Biomedical subjects
Publications and source records attributed to C Lowe.
A series of novel selective phosphodiesterase 4 (PDE4) inhibitors has been developed which displays activity both in vitro and in vivo. These compounds possess good selectivity for the catalytic site of PDE4 over the high affinity Rolipram binding site. In vivo studies demonstrate a reduced propensity to display the emetic side effects which are commonly observed with PDE4 inhibitors.
Neuropsychological test batteries are repeatedly administered to evaluate changes over time or the effects of clinical interventions. Relationships between scores on different tests within batteries are also examined to test models for associations between functional deficits. These comparisons may be misleading unless Test/Re-test reliability for individual tests is satisfactory. Interpretations of repeated measurements also depend on the extent to which improvement with practice varies between tasks and between more and less able individuals. Test/Re-test correlations and practice effects for two neuropsychological test batteries (CANTAB, ISPOCD) and from laboratory tasks commonly used in cognitive assessments of older people were obtained from large groups of healthy elderly. Tests in neuropsychological batteries varied markedly in test/re-test reliability which, in some cases, fell below levels considered methodologically acceptable. Putative measures of 'frontal' or 'executive' function, in which performance may be markedly improved by abrupt discovery of an appropriate strategy, were especially likely to show low reliability. Most tests showed significant practice effects, and on some these are substantial enough to compromise comparisons on repeated testing. On a minority of tests practice effects were counter-intuitive, in that less able showed significantly more gains than more able individuals.
AIM: To discuss the use of Integrated Care Pathways (ICPs) as tools for ensuring cost-effectiveness and high quality patient-focused care. To examine how electronic pathways combining process, practice and audit might be effectively used by Primary Care Groups as a tool to integrate care within the 'New National Health Service'. KEY ISSUES: It has been argued that for doctors pathways lead to 'cook book medicine' and for nurses they move practice away from patient-centred care back to a disease/task-based model. This article explores the origins of pathways, addresses the concerns of critics and goes on to describe a pathway strategy. CONCLUSIONS: Pathway development offers the potential to create a coherent plan of care and treatment (across primary/secondary care) which incorporates evidence-based practice and reduces fragmentation and duplication whilst aiding co-ordination and communication. The incorporation of clinical indicators and measurable goals ensures the audit process is embedded.
Animals have evolved diverse appendages adapted for locomotion, feeding and other functions. The genetics underlying appendage formation are best understood in insects and vertebrates. The expression of the Distal-less (Dll) homeoprotein during arthropod limb outgrowth and of Dll orthologs (Dlx) in fish fin and tetrapod limb buds led us to examine whether expression of this regulatory gene may be a general feature of appendage formation in protostomes and deuterostomes. We find that Dll is expressed along the proximodistal axis of developing polychaete annelid parapodia, onychophoran lobopodia, ascidian ampullae, and even echinoderm tube feet. Dll/Dlx expression in such diverse appendages in these six coelomate phyla could be convergent, but this would have required the independent co-option of Dll/Dlx several times in evolution. It appears more likely that ectodermal Dll/Dlx expression along proximodistal axes originated once in a common ancestor and has been used subsequently to pattern body wall outgrowths in a variety of organisms. We suggest that this pre-Cambrian ancestor of most protostomes and the deuterostomes possessed elements of the genetic machinery for and may have even borne appendages.
Hepatitis A virus (HAV) 3C proteinase is the enzyme responsible for the processing of the viral polyprotein. Although a cysteine proteinase, it displays an active site configuration like those of the mammalian serine proteinases (Malcolm, B. A. Protein Science 1995, 4, 1439). A peptidyl monofluoromethyl ketone (peptidyl-FMK) based on the preferred peptide substrates for HAV 3C proteinase was generated by first coupling the precursor, N,N-dimethylglutamine fluoromethylalcohol, to the tripeptide, Ac-Leu-Ala-Ala-OH, and then oxidizing the product to the corresponding peptidyl-FMK (Ac-LAAQ'-FMK). This molecule was found to be an irreversible inactivator of HAV 3C with a second-order rate constant of 3.3 x 10(2) M-1 s-1. 19F NMR spectroscopy indicates the displacement of fluoride on inactivation of the enzyme by the fluoromethyl ketone. NMR spectroscopy of the complex between the 13C-labeled inhibitor and the HAV 3C proteinase indicates that an (alkylthio)methyl ketone is formed. Studies of polyprotein processing, using various substrates generated by in vitro transcription/translation, demonstrated efficient blocking of even the most rapid proteolytic events such as cleavage of the 2A-2B and 2C-3A junctions. Subsequent ex vivo studies, to test for antiviral activity, show a 25-fold reduction in progeny virus production as the result of treatment with 5 microM inhibitor 24 h post-infection.
