Biomedical subjects

W Nagel

Publications and source records attributed to W Nagel.

At least 19 recordsLinked to original sources

PubMed2005

Effect of dislodging techniques on foliar residue determination for agricultural crops.

The Agricultural Reentry Task Force (ARTF) conducted a study to determine whether different dislodgeable foliar residue (DFR) techniques had any effect on the amount of residues dislodged from the leaves of vegetable crops. Four previously employed techniques were evaluated with two chemicals (carbaryl and methomyl) on two crops (cabbage and lettuce). Carbaryl, a moderately water-soluble chemical, was applied at a rate of 2.0 lbs active ingredient/acre (lb ai/A) while methomyl, a highly water soluble chemical, was applied at a rate of 0.9 lb ai/A. One day after application, leaf punches were collected and the residues were dislodged following one of four techniques that differed in the number of dislodgings per sample, the duration of dislodging, the solution volume, and/or the type of solution used. The results indicated that three of the techniques gave similar results, whereas a fourth gave marginally lower results.

Carbaryl↗

PubMed2004

Ammonium affects tight junctions and the cytoskeleton in MDCK cells.

In the kidney medulla, tubule cells are exposed not only to elevated NaCl but also to high NH(4)Cl concentrations. Although it is well known that long-term exposure to high NaCl concentrations leads to reorganization of the actin-based cytoskeleton and to altered transport properties of renal epithelial cells, there have been no comparable studies on the effects of elevated extracellular NH(4)Cl concentrations. We therefore examined the effect of prolonged (up to 72 h) exposure of Madin-Darby canine kidney (MDCK) cells to increased NH(4)Cl concentrations on the actin-based cytoskeleton, the transepithelial electrical resistance (TER) and the expression and intracellular distribution of the tight junction protein occludin. NH(4)Cl exposure resulted in rarefaction of cytoplasmic stress fibres, formation of intense peripheral actin bands and reduced abundance of both F- and G-actin. While under control conditions occludin staining was restricted to the tight junction region, ample dot-like intracellular staining was apparent after NH(4)Cl exposure. These changes in cell structure were associated with an increase in TER and the enhanced expression of an additional putative, 40-kDa occludin isoform. Exposure to elevated extracellular NH(4)Cl concentrations thus leads to distinct alterations in the architecture and transepithelial transport properties of MDCK cells that may also be relevant for the tubule cells of the renal inner medulla.

Actins↗

PubMed2001

Cytohesin-1 is a dynamic regulator of distinct LFA-1 functions in leukocyte arrest and transmigration triggered by chemokines.

Cytohesin-1 is a regulatory interaction partner of the beta2 integrin alphaLbeta2 (LFA-1) and a guanine exchange factor (GEF) for ADP ribosylation factor (ARF)-GTPases. However, a functional role of cytohesin-1 in leukocyte adhesion to activated endothelium and subsequent transmigration in response to chemokines has not been defined. Overexpression of cytohesin-1 increased LFA-1-dependent arrest of leukocytic cells triggered by chemokines on cytokine-activated endothelium in flow while reducing the fraction of rolling cells. Conversely, a dominant-negative PH domain construct of cytohesin-1 but not a mutant deficient in GEF activity impaired arrest, indicating an involvement of the PH domain while GEF function is not required. Expression of these constructs and a beta2 mutant interrupting the interaction with cytohesin-1 indicated that shape change in flow and transendothelial chemotaxis involve both LFA-1 avidity regulation and GEF activity of cytohesin-1. As a potential downstream target, ARF6 but not ARF1 was identified to participate in chemotaxis. Our data suggest that cytohesin-1 and ARF6 are involved in the dynamic regulation of complex signaling pathways and cytoskeletal remodeling processes governing LFA-1 functions in leukocyte recruitment. Differential effects of cytohesin-1 and ARF6 mutants in our systems reveal that cytohesin-1 with its GEF activity controls both conversion of rolling into firm arrest and transmigration triggered by chemokines, whereas a cyclical activity of ARF6 plays a more important role in diapedesis.

Cell Adhesion Molecules↗

PubMed2001

Controlling small guanine-nucleotide-exchange factor function through cytoplasmic RNA intramers.

