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E Habermann

Publications and source records attributed to E Habermann.

At least 55 records · Page 3Linked to original sources

Inhibition by tetanus and botulinum A toxin of the release of [3H]noradrenaline and [3H]GABA from rat brain homogenate.

Rat brain homogenate was preloaded with [3H]noradrenaline or [3H]GABA and stimulated with high K+. Tetanus toxin and botulinum A neurotoxin partially prevent the evoked [3H]noradrenaline release in the same range of toxin concentrations starting below 10(-10) M. In contrast, release of gamma-amino butyric acid (GABA) is much more sensitive to tetanus than to botulinum A toxin.

Animals↗

Tetanus toxin: biochemical and pharmacological comparison between its protoxin and some isotoxins obtained by limited proteolysis.

Single-chain tetanus toxin (toxin S) was prepared from short-term cultures by lysis under protection with protease inhibitors, precipitation with 40% ammonium sulfate, gel filtration, and chromatography on DEAE ion exchanger. Its limited proteolysis by trypsin, post-arginine cleaving enzyme from mouse submaxillary gland and clostripain led to bichainal derivatives (BT, BA, BCl) consisting of a heavy chain and a larger version of the light chain. The latter was then converted by trypsin into a small version which comigrated with the light chain of bichainal extracellular toxin (BE). The light chain produced by chymotrypsin (BC) and elastase (BE1) was of intermediate size. The nick region serves as substrate for all esteroproteases investigated and comprises between one and two kDa. Limited proteolysis increased the hydrophilicity (BT greater than BE greater than S) in hydrophobic interaction HPLC, and anionic behaviour (BC greater than BE greater than BT greater than S) in DEAE ion exchanger HPLC. The bichainal toxins assessed (BC, BE or BT) were about two times more toxic than toxin S (LD50, mouse s.c. 2 ng/kg vs. 4 ng/kg). They were five to twelve times more potent than toxin S in three in vitro assays measuring the prevention of neurotransmitter release, i.e. on the phrenic nerve-hemidiaphragm preparation of the mouse (acetylcholine, with toxin BE and BT), on primary brain cell cultures from the mouse ([3H]noradrenaline, with toxin BE and BT), and on brain homogenate from rats ([3H]noradrenaline, with toxin BA, BC, BE and BT). Thus single-chain toxin is a less potent precursor of, or protoxin for, various bichainal isotoxins.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Tetanus toxin and botulinum A and C neurotoxins inhibit noradrenaline release from cultured mouse brain.

Primary nerve cell cultures from the brainstem of embryonic mice take up [3H]noradrenaline. Release can be evoked by high K+ or sea anemone toxin II and depends on Ca2+. The cultures allow neurochemical studies on the long-term actions of clostridial neurotoxins. Tetanus and botulinum A and C neurotoxins partially inhibit the absolute and fractional release evoked by high K+, as well as the fractional basal release. The detection limit for the toxins is below 5 pM. Total radioactivity is higher in the poisoned cultures, although the initial velocity of uptake is not measurably influenced by tetanus or botulinum A toxin. Pretreatment with neuraminidase prevents the effects of botulinum A toxin and diminishes those of botulinum C and tetanus toxins. Within 6 days, the cultures partially recover from tetanus toxin poisoning. Antitoxin prevents the actions of the toxin, but only slightly promotes recovery. The data indicate close pharmacological analogies between the clostridial neurotoxins.

Animals↗

Two-year follow-up of the PCA noncemented total hip replacement.

The early results with the PCA total hip replacement have been most gratifying, especially the absence of complications related to the acetabular component. The radiographic evaluation was done critically, and the finding of no progressive acetabular radiolucencies was unexpected. Longer-term evaluation of these interfaces is necessary, but the short-term results have been encouraging. No components have migrated despite the absence of adjunct fixation mechanisms such as screws and flanges. The pressfit achieved with the roughened hemispherical surface has been adequate, and the fixation with the two outrigger pegs appears to have been sufficiently stable to preserve the prosthetic stability and has resulted in successful anchorage of all the components. The results with femoral components are obviously related to technique. In a few early cases when undersized prostheses were used, loosening occurred, and four of these components advanced to detectable loosening. One of these components was revised since this analysis. Attention to detail with maximal filling of the proximal femur apparently led to improved results, with successful anchorage in all subsequent implantations. The application of the dimensional analysis before surgery may indicate those cases in which a tight fitting metaphysis cannot be achieved. In elderly patients who have osteoporotic bone, a substantial mismatch exists between the size of the metaphysis and the diaphysis, and it may be advisable to continue with cement in these cases. By applying the dimensional analysis to preoperative templating, the surgeon may be sufficiently informed to know that a tight fit can be achieved at surgery. No catastrophic failures have occurred. If loosening does occur because of undersizing of the prosthesis, the process appears to be gradual and, although associated with pain, does not result in sudden failure. Despite the prosthesis not being anchored by cement or collar, no sudden subsidence of the components has resulted. Patients' clinical performance has been somewhat slower when compared to cemented series. Performance seems to accelerate once weight bearing occurs, however, and after 2 years no difference exists between this series and a corresponding cemented series. Noncemented total hip arthroplasty appears to offer as good if not better results than cemented total hip arthroplasty, if performed correctly and in the appropriate patient. Successful outcome depends greatly on technique, but when technically adequate implantation has been performed, the results have been gratifying.(ABSTRACT TRUNCATED AT 400 WORDS)

