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Biomedical subjects

R A Clark

Publications and source records attributed to R A Clark.

At least 91 records · Page 5Linked to original sources
PubMed1998

Displacement of the medial rectus pulley in superior oblique palsy.

PURPOSE: The rectus extraocular muscles pass through fibromuscular connective tissue pulleys that stabilize muscle paths and control the direction of muscle pull. The authors investigated whether abnormal forces associated with superior oblique palsy can cause displacement of pulleys and muscle paths. METHODS: Coronal magnetic resonance imaging (MRI) showing significantly reduced superior oblique cross-sectional areas and lack of contractile changes with vertical gaze confirms that seven subjects had superior oblique palsies. Binocular misalignment was quantified using the Hess test. In those seven subjects with palsies and in 18 normal orbits, coronal MRI scans corrected to standardized head position were analyzed digitally to determine muscle paths in primary gaze. Horizontal and vertical coordinates of the pulleys, known histologically to lie just posterior to the equator in primary gaze, were inferred from these muscle paths. RESULTS: Normal pulley coordinates were highly uniform. Compared with both normal orbits and fellow orbits, orbits with superior oblique palsies showed a statistically significant 1.1 mm superior displacement of the medial rectus pulley. No other pulley was displaced significantly from normal. Computer simulation using a biomechanical model of ocular statics showed that, in each case, the pulley position shifts alone were insufficient to reproduce the clinical pattern of strabismus. CONCLUSIONS: The excyclotorsion of the globe that accompanies superior oblique palsy does not systematically displace the pulleys of all the rectus muscles. The only significant rectus muscle path change is for the medial rectus muscle, and it may arise as a mechanical consequence of the atrophy of the adjacent superior oblique muscle belly. Biomechanical modeling suggests that this displacement of the medial rectus pulley alone does not account for the pattern of strabismus observed in superior oblique palsy.

Adolescent↗
PubMed1997

Clinical manifestations and predictors of survival in older women infected with HIV.

To review the natural history of HIV infection in older women, a retrospective review of women enrolled in the HIV Outpatient Program based at the Medical Center of Louisiana in New Orleans was performed. Eighty-four of the women were at least 40 years of age. Older women were more likely to be diagnosed with selected psychosocial illnesses (e.g., injection drug use, alcohol abuse, anxiety, depression, psychosis, dementia) compared with women <40 years of age. There was no association with age and other opportunistic processes or HIV-related symptoms, but cervical dysplasia and chlamydia cervicitis were less common in older women. In a multivariate proportional hazards model, characteristics predictive for death among older women included a CD4 cell count <200 cells/mm3 (relative risk [RR], 2.86; 95% confidence interval [CI], 1.18, 6.86; p < .02), a diagnosis of an opportunistic process (RR, 3.25; 95% CI, 1.24, 8.55; p < .02), antiretroviral combination therapy (RR, 0.36; 95% CI, 0.12, 1.13: p < .08), and hormone replacement therapy (HRT) (RR, 0.28; 95% CI, 0.07, 1.10; p < .06). HRT should be considered in the management of postmenopausal HIV-infected women for its known documented benefits shown in populations of persons not infected with HIV. Prospective studies to better evaluate risks and benefits of HRT in HIV-infected women are warranted.

AIDS Dementia Complex↗
PubMed1997

PU.1 is essential for p47(phox) promoter activity in myeloid cells.

Expression of the phagocyte cytosolic protein p47(phox), a component of NADPH oxidase, is restricted mainly to myeloid cells. To study the cis-elements and trans-acting factors responsible for its gene expression, we have cloned and characterized the p47(phox) promoter. A predominant transcriptional start site was identified 21 nucleotides upstream of the translation initiation codon. To identify the gene promoter sequences, transient transfections of HL-60 human myeloid cells were performed with a series of 5'-deletion p47(phox)-luciferase reporter constructs that extended as far upstream as -3050 bp relative to the transcriptional start site. The -224 and -86 constructs had the strongest p47(phox) promoter activity, whereas the -46 construct showed a major reduction in activity and the -36 construct a complete loss of activity. DNase I footprint analysis identified a protected region from -37 to -53. This region containing a consensus PU.1 site bound specifically both PU.1 present in nuclear extracts from myeloid cells and PU.1 synthesized in vitro. Mutations of this site eliminated PU.1 binding and abolished the ability of the p47(phox) promoter to direct expression of the reporter gene. The p47(phox) promoter was active in all myeloid cell lines tested (HL-60, THP-1, U937, PLB-985), but not in non-myeloid cells (HeLa, HEK293). Finally, PU.1 trans-activated the p47(phox)-luciferase constructs in HeLa cells. We conclude that, similar to certain other myeloid-specific genes, p47(phox) promoter activity in myeloid cells requires PU.1.