ADP-ribosylation factor (ARF) GTPases and their regulatory proteins have been implicated in the control of diverse biological functions. Two main classes of positive regulatory elements for ARF have been discovered so far: the large Sec7/Gea and the small cytohesin/ARNO families, respectively. These proteins harbor guanine-nucleotide-exchange factor (GEF) activity exerted by the common Sec7 domain. The availability of a specific inhibitor, the fungal metabolite brefeldin A, has enabled documentation of the involvement of the large GEFs in vesicle transport. However, because of the lack of such tools, the biological roles of the small GEFs have remained controversial. Here, we have selected a series of RNA aptamers that specifically recognize the Sec7 domain of cytohesin 1. Some aptamers inhibit guanine-nucleotide exchange on ARF1, thereby preventing ARF activation in vitro. Among them, aptamer M69 exhibited unexpected specificity for the small GEFs, because it does not interact with or inhibit the GEF activity of the related Gea2-Sec7 domain, a member of the class of large GEFs. The inhibitory effect demonstrated in vitro clearly is observed as well in vivo, based on the finding that M69 produces similar results as a dominant-negative, GEF-deficient mutant of cytohesin 1: when expressed in the cytoplasm of T-cells, M69 reduces stimulated adhesion to intercellular adhesion molecule-1 and results in a dramatic reorganization of F-actin distribution. These highly specific cellular effects suggest that the ARF-GEF activity of cytohesin 1 plays an important role in cytoskeletal remodeling events of lymphoid cells.

ADP-Ribosylation Factor 1↗

PubMed2001

Measurement of pH gradients using an ion-sensitive vibrating probe technique (IP).

The direct measurement and quantification of proton transport in biological structures are below the detection limit of stationary pH-sensitive microelectrodes. We have thus used a more sensitive system to detect and quantify these small pH gradients: a proton-sensitive vibrating ion probe technique. This technique decreases the noise of the system to less than +/- 15 microV, equivalent to pH gradients below 0.0005 pH units, and can be used to measure pH gradients even in the presence of moderate buffer concentrations. At physiological pH the detection limit, analysed with artificial proton sources, is in the range of 5 pmol.s-1.cm-2. Computer simulation indicates that the spatial resolution is sufficient to localize individual proton sources less than 30 microns apart.

Bicarbonates↗

PubMed2001

Changes in element composition of A6 cells following hypotonic stress.

Cellular element concentrations and dry weight contents were determined in A6 epithelia using electron microprobe analysis. This was done to assess the quantitative contributions of Na, K and Cl to the regulatory volume decrease (RVD) and isovolumetric regulation (IVR) after decreasing the basolateral osmolality from 260 to 140 mosmol/kg in a stepwise or gradual way. Two minutes after inducing acute hypotonic stress the cells behaved almost like ideal osmometers, as indicated by a pronounced increase in cell height and decreases in the cellular dry weight and concentrations of all measured elements by about the same degree. Sixty minutes after inducing acute hypotonic stress the dry weight and concentrations of the impermeant elements P, Mg and Ca had returned approximately to control values, indicating normalized cell volume. Na, K and Cl concentrations, however, remained greatly reduced. The cellular amounts of Na, K and Cl diminished during RVD by approximately 31%, 24% and 46%, respectively. The dry weights and element concentrations measured 60 min after inducing acute hypotonic stress were similar to those obtained after a continuous reduction of basolateral osmolality. The cellular loss of Na and K following hypotonic stress exceeded that of Cl by about 40 mmol/kg wet wt., suggesting the exit of an other anion and/or the titration of fixed negative charges. The contribution of Na, K and Cl to total cellular osmolality increased from about 75% under control conditions to about 85% during RVD and IVR. Since only approximately 70% of the loss of cellular osmolytes necessary for the observed RVD and IVR is accounted for by the cellular exit of Na, K and Cl, other osmolytes, possibly amino acids, must leave the cells following hypotonic stress.

Animals↗

PubMed2001

Signaling by human herpesvirus 8 kaposin A through direct membrane recruitment of cytohesin-1.

