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C Kettner

Publications and source records attributed to C Kettner.

At least 19 recordsLinked to original sources
PubMed2000

Drift ratchet

We consider a silicon wafer, pierced by millions of identical pores with periodically varying diameters but without spatial inversion symmetry (ratchet profile). When a liquid is periodically pumped back and forth through the pores, our theory predicts a net transport of suspended micrometer-sized particles (drift ratchet). The direction of this particle current depends very sensitively on the size of the particles. For typical parameter values of the experiment, two different types of particles at an initially homogeneous 1:1 mixture are spatially separated with a purity beyond 1:1000 on a time scale of a few hours in comparably large quantities. This result is due to the highly parallel architecture of the device. The experimental realization of the setup, presently under construction, thus appears to be a promising new particle separation device, possibly superior to existing methods for particles sizes on the micrometer scale.

Journal Article↗
PubMed1998

Physiological characterization of the yeast plasma membrane outward rectifying K+ channel, DUK1 (TOK1), in situ.

The major voltage-dependent ion channel in the plasma membrane of Saccharomyces cerevisiae, a conspicuous outwardly rectifying K+ channel, was first dubbed YPK1 and later renamed according to its registered gene names (DUK1, TOK1). It has proven novel in both structure and function. Whole-cell patch-clamp studies of the channel directly on yeast protoplasts now extend our earlier description obtained from isolated patches of yeast membrane (Bertl & Slayman, 1992; Bertl et al., 1993), and provide new data both on the contributions of channel properties to yeast physiology and on possible contributions of molecular structure of channel properties. Three recording tactics produce completely equivalent results and thereby allow great flexibility in the design of experiments: whole-cell voltage clamp with sustained voltage steps (approximately 2.5 sec), whole-cell voltage clamp with slow voltage ramps (5 sec, -40 to +100 mV), and time-averaging of single-channel currents. Activation of Duk1 channels under steady-state conditions is dependent upon ATP in the cytoplasmic solution, and the absence of ATP results in channel "rundown"--decreasing numbers of activable channels--over periods of 10 min to 1 hr from the start of patch recording. Several putative serine- and threonine-phosphorylation sites, as well as a variant ATP-binding fold, exist in the molecule as potential mediators of the ATP effects. The channel runs down similarly following cytoplasmic acidification, but is almost completely insensitive to extracellular pH changes (8.0 to 5.5 tested). This remarkable asymmetry may depend on the protein's strongly asymmetric distribution of histidine residues, with 10 out of 12 predicted to lie close to the membrane-cytoplasm interface. Further data confirm the well-recognized observation that changes of K+ concentration, intracellular or extracellular, can shift the gating voltage of Duk1p in the direction of EK. Among the other alkali-metal cations tested, extracellular Rb+ and Cs(+)--but not Na(+)--substitute almost completely for K+. Extracellular TEA+ inhibits whole-cell K+ currents through Duk1p with a KI of 2.8 mM, and does so probably by reducing the single-channel current.

Cations, Divalent↗
PubMed1998

Electrophysiology in the eukaryotic model cell Saccharomyces cerevisiae.

