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Biomedical subjects

D C Winter

Publications and source records attributed to D C Winter.

At least 19 recordsLinked to original sources
PubMed2001

Structure of Arp2/3 complex in its activated state and in actin filament branch junctions.

The seven-subunit Arp2/3 complex choreographs the formation of branched actin networks at the leading edge of migrating cells. When activated by Wiskott-Aldrich Syndrome protein (WASp), the Arp2/3 complex initiates actin filament branches from the sides of existing filaments. Electron cryomicroscopy and three-dimensional reconstruction of Acanthamoeba castellanii and Saccharomyces cerevisiae Arp2/3 complexes bound to the WASp carboxy-terminal domain reveal asymmetric, oblate ellipsoids. Image analysis of actin branches indicates that the complex binds the side of the mother filament, and Arp2 and Arp3 (for actin-related protein) are the first two subunits of the daughter filament. Comparison to the actin-free, WASp-activated complexes suggests that branch initiation involves large-scale structural rearrangements within Arp2/3.

Acanthamoeba↗
PubMed2001

Tumor cell adhesion to endothelial cells is increased by endotoxin via an upregulation of beta-1 integrin expression.

BACKGROUND: Recent studies have demonstrated that metastatic disease develops from tumor cells that adhere to endothelial cells and proliferate intravascularly. The beta-1 integrin family and its ligand laminin have been shown to be important in tumor-to-endothelial cell adhesion. Lipopolysaccharide (LPS) has been implicated in the increased metastatic tumor growth that is seen postoperatively. We postulated that LPS increases tumor cell expression of beta-1 integrins and that this leads to increased adhesion. METHODS: The human metastatic colon cancer cell line LS174T was labeled with an enhanced green fluorescent protein (eGFP) using retroviral transfection. Cell cultures were treated with LPS for 1, 2, and 4 h (n = 6 each) and were subsequently cocultured for 30 or 120 min with confluent human umbilical vein endothelial cells (HUVECs), to allow adherence. Adherent tumor cells were counted using fluorescence microscopy. These experiments were carried out in the presence or absence of a functional blocking beta-1 integrin monoclonal antibody (4B4). Expression of beta-1 integrin and laminin on tumor and HUVECs was assessed using flow cytometric analysis. Tumor cell NF-kappaB activation after incubation with LPS was measured. RESULTS: Tumor cell and HUVEC beta-1 integrin expression and HUVEC expression of laminin were significantly (P < 0.05) enhanced after incubation with LPS. Tumor cell adhesion to HUVECs was significantly increased. Addition of the beta-1 integrin blocking antibody reduced tumor cell adhesion to control levels. LPS increased tumor cell NF-kappaB activation. CONCLUSIONS: Exposure to LPS increases tumor cell adhesion to the endothelium through a beta-1 integrin-mediated pathway that is NF-kappaB dependent. This may provide a target for immunotherapy directed at reducing postoperative metastatic tumor growth.

Cell Adhesion↗
PubMed2001

Inosine attenuates tourniquet-induced skeletal muscle reperfusion injury.

BACKGROUND: Adenosine attenuates skeletal muscle reperfusion injury, but its short half-life in vivo limits potential therapeutic benefits. The aim of this study was to ascertain whether inosine, a stable adenosine metabolite, modulates skeletal muscle reperfusion injury. MATERIALS AND METHODS: C57BL/6 mice were randomized (8-10 per group) to six groups: time controls; inosine (100 mg/kg) before anesthesia; 2 h of bilateral tourniquet hindlimb ischemia; I/R (2 h of bilateral tourniquet hindlimb ischemia, 3 h of reperfusion); inosine (100 mg/kg) before I/R; drug vehicle before I/R. Serum tumor necrosis factor (TNF)-alpha and macrophage inflammatory protein (MIP)-2 were measured before ischemia and at the end of reperfusion. Tissue edema was determined by wet/dry weight ratios. Tissue leucosequestration was assessed by the myeloperoxidase (MPO) content. RESULTS: At the end of reperfusion, inosine pretreatment resulted in lower MPO levels in muscle (P = 0.02) and lung (P = 0.0002) than saline pretreatment. Similarly, muscle (P = 0.04) and lung (P = 0.02) wet/dry ratios were significantly reduced with inosine but not with saline pretreatment. At the end of reperfusion, serum proinflammatory cytokine levels (TNF-alpha and MIP-2) were significantly reduced (P < 0.05) compared to preischemia levels following inosine pretreatment but not saline pretreatment. Ischemia alone did not alter any of the parameters assessed. CONCLUSIONS: These findings demonstrate that pretreatment with inosine attenuates the local and systemic proinflammatory responses associated with skeletal muscle reperfusion injury.

