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Biomedical subjects

J M Clark

Publications and source records attributed to J M Clark.

At least 73 records · Page 4Linked to original sources
PubMed1998

Phospholipase D1 localises to secretory granules and lysosomes and is plasma-membrane translocated on cellular stimulation.

Phospholipase D (PLD) activity has been implicated in the regulation of membrane trafficking [1,2], superoxide generation and cytoskeletal remodelling [3,4]. Several PLD genes have now been identified and it is probable that different isoforms regulate distinct functions. Defining the subcellular localisation of each isoform would facilitate understanding of their roles. Previous PLD localisation studies have been based largely on enzyme activity measurements, which cannot distinguish between isoforms [2,5]. We have cloned the cDNAs encoding human PLD1a and PLD1b from an HL60 cell cDNA library and expressed them as catalytically active fusion proteins with green fluorescent protein (GFP) in COS-1 cells and RBL-2H3 cells, a mast cell model which degranulates upon cross-linking of the high-affinity immunoglobulin E (IgE) receptor. In unstimulated cells, GFP-PLD1b colocalised with secretory granule and lysosomal markers; it was not found at the plasma membrane or nucleus and did not colocalise with markers for the Golgi. Stimulation or RBL-2H3 cells through IgE receptor cross-linking caused plasma membrane recruitment of GFP-PLD1b. Inhibition of IgE-receptor-stimulated, PLD-catalysed phosphatidate formation suppressed secretion of granule and lysosomal contents, but did not affect translocation of GFP-PLD1b. These experiments suggest that PLD1 plays a role in regulated exocytosis rather than endoplasmic reticulum (ER) to Golgi membrane transport.

Animals↗
PubMed1998

Design of potent selective zinc-mediated serine protease inhibitors.

Many serine proteases are targets for therapeutic intervention because they often play key roles in disease. Small molecule inhibitors of serine proteases with high affinity are especially interesting as they could be used as scaffolds from which to develop drugs selective for protease targets. One such inhibitor is bis(5-amidino-2-benzimidazolyl)methane (BABIM), standing out as the best inhibitor of trypsin (by a factor of over 100) in a series of over 60 relatively closely related analogues. By probing the structural basis of inhibition, we discovered, using crystallographic methods, a new mode of high-affinity binding in which a Zn2+ ion is tetrahedrally coordinated between two chelating nitrogens of BABIM and two active site residues, His57 and Ser 195. Zn2+, at subphysiological levels, enhances inhibition by over 10(3)-fold. The distinct Zn2+ coordination geometry implies a strong dependence of affinity on substituents. This unique structural paradigm has enabled development of potent, highly selective, Zn2+-dependent inhibitors of several therapeutically important serine proteases, using a physiologically ubiquitous metal ion.

Animals↗
PubMed1998

Delayed complications of nonoperative management of blunt adult splenic trauma.

OBJECTIVE: To determine the incidence and type of delayed complications from nonoperative management of adult splenic injury. DESIGN: Retrospective medical record review. SETTING: University teaching hospital, level I trauma center. PATIENTS: Two hundred eighty patients were admitted to the adult trauma service with blunt splenic injury during a 4-year period. Men constituted 66% of the population. The mean (+/-SEM) age was 32.2+/-1.0 years and the mean (+/-SEM) Injury Severity Score was 22.8+/-0.9. Fifty-nine patients (21%) died of multiple injuries within 48 hours and were eliminated from the study. One hundred thirty-four patients (48%) were treated operatively within the first 48 hours after injury and 87 patients (31%) were managed nonoperatively. MAIN OUTCOME MEASURES: We reviewed the number of units of blood transfused, intensive care unit length of stay, overall length of stay, outcome, and complications occurring more than 48 hours after injury directly attributable to the splenic injury. RESULTS: Patients managed nonoperatively had a significantly lower Injury Severity Score (P<.05) than patients treated operatively. Length of stay was significantly decreased in both the number of intensive care unit days as well as total length of stay (P<.05). The number of units of blood transfused was also significantly decreased in patients managed nonoperatively (P<.05). Seven patients (8%) managed nonoperatively developed delayed complications requiring intervention. Five patients had overt bleeding that occurred at 4 days (3 patients), 6 days (1 patient), and 8 days (1 patient) after injury. Three patients underwent splenectomy, 1 had a splenic artery pseudoaneurysm embolization, and 1 had 2 areas of bleeding embolization. Two patients developed splenic abscesses at approximately 1 month after injury; both were treated by splenectomy. CONCLUSION: Significant numbers of delayed splenic complications do occur with nonoperative management of splenic injuries and are potentially life-threatening.