Since the mid-1980s, use of the budding yeast, Saccharomyces cerevisiae, for expression of heterologous (foreign) genes and proteins has burgeoned for several major purposes, including facile genetic manipulation, large-scale production of specific proteins, and preliminary functional analysis. Expression of heterologous membrane proteins in yeast has not kept pace with expression of cytoplasmic proteins for two principal reasons: (1) although plant and fungal proteins express and function easily in yeast membranes, animal proteins do not, at least yet; and (2) the yeast plasma membrane is generally regarded as a difficult system to which to apply the standard electrophysiological techniques for detailed functional analysis of membrane proteins. Especially now, since completion of the genome-sequencing project for Saccharomyces, yeast membranes themselves can be seen as an ample source of diverse membrane proteins - including ion channels, pumps, and cotransporters - which lend themselves to electrophysiological analysis, and specifically to patch-clamping. Using some of these native proteins for assay, we report systematic methods to prepare both the yeast plasma membrane and the yeast vacuolar membrane (tonoplast) for patch-clamp experiments. We also describe optimized ambient conditions - such as electrode preparation, buffer solutions, and time regimens - which facilitate efficient patch recording from Saccharomyces membranes. There are two main keys to successful patch-clamping with Saccharomyces. The first is patience; the second is scrupulous cleanliness. Large cells, such as provided by polyploid strains, are also useful in yeast patch recording, especially while the skill required for gigaseal formation is being learned. Cleanliness is aided by (1) osmotic extrusion of protoplasts, after minimal digestion of yeast walls; (2) use of a rather spare suspension of protoplasts in the recording chamber; (3) maintenance of continuous chamber perfusion prior to formation of gigaseals; (4) preparation (pulling and filling) of patch pipettes immediately before use; (5) application of a modest pressure head to the pipette-filling solution before the tip enters the recording bath; (6) optical control for debris at the pipette tip; and (7) discarding of any pipette that does not "work" on the first try at gigaseal formation. Other useful tricks toward gigaseal formation include the making of protoplasts from cells grown aerobically, rather than anaerobically; use of sustained but gentle suction, rather than hard suction; and manipulation of bath temperature and/or osmotic strength. Yeast plasma membranes form gigaseals with difficulty, but these tend to be very stable and allow for long-term cell-attached or whole-cell recording. Yeast tonoplasts form gigaseals with ease, but these tend to be unstable and rarely allow recording for more than 15 min. The difference of stability accrues mainly because of the fact that yeast protoplasts adhere only lightly to the recording chamber and can therefore be lifted away on the patch pipette, whereas yeast vacuoles adhere firmly to the chamber bottom and are subsequently stressed by very slight relative movements of the pipette. With plasma membranes, conversion from cell-attached recording geometry to isolated ISO patch (inside-out) geometry is accomplished by blowing a fine stream of air bubbles across the pipette tip; to whole-cell recording geometry, by combining suction and one high-voltage pulse; and from whole-cell to OSO patch (outside-out) geometry, by sudden acceleration of the bath perfusion stream. With tonoplasts, conversion from the vacuole-attached recording geometry to whole-vacuole geometry is accomplished by application of a large brief voltage pulse; and further conversion to the OSO patch geometry is carried out conventionally, by slow withdrawal of the patch pipette from the vacuole, which usually remains attached to the chamber bottom.

Cell Membrane↗
PubMed1998

Thrombin and human plasma kallikrein inhibition during simulated extracorporeal circulation block platelet and neutrophil activation.

Cardiopulmonary bypass causes hemorrhagic complications, and initiates a chemical and cellular inflammatory response. Contact of blood with synthetic surfaces leads to qualitative and quantitative alterations in platelets, neutrophils, complement, and contact systems. Despite the fact that cardiopulmonary bypass is carried out in the presence of high doses of heparin, there is significant activation of both platelets and neutrophils. Thrombin is protected on cell and fibrin surfaces from antithrombin, even in the presence of high doses of heparin (approximately 5 U/ml). We therefore studied the effect of a small (Mr = 497), highly effective (Ki = 41 pM), reversible tripeptide inhibitor of thrombin, DUP 714 (1 microM), in a well characterized model of simulated extracorporeal circulation. In the absence of DUP 714, platelet counts decreased by 75% 5 min after the start of extracorporeal bypass and increased to 48% at 120 min of recirculation. DUP 714 significantly preserved platelet counts, decreased plasma levels of platelet beta-thromboglobulin levels, but did not prevent a decrease in sensitivity of platelets to adenosine diphosphate. Kallikrein-C1-inhibitor and C1-C1-inhibitor complexes increased progressively from 0.32 U/ml to 0.67 U/ml and from 4.45 U/ml to 7.25 U/ml, respectively, during 120 min of recirculation without DUP 714. Addition of DUP 714 significantly inhibited kallikrein-C1-inhibitor complex formation but did not affect C1-C1-inhibitor complexes. In the absence of DUP 714, human neutrophil elastase levels rose from a baseline of 0.01 +/- 0.00 microg/ml to 1.18 +/- 0.21 microg/ml during 120 min of recirculation. Human neutrophil elastase release at 120 min was significantly inhibited in the presence of DUP 714 to 37% of the value with heparin alone. These results indicated that addition of this novel thrombin (and kallikrein) inhibitor to heparin preserved platelet counts, decreased platelet secretion, and provided the additional benefit of partially blocking neutrophil activation during simulated extracorporeal circulation.

