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E Habermann

Publications and source records attributed to E Habermann.

At least 37 records · Page 2Linked to original sources
PubMed1991

Limited proteolysis of single-chain tetanus toxin by tissue enzymes, in cultured brain tissue and during retrograde axonal to the spinal cord.

Single-chain toxin was investigated in vitro and in vivo for limited proteolysis into the fully active two-chain toxin. Plasmin from serum, elastase and gelatinase from leucocytes, as well as clostripain from C. histolyticum cleaved single-chain toxin and increased by that way its ability to inhibit [3H]noradrenaline release in vitro. Cultured mouse brain generated fragments from 125I-single-chain toxin which were cell-associated. Some of them comigrated in electrophoresis with light and heavy chain after mercaptolysis. When injected i.v. into rats, 125I-single-chain-toxin disappeared from the blood with a half-life of about 11 h without signs of nicking. However, after its injection into the triceps surae muscle both single- and two-chain toxin were found in the ipsilateral ventral horn of the spinal cord. Thus single-chain toxin is subjected to limited proteolysis by enzymes involved in tissue damage, by cultured brain tissue, and during or after its retrograde axonal transport to the spinal cord. Limited proteolysis is necessary for the release of the light chain known to mediate the action of toxin on several systems.

Animals↗
PubMed1991

A comparative biochemical, pharmacological and immunological study of Clostridium novyi alpha-toxin, C. difficile toxin B and C. sordellii lethal toxin.

The three clostridial cytotoxins, i.e. alpha-toxin of C. novyi (Tox alpha-nov), toxin B of C. difficile (ToxB-dif) and lethal toxin of C. sordellii (LT-sor) consist of single peptide chains of about 200,000 (Tox alpha-nov), 250,000 (LT-sor) and 275,000 (ToxB-dif) mol. wts. ToxB-dif and LT-sor but not Tox alpha-nov cross-reacted with rabbit polyclonal antibodies. Toxicity upon i.v. injection in mice was similar (LD50, 100 hr, 50-200 ng/kg) and was characterized by a slowly developing fluid loss into the interstitial space. When injected into the rat paw the toxins caused a delayed local edema lasting for days. In vitro the three toxins provoked a persistent retraction of endothelial cells cultured from pig pulmonary artery. ToxB-dif and Tox alpha-nov triggered the accumulation of F-actin in the perinuclear region at the expense of the tight peripheral bands whereas LT-sor led to a random loss of microfilament structure. The toxins inhibited uridine incorporation into endothelial or chicken embryonic cells whereas T 84 cells responded by an about 10-fold increase of uridine incorporation. Neither toxin ADP-ribosylated actin. The similarities between the three cytotoxins warrant their arrangement into a common group which perturbs the microfilament system.

Adenosine Diphosphate Ribose↗
PubMed1990

Arrangement of disulfide bridges and positions of sulfhydryl groups in tetanus toxin.

Tetanus toxin is a 151-kDa protein. The complete amino acid sequence is known. The mature toxin is made up of two peptide chains and contains 10 half-cystine residues. Treatment with 4-vinylpyridine in the presence of 6 M guanidine converted six of them into S-pyridylethyl cysteine residues as determined by amino acid analysis. When alkylation was preceded by mercaptolysis, all 10 half-cystine residues were recovered in the S-pyridylethylated form. It was therefore concluded that the toxin contains six sulfhydryl groups and two disulfide bonds. The positions of the residues carrying sulfhydryl groups and of those involved in disulfide bridges were determined by labelling of the toxin alternatively with 4-vinylpyridine or with 4-dimethylaminoazobenzene-4'-iodoacetamide (DABIA), directly or after mercaptolysis. The toxin derivatives were cleaved with cyanogen bromide and the elution patterns in reversed-phase HPLC compared. The chromatography components were identified by N-terminal amino acid sequence and amino acid composition. In the chromatography of the non-mercaptolysed, DABIA-treated sample four chromophore-carrying components were detected which could be demonstrated by N-terminal sequence analysis to correspond to six half-cystine-containing cyanogen bromide fragments. In the mercaptolysed, DABIA-treated sample three additional chromophore-carrying components were present, corresponding to two previously disulfide-linked cyanogen bromide fragments and one fragment which had contained an internal disulfide bridge. The HPLC patterns showed characteristic differences as the DABIA-labelled fragments were considerably more hydrophobic than the corresponding vinylpyridine-labelled fragments. It was established that the half-cystine residues in positions 26, 185, 198, 311, 868, and 1300 are present in the sulfhydryl form, that those in positions 438 and 466 are disulfide-bridged, thereby connecting the light and heavy chains of the toxin, and that those in positions 1076 and 1092 are disulfide-bridged, thereby giving rise to a loop in the heavy chain. During the progress of the investigations about 20% of the amino acid sequence previously predicted from DNA analysis was confirmed by protein-chemical methods.

