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Biomedical subjects

J Clarke

Publications and source records attributed to J Clarke.

At least 73 records · Page 4Linked to original sources
PubMed2001

Effect of gonadotrophins on the ovarian interstitial tissue of the wood mouse, Apodemus sylvaticus.

The ovarian interstitial tissue of the wood mouse (Apodemus sylvaticus) is extensively developed. The effect of equine chorionic gonadotrophin (eCG) and human chorionic gonadotrophin (hCG) on ovarian interstitial tissue was investigated in wood mice from a laboratory stock. The tissue was assessed by measuring the relative size of the cells, cell nucleus diameter and (in adults) nuclear shape. Hormone-treated wood mice had larger interstitial cells and larger cell nuclei than untreated animals. In addition, the cell nuclei of adult hormone-treated wood mice had a smooth round or oval outline, whereas those of untreated animals had an irregular outline with spiky projections. Electron microscopy showed that the irregular spiky outline of the cell nuclei in untreated wood mice was caused by distortions of the nuclear membrane by a large number of intracellular lipid droplets; the droplets were less abundant in the hormone-treated animals. These experiments indicate that the cells of the interstitial tissue of the wood mouse are under the control of gonadotrophins, and that these cells are likely to be a site of the synthesis and release of steroid hormones. The methods used in this study to assess the state of the cells could be applied to animals from the field to investigate the role of interstitial tissue in the reproductive biology of wood mice.

Aging↗
PubMed2001

Patients with acute skin loss: are they best managed on a burns unit?

Patients who are critically ill and have large areas of skin loss or breakdown present a difficult management problem. They require the combination of intensive therapy facilities to support failing organs and specialized skin care, sometimes including extensive debridement and reconstruction. The expertise required for both aspects of treatment are found uniquely on a burns unit. We present five patients with large areas of cutaneous loss or damage secondary to a variety of non-burn aetiologies who were managed on a burns unit. We suggest that a burns unit may be the most appropriate place for such patients to be treated during both the acute phase of their illness and the later stages of surgical reconstruction and physical rehabilitation.

Acute Disease↗
PubMed2001

Chlamydia trachomatis: opportunistic screening in primary care.

Following the publication of the Chief Medical Officer's report on chlamydial screening, we identified the sexual health of young women as an area for concern. As part of our review we decided to opportunistically search for chlamydial genital infections using a non-invasive technique. Sexually active women under 25 years of age were invited to submit a first void urine sample for polymerase chain reaction analysis. Over the period of a year we found out that 10.9% of sexually active young women tested positive for chlamydia in our practice.

Adolescent↗
PubMed2001

An incremental approach to a Web-based computerized medical record.

Acceptance of an electronic medical record within physician practices is a challenge that the Everett Clinic and the Wenatchee Valley Clinic in Washington State have chosen to approach incrementally. These two independent clinics, with 180 physicians each, joined forces in 1995 to form an information service organization, CliniTech Information Resources, to consolidate operation of their practice management and lab systems. With that done, in 1997 they asked CliniTech to come up with a solution to give physicians online access to clinical data in a way that didn't interrupt their work-flow or decrease their productivity--not a small order. The first HTML application was On Line Transcription, for viewing chart notes and radiology reports. Next, the On Line Patient Profile was developed to replace the face sheet. On Line Lab Results followed as a way to view results from the clinics' internal and reference lab systems. A single user interface was created in 2000 to link these components to the physician's schedule and clinical references. The new computerized medical record (CMR) has been embraced by the clinics' physicians (currently at 70 percent usage) and will continue to grow with additional results and images. Electronic signature capabilities for transcription and results are also slated for 2001 and will be the first change to how the physician practices.

Clinical Laboratory Information Systems↗
PubMed2001

The drl1 gene, a signal identified by transposon tagging is important for leaf growth.

Control of leaf shape and size was studied at the molecular level by a mutational approach in Arabidopsis thaliana. Mutations that affected leaf growth allowed the identification of genes that are important in that morphological process. The Activator/Dissociation (Ac/Ds) transposable element system of maize was modified to be used as mutagen of the Arabidopsis genome. We isolated the drl 1-2 mutation that is characterized by narrow leaf blades. We describe the mutant phenotype and have cloned and analyzed the gene. These combined analyses will give insight into the molecular mechanisms that control leaf growth.

Arabidopsis↗
PubMed2000

Ultrasensitive magnetic biosensor for homogeneous immunoassay.