Animals↗
PubMed1997

Calreticulin biosynthesis and processing in human myeloid cells: demonstration of signal peptide cleavage and N-glycosylation.

Calreticulin is a soluble endoplasmic reticulum protein comprising the major storage reservoir for inositol trisphosphate-releasable calcium. Although its highly conserved primary structure and a wide range of functions have been well described, less attention has been paid to its biosynthesis, particularly in human tissues. We report analyses of synthesis, proteolytic processing and glycosylation of human calreticulin. In both HL-60 and PLB-985 myeloid cell lines calreticulin was immunoprecipitated as a single 60-kD species without evidence of precursor forms. However, in vitro cell-free synthesis produced a 62-kD primary translation product, which in the presence of microsomal membranes, was processed by cotranslational signal peptide cleavage to a 60-kD species that comigrated with mature calreticulin produced in myeloid cells. Neither tunicamycin treatment of the cells nor endoglycosidase digestion of calreticulin resulted in any forms other than the 60-kD protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, suggesting that the potential site for N-glycosylation at asparagine-327 was unmodified. However, oxidative derivatization of carbohydrate components with digoxigenin showed that human calreticulin produced in either HL-60 cells or Sf9 insect cells is glycosylated, indicating that glycosylated and nonglycosylated human calreticulin have indistinguishable electrophoretic mobilities. Direct measurement by phenol-H2SO4 confirmed the presence of carbohydrate on recombinant human calreticulin. These data show that human myeloid calreticulin undergoes cotranslational signal peptide cleavage and posttranslational N-linked glycosylation. Although glycosylation of calreticulin has been shown in rat liver and bovine liver and brain, it has been reported to be lacking in other tissues including human lymphocytes.

Animals↗
PubMed1997

Tenascin supports lymphocyte rolling.

Tenascin is a large extracellular matrix molecule expressed at specific sites in the adult, including immune system tissues such as the bone marrow, thymus, spleen, and T cell areas of lymph nodes. Tenascin has been reported to have both adhesive and anti-adhesive effects in static assays. We report here that tenascin supports the tethering and rolling of lymphocytes and lymphoblastic cell lines under flow conditions. Binding was calcium dependent and was not inhibited by treatment of lymphocytes with O-glycoprotease or a panel of glycosidases including neuraminidase and heparitinase but was inhibited by treatment of cells with proteinase K. Binding was to the fibrinogen-like terminal domain of tenascin as determined by antibody blocking studies and binding to recombinant tenascin proteins. When compared to rolling of the same cell type on E-selectin, rolling on tenascin was found to be smoother at all shear stresses tested, suggesting that cells formed a larger number of bonds on the tenascin substrate than on the E-selectin substrate. When protein plating densities were adjusted to give similar profiles of cell detachment under increasing shears, the density of tenascin was 8.5-fold greater than that of E-selectin. Binding to tenascin was not dependent on any molecules previously identified as tenascin receptors and is likely to involve a novel tenascin receptor on lymphocytes. We postulate that the ability of tenascin to support lymphocyte rolling may reflect its ability to support cell migration and that this interaction may be used by lymphocytes migrating through secondary lymphoid organs.

Binding Sites↗
PubMed1997

Human fibroblasts bind directly to fibrinogen at RGD sites through integrin alpha(v)beta3.