Amino Acid Sequence↗
PubMed1990

Role of omega-conotoxin-sensitive calcium channels in inositolphosphate production and noradrenaline release due to potassium depolarization or stimulation with carbachol.

omega-Conotoxin GVIA (CTX) has been used to assess the role of voltage-sensitive Ca2+ channels involved in inositolphosphate (InsP) production and in noradrenaline (NA) release from washed brain homogenates. Stimulation was performed by depolarization with high [K+] and by the M-cholinergic agonist carbachol. In chicken brain CTX (1 mumol/l and less) nearly completely inhibited both InsP production and NA release due to high [K+]. The peptide depressed InsP production moderately but NA release largely when evoked with carbachol. In rat brain inhibition by CTX of InsP production or NA release was weak or even absent independent of the mode of stimulation. Independent of species or test system, the dihydropyridine derivative nitrendipine (1 mumol/l and above) was inactive. Because of its preferential CTX sensitivity chicken brain is particularly suitable to study the role of voltage-sensitive Ca2+ channels in presynaptic events. Here InsP production, like NA release, depends on entry of extracellular Ca2+ nearly completely when evoked by depolarization. When evoked by way of cholinergic M-receptor stimulation, InsP production is triggered by a second pathway in addition to the CTX-sensitive Ca2+ entry.

Animals↗
PubMed1990

Microinjection of alpha-toxin from Clostridium novyi type A promotes meiotic maturation in Xenopus laevis oocytes.

Microinjection of purified alpha-toxin into Xenopus laevis oocytes induced meiotic maturation provided insulin was present in the medium. Induction of maturation as indicated by breakdown of the germinal vesicle depended on the amount of intracellular alpha-toxin (with a detection limit of 2 ng/oocyte) and on the concentration of insulin. The hormone concentrations used were inactive when given alone and so was alpha-toxin (1 micrograms/ml) applied from the outside. The results demonstrate an intracellular target for alpha-toxin whereas an extracellular target is apparently lacking in oocytes.

Animals↗
PubMed1990

Inhibition of neurotransmitter release by botulinum neurotoxins and tetanus toxin at Aplysia synapses: role of the constituent chains.

1. The effects on the release of transmitter by botulinum neurotoxins (BoNT; types A, B, E), tetanus toxin (TeTx), constituent chains or fragments were studied on identified cholinergic and non-cholinergic synapses in Aplysia. 2. Cholinergic synapses in the buccal ganglion were found to be greater than 100 fold more sensitive to extracellular application of BoNT than to TeTx whereas in non-cholinergic synapses of the cerebral ganglion the potencies of the toxins were reversed. When intracellularly applied TeTx and BoNT were found nearly equipotent. This disparity in the susceptibilities of BoNT and TeTx to inhibit transmission was attributed to differences in the toxin's acceptors or uptake systems in the two neurone types. 3. Micro-injection into cholinergic neurones of the isolated renatured toxins' chains showed that both light and heavy chains of BoNT are intracellularly required whereas the light chain of TeTx alone is sufficient. 4. The heavy chain of BoNT as well as that of TeTx were found to mediate internalization of active moieties via its amino-terminal half. Furthermore the heavy chain of one toxin could internalize the light chain of the other.

Animals↗
PubMed1990

Chains and fragments of tetanus toxin, and their contribution to toxicity.