Adolescent↗
PubMed1998

Deformation of articular cartilage collagen structure under static and cyclic loading.

Relatively little is known about the morphology of articular cartilage under conditions of normal use, yet a more profound knowledge is both critical to the understanding of cartilage function and helpful for the validation of tissue-engineered cartilage. In this study, the deformation of the articular cartilage of the tibial plateau under compressive static and cyclic loading is characterized. Whole knee joints of rabbits were loaded ex vivo while the knee was held statically or allowed to move against resistance. Load magnitudes of quadriceps were maintained at either three (high) or one (low) times body weight for 30 minutes. For cyclic loading, the tibia was flexed between 70 and 150 degrees relative to the femur at 1 Hz with either a cyclic or constant force. The recovery of cartilage after unloading was examined for each loading condition. At the end of the loading, specimens were cryofixed while under load, freeze-substituted, and prepared for scanning electron microscopy. Morphological examination demonstrated significantly higher deformation of the collagen structure throughout all cartilage zones under static loading conditions compared with cyclic loading conditions in which deformation was limited to the superficial regions. The minimum thickness of the cartilage that remained after loading was dependent on the magnitude of load and was significantly smaller with static loads (54% of the thickness of the unloaded controls) than after cyclic loading or constant-force cyclic loading (78 or 66% of the thickness of the unloaded controls, p < 0.05). Acute bending of the collagen fibers was observed under both loading conditions: in the superficial half of the articular cartilage after static loading and in the superficial quarter after cyclic loading. Complete recovery of all deformation occurred within 30 minutes but was significantly faster after cyclic loading. These data suggest that the structure of the collagen of articular cartilage exhibits a zone-specific deformation that is dependent on the magnitude and type of load.

Animals↗
PubMed1998

Expression and characterization of human tissue kallikrein variants.

Human tissue kallikrein is a serine protease implicated in the pathology of various inflammatory disorders. As one of the two principal enzymes that generate proinflammatory kinin peptides in vivo, tissue kallikrein represents an attractive target for therapeutic intervention in diseases such as asthma, pancreatitis, and rheumatoid arthritis. Three distinct human tissue kallikrein variants, differing in one or two amino acid substitutions, are predicted to exist based on genomic or cDNA nucleotide sequences derived from different tissues. The effects of these substitutions on the biochemical properties of tissue kallikrein are unknown but could, in principle, confer tissue-specific functions on the enzyme or affect the clinical utility of specific kallikrein inhibitors. All three variants, as well as a deglycosylated derivative, were expressed in high yield as recombinant proteins in Pichia pastoris. The recombinant kallikrein variants and natural urinary kallikrein all hydrolyzed synthetic peptides with similar specificity and efficiency and released kallidin from kininogen at comparable rates. Similarly, no significant differences were observed in the interactions between kallikrein variants and protein inhibitors such as SBTI, alpha1-PI, and aprotinin. We conclude that the known tissue kallikrein variants represent allelic variants and are not likely to have tissue-specific activity related to the amino acid substitutions.

Amino Acid Sequence↗
PubMed1998

Efficacy of the carbocyclic 2'-deoxyguanosine nucleoside BMS-200475 in the woodchuck model of hepatitis B virus infection.