Cardiopulmonary Bypass↗
PubMed1997

New inhibitors of thrombin and other trypsin-like proteases: hydrogen bonding of an aromatic cyano group with a backbone amide of the P1 binding site replaces binding of a basic side chain.

Highly effective thrombin inhibitors have been obtained by preparing boronic acid analogues of m-cyano-substituted phenylalanine and its incorporation into peptides. The cyano group enhances binding by several orders of magnitude. For example, Ac-(D)Phe-Pro-boroPheOH binds to thrombin with a Ki of 320 nM and the Ki of Ac-(D)Phe-Pro-boroPhe(m-CN)-OH is 0.79 nM. Protein crystal structure determination of trypsin complexed to H-(D)Phe-Pro-boroPhe(m-CN)-OH indicates that the aromatic side chain is bound in the P1 binding site and that the cyano group can act as a H-bond acceptor for the amide proton of Gly219. Enhanced binding for inhibitors containing the m-cyano group was observed for coagulation factor Xa and for the factor VIIa.tissue factor complex [Ki values of Ac-(D)Phe-Pro-boroPhe(mCN)-OH are 760 and 3.3 nM, respectively]. This result is consistent with the sequence homology of these two enzymes in the P1 binding site. Two enzymes lacking the strict homology in the P1 binding site, pancreatic kallikrein and chymotrypsin, did not exhibit significantly enhanced binding.

Amides↗
PubMed1997

Low barrier hydrogen bond is absent in the catalytic triads in the ground state but Is present in a transition-state complex in the prolyl oligopeptidase family of serine proteases.

High frequency proton NMR spectra for two members of the prolyl oligopeptidase class of serine proteases, prolyl oligopeptidase and oligopeptidase B, showed that resonances corresponding to the active center histidine Ndelta1H and Nepsilon2H generally observed in this region, are absent in these enzymes. However, for both enzymes, as well as with the H652A and H652Q active center variants of oligopeptidase B, there are two resonances observed in this region that could be assigned to two protonated histidines with a noncatalytic function. The results indicate that these two histidines participate in strong hydrogen bonds. The absence of resonances pertinent to the active center histidine resonances suggests the absence of a low barrier hydrogen bond between the Asp and His in these two enzymes in their ground states. Addition of the peptide boronic acid t-butoxycarbonyl-(D)Val-Leu-(L)boroArg to oligopeptidase B resulted in potent, slow binding inhibition of the enzyme and the appearance of a new resonance at 15.8 ppm, whose chemical shift is appropriate for a tetrahedral boronate complex and a low barrier hydrogen bond. The results demonstrate important dissimilarities between the active centers of the prolyl oligopeptidase class of serine proteases and the pancreatic and subtilisin classes both in the ground state and in the transition-state analog complexes.

Animals↗
PubMed1996

Cryptosporidium parvum metalloaminopeptidase inhibitors prevent in vitro excystation.

Cryptosporidium parvum arginine aminopeptidase (RAP) was studied during in vitro excystation. Specific RAP inhibitors were identified by using C. parvum extracts. Amastatin, a series of alpha-aminoboronic acids, and the chelating agents EDTA and 1,10-phenanthrolene, but not endoproteinase inhibitors, blocked enzymatic activity. RAP inhibitors found to be effective in soluble enzymatic assays were then studied for their effect on in vitro excystation. 1,10-Phenanthrolene, amastatin, and H-boronorleucine (pinacol) inhibited excystation by 84, 57, and 61%, respectively, compared with solvent-treated control oocysts. Sporozoites remained viable within the oocyst as determined by propidium iodide and fluorescein diacetate dye uptake, suggesting that alpha-aminoboronic acids were not directly lethal to the parasite.