1. Single-chain toxin is enzymatically converted into two-chain isotoxins which differ from the precursor by their higher pharmacological activity, acidity and hydrophilicity. The interchain disulfide bridge and the disulfide loop within fragment C have been located at the amino acid level. 2. Independent of the enzymes used, the nicking sites are positioned within a region spanning no more than 17 amino acids. The N- and C-termini of the primary gene product are preserved in the two-chain toxin. The chains have been separated by isoelectric focussing and can be reconstituted to functionally intact toxin. 3. Light chain inhibits neurotransmitter release on different systems. First, permeabilized bovine adrenal chromaffin cells and rat pheochromocytoma (PC 12) cells release catecholamines when exposed to micromolar [Ca2+]. Inhibition is achieved with light chain or reduced two-chain toxin, but not with single-chain toxin or heavy chain. Washing away the light chain does not restitute the Ca2(+)-evoked release. The light chains of tetanus and botulinum A toxin act in a apparently similar, however not identical manner. Second, light but not heavy chain inhibits the release of acetylcholine when injected into Aplysia neurones. 4. The pharmacology of heavy chain is quite different. Ganglioside binding is mediated by its fragment C moiety, and modulated by the adjoining beta 2 piece and by light chain. Heavy chain and to a lesser degree its N-terminal beta 2-fragment promote the loss of calcein from liposomes indicating pore formation. Its C-terminal fragment C is inactive in this respect.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein Processing, Post-Translational↗
PubMed1989

The light chain but not the heavy chain of botulinum A toxin inhibits exocytosis from permeabilized adrenal chromaffin cells.

The heavy and light chains of botulinum A toxin were separated by anion exchange chromatography. Their intracellular actions were studied using bovine adrenal chromaffin cells permeabilized with streptolysin O. Purified light chain inhibited the Ca2+-stimulated [3H]noradrenaline release with a half-maximal effect at about 1.8 nM. The inhibition was incomplete. Heavy chain up to 28 nM was neither effective by itself nor did it enhance the inhibitory effect of light chain. It is concluded that the light chain of botulinum A toxin contains the functional domain responsible for the inhibition of exocytosis.

Adrenal Medulla↗
PubMed1989

Light chain of tetanus toxin intracellularly inhibits acetylcholine release at neuro-neuronal synapses, and its internalization is mediated by heavy chain.

The ability of the two-chain form of tetanus toxin (TeTx), its constituent light (LC) or heavy (HC) chains, and papain fragment to block evoked acetylcholine (ACh) release in the buccal ganglia of Aplysia californica was studied electrophysiologically. Extracellularly applied, TeTx or its B fragment (consisting of LC and beta 2, the amino-terminal portion of HC) blocked ACh release, whereas LC, HC, or the beta 2 fragment did not affect it. Toxicity was restored when LC was bath applied together with HC or the beta 2 fragment. When injected into the presynaptic neuron, TeTx, the B fragment or LC, but not HC, induced inhibition of ACh release. These results indicate that the blockade of ACh release by TeTx is mimicked by intracellular action of LC, the internalization of which is mediated by the HC via its amino-terminal moiety.

Acetylcholine↗
PubMed1989

Chains and fragments of tetanus toxin. Separation, reassociation and pharmacological properties.

Tetanus toxin, as obtained from bacterial culture filtrates, consists of two chains. Since their roles in poisoning are unknown, we have made a detailed study of their preparation, reassociation and pharmacological activity. 1. Two-chain tetanus toxin (pI 6.0) was subjected to isoelectric focussing under reducing conditions in 2M urea. Both light (pI 4.8) and heavy (pI 7.2) chains separated as nearly homogeneous proteins of low toxicities. Upon removal of urea and reoxidation, partial homodimerization by formation of disulfide bonds took place in the purified fractions. The toxin was reconstituted nearly quantitatively by covalent heterodimerization of the complementary chains, as shown by SDS/gel electrophoresis, toxicity studies, inhibition of evoked [3H]noradrenaline release and binding to rat brain membranes. 2. Accordingly, fragment B (pI 5.6) resulting from papain hydrolysis, was separated into a light chain and the N-terminal moiety of the heavy chain, called fragment beta 2 (pI 7.1 and 6.8, two maxima). Removal of urea and reoxidation led to reconstitution of fragment B. Covalent linkage did not occur between the two parts of the heavy chain, or between the light chain and the C-terminal part of the heavy chain. 3. The heavy chain alone inhibited K+-evoked [3H]noradrenaline release from a rat brain homogenate. However, the concentration-response ratio was flat and 10-100-fold higher concentrations were required than with native or reconstituted two-chain toxin. The light chain was inactive. Purified heavy chain but not light chain decreased the [3H]noradrenaline content, whereas the two-chain toxin increased it. Binding to rat brain membranes was assessed by competition with 125I-labelled two-chain toxin. In hypotonic buffer, the heavy chain, the papain fragment C and native and reconstituted two-chain toxin had comparable affinities to membranes. In isotonic buffer the heavy chain displayed an about 1000-fold lower affinity than native or reconstituted two-chain toxin. The light chain did not bind to membranes in either test. Our data indicate that (a) the light chain and the N-terminal part of the heavy chain are held together not only by one disulfide bond but also by hydrogen bonds and ionic forces to yield a two-chain toxin or fragment B and (b) both chains contribute to the actions of the toxin in vivo and in vitro, and to its binding.