Daily oral treatment with the cyclopentyl 2'-deoxyguanosine nucleoside BMS-200475 at doses ranging from 0.02 to 0.5 mg/kg of body weight for 1 to 3 months effectively reduced the level of woodchuck hepatitis virus (WHV) viremia in chronically infected woodchucks as measured by reductions in serum WHV DNA levels and endogenous hepadnaviral polymerase activity. Within 4 weeks of daily therapy with 0.5 or 0.1 mg of BMS-200475 per kg, endogenous viral polymerase levels in serum were reduced about 1,000-fold compared to pretreatment levels. Serum WHV DNA levels determined by a dot blot hybridization technique were comparably decreased in these treated animals. In the 3-month study, the sera of animals that had undetectable levels of WHV DNA by the dot blot technique were further analyzed by a highly sensitive semiquantitative PCR assay. The results indicate that BMS-200475 therapy reduced mean WHV titers by 10(7)- to 10(8)-fold, down to levels as low as 10(2) to 10(3) virions/ml of serum. Southern blot hybridization analysis of liver biopsy samples taken from animals during and after BMS-200475 treatment showed remarkable reductions in the levels of WHV DNA replicative intermediates and in the levels of covalently closed circular viral DNA. WHV viremia in BMS-200475-treated WHV carriers eventually returned to pretreatment levels after therapy was stopped. These results indicate that BMS-200475 should be evaluated in clinical trials for the therapy of chronic human hepatitis B virus infections.

Animals↗
PubMed1998

Hypoxic ventilatory sensitivity in men is not reduced by prolonged hyperoxia (Predictive Studies V and VI).

Potential adverse effects on the O2-sensing function of the carotid body when its cells are exposed to toxic O2 pressures were assessed during investigations of human organ tolerance to prolonged continuous and intermittent hyperoxia (Predictive Studies V and VI). Isocapnic hypoxic ventilatory responses (HVR) were determined at 1.0 ATA before and after severe hyperoxic exposures: 1) continuous O2 breathing at 1.5, 2.0, and 2.5 ATA for 17.7, 9.0, and 5.7 h and 2) intermittent O2 breathing at 2.0 ATA (30 min O2-30 min normoxia) for 14.3 O2 h within 30-h total time. Postexposure curvature of HVR hyperbolas was not reduced compared with preexposure controls. The hyperbolas were temporarily elevated to higher ventilations than controls due to increments in respiratory frequency that were proportional to O2 exposure time, not O2 pressure. In humans, prolonged hyperoxia does not attenuate the hypoxia-sensing function of the peripheral chemoreceptors, even after exposures that approach limits of human pulmonary and central nervous system O2 tolerance. Current applications of hyperoxia in hyperbaric O2 therapy and in subsea- and aerospace-related operations are guided by and are well within these exposure limits.

Adult↗
PubMed1997

Production of a soluble cyclin B/cdc2 substrate for cdc25 phosphatase.

Study of the function and regulation of the important cell cycle regulator cdc25 phosphatase has been hampered by the lack of a sensitive and specific substrate and assay. Here we report the production of a specific and sensitive substrate for the cdc25 phosphatase. The substrate is human cyclin B1/cdc2 phosphorylated on the inhibitory Thr14 and Tyr15 residues and activating Thr161 on cdc2, and is relatively simple to produce from readily available materials. The assay is based on the cdc25-specific dephosphorylation and activation of the phosphorylated cyclin B1/cdc2 substrate (PY15), using the increased histone H1 kinase activity of the activated PY15 as a read-out of cdc25 activity.

Animals↗
PubMed1997

Hyperphosphorylation of the N-terminal domain of Cdc25 regulates activity toward cyclin B1/Cdc2 but not cyclin A/Cdk2.