The induction of a transformed cellular phenotype by viruses requires the modulation of signaling pathways through viral proteins. We show here that the phenotypic changes induced by the kaposin A protein of human herpesvirus 8 are mediated through its direct interaction with cytohesin-1, a guanine nucleotide exchange factor for ARF GTPases and regulator of integrin-mediated cell adhesion. Focus formation, stress fiber dissolution, and activation of the ERK-1/2 MAP kinase signal cascade were reverted by the cytohesin-1 E157K mutant, which is deficient in catalyzing guanine nucleotide exchange. Furthermore, liposome-embedded kaposin A specifically stimulates cytohesin-1 dependent GTP binding of myristoylated ARF1 in vitro. These results suggest a previously unknown involvement of ARF GTPases in the control of cellular functions by herpesviruses.

3T3 Cells↗

PubMed2001

Selective inhibition of Cl(-) conductance in toad skin by blockers of Cl(-) channels and transporters.

We compared the effects exerted by two classes of Cl(-) transport inhibitors on a Cl(-)-selective, passive anion transport route across the skin of Bufo viridis, the conductance (G(Cl)) of which can be activated by transepithelial voltage perturbation or high cAMP at short circuit. Inhibitors of antiporters (erythrosine, eosin) or cotransporters (furosemide) reduced voltage-activated G(Cl) with IC(50) of 6 +/- 1, 54 +/- 12, and 607 +/- 125 microM, respectively; they had no effect on the cAMP-induced G(Cl). The voltage for half-maximal activation of G(Cl) (V(50)) increased compared with controls, but effects on the maximal G(Cl) at more positive clamp potentials were small. Cl(-) channel blockers from the diphenylamino-2-carboxylic acid (DPC) family [dichloro-DPC, niflumic acid, flufenamic acid, and 5-nitro-2-(3-phenylpropylamino)benzoic acid] reduced the voltage-activated G(Cl) with IC(50) of 8.3 +/- 1.2, 10.5 +/- 0.6, 16.5 +/- 3.4, and 36.5 +/- 11.4 microM, respectively, and also inhibited the cAMP-induced G(Cl), albeit with slightly larger IC(50). V(50) was not significantly changed compared with controls; the maximal G(Cl) was strongly reduced. We conclude that the pathway for Cl(-) is composed of the conductive pore proper, which is blocked by the derivatives of DPC, and a separate, voltage-sensitive regulator, which is influenced by blockers of cotransporters or antiporters. This influence is partly overcome by increasing the clamp potential and removed by high concentrations of cAMP, which renders the pathway insensitive to voltage.

Angiogenesis Inhibitors↗

PubMed2000

Cytohesin-1 regulates beta-2 integrin-mediated adhesion through both ARF-GEF function and interaction with LFA-1.

Intracellular signaling pathways, which regulate the interactions of integrins with their ligands, affect a wide variety of biological functions. Here we provide evidence of how cytohesin-1, an integrin-binding protein and guanine-nucleotide exchange factor (GEF) for ARF GTPases, regulates cell adhesion. Mutational analyses of the beta-2 cytoplasmic domain revealed that the adhesive function of LFA-1 depends on its interaction with cytohesin-1, unless the integrin is activated by exogenous divalent cations. Secondly, cytohesin-1 induces expression of an extracellular activation epitope of LFA-1, and the exchange factor function is not essential for this activity. In contrast, LFA-1-mediated cell adhesion and spreading on intercellular cell adhesion molecule 1 is strongly inhibited by a cytohesin-1 mutant, which fails to catalyze ARF GDP-GTP exchange in vitro. Thus, cytohesin-1 is involved in the activation of LFA-1, most probably through direct interaction with the integrin, and induces cell spreading by its ARF-GEF activity. We therefore propose that both direct regulation of the integrin and concomitant changes in the membrane topology of adherent T cells are modulated by dissectable functions of cytohesin-1.

ADP-Ribosylation Factors↗

PubMed2000

Cyanide inhibition of chloride conductance across toad skin.