Fibroblast migration into the blood clot initially filling a wound requires close interaction between fibroblasts and the matrix of the fibrin clot. However, very little is known about the specific receptor-ligand interactions that mediate fibroblast attachment to fibrin. Using an attachment assay developed to measure even relatively weak interactions, we demonstrate here that normal human dermal fibroblasts can attach to substrates coated with fibrinogen, fibrin, or the fibrinogen breakdown product I-9D. Fibroblast attachment to these ligands did not require the presence of fibronectin on the cell surface or as a component of the substrate. Cells treated with cycloheximide and monensin, to limit the synthesis and secretion of endogenous fibronectin, attached as well as untreated cells. The synthetic peptide GRGDS inhibited adhesion to fibrinogen, fibrin, and fibrinogen I-9D by about 60%, while the control peptide GRGES had no substantial effect. We conclude that attachment to these ligands is mediated at least partially by direct interactions between the substrates and one specific receptor, the integrin alpha(v)beta3. Affinity chromatography demonstrated that alpha(v)beta3 from detergent lysates of fibroblasts bound to a fibrinogen matrix and was eluted with EDTA. Furthermore, antibodies against the alpha(v)beta3 complex or against the alpha(v) subunit inhibited fibroblast attachment to fibrinogen and fibrin by 50-70%. An inhibitory antibody against the integrin beta1 subunit had no effect. The observation that integrin antagonists could not produce complete inhibition suggests that there may be other fibroblast cell surface proteins that can bind directly to fibrinogen.

Cell Adhesion↗
PubMed1997

A three-dimensional collagen lattice induces protein kinase C-zeta activity: role in alpha2 integrin and collagenase mRNA expression.

A three-dimensional collagen lattice can provide skin fibroblasts with a cell culture environment that simulates normal dermis. Such a collagen matrix environment regulates interstitial collagenase (type I metalloproteinase [MMP-1], collagenase-1) and collagen receptor alpha2 subunit mRNA expression in both unstimulated or platelet-derived growth factor-stimulated dermal fibroblasts (Xu, J., and R.A.F. Clark. 1996. J. Cell Biol. 132:239-249). Here we report that the collagen gel can signal protein kinase C (PKC)-zeta activation in human dermal fibroblasts. An in vitro kinase assay demonstrated that autophosphorylation of PKC-zeta immunoprecipitates was markedly increased by a collagen matrix. In contrast, no alteration in PKC-zeta protein levels or intracellular location was observed. DNA binding activity of nuclear factor kappaB (NF-kappaB), a downstream regulatory target of PKC-zeta, was also increased by fibroblasts grown in collagen gel. The composition of the NF-kappaB/Rel complexes that contained p50, was not changed. The potential role of PKC-zeta in collagen gel-induced mRNA expression of collagen receptor alpha2 subunit and human fibroblast MMP-1 was assessed by the following evidence. Increased levels of alpha2 and MMP-1 mRNA in collagen gel-stimulated fibroblasts were abrogated by bisindolylmaleimide GF 109203X and calphostin C, chemical inhibitors for PKC, but retained when cells were depleted of 12-myristate 13-acetate (PMA)-inducible PKC isoforms by 24 h of pretreatment with phorbol PMA. Antisense oligonucleotides complementary to the 5' end of PKC-zeta mRNA sequences significantly reduced the collagen lattice-stimulated alpha2 and MMP-1 mRNA levels. Taken together, these data indicate that PKC-zeta, a PKC isoform not inducible by PMA or diacylglycerol, is a component of collagen matrix stimulatory pathway for alpha2 and MMP-1 mRNA expression. Thus, a three-dimensional collagen lattice maintains the dermal fibroblast phenotype, in part, through the activation of PKC-zeta.

Antigens, CD↗
PubMed1997

TGF-beta 1 stimulates cultured human fibroblasts to proliferate and produce tissue-like fibroplasia: a fibronectin matrix-dependent event.

During wound repair, fibroblasts accumulate in the injured area until any defect is filled with stratified layers of cells and matrix. Such fibroplasia also occurs in many fibrotic disorders. Transforming growth factor-beta (TGF-beta), a promotor of granulation tissue in vivo and extracellular matrix production in vitro, is expressed during the active fibroplasia of wound healing and fibroproliferative diseases. Under usual tissue culture conditions, normal fibroblasts grow to confluence and then cease proliferation. In this study, culture conditions with TGF-beta 1 have been delineated that promote human fibroblasts to grow in stratified layers mimicking in vivo fibroplasia. When medium supplemented with serum, ascorbate, proline, and TGF-beta was added thrice weekly to normal human dermal fibroblasts, the cells proliferated and stratified up to 16 cell layers thick within the culture dish, producing a tissue-like fibroplasia. TGF-beta stimulated both DNA synthesis as measured by 3H-thymidine uptake and cell proliferation as measured by a Hoechst dye DNA assay in these postconfluent cultures. The stratification was dependent on fibronectin assembly, as demonstrated by anti-fibronectin antibodies which inhibited both basal and TGF-beta-stimulated cell proliferation and stratification. Suppression of collagen matrix assembly in cell layers with beta-amino-proprionitrile (BAPN) did not inhibit basal or TGF-beta stimulated in vitro fibroplasia. BAPN did not interfere with fibronectin matrix assembly as judged by immunofluorescence microscopy. Thus, in concert with serum factors, TGF-beta stimulates postconfluent, fibronectin matrix-dependent, fibroblast growth creating a fibroplasia-like tissue in vitro.