Cdc25 regulates entry into mitosis by regulating the activation of cyclin B/cdc2. In humans, at least two cdc25 isoforms have roles in controlling the G2/M transition. Here we show, using bacterially expressed recombinant proteins, that two cdc25B splice variants, cdc25B2 and cdc25B3, are capable of activating cyclin A/cdk2 and cyclin B/cdc2, but that mitotic hyperphosphorylation of these proteins increases their activity toward only cyclin B1/cdc2. Cdc25C has only very low activity in its unphosphorylated form, and following hyperphosphorylation it will efficiently catalyze the activation of only cyclin B/cdc2. This was reflected by the in vivo activity of the immunoprecipitated cdc25B and cdc25C from interphase and mitotic HeLa cells. The increased activity of the hyperphosphorylated cdc25s toward cyclin B1/cdc2 was in large part due to increased binding of this substrate. The substrate specificity, activities, and timing of the hyperphosphorylation of cdc25B and cdc25C during G2 and M suggest that these two mitotic cdc25 isoforms are activated by different kinases and perform different functions during progression through G2 into mitosis.

CDC2 Protein Kinase↗
PubMed1997

Production of crystallizable human chymase from a Bacillus subtilis system.

A Bacillus subtilis strain deficient in seven extracellular proteases was used to produce human mast cell chymase and is a viable expression system for serine proteases and other classes of proteins. Chymase is produced at 0.3-0.5 mg/l and is purified by three chromatography steps. Two crystal forms of PMSF-treated chymase were optimized. The first is C2 with a=47.94 A, b=85.23 A, c=174.18 A, beta=96.74 degrees, and diffracts to at least 2.1 A, while the second is P212121, with cell dimensions a=43.93 A, b=58.16 A, and c=86.09 A, and a diffraction limit of approximately 1.9 A. The first crystal form has either three or four molecules/asymmetric unit, while the second has one molecule/asymmetric unit.

Amino Acid Sequence↗
PubMed1997

Ultraviolet light-induced G2 phase cell cycle checkpoint blocks cdc25-dependent progression into mitosis.

In response to low doses of ultraviolet (U.V.) radiation, cells undergo a G2 delay. In this study we have shown that the G2 delay results in the accumulation of inactive forms of cyclin B1/cdc2 and both the G2 and mitotic complexes of cyclin A/cdk. This appears to be through a block in the cdc25-dependent activation of these complexes. The expression and localisation of cyclin A and cyclin B1/cdk complexes are similar in U.V.-induced G2 delay and normal early G2 phase cells. Cdc25B and cdc25C also accumulate to normal G2 levels in U.V. irradiated cells, but the mitotic phosphorylation associated with increased activity of both cdc25B and cdc25C is absent. The cdc25B accumulates in the nucleus of U.V. irradiated cells and in normal G2 phase cells. Thus the block in cyclin B/cdc2 activation is in part due to the physical separation of cyclin B/cdc2, localised in the cytoplasm, from the cdc25B and cdc25C phosphatases localised in the nucleus. The data positions the U.V.-induced G2 checkpoint at either the S/G2 transition or early G2 phase, prior to the activation of cyclin A/cdk2.

CDC2 Protein Kinase↗
PubMed1997

Scanning electron microscopy of "fibrillated" and "malacic" human articular cartilage: technical considerations.

