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H Faulstich

Publications and source records attributed to H Faulstich.

At least 55 records · Page 3Linked to original sources
PubMed1988

The interaction of triethyl lead with tubulin and microtubules.

The impact of triethyl lead chloride was studied on: (i) the in vitro assembly and disassembly of microtubules from porcine brain by turbidometry and electron microscopy, (ii) the microtubule system of living mammalian cells using immunofluorescence microscopy, (iii) cell motility and chemotaxis employing the methods of phagokinetic track formation and the Boyden chamber assay, respectively, and (iv) thiol groups of the protein tubulin by their titration in the presence and absence of the organic lead compound. Triethyl lead chloride inhibited microtubule assembly and depolymerized preformed microtubules in vitro and in living cells. Random motility of cells was not markedly inhibited by triethyl lead chloride, whereas chemotaxis (directed cellular movement) was strongly inhibited. Triethyl lead chloride was found to interact with 2 thiol groups of the tubulin dimer. The interaction of triethyl lead chloride with the tubulin/microtubule system in vivo likely causes aneuploidy and is at least partly responsible for the cytotoxicity of the drug.

Animals↗
PubMed1988

Strongly enhanced toxicity of the mushroom toxin alpha-amanitin by an amatoxin-specific Fab or monoclonal antibody.

A monoclonal antibody, with high affinity against the mushroom toxin alpha-amanitin, was prepared. Administration of the Fab fragment of the monoclonal antibody to mice caused a 50-fold increase in alpha-amanitin toxicity. Electron micrographs showed normal appearance of hepatocytes but typical, amanitin-induced lesions in cells of the proximal convoluted tubules of the kidney. The pronounced nephrotoxicity is mainly explained by glomerular filtration and tubular reabsorption of the Fab-amatoxin complex and, to a lesser extent, of the immunoglobulin-amatoxin complex, which is still c. Twice as toxic as free alpha-amanitin. To our knowledge this is the first reported case where immunoglobulins or their fragments enhance rather than decrease the activity of a toxin. Accordingly, immunotherapy of Amanita mushroom poisoning in humans does not appear promising.

Amanitins↗
PubMed1988

Coumarin-phalloidin: a new actin probe permitting triple immunofluorescence microscopy of the cytoskeleton.

7-Diethylamino-3-(4-isothiocyanotophenyl)-4-methylcoumarin (CPITC) was coupled to amino-methyldithiolanophalloidin to produce a new phalloidin derivative, coumarin-phalloidin, fluorescent in the blue region of the spectrum. Coumarin-phalloidin binds to actin with around 100-fold less affinity than unconjugated phalloidin, but with enough avidity to make it a useful stain for actin filaments. Appropriate filter combinations permit triple immunofluorescence microscopy of the cytoskeleton with fluorescein and rhodamine conjugates together with coumarin-phalloidin.

Actins↗
PubMed1987

Changes in OD at 235 nm do not correspond to the polymerization step of actin.

Discrepancies were observed when the polymerization of rabbit muscle actin was monitored by delta OD235 and viscometry (eta). For example, in the presence of (beta,gamma)-methyleno ATP, the delta OD signal was as large as with ATP although polymerization was very poor (eta 1.1, compared with eta = 1.7 in the presence of ATP). Furthermore, when monomeric actin, kept for 1 h in the presence of a stoichiometric equivalent of ADP, was exposed to conditions favoring polymerization (addition of MgCl2), a considerable delta OD235 signal appeared, although the actin had completely lost its polymerizability (eta = 1.0). We conclude that the observed changes in OD235 cannot reflect polymerization itself, but must be caused by another reaction preceding the assembly. Under normal conditions, this reaction is supposed to be the slowest step of filament formation and so to determine the velocity of the whole process. In conclusion, monitoring of actin polymerization by delta OD235 is a valid method only when polymerization has been assessed by another, independent method.

Actins↗
PubMed1986

Characterization of a transporting system in rat hepatocytes. Studies with competitive and non-competitive inhibitors of phalloidin transport.