Activities of Daily Living↗

Involvement of (Na+ + K+)-ATPase in binding and actions of palytoxin on human erythrocytes.

Palytoxin (about 1 pM) increases the permeability of human erythrocytes. We now report its radiolabeling with 125I, followed by affinity purification on porcine kidney membranes. The resulting ligand binds fast and reversibly to intact erythrocytes. The Kd from velocity and equilibrium measurements is 2 X 10(-11) M, and the number of binding sites about 200 per cell. Binding is promoted by divalent cations (Ca2+ greater than Sr2+ greater than Ba2+) and by borate. It is inhibited by K+ (IC50 2 mM), ouabain (IC50 3 X 10(-9) M) and ouabagenin (IC50 6 X 10(-6) M). Conversely, [3H]ouabain is displaced by the substances and concentrations mentioned, and also by palytoxin (Ki 3 X 10(-11) M). Dog erythrocytes, which are known to possess a very low (Na+ + K+)-ATPase activity, are resistant to and lack specific binding sites for palytoxin. Binding of 125I-palytoxin, like that of [3H]ouabain, depends on the state of (Na+ + K+)-ATPase. ATP depletion decreases binding of both ligands to erythrocytes. Binding of 125I-palytoxin and [3H]ouabain to red cell stroma is partially restored by ATP. In contrast to [3H]ouabain, binding of 125I-palytoxin to red cell stroma is not promoted by Mg2+ and Pi. The data show that (a) all known promoters and inhibitors of palytoxin action on human red cells do so by enhancing or decreasing its binding, (b) (Na+ + K+)-ATPase serves as a receptor for palytoxin, and (c) the antagonism by ouabain is competitive at the receptor level. They support our previous hypothesis that palytoxin increases human erythrocyte permeability by formation of pores through (Na+ + K+)-ATPase or its close vicinity.

Acrylamides↗

Affinity of heavy metal ions to intracellular Ca2+-binding proteins.

Parvalbumin, troponin C and vitamin D dependent Ca2+-binding proteins (CaBP type I and II) share the property of calmodulin to interact with some heavy metal ions. In flow dialysis and in spot tests the affinities of Cd2+ and Pb2+ to these proteins were comparable to those of Ca2+. The relative affinities were for calmodulin: Pb2+ greater than Ca2+ greater than Cd2+, for troponin C: Ca2+ greater than Cd2+ greater than Pb2+, for CaBP I: Ca2+ approximately Pb2+, for CaBP II; Ca2+ greater than Pb2+ greater than Cd2+, and for parvalbumin: Cd2+ approximately Ca2+ greater than Pb2+. Upon gel filtration of the supernatant of a pig mucosal homogenate, binding for both Pb2+ and Ca2+ appeared in the MW range of 10,000, together with CaBP II. We conclude that the investigated proteins bind heavy metal ions, in particular Pb2+ and Cd2+, similar to calmodulin. Their role in transport, storage and toxicity remains to be assessed.

Animals↗

Tetanus toxin: primary structure, expression in E. coli, and homology with botulinum toxins.

A pool of synthetic oligonucleotides was used to identify the gene encoding tetanus toxin on a 75-kbp plasmid from a toxigenic non-sporulating strain of Clostridium tetani. The nucleotide sequence contained a single open reading frame coding for 1315 amino acids corresponding to a polypeptide with a mol. wt of 150,700. In the mature toxin molecule, proline (2) and serine (458) formed the N termini of the 52,288 mol. wt light chain and the 98,300 mol. wt heavy chain, respectively. Cysteine (467) was involved in the disulfide linkage between the two subchains. The amino acid sequences of the tetanus toxin revealed striking homologies with the partial amino acid sequences of botulinum toxins A, B, and E, indicating that the neurotoxins from C. tetani and C. botulinum are derived from a common ancestral gene. Overlapping peptides together covering the entire tetanus toxin molecule were synthesized in Escherichia coli and identified by monoclonal antibodies. The promoter of the toxin gene was localized in a region extending 322 bp upstream from the ATG codon and was shown to be functional in E. coli.

Amino Acid Sequence↗

Depolarization increases inositolphosphate production in a particulate preparation from rat brain.