A technique is described for specific, sensitive, quantitative, and rapid detection of biological targets by using superparamagnetic nanoparticles and a "microscope" based on a high-transition temperature dc superconducting quantum interference device (SQUID). In this technique, a mylar film to which the targets have been bound is placed on the microscope. The film, at room temperature and atmospheric pressure, is typically 40 micrometer from the SQUID, which is at 77 K in a vacuum. A suspension of magnetic nanoparticles carrying antibodies directed against the target is added to the mixture in the well, and 1-s pulses of magnetic field are applied parallel to the SQUID. In the presence of this aligning field the nanoparticles develop a net magnetization, which relaxes when the field is turned off. Unbound nanoparticles relax rapidly by Brownian rotation and contribute no measurable signal. Nanoparticles that are bound to the target on the film are immobilized and undergo Néel relaxation, producing a slowly decaying magnetic flux, which is detected by the SQUID. The ability to distinguish between bound and unbound labels allows one to run homogeneous assays, which do not require separation and removal of unbound magnetic particles. The technique has been demonstrated with a model system of liposomes carrying the FLAG epitope. The SQUID microscope requires no more than (5 +/- 2) x 10(4) magnetic nanoparticles to register a reproducible signal.

Biosensing Techniques↗
PubMed2000

A randomised, double-blind, controlled trial of the immunogenicity and tolerability of a meningococcal group C conjugate vaccine in young British infants.

A double-blind, randomised, controlled trial was conducted in 248 British infants to assess the immunogenicity and tolerability of three doses of a meningococcal group C/CRM (197) conjugate vaccine (Lederle Laboratories, USA) given at 2, 3 and 4 months. Control children received three doses of Hepatitis B vaccine (Engerix B(R); SmithKline Beecham). At 5 months of age, 100% of children receiving the conjugate vaccine had specific immunoglobulin G concentrations >2.0 microg/ml (n=116) compared with only 4% of control children (n=121). Those receiving the conjugate also had 2.5- and 1.6-fold higher geometric mean concentrations of PRP and diphtheria antibodies, respectively. The vaccine was well tolerated.

Antibodies, Bacterial↗
PubMed2000

Two proteins with the same structure respond very differently to mutation: the role of plasticity in protein stability.

As part of a systematic study of the folding of protein structural families we compare the effect of mutation in two closely related fibronectin type III (fnIII) domains, the tenth fnIII domain of human fibronectin (FNfn10) and the third fnIII domain of human tenascin (TNfn3). This comparison of the two related proteins allows us to distinguish any anomalous response to mutation. Although they have very similar structures, the effect of mutation is very different. TNfn3 behaves like a "typical" protein, with changes in free energy correlated to the number of contacts lost on mutation. The loss of free energy upon mutation is significantly lower for FNfn10, particularly mutations of residues in the A, B and G strands. Remarkably, some of the residues involved are completely buried and closely packed in the core. In FNfn10 the regions of the protein that can accommodate mutation have previously been shown to be mobile. We propose that there is a "plasticity" in the peripheral regions of FNfn10 that allows it to rearrange to minimise the effect of mutations. This study emphasises the difficulties that might arise when making generalisations from a single member of a protein family.

Amino Acid Sequence↗
PubMed2000

Atomic force microscopy reveals the mechanical design of a modular protein.

Tandem modular proteins underlie the elasticity of natural adhesives, cell adhesion proteins, and muscle proteins. The fundamental unit of elastic proteins is their individually folded modules. Here, we use protein engineering to construct multimodular proteins composed of Ig modules of different mechanical strength. We examine the mechanical properties of the resulting tandem modular proteins by using single protein atomic force microscopy. We show that by combining modules of known mechanical strength, we can generate proteins with novel elastic properties. Our experiments reveal the simple mechanical design of modular proteins and open the way for the engineering of elastic proteins with defined mechanical properties, which can be used in tissue and fiber engineering.

Biomechanical Phenomena↗
PubMed2000

The folding of an immunoglobulin-like Greek key protein is defined by a common-core nucleus and regions constrained by topology.

TNfn3, the third fibronectin type III domain of human tenascin, is an immunoglobulin-like protein that is a good model for experimental and theoretical analyses of Greek key folding. The third fibronectin type III domain of human tenascin folds and unfolds in a two-state fashion over a range of temperature and pH values, and in the presence of stabilising salts. Here, we present a high resolution protein engineering analysis of the single rate determining transition state. The 48 mutations report on the contribution of side-chains at 32 sites in the core and loop regions. Three areas in the protein exhibit high Phi-values, indicating that they are partially structured in the transition state. First, a common-core ring of four positions in the central strands B, C, E and F, that are in close contact, form a nucleus of tertiary interactions. The two other regions that appear well-formed are the C' region and the E-F loop. The Phi-values gradually decrease away from these regions such that the very ends of the two terminal strands A and G, have Phi-values of zero. We propose a model for the folding of immunoglobulin-like proteins in which the common-core "ring" forms the nucleus for folding, whilst the C' and E-F regions are constrained by topology to pack early. Folding characteristics of a group of structurally related proteins appear to support this model.

Amino Acid Motifs↗
PubMed2000

Towards a complete description of the structural and dynamic properties of the denatured state of barnase and the role of residual structure in folding.