Specimens of articular cartilage from human knees with gross evidence of malacia (dull appearance and/or softness) or fibrillation (exposed fibrous strands and/or staining with India ink) were prepared for scanning electron microscopy (SEM) and compared to cartilage from apparently intact regions. Vertical cryofractures were made through the center of each specimen, so the matrix collagen structure and its relationship to surface features could be examined. Soft, dull, malacic cartilage was characterized by the presence of numerous clefts among the collagen fibers within the most superficial region of the cartilage. In one form of this condition, these clefts did not extend through the articular surface. In a second form, usually observed where the tangential zone was normally thin or absent, the free ends of radial collagen fibers were exposed, but the deeper layers were intact. Two forms of fibrillation were also identified. The first is created by separation of the superficial lamellae which curl up from the tangential layer and form frondlike projections above the normal plane of the joint surface. In the second, deep radial fibers are exposed by vertical fissures. This second form is associated with advanced damage to the joint. The early stages of cartilage failure are characterized by debonding among the major collagen fiber tracts. This process may initiate in the deep tangential zone where the radial fibers cross into the surface. The patterns of the degenerative changes are dictated by the original architecture of the collagen matrix. The microscopic findings do not correlate adequately with conventional gross grading. SEM provides useful information about injured articular cartilage.

Adolescent↗
PubMed1997

Ultrastructural localization of relaxin in the corpus luteum of the pregnant and early lactating tammar wallaby, Macropus eugenii.

Electron-microscope immunocytochemistry was used to determine the subcellular distribution and presence of immunoreactive relaxin throughout pregnancy and early lactation in the corpus luteum of a marsupial, the tammar wallaby. Membrane-bound, electron-dense granules were a prominent feature of the luteal cell cytoplasm. The highest numbers of granules were observed between days 20 and 24 of the 26-day gestation, with a rapid clearance immediately after birth. Relaxin immunogold particles were present only in small, electron-dense granules (200-350 nm in diameter), with no particles observed in larger granules (>400 nm diameter), nuclei or mitochondria. Relaxin immunoreactivity was low throughout early and mid pregnancy but increased markedly between days 21 and 22 and remained high over the last 4 days of pregnancy. The number of granules containing relaxin immunogold particles and the density of immunostaining were both reduced on the day of expected births (day 26). Our data demonstrate that electron-dense granules in the luteal cell cytoplasm of a pregnant marsupial contain relaxin. The peptide is produced in greatest amounts at the end of pregnancy, consistent with a role in parturition.

Animals↗
PubMed1997

Project 2000: perceptions of the philosophy and practice of nursing: shifting perceptions--a new practitioner?

This is the first of two papers which address aspects of the findings of a large scale study commissioned by the English National Board which set out to examine the impact of Project 2000 on perceptions of the philosophy and practice of nursing. The findings presented here suggest that there have been fundamental shifts in perceptions of the nature and discipline of nursing. Students and diplomates of the course perceive themselves as knowledgeable doers, with their practice well grounded in theory and research. They value the interpersonal skills teaching and place the patient firmly at the centre of care delivery, viewing the patient holistically and being prepared to be fierce patient advocates where necessary. It is difficult to determine the extent to which these shifts can be attributed to the Project 2000 course, although the Project 2000 approach to education appears to be an important factor.

Attitude of Health Personnel↗
PubMed1997

Project 2000. Perception of the philosophy and practice of nursing: preparation for practice.

As part of a comprehensive study of perceptions of the philosophy and practice of nursing Project 2000 students and newly qualified diplomates were asked to reflect on their Project 2000 course as a preparation for professional practice. Data were collected from both students and diplomates through self-completion questionnaires and a small number of interviews with diplomates in two study centres, one in the north and the other in the south of England. Nurse managers and G grade practitioners were invited in focus group interviews to discuss the Project 2000 preparation and assess the diplomates 'fitness for purpose'. Findings suggest that the majority of students and diplomates acknowledged that the course had prepared them well. They acknowledged the importance of the inter-personal skills component of the course, together with the life sciences input. The diplomates particularly valued the theoretical aspect of the course, being well versed in research and keen to continue their learning. Perceived limitations of the course were the management preparation and course organization. Practical skill deficits were perceived as initial skills deficits only. Managers and practitioners were keen to employ the diplomates, whilst acknowledging their need for support and preceptorship. Three important issues for nursing and nurse education are discussed, including the balance of the Project 2000 course, preceptorship and support, and preparation for a role in the community.

Attitude of Health Personnel↗