Aminopropionitrile↗
PubMed1997

Quantitative measurements of released amines from individual exocytosis events.

Chemical analysis of single cells is an area of great interest in the biological sciences. Single-cell systems are being utilized as a model to understand in vivo processes better. One method that is moving to the forefront in cellular analysis is electrochemistry. Owing to their rapid response time and small dimensions, voltammetric microelectrode techniques, such as amperometry and fast-scan voltammetry, have made it possible to monitor minute amounts of biological compounds and transiently occurring chemical events in cellular systems. The application of these methods to the quantitation of individual vesicular release events from single cells is overviewed here. The application of electrochemical monitoring to several types of cultured cells, including bovine adrenal chromaffin cells, rat pheochromocytoma (PC12) cells, beige mouse mast cells, superior cervical ganglion neurons, and human pancreatic beta-cells, as well as to the invertebrate systems, the leech Hirudo medicinalis, and pond snail Planorbis corneus has provided a wealth of new information concerning exocytosis. Results obtained from the studies highlight the potential of electrochemical techniques in cellular analysis to contribute to our understanding of molecular and pharmacological effects on exocytosis. This article overviews work done on all the above cell types with an emphasis on PC12 cells.

Adrenal Gland Neoplasms↗
PubMed1997

How general practitioners manage acute asthma attacks.

BACKGROUND: The management of asthma attacks by general practitioners in 1992/93 was compared with 1991/92 in relation to recommended guidelines. METHODS: The findings from a postal survey of 299 UK general practitioners who managed 2332 patients with an asthma attack in 1992/93 were compared with those from 218 practitioners who managed 1805 attacks in 1991/92. Management by a subgroup of practitioners from a special interest group was analysed separately. RESULTS: In 1992/93 2031 (87%) of attacks were managed entirely by general practitioners, 251 (11%) were referred for admission to hospital, and 50 (2%) were managed by an accident and emergency department. There was no change in the pattern of management relative to 1991/92. Compared with recommended guidelines there was underuse of systemic steroids and nebulised bronchodilators. Between 1991/92 and 1992/93 use of systemic steroids in acute attacks increased from 56% to 71%, nebulised bronchodilators from 31% to 32%, and an increase in prophylactic medication after an acute attack from 41% to 49%. Contrary to guidelines, antibiotic use increased from 32% to 40% of attacks. Practitioners from a special interest asthma group gave more of their patients systemic steroids and nebulised bronchodilators both in 1991/92 and 1992/93. CONCLUSIONS: Some aspects of the management of asthma attacks by general practitioners has changed in line with guidelines, but there is still a large gap between actual and recommended management.

Acute Disease↗
PubMed1997

What determines levels of passive smoking in children with asthma?

BACKGROUND: Children with parents who smoke are often exposed to high levels of environmental tobacco smoke, and children with asthma are particularly susceptible to the detrimental effects of passive smoking. Data were collected from parents who smoke and from their asthmatic children. The families are currently taking part in a randomised controlled trial to test an intervention designed to reduce passive smoking in children with asthma. This paper reports on the baseline data. Questionnaire data and cotinine levels were compared in an attempt to assess exposure and to identify factors which influence exposure of the children. The aim of the study was to identify the scope for a reduction in passive smoking by these children. METHODS: A sample of 501 families with an asthmatic child aged 2-12 years was obtained. Factors influencing passive smoking were assessed by interviewing parents. Cotinine levels were measured from saliva samples using gas liquid chromatography with nitrogen phosphorous detection. RESULTS: Cotinine levels in children were strongly associated with the age of the child, the number of parents who smoked, contact with other smokers, the frequency of smoking in the same room as the child, and crowding within the home. Parental cotinine levels, the amount smoked in the home, and whether the home had a garden also exerted an independent effect on cotinine levels in the children. CONCLUSIONS: Many children are exposed to high levels of environmental tobacco smoke and their cotinine levels are heavily dependent upon proximity to the parent who smokes. Parents who smoke have a unique opportunity to benefit their child's health by modifying their smoking habits within the home.