Animals↗
PubMed2001

Adenosine inhibits neutrophil vascular endothelial growth factor release and transendothelial migration via A2B receptor activation.

The effects of adenosine on neutrophil (polymorphonuclear neutrophils; PMN)-directed changes in vascular permeability are poorly characterized. This study investigated whether adenosine modulates activated PMN vascular endothelial growth factor (vascular permeability factor; VEGF) release and transendothelial migration. PMN activated with tumour necrosis factor-alpha (TNF-alpha, 10 ng/mL) were incubated with adenosine and its receptor-specific analogues. Culture supernatants were assayed for VEGF. PMN transendothelial migration across human umbilical vein endothelial cell (HUVEC) monolayers was assessed in vitro. Adhesion molecule receptor expression was assessed flow cytometrically. Adenosine and some of its receptor-specific analogues dose-dependently inhibited activated PMN VEGF release. The rank order of potency was consistent with the affinity profile of human A2B receptors. The inhibitory effect of adenosine was reversed by 3,7-dimethyl-1-propargylxanthine, an A2 receptor antagonist. Adenosine (100 microM) or the A2B receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA, 100 microM) significantly reduced PMN transendothelial migration. However, expression of activated PMN beta2 integrins and HUVEC ICAM-1 were not significantly altered by adenosine or NECA. Adenosine attenuates human PMN VEGF release and transendothelial migration via the A2B receptor. This provides a novel target for the modulation of PMN-directed vascular hyperpermeability in conditions such as the capillary leak syndrome.

Adenosine↗
PubMed2000

Rapid activation of basolateral potassium transport in human colon by oestradiol.

1. We investigated the effect of oestradiol on basolateral potassium channels in human colonic epithelium. 2. Ion transport was quantified using short circuit current (I:(sc)) measurements of samples mounted in Ussing chambers. Serosal K transport was studied using nystatin permeabilization of the apical membrane. Intracellular pH changes were quantified using spectroflouresence techniques. 3. Experiments were performed with either 10 nM or 1 microM Ca(2+) in the apical bathing solution. With 10 nM Ca(2+) in the apical bathing solution addition of oestradiol (1 nM) to the basolateral bath produced a rapid increase in current (delta I(K)=11.2+/-1.2 microA.cm(-2), n=6). This response was prevented by treatment of the serosal membrane with tolbutamide (1 microM). With 1 microM Ca(2+) in the apical bathing solution addition of oestradiol produced a rapid fall in current (delta I(K)=-12.8+/-1.4 microA.cm(-2)), this response was prevented by treatment of the basolateral membrane with tetra-pentyl-ammonium (TPeA). These responses were rapid and occurred independently of protein synthesis. 4. Inhibition of basolateral Na(+)/H(+) exchange with either amiloride or a low sodium bathing solution prevented this response. These responses were prevented by inhibition of protein kinase C (PKC) with bis-indolyl-maleimide. 5. Oestradiol (1 nM) produced a rapid intracellular alkanization (mean increase=0.11 pH units; n=6; P<0.01). 6. These results suggest that oestradiol rapidly modulates serosal K transport in human colon. These effects depend upon intact Na(+)/H(+) exchange and protein kinase C. We propose a non-classical, possibly membrane linked, mechanism for oestradiol action in human colonic epithelium.

Calcium↗
PubMed2000

Mitogenic effects of oestrogen mediated by a non-genomic receptor in human colon.

