cells.
A polyclonal antibody was raised in rabbits against a fusion protein immunogen consisting of bacterial maltose-binding protein coupled to a 92-amino acid C-terminal fragment of the rat AT1b angiotensin II (Ang II) receptor. The antibody immunoprecipitated the photoaffinity-labeled bovine AT1 receptor (AT1-R), but not the rat AT2 receptor, and specifically stained bovine adrenal glomerulosa cells and AT1a receptor-expressing Cos-7 cells, as well as the rat adrenal zona glomerulosa and renal glomeruli. The antibody was employed to analyze Ang II-induced phosphorylation of the endogenous AT1-R immunoprecipitated from cultured bovine adrenal glomerulosa cells. Receptor phosphorylation was rapid, sustained for up to 60 min, and enhanced by pretreatment of the cells with okadaic acid. Its magnitude was correlated with the degree of ligand occupancy of the receptor. Activation of protein kinase A and protein kinase C (PKC) also caused phosphorylation of the receptor, but to a lesser extent than Ang II. Inhibition of PKC by staurosporine augmented Ang II-stimulated AT1-R phosphorylation, suggesting a negative regulatory role of PKC on the putative G protein-coupled receptor kinase(s) that mediates the majority of AT1-R phosphorylation. The antibody should permit further analysis of endogenous AT1-R phosphorylation in Ang II target cells.
BACKGROUND: The cornerstone of the National Health Service (NHS) reforms was the establishment of an internal market, which separated purchasing and providing roles. As purchasers of care, general practice fundholders were seen as a pivotal part of the 'new patient-led NHS', which was intended to lead to improved cost-containment and cost-effectiveness, quality of care, and patient choice and empowerment. AIM: To review published evidence of the extent to which these objectives may have been achieved over the past six years. METHOD: Keyword search of on-line databases (MEDLINE and Econ-lit) from 1990 to 1996, plus manual search of references within those articles identified. RESULTS: In the absence of any formal evaluation of fundholding, it is difficult to assess the overall success of this reform. However, in terms of cost-containment and cost-effectiveness, there is mixed evidence. In some areas, such as prescribing, the evidence suggests cost-savings, although the evidence is less clear on reductions or changes in referrals. There is also evidence that suggests that improvements in prescribing may have been achieved at substantial additional administration and transaction costs. With respect to quality of care, the evidence suggests that, although quality in the procedural aspects of health provision has improved, there is little evidence about how health outcomes may have been affected. In terms of patient choice and empowerment, the evidence suggests that, whilst general practitioner choice of secondary providers has improved, little progress has been made with regard to increased consumer choice. CONCLUSION: Evidence concerning the success or otherwise of general practice fundholding over the past six years is incomplete and mixed. The major deficiency concerns any effect on health outcomes that may be the result of fundholding. Until such research is conducted, the jury will have to remain out on whether fundholding has secured improved efficiency in the delivery of health care.
Phosphorylation of brain Na+ channel alpha subunits by cAMP-dependent protein kinase (PKA) decreases peak Na+ current in cultured brain neurons and in mammalian cells and Xenopus oocytes expressing cloned brain Na+ channels. We have studied PKA regulation of Na+ channel function by activation of D1-like dopamine receptors in acutely isolated hippocampal neurons using whole-cell voltage-clamp recording techniques. The D1 agonist SKF 81297 reversibly reduced peak Na+ current in a concentration-dependent manner. No changes in the voltage dependence or kinetics of activation or inactivation were observed. This effect was mediated by PKA, as it was mimicked by application of the PKA activator Sp-5, 6-dichloro-1-beta-D-ribofuranosylbenzimidazole-3', 5'-monophosphorothioate(cBIMPS) and was inhibited by the specific PKA inhibitor peptide PKAI5-24. cBIMPS had similar effects on type IIA brain Na+ channel alpha subunits expressed in tsA-201 cells, but no effect was observed on a mutant Na+ channel alpha subunit in which serine residues in five PKA phosphorylation sites in the intracellular loop connecting domains I and II (LI-II) had been replaced by alanine. A single mutation, S573A, similarly eliminated cBIMPS modulation. Thus, activation of D1-like dopamine receptors results in PKA-dependent phosphorylation of specific sites in LI-II of the Na+ channel alpha subunit, causing a reduction in Na+ current. Such modulation is expected to exert a profound influence on overall neuronal excitability. Dopaminergic input to the hippocampus from the mesocorticolimbic system may exert this influence in vivo.
Peak spectral amplitudes for choline moieties (Cho), creatine (Cr), N-acetylaspartate (NAA), and myo-inositol (mI) were examined using proton magnetic resonance spectroscopy in 19 social-phobia patients and 10 controls. Compared with controls, social phobics demonstrated significantly lower NAA/Cho and higher Cho/Cr, mI/Cr, and mI/NAA amplitudes in cortical gray matter. Higher mI/Cr and mI/NAA amplitudes for social phobics were also observed in subcortical gray matter. Minimal between-group differences were noted for white matter. Symptom severity correlated inversely with amplitudes primarily in subcortical gray matter, and to a lesser extent in cortical gray matter. Inclusion of age and sex in statistical modeling strengthened comparisons with controls but eliminated those for symptom severity. No changes were observed in any amplitude measure following at least 8 weeks of clonazepam treatment, nor was a relationship observed between amplitudes and cumulative clonazepam dose. The possible psychobiological implications of our findings in social phobia are discussed.