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Despite the profusion of promising new vaccines against illnesses prevalent in developing countries, uncertainties about the balance between costs and benefits of new vaccines have retarded their use in public health practice. Conventional prelicensure trials of vaccine protection exacerbate these uncertainties by focusing on measurement of vaccine efficacy--the performance of a vaccine under idealized conditions. Vaccine effectiveness trials provide a more pragmatic perspective by addressing the performance of a vaccine under the ordinary conditions of a public health program, by capturing direct as well as indirect effects of vaccination, and by comprehensively addressing outcomes of public health concern. The use of effectiveness trials should enable more rational triaging of new vaccines for developing countries and may accelerate the introduction of new vaccines into public health practice by resolving speculative debates about practical costs and benefits.
Inflammatory bowel disease (IBD) symptoms may be due not only to the effects of bowel inflammation, but also can result from many non-inflammatory consequences of the disease. Clinical scores and health questionnaire ratings produce a global assessment which is useful in the overall evaluation of the impact of the illness on the patient, and the effect of treatment. However many studies have failed to recognize the need to separately assess changes in the degree of bowel inflammation, in addition to a global clinical response. Radiolabelled white cell scanning using 111-indium has been shown to provide an accurate, quantitative and non-invasive method for assessing the degree of bowel inflammation in IBD and its response to therapy, using faecal collection or total body counts of radioactivity. More recently 99mTc hexamethyl propylene amine oxime (HMPAO) labelled white cell scanning has been introduced, which has advantages over the 111-indium method, including reduced radiation dosimetry and enhanced image quality. 99mTc-HMPAO scanning using three-dimensional white cell scanning (single photon emission computerized tomography; SPECT) allows visualization of the entire bowel separate from overlying structures. 99mTc-HMPAO white cell scanning with SPECT has now been computerized and automated to permit measurement of segmental and total bowel radiolabelled white cell infiltrate. This method could potentially provide the gold standard for objective assessment of the response of disease activity in Crohn's disease and ulcerative colitis, and merits application in clinical trials of novel therapies for IBD.
Kinematics and critical swimming speed (Ucrit) of juvenile scalloped hammerhead sharks Sphyrna lewini were measured in a Brett-type flume (635 l). Kinematic parameters were also measured in sharks swimming in a large pond for comparison with those of sharks swimming in the flume. Sharks in the flume exhibited a mean Ucrit of 65±11 cm s-1 (± s.d.) or 1.17±0.21 body lengths per second (L s-1), which are similar to values for other species of sharks. In both the flume and pond, tailbeat frequency (TBF) and stride length (LS) increased linearly with increases in relative swimming speed (Urel=body lengths traveled per second). In the flume, tailbeat amplitude (TBA) decreased with increasing speed whereas TBA did not change with speed in the pond. Differences in TBF and LS between sharks swimming in the flume and the pond decreased with increases in Urel. Sharks swimming at slow speeds (e.g. 0.55 L s-1) in the pond had LS 19 % longer and TBF 21 % lower than sharks in the flume at the same Urel. This implies that sharks in the flume expended more energy while swimming at comparable velocities. Comparative measurements of swimming kinematics from sharks in the pond can be used to correct for effects of the flume on shark swimming kinematics and energetics.
Picornaviral 3C proteinases are a group of closely related thiol proteinases responsible for processing of the viral polyprotein into its component proteins. These proteinases adopt a chymotrypsin-like fold [Allaire et al. (1994) Nature 369, 72-77; Matthews et al. (1994) Cell 77, 761-771] and a display an active-site configuration like those of the serine proteinases. Peptide-aldehydes based on the preferred peptide substrates for hepatitis A virus (HAV) 3C proteinase were synthesized by reduction of a thioester precursor. Acetyl-Leu-Ala-Ala-(N,N'-dimethylglutaminal) was found to be a reversible, slow-binding inhibitor for HAV 3C with a Ki* of (4.2 +/- 0.8) x 10(-8) M. This inhibitor showed 50-fold less activity against the highly homologous human rhinovirus (strain 14) 3C proteinase, whose peptide substrate specificity is slightly different, suggesting a high degree of selectivity. NMR spectrometry of the adduct of the 13C-labeled inhibitor with the HAV-3C proteinase indicate that a thiohemiacetal is formed between the enzyme and the aldehyde carbon as previously noted for peptide-aldehyde inhibitors of papain [Lewis & Wolfenden (1977) Biochemistry 16,4890-4894; Gamcsik et al. (1983) J. Am. Chem. Soc. 105, 6324-6325]. The adduct can also be observed by electrospray mass spectrometry.
