Biomedical subjects
Publications and source records attributed to S Jones.
Sarah Jones provides a practical aide-memoire for care staff on identifying reasons for incontinence and ideas on promoting continence in older people. She points out that healthcare professionals whose own continence is compromised may not make the connection between their own symptoms and those of older people.
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STUDY DESIGN: The psychometric properties and clinical use of a battery of physical performance measures were tested on 44 patients with low back pain and 48 healthy, pain-free control subjects. OBJECTIVES: Reliability, validity, and clinical use of nine physical performance measures were evaluated. SUMMARY OF BACKGROUND DATA: Although physical performance measures have potential use in evaluation, treatment planning, and determination of treatment outcome, there is sparse systematic investigation of their reliability, validity, and clinical use. METHODS: Forty-four subjects with low back pain and 48 healthy pain-free subjects participated. The following physical performance measures were tested: distance walked in 5 minutes; 50-foot walk at fastest speed; 50-foot walk at preferred speed; 5 repetitions of a sit-to-stand task; 10 repetitions of a repeated trunk flexion task; timed up-and-go task; unloaded forward reach task; loaded forward reach task; and Sorensen fatigue test. Subjects were assessed twice on 2 days. RESULTS: All measures had excellent intertester reliability (intraclass correlation coefficient [ICC]1,1 > 0.95). Test-retest (within session) reliability was adequate for all measures (ICC1,1 > 0.83) except repeated trunk flexion (ICC1,1 > 0.45) in the low back pain group. Test-retest (day-to-day) reliability ranged between 0.59 and 0.88 in the low back pain group and between 0.46 and 0.76 in the control group. Day-to-day reliability improved when the averages of two trials of repeated trunk flexion and sit-to-stand were used (0.76-0.91 low back pain group and 0.62-0.89 control group). Results of a multivariate analysis of variance showed a significant effect of group (F10,65 = 3.52, P = 0.001). Results of univariate analyses showed significant group differences on all measures except the 50-foot walk at preferred speed and unloaded forward reach. Self-report of disability was moderately correlated with the performance tasks (r = 0.400 to -0.603). CONCLUSIONS: The results provide support for the use of these physical performance measures as a complement to patient self-report.
Cathepsin K is a recently identified lysosomal cysteine proteinase. It is abundant in osteoclasts, where it is believed to play a vital role in the resorption and remodeling of bone. Pycnodysostosis is a rare inherited osteochondrodysplasia that is caused by mutations of the cathepsin-K gene, characterized by osteosclerosis, short stature, and acroosteolysis of the distal phalanges. With a view to delineating the role of cathepsin K in bone resorption, we generated mice with a targeted disruption of this proteinase. Cathepsin-K-deficient mice survive and are fertile, but display an osteopetrotic phenotype with excessive trabeculation of the bone-marrow space. Cathepsin-K-deficient osteoclasts manifested a modified ultrastructural appearance: their resorptive surface was poorly defined with a broad demineralized matrix fringe containing undigested fine collagen fibrils; their ruffled borders lacked crystal-like inclusions, and they were devoid of collagen-fibril-containing cytoplasmic vacuoles. Assaying the resorptive activity of cathepsin-K-deficient osteoclasts in vitro revealed this function to be severely impaired, which supports the contention that cathepsin K is of major importance in bone remodeling.
The determination of protein structures has furthered our understanding of how various proteins perform their functions. With the large number of structures currently available in the PDB, it is necessary to be able to easily study these proteins in detail. Here new software tools are presented which aim to facilitate this analysis; these include the PDBsum WWW site which provides a summary description of all PDB entries, the programs TOPS and NUCPLOT to plot schematic diagrams representing protein topology and DNA-binding interactions, SAS a WWW-based sequence-analysis tool incorporating structural data, and WWW servers for the analysis of protein-protein interfaces and analyses of over 300 haem-binding proteins.