Animals↗
PubMed1989

Effects of botulinum A toxin on presynaptic modulation of evoked transmitter release.

A possible influence of botulinum A toxin on the modulation of evoked neurotransmitter release was investigated in hippocampus tissue. Rabbit hippocampal slices prelabelled with [3H]noradrenaline ([3H]NA), [3H]5-hydroxytryptamine ([3H]5-HT) or [3H]choline were superfused with physiological medium and were stimulated electrically during superfusion. The evoked release of [3H]NA, [3H]5-HT and [3H]acetylcholine [( 3H]ACh) was inhibited by botulinum A toxin in a concentration- and time-dependent manner. Neither the inhibition of release of [3H]NA and [3H]5-HT by the alpha 2-adrenoceptor agonist clonidine nor facilitation of release in the presence of alpha 2-antagonists were influenced by pretreatment of the tissue with botulinum toxin. The toxin caused no [32P]ADP ribosylation of synaptosomal proteins of hippocampus. The facilitation of the stimulation-induced [3H]NA and [3H]5-HT release by the specific protein kinase C (PKC) activator 4 beta-phorbol-12,13-dibutyrate (PDB) was significantly diminished by botulinum A toxin. These results show that the evoked transmitter release is inhibited by botulinum A toxin by a mechanism which does not involve ADP ribosylation or an interaction with the alpha 2-adrenoceptor mechanism.

Acetylcholine↗
PubMed1989

[Oxoferin and sodium chlorite--a comparison].

Oxoferin, a preparation approved for wound treatment, has been subjected to an in vitro analysis. 1. Oxoferin produces methaemoglobin even if diluted 500 fold, and leads to additional alterations if added in high concentrations to red cells. Sodium chlorite (NaClO2, 15 mM) is equivalent to undiluted Oxoferin. 2. Oxoferin in fiftyfold dilution damages fibroblasts in cell culture slowly and persistently. This dilution is equieffective with a 200 microM solution of sodium chlorite. Oxoferin and sodium chlorite also damage vascular endothelial cells. 3. The oxidation equivalent of Oxyoferin is 12.3 mM sodium chlorite. Our data indicate that Oxoferin may be equated essentially with aqueous sodium chlorite.

Animals↗
PubMed1989

The tetanus toxin light chain inhibits exocytosis.

The intracellular action on exocytosis of various forms of tetanus toxin was studied using adrenal medullary chromaffin cells, the membrane barrier of which has been removed by permeabilization with streptolysin O. Such cells still release catecholamines on stimulation with calcium. The two-chain form of tetanus toxin (67 nmol/l) strongly inhibited exocytosis, but only if dithiothreitol was present as a reducing agent. Purified light chain completely prevented [3H]noradrenaline release with a half-maximal effect at about 5 nmol/l. Heavy chain (up to 11 nmol/l) and unprocessed single-chain toxin (up to 133 nmol/l) were without effect. It is concluded that the original single-chain form of tetanus toxin has to be processed by proteolysis and reduction to yield a light chain which inhibits transmitter release.

Adrenal Medulla↗
PubMed1989

Palytoxin acts through Na+,K+-ATPase.