BACKGROUND: Oestrogens are important mitogens in epithelial cancers, particularly where tumours express complementary receptors. While the traditional model of oestrogen action involves gene-directed (genomic) protein synthesis, it has been established that more rapid, non-genomic steroid hormone actions exist. This study investigated the hypothesis that oestrogen rapidly alters cell membrane activity, intracellular pH and nuclear kinetics in a mitogenic fashion. METHODS: Crypts isolated from human distal colon and colorectal cancer cell lines were used as robust models. DNA replication and intracellular pH were measured by radiolabelled thymidine incorporation (12 h) and spectrofluorescence imaging respectively. Genomic protein synthesis, sodium-hydrogen exchanger (NHE) and protein kinase C (PKC) activity were inhibited with cycloheximide, ethylisopropylamiloride and chelerythrine chloride respectively. RESULTS: Oestrogen induced a rapid (less than 5 min) cellular alkalinization of crypts and cancer cells that was sensitive to NHE blockade (P < 0.01) or PKC inhibition (P < 0.01). Oestrogen increased thymidine incorporation by 44 per cent in crypts and by up to 38 per cent in cancer cells (P < 0.01), and this was similarly reduced by inhibiting the NHE (P < 0.01) or PKC (P < 0.05). CONCLUSION: Oestrogen rapidly activates cell membrane and nuclear kinetics by a non-genomic mechanism mediated by PKC but not gene-directed protein synthesis.

Cell Membrane↗
PubMed2000

Biological behaviour and clinical implications of micrometastases.

BACKGROUND: The most important prognostic determinant in cancer is the identification of disseminated tumour burden (metastases). Micrometastases are microscopic (smaller than 2 mm) deposits of malignant cells that are segregated spatially from the primary tumour and depend on neovascular formation (angiogenesis) to propagate. METHODS: The electronic literature (1966 to present) on micrometastases and their implications in malignant melanoma and epithelial cancers was reviewed. RESULTS: Immunohistochemical techniques combined with serial sectioning offer the best accuracy for detection of nodal micrometastases. Molecular techniques should be reserved for blood samples or bone marrow aspirates. Detection of micrometastases in regional lymph nodes and/or bone marrow confers a poor prognosis in epithelial cancers. The concept of sentinel node biopsy combined with serial sectioning and dedicated screening for micrometastases may improve staging procedures. Strategies against angiogenesis may provide novel therapies to induce and maintain micrometastatic dormancy. CONCLUSION: The concept of micrometastases has resulted in a paradigm shift in the staging of epithelial tumours and our overall understanding of malignant processes.

Biopsy, Needle↗
PubMed2000

Hypertonic saline attenuates end-organ damage in an experimental model of acute pancreatitis.

BACKGROUND: Hypertonic saline (HTS) has been noted previously to reduce neutrophil activation. The aim of this study was to elucidate the effect of hypertonic resuscitation on the development of end-organ damage in an animal model of pancreatitis. METHODS: Pancreatitis was induced in Sprague-Dawley rats by intraperitoneal injection of 20 per cent L-arginine. Animals were randomized into four groups (each n = 8): controls; pancreatitis without intervention; pancreatitis plus intravenous resuscitation with normal saline (0.9 per cent sodium chloride 2 ml/kg) at 24 and 48 h; or HTS (7.5 per cent sodium chloride 2 ml/kg) at these time points. Pulmonary endothelial leakage was assessed by measurement of lung wet : dry ratios, bronchoalveolar lavage protein and myeloperoxidase activity. RESULTS: Animals that received HTS showed less pancreatic damage than those resuscitated with normal saline (1.0 versus 3.0; P = 0.04). Lung injury scores were also significantly diminished in the HTS group (1.0 versus 3.5; P = 0.03). Pulmonary neutrophil sequestration (myeloperoxidase activity 1.80 units/g) and increased endothelial permeability (bronchoalveolar lavage protein content 1287 microgram/ml) were evident in animals resuscitated with normal saline compared with HTS (1.22 units/g and 277 microgram/ml respectively; P < 0.02). CONCLUSION: HTS resuscitation results in a significant attenuation of end-organ injury following a systemic inflammatory response to severe pancreatitis.

Animals↗
PubMed1999

Basolateral K+ channel involvement in forskolin-activated chloride secretion in human colon.