We have studied the accumulation of inositol phosphates (InsP) due to depolarization. A particulate preparation of rat brain was introduced to rule out transmitter activated mechanisms and to allow free access for drugs of high molecular weights. Potassium depolarization doubled InsP within a few minutes. InsP accumulation depended on time and K+ concentration, and was affected neither by tetrodotoxin nor by atropine. Radioactive metabolites co-eluted with inositol mono-phosphate and inositol bis-phosphate, whereas only minor amounts appeared with inositol tris-phosphate. The content in phosphatidylinositols was decreased. No evidence was found for the involvement of a neurotransmitter. Sea anemone toxin II (around 1 mumol/l), which keeps the Na+-channels open, promoted the InsP accumulation in an atropine-resistant manner. Tetrodotoxin prevented it when given before, and inhibited it when given after initiation by sea anemone toxin II. Moreover the K+ channel blockers 4-aminopyridine, dendrotoxin and tetraethylammonium all caused InsP accumulation. Palytoxin was by far the most potent promoter of InsP accumulation with a detection limit below 10 pmol/l, and displayed a unique bell-shaped concentration-effect correlation. Ouabain (3 mumol/l and above) also elicited the InsP accumulation. The response to carbachol was not only inhibited completely by atropine, but also partially (more than 50%) by tetrodotoxin, which indicates the involvement of voltage-dependent sodium channels in the receptor-triggered InsP accumulation. Thus independent of the causative agent, depolarization promotes an InsP accumulation. We conclude that degradation of phosphatidylinositols is mediated not only by receptor occupation but also by a positive shift in membrane voltage.

Acrylamides↗

Quantitative comparison between tetanus toxin, some fragments and toxoid for binding and axonal transport in the rat.

The fragments BIIb and C of tetanus toxin, which contain its binding domain, were quantitatively compared with native toxin and a toxoid with respect to axonal transport from the gastrocnemius muscle to the spinal cord in rats. Against 125I-toxin, the dose-ascent curve of labelled toxoid was shifted by a factor of 3-5 to higher concentrations, whereas the ascent of the labelled binding fragments was at least 50-100 times less. The binding fragments also differed from tetanus toxin by their very low affinity to rat brain membranes buffered to pH 7.5 in saline, but were equivalent with the toxin in buffer of low molarity and low pH. We conclude that additional parts of the toxin molecule have to complement the binding domain for expression of the full binding and transport characteristics of the toxin.

Animals↗

Interaction between tetanus toxin and rabbit kidney: a comparison with rat brain preparations.

125I-Tetanus toxin is bound by basolateral membranes from rabbit kidneys. Fixation is specific, as it is minimally inhibited by the nonbinding (fragment B) moiety of tetanus toxin, whereas the binding moiety (fragment C) is equivalent to the native toxin in inhibiting fixation. Competition is also pronounced with mildly toxoided toxin. Association and dissociation of 125I-toxin are delayed in kidney when compared to brain membranes. The binding sites in kidney membranes are partially sensitive to neuraminidase and resist heating to 56 degrees C, in contrast to those in brain membranes which are very sensitive to both treatments. The binding sites of the two preparations can be discriminated further by variation of the ionic environment. Sodium dodecyl sulfate-disc gel electrophoresis followed by transfer to nitrocellulose, and TLC with consecutive overlay indicate that tetanus toxin exclusively binds to long-chain gangliosides from rat brain. Binding sites in kidney membranes from rabbits and rats can be made visible by the overlay technique. They are apparently heterogeneous and more hydrophobic. We conclude that rabbit kidney contains binding sites for tetanus toxin which resemble gangliosides but differ from the major gangliosides in brain both chemically and with respect to their interaction with tetanus toxin.

Animals↗

The interaction of aluminum and other metal ions with calcium-calmodulin-dependent phosphodiesterase.

In the search for the mode of action of aluminum ions in dialysis encephalopathy, their interaction with calmodulin has been assessed, and compared with those of Ca2+, Pb2+, Mn2+, Hg2+ and Cd2+. The basal and calmodulin-dependent activity of phosphodiesterase was measured in the presence of the ions, and their binding to calmodulin was assessed by competition with 45Ca2+ in flow dialysis. Al3+, Mn2+, Hg2+, and Cd2+ cannot be regarded as exclusive calmodulin antagonists or agonists, but rather interact with the phosphodiesterase itself. Pb2+ however, mimicks Ca2+ in every system tested, and is active in the same concentration range as Ca2+. Our results indicate that the assumed role of Al3+ ions in dialysis encephalopathy or other neurological disturbances is not linked with calmodulin.

Aluminum↗

Electrophysiological and neurobiochemical evidence for the blockade of a potassium channel by dendrotoxin.