Aminopeptidases↗
PubMed1995

Selective kallikrein inhibitors alter human neutrophil elastase release during extracorporeal circulation.

Cardiopulmonary bypass causes hemorrhagic complications and initiates a biochemical and cellular "whole body inflammatory response." This study investigates whether a variety of selective inhibitors of the contact pathway of intrinsic coagulation modulate complement and neutrophil activation during simulated extracorporeal circulation. After 60 min of recirculation in the presence of the slow tight-binding boronic acid inhibitor, Bz-Pro-Phe-boroArg-OH (10.7 microM), complete inhibition of kallikrein-C1-inhibitor complex formation and marked inhibition of C1-C1-inhibitor complex formation and the release of human neutrophil elastase were observed. Arg15-aprotinin (3.1 microM), Ala357,Arg358 alpha 1-antitrypsin (2.6 microM), and soybean trypsin inhibitor (48.0 microM) either completely or partially inhibited the generation of kallikrein-C1-inhibitor complexes but were less effective inhibitors of human neutrophil elastase release. The second-order rate constants for the inhibition of kallikrein in purified systems are consistent with the order of effectiveness of the inhibitors in blocking human neutrophil elastase release in heparinized blood. Our results suggest that low-molecular-weight selective inhibitors of kallikrein may be effective agents in the attenuation of the contact-mediated inflammatory response in cardiopulmonary bypass.

Amino Acid Sequence↗
PubMed1990

The selective inhibition of thrombin by peptides of boroarginine.

Peptides containing alpha-aminoboronic acids with neutral side chains are highly effective reaction intermediate analog inhibitors of the serine proteases leukocyte elastase, pancreatic elastase, and chymotrypsin. A protocol has been developed for the synthesis of peptides containing alpha-aminoboronic acids with a basic, 3-guanidinopropyl side chain (boroArg) to extend the range of these compounds to trypsin-like proteases. Ac-(D)Phe-Pro-boroArg-OH, Boc-(D)Phe-Pro-boroArg-OH, and H-(D)Phe-Pro-boroArg-OH were prepared as inhibitors of thrombin based on earlier observations that it has a high affinity for this sequence. All three boronic acids are highly effective, slow-binding inhibitors of thrombin, inhibiting it with final inhibition constants and association rates of: 41 pM, 5.5 x 10(6) M-1 s-1; 3.6 pM, 9.3 x 10(6) M-1 s-1; less than 1 pM, 8.0 x 10(6) M-1 s-1, respectively. Comparison of their binding at equilibrium to thrombin, plasma kallikrein, factor Xa, plasmin, and two-chain tissue plasminogen activator has shown that all three inhibitors have at least 2 orders of magnitude greater affinity for thrombin, with the exception of the acetyl derivative which has a 40-fold greater affinity for thrombin than kallikrein. The boroarginine peptides are effective in inhibiting the action of thrombin in rabbit plasma against its physiological substrates. Activated partial thromboplastin time was significantly prolonged in vitro by all of the inhibitors at concentrations of 50-200 nM. Prolongations of activated partial thromboplastin time were also observed in rabbits after intravenous (40-80 micrograms/kg or subcutaneous (0.20-2 mg/kg) injections of Ac-(D)Phe-Pro-boroArg-OH. Results indicate that this new class of synthetic thrombin inhibitors may be clinically useful as antithrombotic agents.

Blood Coagulation↗
PubMed1986

Effects of dosage and timing of administration of a peptide boronic acid inhibitor on lung mechanics and morphometrics in elastase-induced emphysema in hamsters.