1. In this study we investigated the role of basolateral potassium transport in maintaining cAMP-activated chloride secretion in human colonic epithelium. 2. Ion transport was quantified in isolated human colonic epithelium using the short-circuit current technique. Basolateral potassium transport was studied using nystatin permeabilization. Intracellular calcium measurements were obtained from isolated human colonic crypts using fura-2 spectrofluorescence imaging. 3. In intact isolated colonic strips, forskolin and prostaglandin E2 (PGE2) activated an inward transmembrane current (ISC) consistent with anion secretion (for forskolin DeltaISC = 63.8+/-6.2 microA cm(-2), n = 6; for PGE2 DeltaISC = 34.3+/-5.2 microA cm(-2), n = 6). This current was inhibited in chloride-free Krebs solution or by inhibiting basolateral chloride uptake with bumetanide and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid DIDS). 4. The forskolin- and PGE2-induced chloride secretion was inhibited by basolateral exposure to barium (5 mM), tetrapentylammonium (10 microM) and tetraethylammonium (10 mM). 5. The transepithelial current produced under an apical to serosal K+ gradient in nystatin-perforated colon is generated at the basolateral membrane by K+ transport. Forskolin failed to activate this current under conditions of high or low calcium and failed to increase the levels of intracellular calcium in isolated crypts 6. In conclusion, we propose that potassium recycling through basolateral K+ channels is essential for cAMP-activated chloride secretion.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1999

Rapid effects of aldosterone on sodium-hydrogen exchange in isolated colonic crypts.

Aldosterone plays a central role in the homeostatic regulation of extracellular fluid volume by stimulating transepithelial electrolyte transport. These effects involve binding to an intracellular receptor, modification of genomic events and protein synthesis. Rapid cellular responses to steroid hormones have been observed in a variety of nonepithelial tissues. The term "nongenomic" has been proposed for these fast steroid responses since they are unaffected by inhibitors of protein synthesis. We hypothesized that colonic crypts, recently demonstrated to absorb fluid, would respond rapidly to aldosterone. Cytoplasmic pH changes in crypts loaded with a pH-sensitive, fluorescent dye (BCECF) were recorded with confocal laser imaging. An intracellular alkalization of colonic crypts was observed within one minute of aldosterone application that was inhibited by ethylisopropylamiloride or the absence of extracellular sodium, yet unaffected by inhibitors of protein synthesis. The genesis of this rapid and distinct steroid action involves a signal transduction pathway that involves G proteins, protein kinase C, and prostaglandins. We have identified, by real-time imaging, a nongenomic upregulation of sodium-hydrogen exchange in colonic crypts by aldosterone that occurs independent of the traditional receptor. This distinct, rapid onset effect of aldosterone on epithelial ion transport has major implications for our understanding of fluid and electrolyte homeostasis in health and disease.

Aldosterone↗
PubMed1999

Genetic dissection of the budding yeast Arp2/3 complex: a comparison of the in vivo and structural roles of individual subunits.

In previous work, we identified the yeast Arp2/3 complex, which localizes to cortical actin patches and is required for their motility and integrity in vivo. This complex contains proteins homologous to each subunit of the Acanthamoeba and human Arp2/3 complex except for a 40-kDa subunit (p40), which was missing from the purified yeast complex. Here, we demonstrate by using immunoprecipitation and gel-filtration analysis that Arc40p, the homolog of p40 identified from the yeast genome database, associates with the yeast Arp2/3 complex. We have carried out gene disruptions of each subunit of the yeast Arp2/3 complex to study each subunit's role in the function of the complex. Surprisingly, we find that only ARC40 is fully essential for cell viability. Strains lacking each of the other subunits exhibit varying degrees of defects in cell growth and viability and in assembly and polarization of cortical actin patches. We have also examined each subunit's role in maintaining the structural integrity of the Arp2/3 complex. Arp2p, Arp3p, and Arc40p fall into the monomer pool in Deltaarc19 and Deltaarc35 cells, suggesting that Arc19p and Arc35p are the central scaffolding components of the complex. Arp2p and Arp3p do not have major roles in maintaining complex integrity, and Arc15p is required for association of Arp2p and Arc40p, but not other subunits, with the complex. These results provide evidence that each subunit contributes differently to the assembly and function of the Arp2/3 complex.

Acanthamoeba↗
PubMed1999

Direct effects of dopamine on colonic mucosal pH: implications for tonometry.