The effects of dendrotoxin (DTX), a toxic peptide from Dendroaspis angusticeps venom, were studied electrophysiologically on peripheral frog nerve fibres, and biochemically on large synaptosomes from rat brain. On nerve fibres, DTX reduced the amplitude and prolonged the duration of the action potential; even at 0.1 nmol/l DTX produced significant effects. Maximum block of potassium currents occurred at about 30 nmol/l. Turning on of the remaining current was slowed. Reversibility was incomplete. The reduction of potassium currents was between 31% and 85% at 85 nmol/l DTX (n = 8). The remainder appeared to be resistant to DTX. Sodium channels were not affected. On large synaptosomes DTX (above 1 nmol/l) produced a slight depolarization, indicated by an outward shift of the lipophilic cation tetraphenylphosphonium, and promoted the release of radioactivity after preloading with [3H] GABA. DTX had similar potency but lower efficacy in this respect than sea anemone toxin II (ATX II). In contrast to the effects of ATX II, those due to DTX were only partially inhibited by tetrodotoxin. The actions of 4-aminopyridine resembled those of DTX, but the latter was about 500 times more potent. The electrophysiological data provide direct evidence for blockade of a potassium channel by DTX. This action is sufficient to explain the biochemical observations, although additional effects on synaptosomes cannot be excluded.

Action Potentials↗

Enzymatic hydrolysis of tetanus toxin by intrinsic and extrinsic proteases. Characterization of the fragments by monoclonal antibodies.

Enzymatic fragments of tetanus toxin were characterized by immunoblotting using a set of previously characterized antibodies (Ahnert-Hilger et al. (1983) and a set of novel antibodies. The selected antibodies recognized the light chain, fragment C (beta 1), and the complementary piece (beta 2) of the heavy chain when blotted on nitrocellulose. All toxin preparations contained intrinsic esteroprotease activity which became manifest in the presence of urea. The main split product was a fragment (MW 100 000) reacting with anti-fragment C and anti-beta 2 antibodies. Toxicity does not depend on this protease activity. Some crude preparations of tetanus toxin contain another split product (MW 47 000) which resembles fragment C. The main product of papain hydrolysis is fragment C, which appears as a double band under nonreducing conditions but is homogeneous when reduced. Chymotryptic digestion hydrolyses the heavy chain well but leaves the light chain largely intact. Tetanus toxin is very resistant against trypsin as compared with other proteases, although this enzyme splits numerous different links. Our data show the usefulness of immunoblotting with monoclonal antibodies in analytical work with tetanus toxin, and the relevance of intrinsic proteases.

Antibodies, Monoclonal↗

Interaction of solid-phase gangliosides with tetanus toxin and toxoid.

Tetanus toxin has been fractionated on a column of silica beads coated with a ganglioside mixture. Toxicity and affinity of the fractions for brain membranes increased with their affinity for the column. The toxicity recovered with the nonabsorbed fraction was less than 5% with respect to the unfractionated toxin or its firmly bound moiety. Tetanus toxoid yielded less toxic or nontoxic fractions with different affinities for the column. Accordingly, binding of tetanus toxoid to brain membranes was partially preserved under mild toxoiding with formaldehyde. It is concluded that binding to gangliosides is a necessary, but not a sufficient, precondition for the pharmacological activity of tetanus toxin and its derivatives.

Animals↗

The effects of apamin in rats with pretrigeminal or high spinal transsection of the central nervous system.

Rats were injected in one lateral cerebral ventricle (i.c.v.) with apamin (100 ng per animal). The resulting desynchronisation pattern in the electrocorticogram (ECoG) and the symptoms of poisoning were monitored before and after transsection at different levels, and following morphine. Apamin acts primarily on the brain stem and spinal cord, i.e. structures possessing a sensory input, and then indirectly on the higher integrating systems. There is no general parallelism between receptor density and locus of action.

Animals↗

Palytoxin binds to and inhibits kidney and erythrocyte Na+, K+-ATPase.

Hog kidney Na+, K+-ATPase, purified to the microsomal stage and activated with detergent, binds palytoxin, as shown by the nearly complete competition of the toxin with 3H-ouabain. The Ki-values of palytoxin, but not of ouabain, depend on the protein concentration; this indicates additional binding sites for the toxin on kidney membranes. - Palytoxin inhibits the enzymatic activity of the detergent-activated preparation nearly completely (IC50 8 X 10(-7) mol/l). Inhibition of ATPase activity and of ouabain binding are promoted by borate, a known activator of palytoxin. - Palytoxin also inhibits the Na+,K+-ATPase of erythrocyte ghosts in the same dose range. The data are discussed in context with the hypothesis (Chhatwal et al. 1983) that palytoxin raises the cellular permeability by altering the state of Na+,K+-ATPase or its environment.

Acrylamides↗

Apamin.

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Amino Acid Sequence↗