Primary cultures of rat hepatocytes were used for assaying several drugs not previously known for inhibiting the transport of phalloidin. In order to have 50% inhibition (IC50) of the entrance of a tritiated phallotoxin derivative ([3H]demethylphalloin, 1 microM) from the medium into the cells the following concentrations (microM) of the various inhibitors were determined: cyclolinopeptide (0.5), Nocloprost (5.0), Nileprost (7.0), beta-estradiol (42), Verapamil (70). For comparison, the corresponding IC50 values of some known antagonists of phalloidin toxicity were determined by the same method. Moreover, we studied several natural and synthetic phallotoxins and alpha-amanitin for their ability to displace [3H]demethylphalloin from the transporting system. Lineweaver-Burk plots made it obvious that two groups of inhibitors exist. Competitive inhibitors are, for example, antamanide, beta-estradiol, silybin, Nileprost, taurocholate, and the cyclic somatostatin analog cyclo[Phe-Thr-Lys-Trp-Phe-D-Pro], whereas Verapamil and monensin inhibit phallotoxin uptake in a non-competitive way. Considering the very different chemical features of the competitive inhibitors, we tentatively conclude that the phallotoxin transport system selects compounds not on the basis of their chemical features, but rather their physical properties. The physical properties of a typical substrate are low molecular mass, lipophilic nature, and, possibly the presence of rigid ring structures. Negative charges accelerate the transport of a substrate, while positive charges have the opposite effect. The phalloidin-transporting system may represent part of a hepatic equipment which clears portal blood from, for example, bile acids, lipophilic hormones, or xenobiotics. By chance, the transporting system incorporates phallotoxins into the hepatocytes leading to the death of these cells.

Amanitins↗
PubMed1986

Purification of amatoxin-specific antibodies from rabbit sera by affinity chromatography, their characterization and use in toxicological studies.

Rabbits were immunized with fetuin-beta-amanitin. They produced amatoxin-specific immunoglobulins of various classes, predominantly IgG. The crude IgG fraction was isolated by gel filtration on Sephacryl S-300. By polyethylene glycol precipitation in the presence of tritiated amatoxin, as well as by a solid phase radioimmunoassay technique, the portion of amatoxin-specific antibodies in the IgG fraction was determined to be 5-13%. The affinity of the amatoxin-specific IgG for a tritiated amatoxin derivative was measured by equilibrium dialysis and calculated according to the method of Scatchard. The dissociation constant was 2.6 X 10(-9) M. The amatoxin-specific immunoglobulins were extracted by their affinity to alpha-amanitin-Sepharose 4B and eluted with excess alpha-amanitin. The complex was isolated in 95% yield with a ratio immunoglobulin:toxin of c. 2:1. Alternatively, the uncomplexed IgG could be eluted from the alpha-amanitin-Sepharose 4B with 5 M guanidine hydrochloride; this treatment, however, decreased the binding capacity for amatoxin by 30% (ratio 1.4:1). The purified amatoxin-specific IgG was assayed for its therapeutic efficiency in mice poisoned with alpha-amanitin, but was unable to neutralize the toxic effects of the mushroom toxin. On the contrary, equimolar amounts of the amatoxin-specific immunoglobulins enhanced the toxicity of alpha-amanitin in the mouse by a factor 2.

Amanitins↗
PubMed1986

A soluble biotinyl-alpha-amanitin affinity probe for the isolation of RNA polymerase B.

Utilizing the 6'-hydroxyindole moiety of alpha-amanitin for substitution, biotinyl-alpha-amanitin has been synthesized to use as a soluble affinity probe for the isolation of RNA polymerase B from mammalian cell culture. The synthetic biotinyl-alpha-amanitin remains a potent inhibitor of RNA polymerase B having a Ki of 4.1 X 10(-8) M as compared with a Ki of 5 X 10(-9) M for natural alpha-amanitin. RNA polymerase B complexed with biotinyl-alpha-amanitin can be isolated on Bio-Gel P300 polyacrylamide gel beads to which avidin has been attached. RNA polymerase B may then be released from the complex by treatment with sodium dodecyl sulfate or by monochromatic irradiation at 314 nm which destroys the anatoxin moiety. We have used this affinity probe to analyze the subunit composition of RNA polymerase B from various mouse myeloma cell lines. We believe that the biotinyl-alpha-amanitin may be very useful for the isolation of factors which associate with RNA polymerase B; e.g., we have substantiated that actin can be associated with RNA polymerase.

Actins↗
PubMed1985

Phallolysin. A mushroom toxin, forms proton and voltage gated membrane channels.