The effect of cyanide (CN(-)) on voltage-activated or cAMP-induced passive chloride conductance (G(Cl)) was analyzed in isolated toad skin. Comparatively low concentrations of CN(-) inhibited G(Cl) almost completely and fully reversibly, regardless of whether it was applied from the mucosal or serosal side. The IC(50) was 180 +/- 12 microm for voltage-activated G(Cl) and 305 +/- 30 microm for the cAMP-inducted conductance. At [CN] <100 microm, the initial inhibition frequently declined partly in the continuous presence of CN(-). Inhibition was independent of the presence of Ca(2+). Inhibition was stronger at more alkaline pH, which suggests that dissociated CN(-) is the effective inhibitor. The onset of the inhibition of voltage-activated or cAMP-induced G(Cl) by CN(-) occurred with half-times of 34 +/- 10 sec, whereas reversibility upon washout was twice as fast (18 +/- 7 sec). If [CN(-)] <200 microm was applied under inactivating conditions (serosa -30 mV), the reduction of G(Cl) was stronger upon subsequent voltage-activation than under steady-state activated conditions. This effect was essentially complete less than 30 sec after apical addition of CN(-), but G(t) recovered thereafter partially in the continuous presence of CN(-). Dinitrophenol inhibited G(Cl) similarly, while omission of oxygen did not affect it. These observations, as well as the time course of inhibition and the full reversibility, suggest that interference of CN(-) with oxidative phosphorylation and subsequent metabolic depletion is not the reason for the inhibition of G(Cl). We propose that the inhibition is directly on G(Cl), presumably by competition with Cl(-) at a rate-limiting site in the pathway. Location and molecular nature of this site remain to be identified.

Animals↗

PubMed2000

Anergic T lymphocytes selectively express an integrin regulatory protein of the cytohesin family.

It has been proposed that the maintenance of T cell anergy depends on the induction of negative regulatory factors. Differential display of reverse transcribed RNA was used to identify novel genes that might mediate this function in anergic Th1 clones. We report that anergic Th1 clones do indeed express a genetic program different from that of responsive T cells. Moreover, one gene, the general receptor of phosphoinositides 1 (GRP1), was selectively induced in anergic T cells. The GRP1, located in the plasma membrane, regulated integrin-mediated adhesion and was invariably associated with unresponsiveness in multiple models of anergy. T cells expressing retrovirally transduced GRP1 exhibited normal proliferation and cytokine production. However, GRP1-transduced T cells were not stable and rapidly lost GRP1 expression. Thus, although GRP1 may not directly mediate T cell anergy, it regulates cell expansion and survival, perhaps through its integrin-associated activities.

Amino Acid Sequence↗

PubMed2000

Transepithelial chloride conductance in amphibian skin: regulatory mechanisms and localization.

The transepithelial transport of Na+ by amphibian skin must be accompanied by the corresponding anion, Cl-, and much effort has been devoted to the characterization of Cl- transport. The transepithelial Cl- conductance, G(Cl), is activated by voltage and adenosine 3',5'-cyclic monophosphate (cAMP), shows rectification, requires the presence of Cl- in the pathway and is influenced by factors modifying intracellular signalling cascades and by metabolic poisons such as cyanide (CN-). Until recently, these findings were interpreted as strong evidence for a transcellular path, for which, given the impermeability of the principal cells for Cl-, the mitochondria-rich cells (MRC) are the only candidate. This was supported by the apparent parallelism between G(Cl) and the density of MRC (D(mrc)). Data accumulated in recent years, however, raise serious doubts as to the validity of this concept. The single-channel conductance derived from various techniques is too small by an order of magnitude to account for the observed G(Cl), the very slow time course of conductance activation is not reconcilable with any known membrane channel gating processes, a more thorough examination of the relationship between G(Cl) and D(mrc) fails to show any consistent pattern and analysis of current density immediately above the transporting epithelium using the vibrating voltage probe shows current peaks associated with only a small fraction of MRC, and even so, these current peaks account for about 20% of the transepithelial current. The remaining 80% of the current cannot be localized to specific structures. Given the increasing evidence for close cellular control of tight-junction function, the foregoing findings are equally consistent with an additional, major, paracellular pathway for Cl-. A comprehensive description of Cl- transport must await the final resolution of the transport pathway(s).

Amphibians↗

PubMed2000

A novel brain trauma model in the mouse: effects of dexamethasone treatment.