A number of cytokines have been shown to exert their effects via a recently discovered signaling cascade. One step in this pathway is mediated by a family of nonreceptor protein tyrosine kinases, the Janus kinases or JAK kinases, which become phosphorylated upon ligand-receptor binding and receptor phosphorylation. This in turn is followed by phosphorylation of certain members of a family of latent transcription factors, called signal transducers and activators of transcription (STATs), which subsequently enter the nucleus, bind to DNA in a sequence-specific fashion, and modulate transcription. In view of the apparent role of leukemia inhibitory factor (LIF) in bone remodeling, we sought to determine which, if any, of the JAK/STAT family members are involved in mediating the actions of LIF using the MC3T3-E1 cell line (a spontaneously immortalized osteoblast) and normal murine calvarial osteoblasts. We report here rapid and transient phosphorylation of the LIF receptor, and similarly, we detect phosphorylation of predominantly JAK1 and to a minor extent JAK2 in response to LIF treatment in MC3T3-E1 cells. In these experiments we also detect phosphorylation of STAT1 and to a much lesser degree STAT3 upon addition of LIF. Phosphorylation of the STAT1 proteins correlates directly with their ability to bind DNA in a gel mobility shift assay in MC3T3-E1 and in normal calvarial osteoblasts. These studies suggest that LIF action in these cells, as in other cell types, is mediated in part via specific members of the JAK/STAT pathway.
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A role for pp60c-src) tyrosine kinase in bone resorption was recently demonstrated in vivo. However, it is not known whether expression of this ubiquitous tyrosine kinase is essential in osteoblasts or osteoclasts. In this work, we have examined the expression of c-src in osteoblast-like cells. We found that c-src was expressed in the MG-63 osteoblastic osteosarcoma cell line as assessed by immunocytochemistry. When MG-63 cells were treated for 30 minutes with parathyroid hormone (PTH), the levels of tyrosine phosphorylation of c-src were increased in comparison with the untreated control. On the other hand, no changes in total c-src protein were observed after PTH treatment. The increase in c-src tyrosine phosphorylation due to PTH treatment was paralleled by an increase in c-src tyrosine kinase activity. Calcitonin had no effect on c-src phosphorylation, c-src protein level, or tyrosine kinase activity. To determine if c-src tyrosine kinase was required for normal bone cell function and PTH responsiveness, osteoblastic cells were isolated from the calvaria of a src-deficient mouse. The cyclic AMP response to PTH in this cell was identical to that seen in freshly isolated calvarial cells from normal mice. These results suggest that the activity of c-src in MG-63 cells is regulated by PTH as a result of tyrosine phosphorylation. Expression of src is not required for PTH responsiveness in osteoblastic cells as assessed by adenylate cyclase activation. The mode of signal transduction from the PTH receptor to nonreceptor c-src tyrosine kinase is not known at present.
Osteopetrosis is a bone modeling disorder resulting in excessive accumulation of bone matrix due to defective function of osteoclasts, the cells that resorb bone. Mice carrying a targeted disruption of the gene Src that encodes pp60c-src (Src), a nonreceptor protein tyrosine kinase, develop this phenotype but do not exhibit other overt defects despite the fact that the kinase is normally present in a broad variety of cell types. Because Src is expressed in osteoblasts as well as in osteoclasts and both are required for normal bone resorption, the basic defect could occur in either cell type. In this study we have used in vitro approaches and fetal liver transplantation into irradiated Src- recipients to demonstrate that the inherent defect is with osteoclasts and autonomous of the bone marrow microenvironment. This result (i) identifies a cell type in which Src function is essential and cannot be replaced by other related kinases and (ii) should allow the isolation of a substrate that is specific to Src.