Tonometric measurements of colonic and gastric mucosa pH are used as indirect determinants of splanchnic perfusion in shocked patients or those undergoing aortic cross-clamp. Mucosal acidification in response to splanchnic vasodilators such as dopamine has been assumed to signify ischemia. However, cellular acidification may occur independent of oxygenation and the direct effects of dopamine on mucosal acid-base are unknown. We examined the effects of dopamine on cellular pH (independent of oxygenation) of intestinal mucosa in vitro. Crypts isolated from the distal colon of Sprague-Dawley rats were loaded with a pH-sensitive fluorescent probe, perfused with a Hepes-buffered Ringers solution, and imaged with confocal laser scanning microscopy. In separate experiments, crypts were loaded with a calcium-sensitive probe (Fura-2) and concentrations of free cytosolic calcium were measured with fluorescence imaging. Dopamine perfusion produced a reversible cytosolic acidification of crypts which was not significantly affected by (i) the nominal absence of bicarbonate, (ii) alpha- and beta-adrenergic receptor blockade, or (iii) protein kinase C inhibition. Dopamine did not significantly affect intracellular calcium concentrations. However, dopamine-induced acidification was inhibited by (a) blocking sodium-hydrogen exchange with amiloride, (b) prior exposure to adenosine 3', 5'-cyclic monophosphate (cAMP), or (c) protein kinase A blockade (all P < 0.01). Dopamine directly acidifies mucosal crypt cells in a mechanism that involves a cAMP-mediated inhibition of sodium-hydrogen exchange. This finding accounts for the acidification of intestinal mucosa during low-dose dopamine infusion despite a demonstrable improvement in splanchnic perfusion. Direct mucosal effects of pharmacological agents must be considered in the evaluation of perfusion parameters based on tonometric data.

Animals↗
PubMed1999

Berberine inhibits ion transport in human colonic epithelia.

The effects of berberine on ion transport in both human colonic mucosal epithelia and an intestinal epithelial cell line (T84) were examined. Berberine (concentration range 0-500 microM) reduced both basal and stimulated ion transport responses in human colonic mucosae in a manner which was non-specific for Ca2+ -or cAMP-mediated signals. Similarly, in cultured intestinal epithelial monolayers, berberine inhibited Ca2+ -and cAMP-mediated responses indicating an inhibitory activity directly at the level of the epithelium rather than an indirect effect through other mucosal element(s). Berberine did not alter the rate of generation of cAMP by adenylyl cyclase or the activity of protein kinase A, the effector enzyme of the cAMP pathway. Berberine inhibited carbachol-stimulated 86Rb+ efflux from T84 monolayers. Berberine also inhibited K+ conductance in apically-permeabilised re-sected mucosae. These results indicate i) that berberine exerts an anti-secretory action directly upon epithelial cells and ii) the mechanism of action may be at the level of blockade of K+ channels.

Berberine↗
PubMed1999

Nitric oxide synthetase and Helicobacter pylori in patients undergoing appendicectomy.

BACKGROUND: This study was designed to determine whether Helicobacter pylori forms part of the normal microenvironment of the appendix, whether it plays a role in the pathogenesis of acute appendicitis, and whether it is associated with increased expression of inducible nitric oxide synthetase (iNOS) in appendicular macrophages. METHODS: Serology for H. pylori was performed on 51 consecutive patients undergoing emergency appendicectomy. Appendix samples were tested for urease activity, cultured and stained for H. pylori, graded according to the degree of inflammatory infiltrate, and probed immunohistochemically for iNOS expression. RESULTS: The mean age of the patients was 21 (range 7-51) years. Seventeen patients (33 per cent) were seropositive for H. pylori but no evidence of H. pylori was found in any appendix specimen. However, an enhanced inflammatory cell infiltration was observed in seropositive patients (P < 0.04) and the expression of macrophage iNOS in the mucosa of normal and inflamed appendix specimens was increased (P < 0.01). CONCLUSION: H. pylori does not colonize the appendix and is unlikely to be a pathogenic stimulus for appendicitis. Priming effects on mucosal immunology downstream from the foregut may occur after infection with H. pylori.

Adolescent↗
PubMed1997

Chylothorax.

BACKGROUND: Chylothorax is a condition that is debilitating to the point of threatening life. There is controversy over its management, in particular the relative merits of conservative measures and the timing of surgical intervention. METHOD: The literature is reviewed from the basic sciences of chyle composition and flow, to diagnostic approaches, the complications of chyle loss and appropriate management strategies. RESULTS AND CONCLUSION: Prompt diagnosis is essential to institute an effective therapeutic regimen. Surgery achieves fast, safe and effective reversal of this dire situation. Minimally invasive thoracoscopic techniques are gaining wide recognition. Early intervention, which should be aggressive and complete to avoid the immune and nutritional consequences of extended chyle depletion, is recommended.

Chylothorax↗