Phallolysin, a water soluble protein of Mr 34,000 produced by the poisonous mushroom Amanita phalloides, causes lysis of various mammalian cell types. Lysis is thought to be initiated by the formation of ion permeable membrane channels. We therefore studied the interaction of phallolysin with solvent-free planar lipid bilayers. In the presence of low phallolysin concentrations (10-100 nM) single channel current fluctuations were observed. Unit channel conductances are 44 pS in 500 mM NaCl and 77 pS in 1 M NaCl. Although the channel does not significantly discriminate between alkali cations, its permeability to Cl- is lower (PK+/PCl- = 4/1). Gating kinetics display a pronounced bursting behavior and a dependence on membrane voltage, cis side pH-value, and on membrane lipid composition. An equivalence relation between membrane voltage and proton concentration was found, i.e. a pH change of one unit is equivalent to a corresponding voltage change of 130 mV. Dependence on the amount of negatively charged lipids is explained by changes of the actual pH due to surface charge effects.

Amanitins↗
PubMed1985

Toxicokinetics of labeled amatoxins in the dog.

Radioactivities were measured in serum, urine, and bile of dogs at different times after intravenous injection of 14C-methyl-gamma-amanitin (14C-A) and 3H-O-methyl-dehydroxymethyl-alpha-amanitin (3H-A). For either substance, the relation between the specific plasma activity C and the time t could be best described with the function C = C1 X e- lambda 1 X t + C2 X e- lambda 2 X t. Therefore the linear open two-compartment system was selected as an adequate toxicokinetic model. Most important, the distribution volumes (in the steady state) were in the range of the extracellular space, and the total body clearances were in the range of the dog creatinine clearance. In accordance with former findings for 3H-A, 14C-A was not bound to plasma proteins. More than 80% of 14C-A was eliminated in the urine; less than 10% was found in the bile. From these data, two suggestions may be derived for the therapy of Amanita intoxication in man. First, detection in the urine of amatoxins 2 or 3 days after mushroom ingestion points to an ongoing amatoxin absorption or reabsorption from the intestine, and should lead to therapy with adsorbents and, in the absence of diarrhea, with laxatives. Second, hemoperfusion will remove significant amounts of amatoxins during the time of ongoing absorption or reabsorption and a few hours thereafter.

Amanitins↗
PubMed1984

The molecular mechanism of interaction of Et3Pb+ with tubulin.

Triethyllead ion (Et3Pb+) was found to interact with 2 out of 18 thiol groups present in tubulin dimers. Specificity of the interaction was shown by the high affinity of Et3Pb+ to tubulin, by the fact that the 16 residual thiol groups in tubulin remained unaffected, and by the observation that other proteins with exposed thiol groups, e.g., actin, did not react with Et3Pb+. After complexation of the two thiol groups, tubulin in vitro had lost its capability for microtubule assembly. Likewise, polymerized tubulin disassembled on addition of the lead compound.

Actins↗
PubMed1984

Nucleotide in monomeric actin regulates the reactivity of the thiol groups.

A new thiol reagent, 2,4-dinitrophenyl glutathionyl disulfide, allowed the characterization of four thiol groups in monomeric actin by stoichiometric reaction. The number of thiol groups exposed to the reagent was found to depend on the nucleotide bound. In the absence of ATP, G-actin exposed four thiol groups ( G4s ). On the addition of ATP (1 equiv), three of them were shielded. The resulting actin with one thiol group exposed ( G1s ) is the form of monomeric actin normally produced by depolymerization of F-actin in buffers containing ATP. G1s is stable over hours, while G4s , i.e., monomeric actin in ATP-free solution, is not. This must be concluded from the fact that the shielding effect of thiol groups induced by addition of ATP was lost within ca. 30 min probably due to denaturation of G4s to G4s *. Therefore, denaturation of monomeric actin must be understood in terms of loss of thiol shielding, rather than by oxidation of the thiol groups. Addition of equimolar amounts of Ca2+ significantly retarded the denaturation process. ADP (50 equiv) shielded only ca. two of the four thiol groups but, similar to ATP, protected actin from denaturation. Three ATP analogues (10 equiv) were tested but had no shielding effect. In the presence of these analogues actin ( G4s ) rapidly denatured (to G4s *) as in the absence of added nucleotides. It was shown that the thiol-shielding activity and the protective capacity of a nucleotide are interrelated with its binding capability to monomeric actin. G1s was found to be polymerizable as was G approximately 2s on the addition of ATP. No polymerization could be detected for G4s or G4s *.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗
PubMed1984

Affinity isolation of RNA polymerase II on amanitin-Sepharose.