Since absence of expression of the c-src gene product in mice indicates that the pp60c-src tyrosine kinase is required and essential for osteoclastic bone resorption, we tested the effects of the antibiotic herbimycin A, which is an inhibitor of pp60c-src on osteoclastic bone resorption in vitro and on hypercalcemia in vivo. We examined the effects of herbimycin A on the formation of bone resorbing osteoclasts in mouse long-term marrow cultures, on isolated rodent osteoclasts and on bone resorption in organ cultures of fetal rat long bones stimulated by parathyroid hormone. We found that herbimycin A in concentrations of 1-100 ng/ml inhibited bone resorption in each of these systems. We determined the effects of herbimycin A (100 ng/ml) on src tyrosine kinase activity in mouse marrow cultures and found that it was decreased. Herbimycin A also decreased elevated blood calcium levels that were induced either by repeated subcutaneous injections of recombinant human interleukin-1 alpha or by a human tumor. There was no evidence for toxicity in any of these culture systems or in mice treated with herbimycin A. A different tyrosine kinase inhibitor that does not inhibit pp60c-src was used as a control and caused none of these effects. These data suggest that pp60c-src tyrosine kinase inhibitors may be useful pharmacologic inhibitors of osteoclastic bone resorption and hypercalcemia.
Recent studies have shown that the protooncogene c-src is required for normal osteoclastic bone resorption. In this study we examined the expression and regulation of pp60c-src in murine bone marrow cultures in which bone-resorbing multinucleated osteoclasts form over 6 days of culture. We found that both pp60c-src protein expression and pp60c-src tyrosine kinase activity correlated closely with the numbers of active osteoclasts in the cultures. PTH increased the numbers of osteoclasts, pp60c-src tyrosine kinase activity, and src protein, whereas calcitonin decreased the numbers of osteoclasts, src protein, and tyrosine kinase activity. However, when calcitonin was incubated for short periods (< 2 h) with the active osteoclasts present after 6 days of culture, there was a decrease in pp60c-src tyrosine kinase activity and the phosphorylation state, but not in total pp60c-src protein. These data suggest that pp60c-src is expressed in cultures of osteoclasts in parallel with the number of active bone-resorbing osteoclasts. They indicate that pp60c-src activity in osteoclast cultures depends on the activity and numbers of osteoclasts and is hormone regulated. As calcitonin receptors are detectable only in osteoclasts in these cultures, the inhibitory effects of calcitonin suggest that the critical site for pp60c-src expression in these cultures is in osteoclasts.
Chronic glucocorticoid excess is associated with the development of osteoporosis and, in human subjects, there is histomorphometric evidence of increased bone resorption. Paradoxically, most in vitro studies have suggested that glucocorticoids inhibit bone resorption but recently two groups have demonstrated increased osteolysis in glucocorticoid-treated bone organ cultures. The present study reexamines the effect of cortisol on basal bone resorption in neonatal mouse calvaria with particular emphasis on the effect of serum supplementation of the media. In the absence of serum, 45Ca release was significantly stimulated by 10(-7) M cortisol (treatment/control 1.37 +/- 0.06, P less than 0.005) and by 10(-6) M cortisol (treatment/control 1.27 +/- 0.08, P less than 0.005). The stimulation of resorption by 10(-7) M hydrocortisone was progressive from 24 to 96 hours of incubation. In contrast, when calvaria were incubated in the presence of 5% serum, bone resorption was not increased by cortisol (10(-8) M-10(-6) M). In the presence of 5% charcoal-stripped, heat-inactivated serum, there was a small stimulation of 45Ca release at 10(-6) M hydrocortisone only (treatment/control 1.19 +/- 0.06, P less than 0.01). Incubation of bones with indomethacin did not modify the effect of cortisol in either the presence or absence of serum. In serum-free conditions, cortisol 10(-8) M significantly inhibited the rate of thymidine incorporation, though at higher concentrations this effect was not seen. Cortisol produced a dose-related inhibition of serum-stimulated thymidine incorporation. It is concluded that the presence of serum substantially modifies the effect of cortisol on basal bone resorption.(ABSTRACT TRUNCATED AT 250 WORDS)
Targeted disruption of the c-src proto-oncogene in mice has shown that src expression is required for normal bone resorption, since the src-deficient mutants develop osteopetrosis. To evaluate the mechanisms by which src-deficiency affects osteoclast function, we treated src-deficient mice with the stimulants of bone resorption, IL-1, parathyroid hormone, and parathyroid hormone-related protein, and analyzed the effects by quantitative bone histomorphometry and electron microscopy. Increased numbers of multinucleated cells with the morphological characteristics of osteoclasts appeared on bone surfaces, but these cells did not form ruffled borders or normal resorption lacunae. To confirm these in vivo findings, we cultured src-mutant bone marrow cells on dentine slices in the presence of 1,25 dihydroxyvitamin D3. Increased numbers of multinucleated cells were formed, but unlike normal murine bone marrow cells, they did not form resorption pits. These results indicate that osteoclasts appear in the absence of pp60c-src, but that pp60c-src expression is required for mature osteoclasts to form ruffled borders and resorb bone.