The CATH database of protein domain structures classifies structures according to their (C)lass, (A)rchitecture, (T)opology or fold and (H)omologous family (http://www.biochem.ucl.ac.uk/bsm/cath). Although the protocol used is mostly automatic, manual inspection is used to check assignments at some critical stages, such as the detection of very distantly related homologues and anologues and the assignment of novel architectures. Described in this article is a recently established facility to search the database with the coordinates of a newly determined structure. The CATH server first locates domain boundaries and then uses automatic sequence and structure comparison methods to assign this new structure to one or more of the domain families within CATH. Diagnostic reports are generated, together with multiple structural alignments for close relatives. The Server can be accessed over the World Wide Web (WWW) and mirror sites are planned to improve access.
Homologous recombination was used to construct a series of hybrid chaperonin genes, containing various lengths of Escherichia coli groEL replaced by the equivalent region from the homologous cpn60-1 gene of Rhizobium leguminosarum. Analysis of proteins produced by these hybrids showed that many of them formed structures with properties consistent with their being single heptameric rings under some conditions, as opposed to the double ring form in which both the GroEL and the Cpn60-1 proteins are found. By determining precise cross-over points, two regions in Cpn60-1 were defined which appeared to be critical for ring-ring interactions. Within one of these regions is a highly conserved arginine residue (Arg101), which we hypothesised to interact with a residue or residues toward the C terminus of the protein, this contact being required for double rings to form. To test this hypothesis, we mutagenised this residue from arginine to threonine in chaperonin genes from two different species of Rhizobium. In both cases, proteins which ran on non-denaturing gels as single rings were produced. Conversion of Arg101 to serine also had the same effect, whereas conversion of Arg101 to lysine did not. Two different single rings created by homologous recombination could be converted back to double rings by changing the threonine, which naturally occurs at this position in E. coli GroEL, back to arginine. The in vivo properties of the proteins were investigated by complementation following deletion of the chromosomal copy of the groEL gene, and by monitoring the ability of cells expressing the hybrid proteins to plate bacteriophage. Most of the hybrid and mutant proteins were functional in these assays, despite their altered properties compared to wild-type GroEL.
BACKGROUND: The recent rapid increase in the number of available three-dimensional protein structures has further highlighted the necessity to understand the relationship between biological function and structure. Using structural classification schemes such as SCOP, CATH and DALI, it is now possible to explore global relationships between protein fold and function, something which was previously impractical. RESULTS: Using a relational database of CATH data we have generated fold distributions for arbitrary selections of proteins automatically. These distributions have been examined in the light of protein function and bound ligand. Different enzyme classes are not clearly reflected in distributions of protein class and architecture, whereas the type of bound ligand has a much more dramatic effect. CONCLUSIONS: The availability of structural classification data has enabled this novel overview analysis. We conclude that function at the top level of the EC number enzyme classification is not related to fold, as only a very few specific residues are actually responsible for enzyme activity. Conversely, the fold is much more closely related to ligand type.
1. The kinetics of desensitization of the 5-HT3 receptor (5-HT3R)-gated ion channel were investigated using whole-cell and perforated-patch recording techniques in NG108-15 cells. 2. Rapid application of 5-HT (50 microM) elicited a 5-HT3R-mediated inward current response that desensitized completely in the continued presence of agonist. In the whole-cell recording configuration (holding potential of -70 mV) while buffering internal calcium (Cai2+) with 5 mM EGTA (0.5 mM added Ca2+; with an estimated free [Ca2+] of 30 nM), the rate of desensitization was initially rapid (with a half-time of approximately 230 ms), but dramatically slowed with time by 1120 +/- 160%. 3. This slowing in the rate of desensitization was reduced by stronger Ca2+ buffering (20 mM BAPTA, without added Ca2+), or by the bath application of cadmium (100 microM) to block voltage-gated Ca2+ channels. The rate of desensitization was also dependent on membrane potential. 4. In perforated-patch recordings, the rate of desensitization remained constant. However, a slowing in the desensitization rate could be induced by depolarizing cells immediately prior to the application of 5-HT. 5. The depolarization-induced slowing was blocked by incubating cells with BAPTA-AM (a membrane-permeant analogue of BAPTA) or by the bath application of cadmium. 6. These data suggest that Ca2+ influx through a cadmium-sensitive voltage-gated Ca2+ channel increases the cytoplasmic Ca2+ concentration ([Ca2+]i) and induces a dramatic slowing in the kinetics of desensitization of the 5-HT3R channel. These data provide evidence for cross-talk between voltage-gated Ca2+ channels and 5-HT3Rs in NG108-15 cells.