Palytoxin is the most potent animal toxin, with a unique structure. The author's group has searched for its mode of action with the following results: 1. Palytoxin (1 pM and less) causes a fast K+ outflow from erythrocytes; 2. Extracellular Ca2+ and borate, and intracellular ATP enhance, but ouabain potently inhibits the palytoxin effects; 3. Palytoxin increases the permeability for Na+ and K+ but not for Ca2+; 4. Palytoxin in comparatively high concentrations (100 nM and above) inhibits Na+,K+-ATPase; 5. Palytoxin can be radiolabeled with 125I. Its receptor is very similar to, but not identical to that of ouabain. A reaction scheme has been delineated which allows an explanation to be obtained for all the known actions of palytoxin. It centers on the hypothesis that palytoxin binds to Na+,K+-ATPase and converts the enzyme or its close vicinity into an open channel with the permselectivity measured on erythrocytes. Patch clamp data from myocytes were obtained in other laboratories. They prove the presence of the predicted palytoxin channel.

Acrylamides↗
PubMed1989

Palytoxin promotes potassium outflow from erythrocytes, HeLa and bovine adrenomedullary cells through its interaction with Na+, K+ -ATPase.

Erythrocytes from four mammalian species were compared with regard to K+ loss triggered by palytoxin, to Na+, K+ -ATPase activity, and to ouabain sensitivity of both events. Palytoxin sensitivity (EC50) decreased in the order rat, man (approximately equal to 1 pM) greater than cattle (approximately equal to 500 pM) greater than dog (greater than 10 nM). Na+, K+ -ATPase activity, as measured by Rb uptake, was in the series rat greater than man greater than cattle greater than dog. The glycoside potently inhibited both palytoxin action and ATPase activity in man, cattle and dog erythrocytes, but weakly in those from rats. Ca2+ promoted the palytoxin effects on all erythrocytes. As shown for human erythrocytes, Sr2+ and Ba2+ but not Mg2+ can substitute for Ca2+, and sucrose can substitute for sodium chloride. Human HeLa and bovine adrenomedullary cells also lost their K+ within a few min when exposed to palytoxin (1-10 pM). Ouabain acted as a palytoxin antagonist on both cell types. We conclude that: (a) the ouabain binding site of Na+, K+ -ATPase is part of the palytoxin receptor in every cell type tested, (b) high palytoxin sensitivity is not necessarily accompanied by high ouabain sensitivity, and (c) active ion transport is not a precondition for the action of palytoxin or for its inhibition by ouabain.

Acrylamides↗
PubMed1989

Pharmacological and biochemical studies of cytotoxicity of Clostridium novyi type A alpha-toxin.

The actions of apparently homogeneous alpha-toxin from Clostridium novyi type A were studied in order to develop an in vitro system which closely mimics its in vivo effects and to search for the mode of poisoning. Time to death (by intravenous injection of mice) was inversely related to dose, with a detection limit of about 200 ng/kg of body weight at 100 h. Injections of 2.5 ng or more into the rat paw led to a slowly (maximum after about 30 h) developing, dose-dependent edema which was useful as a quantitative in vivo assay based on volumetry. Vascular leakage was due to gap formation between endothelial cells. Similarly, endothelial cells cultured from pig pulmonary artery lost their "cobblestone" arrangement after a dose-dependent lag period of some hours after poisoning. The morphological changes were accompanied by depression of uptake or incorporation of [3H]uridine. A quantitative in vitro assay was established on the inhibition of [3H]uridine incorporation. As in animals, the action of alpha-toxin started with a few nanograms per milliliter and proceeded slowly for at least 1 day but became resistant to antitoxin within 2 h of exposure. The toxin action is not limited to endothelial cells, since chicken embryonic cells, a mouse fibroblast line (L-929), and a rat phaeochromocytoma line (PC-12) behaved similarly. Alpha-toxin was found to differ from other bacterial toxins investigated whose modes of action are already known.

Adenosine Diphosphate Ribose↗
PubMed1989

Hemophilus parainfluenzae complicating total knee arthroplasty. A case report.

Infections are an unfortunate but well-known complication of prosthetic joint replacements. Prophylactic antibiotics have been prescribed to help combat infections in total joint arthroplasties. A 78-year-old woman with a cemented total knee replacement developed a Hemophilus parainfluenzae infection following routine root canal dental surgery. The infection was successfully treated with arthroscopic irrigation and antibiotic therapy.

Abscess↗