We report here the first case of an affinity isolation of eukaryotic RNA polymerase II. The procedure employs an affinity matrix composed of alpha-amanitin coupled to Sepharose 4B via a ten atom spacer. RNA polymerase II from either calf thymus or wheat germ binds to the amanitin-Sepharose, as indicated by subsequent elution with sodium dodecylsulfate-containing buffer and analysis by polyacrylamide gel electrophoresis. The specificity of binding is demonstrated by the fact that when the enzyme is preincubated with 1 microgram/ml of free alpha-amanitin, subsequent binding to the amanitin-Sepharose is abolished. Elution methods that should permit the recovery of active enzyme from the column are discussed.

Adsorption↗
PubMed1984

Phalloidin depletes the mitochondrial Ca2+ compartment of hepatocytes.

In 3 h of incubation, primary cultures of rat hepatocytes attach to the substratum and exchange about 2.2 nmol 45Ca2+ per mg protein. In the presence of 1 microM phalloidin, the exchanged amount of 45Ca2+ was found to be decreased by about 30%. Using the uncoupling agent FCCP and the ionophore A23187 for further characterisation we determined that the 45Ca2+ deficit caused by phalloidin occurs in the FCCP-sensitive compartment, i.e., the mitochondria.

Animals↗
PubMed1984

High cytotoxicity and membrane permeability of Et3Pb+ in mammalian and plant cells.

Cells of mammalian origin as well as those of higher plants appear to be very sensitive to triethyllead ion (Et3Pb+). Neuroblastoma cells kept in the presence of 1 microM Et3Pb+ lost their viability within 6 h. Growth of suspension culture cells of soybean (G. max(L.)Merr.) was inhibited by 1 microM Et3Pb+, and finally the cells died. Morphologically, Et3Pb+ caused the complete breakdown of microtubular structures in neuroblastoma cells; thus microtubules appeared to be the main target for the toxin. While in a previous study the effect of Et3Pb+ on microtubules has been well documented at concentrations of 50-200 microM 1, the present study demonstrates that the formation of microtubules from pig brain tubulin is disturbed at concentrations of Et3Pb+ as low as 0.5 to 1 microM. We conclude from these data that Et3Pb+ freely permeates the plasma membranes of mammalian as well as plant cells.

Animals↗
PubMed1983

Physical properties and function of phallolysin.

Phallolysin, a mixture of two to three cytolytic proteins (all of Mr 34 000), has been isolated from Amanita phalloides mushrooms and purified to homogeneity (specific activity 24 000 hemolytic units/mg of protein). After separation by isoelectric focusing, the amino acid composition of two of these proteins has been determined. They are rich in water-soluble amino acids and contain one tryptophan residue each, but no cysteine or methionine. Mr was determined to be 34 000 in the native form as well as under denaturing conditions, indicating that the native proteins exist as monomers. Many of the physical properties of phallolysin are strikingly similar to those of staphylococcal alpha-toxin, e.g., molecular weight, existence of multiple forms, pI values, amino acid composition, and thermolability (60 degrees C). Pure phallolysin allowed us to prepare a radioactively labeled toxin. Labeling was achieved by reaction with formaldehyde, followed by reduction with sodium [3H]borohydride. With the labeled toxin (specific activity 7-14 Ci/mmol, ca. 60% biological activity), we investigated its binding to human A2 erythrocytes. We determined the number of receptors on these cells (2 X 10(4) per cell) as well as their affinity to the toxin (KD = 4 X 10(-9) M). In studies on the mechanism of cytolytic activity, we were able to distinguish at least three sequential events: binding of the toxin to human erythrocytes, K+ release, and membrane rupture (hemoglobulin release). These steps could be characterized by different kinetics as well as by different temperature dependencies. Again, the kinetic data for phallolysin are very closely related to those obtained for staphylococcal alpha-toxin.(ABSTRACT TRUNCATED AT 250 WORDS)

Amanita↗