The peptide boronic acid, MeOSuc-Ala-Ala-Pro-boroVal-pinacol (AAPbV), is an effective inhibitor of both pancreatic and leukocyte elastase. Initial work showed that AAPbV diminishes the effect of emphysema induced by pancreatic elastase. This initial work has been expanded to show that AAPbV provides a high degree of protection against elastase-induced increases in lung volume and mean linear intercept when given intratracheally at 200 mg/kg either 15 min before, simultaneous with, or 15 min after instilling elastase. Intraperitoneal administration, although less effective, is dose dependent and dependent on the time of treatment. We conclude that a reversible protease inhibitor can be used to prevent aberrant proteolysis in vivo.

Animals↗
PubMed1986

A new peptide boronic acid inhibitor of elastase-induced lung injury in hamsters.

A highly effective, reversible elastase inhibitor, MeOSuc-Ala-Ala-Pro-boroVal-OH, was tested for its ability to prevent emphysema induced by intratracheally administered elastase in hamsters. Anesthetized hamsters were given elastase intratracheally with or without the inhibitor or were given elastase intratracheally and the inhibitor intraperitoneally. Two weeks after administration, lungs were removed, and static air pressure volume curves were performed followed by intratracheal fixation and morphometric determination of mean linear intercepts. The results indicate significant preservation of structure and function whether the inhibitor is given intratracheally or intraperitoneally and suggest that this inhibitor may be useful in controlling diseases arising from aberrant proteolysis by elastolytic enzymes.

Animals↗
PubMed1982

Kallikrein-like activity of crotalase, a snake venom enzyme that clots fibrinogen.

During the amino acid sequence determination of crotalase (EC 3.4.21.30), the thrombin-like enzyme from the venom of Crotalus adamanteus (eastern diamondback rattlesnake), we found that, in addition to the expected structural homology with bovine thrombin (EC 3.4.21.5), there was even greater homology with porcine pancreatic kallikrein (EC 3.4.21.8). In exploring further the similarities between crotalase and kallikrein, several striking observations were made. First, crotalase was rapidly and specifically inhibited by the tripeptide affinity labeling derivative prolylphenylalanylarginine chloromethyl ketone, which is known to be a specific inhibitor of kallikrein. Second, NaDodSO4/acrylamide gel electrophoresis revealed that crotalase cleaves the plasma kallikrein-susceptible bonds in human high molecular weight kininogen, producing an intermediate with procoagulant activity. Crotalase-catalyzed cleavage of high molecular weight kininogen also liberates kinin as evidenced by rat blood pressure bioassay. Finally, crotalase exhibits substrate specificity not only for the thrombin chromogenic substrate S-2238 but also for the kallikrein substrates S-2302 and S-2266. Interestingly, one of the other reactions catalyzed by plasma kallikrein, the activation of plasminogen, was not one of the activities exhibited by crotalase.

Animals↗
PubMed1981

Cofactor dependence of factor Xa incorporation into the prothrombinase complex.

The blood coagulation serine protease, factor Xa, was dansylated with the active site-directed inhibitor, dansyl-glutamyl-glycyl-arginyl chloromethyl ketone. The Ca2+-dependent interactions of the inactivated factor Xa with its cofactors, phospholipid and activated factor V (factor Va), were studied through alterations of fluorescence polarization values of dansyl moiety of the modified enyme. In the presence of phospholipid and Ca2+, factor Va and factor Xa interacted with 1:1 stoichiometry, an interaction characterized by markedly enhanced polarization. The factor Va-independent interaction of factor Xa with phospholipid was also observed, characterized by dissociation constant Kd=2.7 x 10-6 M and stoichiometry of 66 mol of phospholipid/mol of factor Xa. The interaction of factor Xa with vesicles in the absence of factor Va exhibited considerably lower polarization values than in the presence of factor Va. These data obtained by direct spectral measurements are in agreement with the inferences drawn previously from studies of kinetics that the prothrombinase complex consists of 1:1 stoichiometric complex of factor Xa and phospholipid-bound factor Va, and that the enzymatic complex assembles in the absence of the natural substrate, prothrombin.

Amino Acid Chloromethyl Ketones↗