The detailed characterization of denatured proteins remains elusive due to their mobility and conformational heterogeneity. NMR studies are beginning to provide clues regarding residual structure in the denatured state but the resulting data are too sparse to be transformed into molecular models using conventional techniques. Molecular dynamics simulations can complement NMR by providing detailed structural information for components of the denatured ensemble. Here, we describe three independent 4 ns high-temperature molecular dynamics simulations of barnase in water. The simulated denatured state was conformationally heterogeneous with respect to the conformations populated both within a single simulation and between simulations. Nonetheless, there were some persistent interactions that occurred to varying degrees in all simulations and primarily involved the formation of fluid hydrophobic clusters with participating residues changing over time. The region of the beta(3-4) hairpin contained a particularly high degree of such side-chain interactions but it lacked beta-structure in two of the three denatured ensembles: beta(3-4) was the only portion of the beta-structure to contain significant residual structure in the denatured state. The two principal alpha-helices (alpha1 and alpha2) adopted dynamic helical structure. In addition, there were persistent contacts that pinched off core 2 from the body of the protein. The rest of the protein was unstructured, aside from transient and mostly local side-chain interactions. Overall, the simulated denatured state contains residual structure in the form of dynamic, fluctuating secondary structure in alpha1 and alpha2, as well as fluctuating tertiary contacts in the beta(3-4) region, and between alpha1 and beta(3-4), in agreement with previous NMR studies. Here, we also show that these regions containing residual structure display impaired mobility by both molecular dynamics and NMR relaxation experiments. The residual structure was important in decreasing the conformational states available to the chain and in repairing disrupted regions. For example, tertiary contacts between beta(3-4) and alpha1 assisted in the refolding of alpha1. This contact-assisted helix formation was confirmed in fragment simulations of beta(3-4) and alpha1 alone and complexed, and, as such, alpha1 and beta(3-4) appear to be folding initiation sites. The role of these sites in folding was investigated by working backwards and considering the simulation in reverse, noting that earlier time-points from the simulations provide models of the major intermediate and transition states in quantitative agreement with data from both unfolding and refolding experiments. Both beta(3-4) and alpha1 are dynamic in the denatured state but when they collide and make enough contacts, they provide a loose structural scaffold onto which further beta-strands pack. The beta-structure condenses about beta(3-4), while alpha1 aids in stabilizing beta(3-4) and maintaining its orientation. The resulting beta-structure is relatively planar and loose in the major intermediate. Further packing ensues, and as a result the beta-sheet twists, leading to the major transition state. The structure is still expanded and loops are not well formed at this point. Fine-tuning of the packing interactions and the final condensation of the structure then occurs to yield the native state.

Amino Acid Sequence↗
PubMed2000

Conservation of folding and stability within a protein family: the tyrosine corner as an evolutionary cul-de-sac.

What are the selective pressures on protein sequences during evolution? Amino acid residues may be highly conserved for functional or structural (stability) reasons. Theoretical studies have proposed that residues involved in the folding nucleus may also be highly conserved. To test this we are using an experimental "fold approach" to the study of protein folding. This compares the folding and stability of a number of proteins that share the same fold, but have no common amino acid sequence or biological activity. The fold selected for this study is the immunoglobulin-like beta-sandwich fold, which is a fold that has no specifically conserved function. Four model proteins are used from two distinct superfamilies that share the immunoglobulin-like fold, the fibronectin type III and immunoglobulin superfamilies. Here, the fold approach and protein engineering are used to question the role of a highly conserved tyrosine in the "tyrosine corner" motif that is found ubiquitously and exclusively in Greek key proteins. In the four model beta-sandwich proteins characterised here, the tyrosine is the only residue that is absolutely conserved at equivalent sites. By mutating this position to phenylalanine, we show that the tyrosine hydroxyl is not required to nucleate folding in the immunoglobulin superfamily, whereas it is involved to some extent in early structure formation in the fibronectin type III superfamily. The tyrosine corner is important for stability, mutation to phenylalanine costs between 1.5 and 3 kcal mol(-1). We propose that the high level of conservation of the tyrosine is related to the structural restraints of the loop connecting the beta-sheets, representing an evolutionary "cul-de-sac".

Amino Acid Sequence↗
PubMed2000

Cannula related suppurative thrombophlebitis in the burned patient.

Suppurative thrombophlebitis is a well recognised and potentially fatal complication of intravenous cannulation in burns patients. We report a case of an Afro-Caribbean patient with noninsulin-dependent diabetes who developed signs of systemic sepsis two weeks after a 14% total body surface area flame burn. Despite an initial paucity of clinical signs at the cannulation site, exploratory venotomy revealed frank suppuration within the long saphenous vein from the ankle to the groin. This was treated successfully by total excision of the vein and its tributaries and delayed wound closure. Following this, a retrospective analysis of the measured clinical parameters and blood tests revealed no obvious, missed pointers to the impending sepsis other than a dramatic increase in the overall daily insulin requirement. This had doubled over a 48-h period, preceding the clinical diagnosis by three days. The relevant literature and guidelines for management are reviewed.

Adult↗