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Current clinical gene therapy protocols for the treatment of human immunodeficiency virus type 1 (HIV-1) infection often involve the ex vivo transduction and expansion of CD4+ T cells derived from HIV-positive patients at a late stage in their disease (CD4 count <400). These protocols involve the transduction of T cells by murine leukemia virus (MLV)-based vectors encoding antiviral constructs such as the rev m10 dominant negative mutant or a ribozyme directed against the CAP site of HIV-1 RNA. We examined the efficiency and stability of transduction of CD4+ T cells derived from HIV-infected patients at different stages in the progression of their disease, from seroconversion to AIDS. CD4+ T cells from HIV-positive patients and uninfected donors were transduced with MLV-based vectors encoding beta-galactosidase and an intracellular antibody directed against gp120 (sFv 105) or Tat. (sFvtat1-Ckappa). The expression of marker genes and the effects of the antiviral constructs were monitored in vitro in unselected transduced CD4+ T cells. Efficiency and stability of transduction varied during the course of HIV infection; CD4+ T cells derived from asymptomatic patients were transducible at higher efficiencies and stabilities than CD4+ T cells from patients with acquired immunodeficiency syndrome (AIDS). Expression of the anti-tat intracellular antibody was more effective at stably inhibiting HIV-1 replication in transduced cells from HIV-infected individuals than was sFv 105. The results of this study have important implications for the development of a clinically relevant gene therapy for the treatment of HIV-1 infection.
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A consensus approach for the assignment of structural domains in proteins is presented. The approach combines a number of previously published algorithms, and takes advantage of the elevated accuracy obtained when assignments from the individual algorithms are in agreement. The consensus approach is tested on a data set of 55 protein chains, for which domain assignments from four automated methods were known, and for which crystallographers assignments had been reported in the literature. Accuracy was found to increase in this test from 72% using individual algorithms to 100% when all four methods were in agreement. However a consensus prediction using all four methods was only possible for 52% of the dataset. The consensus approach [using three publicly available domain assignment algorithms (PUU, DETECTIVE, DOMAK)] was then used to make domain assignments for a data set of 787 protein chains from the Protein Data Bank. Analysis of the assignments showed 55.7% of assignments could be made automatically, and of these, 13.5% were multi-domain proteins. Of the remaining 44.3% that could not be assigned by the consensus procedure 90.4% had their domain boundaries assigned correctly by at least one of the algorithms. Once identified, these domains were analyzed for trends in their size and secondary structure class. In addition, the discontinuity of each domain along the protein chain was considered.
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Explant cultures of adult rabbit bone were obtained from a variety of skeletal sites, with the intention of using the cells in a new type of bone graft. The explant cultures contained large numbers of esterase-positive, alkaline phosphatase-negative, TRAP-negative macrophage-like cells (MP), as well as osteoblast-like osteoprogenitor (OP) cells (alkaline phosphatase-positive). OP numbers were assessed by cell counts and MTT assay. The presence of the MP cells appeared to give rise to a reduction in OP numbers in culture, through cell detachment and cell lysis. In addition, after passaging, many OP were unable to reattach to the culture vessels in the presence of MP, depending upon the surface area available for reattachment. The presence of tumor necrosis factor alpha (TNFalpha) in culture medium from these cell cultures was demonstrated by a specific enzyme-linked immunosorbent assay (ELISA). A direct relationship was demonstrated between MP numbers and TNFalpha concentration and an inverse relationship between MP numbers and OP numbers in co-cultures. This was also found when OP cells were exposed to different concentrations of rTNFalpha, in place of the MP. Incubation with anti-TNFalpha inhibited the effect of MP and TNFalpha on OP. These results suggest that MP are able to cause detachment and lysis of OP cells, probably by secretion of a soluble factor in vitro which may be TNFalpha.