Voltage-gated sodium channels respond to excitatory inputs in nerve cells, generating spikes of depolarization at axon hillock regions and propagating the initial rising phase of action potentials through axons. It previously has been shown that protein kinase A (PKA) attenuates sodium current amplitude 20-50% by phosphorylating serines located in the I-II linker of the sodium channel. We have tested the individual contributions of five PKA consensus sites in the I-II linker by measuring sodium currents expressed in Xenopus oocytes during conditions of PKA induction. PKA was induced by perfusing oocytes with a cocktail that contained forskolin, chlorophenylthio-cAMP, dibutyryl-cAMP, and 3-isobutyl-1-methylxanthine. Phosphorylation at the second PKA site (serine-573) was necessary and sufficient to diminish sodium current amplitude. Phosphorylation at the third and fourth positions (serine-610 and serine-623) reduced current amplitude, but the effect was considerably smaller at those positions. Introduction of a negative charge at site 2 by substitution of serine-573 with an aspartate constitutively reduced the basal level of sodium current, indicating that the attenuation of sodium current by phosphorylation of site 2 by PKA results from the introduction of a negative charge at this site.
A generally applicable algorithm has been developed to allow base composition of polymerase chain reaction (PCR) products to be determined from mass spectrometrically measured molecular weights and the complementary nature of DNA. Mass measurements of arbitrary precision for single-stranded DNA species are compatible with an increasingly large number of possible base compositions as molecular weight increases. For example, the number of base compositions that are consistent with a molecular weight of 35,000 is approximately 6000, based on a mass measurement precision of 0.01%. However, given the low misincorporation rate of standard DNA polymerases, mass measurement of both of the complementary single strands produced in the PCR reduces the number of possibilities to less than 100 at 0.01% mass precision, and base composition is uniquely defined at 0.001% mass precision. Taking into account the low misincorporation rate of standard DNA polymerases and the fact that the final PCR product also contains primers of known sequence (generally 15-20-mer in size, which flank the targeted region), this reduces the number of possible base combinations to only approximately 3 at MW = 35,000. In addition, the number of base pairs (i.e., length of the DNA molecule) is uniquely defined. We show that the use of modified bases in PCR or post-PCR modification chemistry allows unique solutions for the base composition of the PCR product with only modest mass measurement precision.
A new arrangement consisting of two separate Fourier transform ion cyclotron resonance (FTICR) ion traps was used to develop methods for the manipulation of the ions produced by an electrospray ionization source (ESI). A first, "accumulation" trap, is generally maintained at a higher pressure than the second, high-performance "analyzer" trap. The manipulations developed and demonstrated include the following: (1) mass-selective ion transfers between the traps; (2) mass-selective step-wise accumulation of low-abundance ions of different mass-to-charge ratios transferred from the first trap to the analyzer trap; (3) simultaneous detection of ions in the analyzer trap and ion accumulation in the source trap; (4) simultaneous ion detection in the accumulation trap and ion storage in the analyzer trap; (5) sequential multiple transfers of the ions into the analyzer trap from the same ion population stored in the accumulation trap; (6) collision-induced dissociation of ions stored in the accumulation trap followed by mass-selective transfer of the product ions into the analyzer trap; (7) sequential transfer of the ions of different mass-to-charge ratios into the analyzer trap from the same ion population stored in the accumulation trap followed by the collision-induced dissociation of transferred ions in the analyzer trap. These ion manipulations benefit multistage studies and are projected to be useful in many biochemical applications of ESI-FTICR, including structural determination of biopolymers and study of noncovalent complexes.
OBJECTIVE: To compare the value of synthetic serum substitute and fetal cord serum (FCS) as protein supplements in the media used for IVF. DESIGN: Prospective, randomized study of 45 patients undergoing IVF. SETTING: Private infertility practice. INTERVENTION(S): The protein supplement for use in IVF medium was randomized prospectively to synthetic serum substitute or FCS. MAIN OUTCOME MEASURE(S): Oocyte fertilization rate, incidence of embryo arrest, number of embryos transferred, ongoing pregnancy rate, and implantation rate were assessed. RESULT(S): There was no significant difference in the fertilization rate, incidence of embryo arrest, number of embryos transferred, ongoing pregnancy rate, and implantation rate. CONCLUSION(S): Synthetic serum substitute is a better alternative to FCS as a protein supplement for IVF because of its availability and cost effectiveness. Synthetic serum substitute can be used in culture medium for sperm washing, insemination, culture, and transfer translating to decreased technician time without a significant difference in fertilization, pregnancy, or implantation rate.
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