We describe a novel methodological approach for inducing cold lesion in the mouse as a model of human cortical contusion trauma. To validate its reproducibility and reliability, dexamethasone (Dxm) was repeatedly applied to demonstrate possible antioedematous drug effects. Following the induction of anaesthesia with halothane, the dura was exposed via trephination. Using a micromanipulator a pre-cooled (-78 degrees C) copper cylinder, 3 mm in diameter, was pressed down to a depth of 1 mm onto the dura for 30 s under microscopic control. The body temperature was held constant at 37 degrees C throughout the procedure. Blood pressure (BP), measured by a modified photosensor-monitored tail-cuff method, and acid-base status were not significantly different when analysed before and after cold lesion and prior to sacrifice. However, there was a marginal mixed respiratory and metabolic acidosis. The antioedematous action of Dxm was studied in four standard pre-and post-treatment paradigms: 2x0.5 mg/kg (II), 2x12.5 mg/kg (III) and 4x6.25 mg/kg (IV: 3x pre-, 1x post-treatment: V: 1x pre-, 3x post-treatment). Physiological saline injections served as controls. High doses of Dxm (III-V) significantly attenuated the cold-lesion-induced loss of body mass. Dxm treatment also resulted in a reduction of brain water content (III; P<0.05), and brain swelling (IV; P<0.05) in the lesioned hemisphere, relative to controls. In conclusion, we have characterized a novel cold lesion model in the mouse to mimic traumatic brain injury and the beneficial effect of Dxm treatment on the extent of brain oedema.

Acid-Base Equilibrium↗

PubMed2000

An integral-geometric approach for the euler-Poincare characteristic of spatial images

The determination of the Euler-Poincare characteristic of a set can be based on observations of a digitized image of that set. In the present paper the correctness of the method is proved due to a strict integral-geometric approach. Our result also provides a link to the methods which are used in image analysis and are based on graph theory.

Journal Article↗

PubMed2000

Phosphoinositides determine specificity of the guanine-nucleotide exchange activity of cytohesin-1 for ADP-ribosylation factors derived from a mammalian expression system.

ADP-ribosylation factors (ARFs) are small Ras-like GTPases which play important roles in intracellular vesicle transport and in the remodeling of the actin cytoskeleton. Guanine nucleotide exchange factors (GEFs) for ARFs have recently been identified. One of them, cytohesin-1, a 47-kDa cytoplasmic protein acts as an inside-out signaling molecule and regulates binding of the beta2 integrin leukocyte function antigen 1 (LFA-1) to its ligand intercellular adhesion molecule 1 (ICAM-1). In this study, we address the regulation of the GEF activity of cytohesin-1 by phosphoinositides, using mammalian expression of functional ARF-Ig chimeras. The fusion proteins, which can be quantitatively immunoprecipitated on protein A-Sepharose, target to the expected intracellular compartments, and they are readily induced to bind GTP in vitro. We show that both ARF1-Ig and ARF6-Ig chimeras are activated in vitro by cytohesin-1. However, GEF activity towards ARF6 is strongly suppressed by phosphatidylinositol-(3,4,5)-trisphosphate (PtdInsP3). In contrast, cytohesin-1-dependent GTP binding of ARF1 is significantly enhanced by PtdInsP3. We conclude that the membrane phospholipid PtdInsP3 determines the specificity of the GEF activity of cytohesin-1.

ADP-Ribosylation Factor 1↗

PubMed2000

Voltage clamping single cells in intact malpighian tubules of mosquitoes.

Principal cells of the Malpighian tubule of the yellow fever mosquito were studied with the methods of two-electrode voltage clamp (TEVC). Intracellular voltage (V(pc)) was -86.7 mV, and input resistance (R(pc)) was 388.5 kOmega (n = 49 cells). In six cells, Ba(2+) (15 mM) had negligible effects on V(pc), but it increased R(pc) from 325.3 to 684.5 kOmega (P < 0.001). In the presence of Ba(2+), leucokinin-VIII (1 microM) increased V(pc) to -101.8 mV (P < 0.001) and reduced R(pc) to 340.2 kOmega (P < 0.002). Circuit analysis yields the following: basolateral membrane resistance, 652. 0 kOmega; apical membrane resistance, 340.2 kOmega; shunt resistance (R(sh)), 344.3 kOmega; transcellular resistance, 992.2 kOmega. The fractional resistance of the apical membrane (0.35) and the ratio of transcellular resistance and R(sh) (3.53) agree closely with values obtained by cable analysis in isolated perfused tubules and confirm the usefulness of TEVC methods in single principal cells of the intact Malpighian tubule. Dinitrophenol (0.1 mM) reversibly depolarized V(pc) from -94.3 to -10.7 mV (P < 0.001) and reversibly increased R(pc) from 412 to 2,879 kOmega (P < 0.001), effects that were duplicated by cyanide (0.3 mM). Significant effects of metabolic inhibition on voltage and resistance suggest a role of ATP in electrogenesis and the maintenance of conductive transport pathways.