Age Factors↗
PubMed1997

A repeat audit of hospital discharge letters in patients admitted with acute asthma.

Subsequent to the implementation of recommendations from a previous audit of acute asthma admission discharge letters from our specialist respiratory unit, a repeat audit of typed discharge letters of 86 patients (33 male, mean age 29 SD 9 years) admitted with acute asthma to the same unit over a 12 month period was performed. There was significant improvement in the discharge letter documentation of precipitating factors (p < 0.001), previous admissions with acute asthma (p < 0.01), admission arterial blood gas analysis (p < 0.001), admission peak flow rates (p < 0.05), discharge peak flow rates (p < 0.001), corticosteroid (p < 0.01) and inhaled beta 2 agonist (p < 0.01) prescription on discharge and on the specification of inhaler delivery device on discharge (p < 0.001). No significant differences in discharge letters were found in the documentation of acute therapy or post discharge follow up plan. The improvement in discharge letter quality was attributed to closing the feed back loop from the previous audit though continuing deficiencies in discharge letter contents have been identified again. These deficiencies need to be rectified and the results reaudited.

Acute Disease↗
PubMed1997

Reaudit of acute asthma admissions using a severity marker stamp and determinants of an outcome measure.

Subsequent to the implementation of a severity marker stamp in case notes, an audit was performed in 86 admissions with acute asthma to a specialist centre over a 12 month period. Compared to previous audit the documentation of severity markers was significantly better (PEFR: 52% vs 83% p = 0.001, Respiratory rate: 44% vs 81% p = 0.001, ABG: 72% vs 80% p = 0.04, air entry: 58% vs 86% p = 0.001, speech: 27% vs 86% p = 0.001, exhaustion: 4% vs 86% p = 0.001). In contrast to the previous audit where no patient received FiO2 > 0.35, 66% of the cases in the repeat audit received FiO2 0.60 (p = 0.001). The mean duration of admission was five days and showed highest partial correlation (r = 0.6) to the time in hours for the pulse to fall to 80/min. Multiple linear regression showed that this was the only variable best predicting the duration of admission (R2 = 0.3). Admission pulse rate (p = 0.04) and serum K+ (p = 0.04) best discriminated between patients admitted for over and under five days. Logistic regression identified only the admission pulse as significant in calculating the odds of the patient staying in the hospital for > 5 days.

Adult↗
PubMed1997

Fibronectin provides a conduit for fibroblast transmigration from collagenous stroma into fibrin clot provisional matrix.

After injury, the wound space is filled with a fibrin/fibronectin clot containing growth factors released by platelets and monocytes. In response to these factors, fibroblasts migrate into the fibrin clot and contribute to the formation of granulation tissue. The functional mechanisms allowing fibroblasts to leave the collagenous matrix of normal connective tissue and invade the provisional matrix of the fibrin clot have not been fully defined. To investigate these mechanisms we established a new in vitro model which simulates specific aspects of early wound healing, that is, the migration of fibroblasts from a three-dimensional collagen matrix into a fibrin clot. This transmigration could be induced by physiological concentrations of platelet releasate or platelet-derived growth factor BB (PDGF-BB) in a concentration-dependent manner. At 24 hours irradiated fibroblasts invaded the fibrin gel almost as well as non-irradiated cells, indicating that transmigration was independent of proliferation. Plasminogen and its activators appear to be necessary for invasion of the fibrin clot since protease inhibitors decreased the amount of migration. These serine proteases, however, were not necessary for exit from the collagen gel as fibroblasts migrated out of the collagen gel onto a surface coated with fibrin fibrils even in the presence of inhibitors. Removal of fibronectin (FN) from either the collagen gel or the fibrin gel markedly decreased the number of migrating cells, suggesting that FN provides a conduit for transmigration. Cell movement in the in vitro model was inhibited by RGD peptide, and by monoclonal antibodies against the subunits of the alpha5 beta1 and alpha v beta3 integrin receptor. Thus, the functional requirements for fibroblast transmigration from collagen-rich to fibrin-rich matrices, such as occurs in early wound healing, have been partially defined using an in vitro paradigm of this important biologic process.

Antigens, CD↗