Animals↗

PubMed2000

Central role of the apical membrane H+-ATPase in electrogenesis and epithelial transport in Malpighian tubules.

The effects of bafilomycin A(1), a blocker of V-type H(+)-ATPases, were investigated in Malpighian tubules of Aedes aegypti. Bafilomycin A(1) reduced rates of transepithelial fluid secretion and the virtual short-circuit current (vI(sc)) with an IC(50) of approximately 5 micromol l(-)(1). As vI(sc) decreased, the electrical resistance increased across the whole epithelium and across the apical membrane, indicating effects on electroconductive pathways. Bafilomycin A(1) had no effect when applied from the tubule lumen, pointing to the relative impermeability of the apical membrane to bafilomycin A(1). Thus, bafilomycin A(1) must take a cytoplasmic route to its blocking site in the proton channel of the H(+)-ATPase located in the apical membrane of principal cells. The inhibitory effects of bafilomycin A(1) were qualitatively similar to those of dinitrophenol in that voltages across the epithelium (V(t)), the basolateral membrane (V(bl)) and the apical membrane (V(a)) depolarized towards zero in parallel. Moreover, V(bl )always tracked V(a), indicating electrical coupling between the two membranes through the shunt. Electrical coupling allows the H(+)-ATPase to energize not only the apical membrane, but also the basolateral membrane. Furthermore, electrical coupling offers a balance between electroconductive entry of cations across the basolateral membrane and extrusion across the apical membrane to support steady-state conditions during transepithelial transport.

2,4-Dinitrophenol↗

PubMed1999

Bradykinin-stimulated Cl- secretion in T84 cells. Role of Ca2+-activated hSK4-like K+ channels.

Bradykinin (BK)-stimulated colonic Cl- secretion was studied in T84 colonic adenocarcinoma cells by measuring BK (50 nM)-evoked changes in cytosolic free [Ca2+] ([Ca2+]i), membrane conductance and transepithelial ion transport. In T84 cells grown on impermeable supports, BK stimulated a transient increase in [Ca2+]i as assessed by fura-2 ratio imaging. In cell-attached, patch-clamp recordings, BK transiently activated low-conductance K channels. These channels were activated/inactivated reversibly in inside-out patches by switching [Ca2+]i in the bath between 30 nM and 100 nM. Excised channels recorded with 160 mM [K+] in bath and pipette exhibited an inwardly rectifying current/voltage-relation, conductances of 10+/-1 pS and 34+/-4 pS (n=10) at positive and negative voltages, respectively, and a 15-fold lower permeability for Na+ than for K+. The mean open probability of these channels did not depend on voltage but increased with increasing [Ca2+]i with an apparent concentration for a half-maximal response (EC50) of 110 nM, resembling that of hSK4 K+ channels. Application of the reverse transcriptase-polymerase chain reaction technique showed hSK4 messenger ribonucleic acid (mRNA) to be expressed in T84 cells. Macroscopic currents in T84 cells showed a similar dependence on [Ca2+]i. Whole cell conductance (in nS/10pF) increased from 0.5+/-0. 1 (n=6) at 10 nM [Ca2+]i in the pipette solution to 1.5+/-0.2 (n=7) at 100 nM, and to 2.0+/-0.5 (n=7) at 1 microM due to activation of a K+ conductance. In Ussing-chambered T84 monolayers grown on filters, BK did not evoke a short-circuit current (Isc). When, however, the monolayers were pre-stimulated by forskolin (1 microM), BK further enhanced Cl-secretion (DeltaIsc=21+/-5 microA/cm2, n=10) transiently and biphasically. In conclusion, BK enhances cyclic adenosine monophosphate-stimulated Cl- secretion in T84 cells, probably via basolateral, Ca2+-liganded activation of low-conductance hSK4-type K+ channels.

Adenocarcinoma↗