Xeomin: an innovative new botulinum toxin type A.

Botulinum toxin type A (BoNT/A) is a well-established treatment for conditions characterized by muscle and autonomic nerve terminal overactivity, such as cervical dystonia and blepharospasm, and hyperhidrosis, respectively. BoNT/A is not digested in the gastrointestinal tract as it forms a complex with several proteins that protect and stabilize the neurotoxin. However, the pure neurotoxin is solely responsible for the therapeutic effect, and the complexing proteins have been shown to exhibit immunostimulating activity. The complexing proteins are not required for the stabilization of the neurotoxin in a formulation; the complexing proteins immediately dissociate from the neurotoxin at a physiologic pH, so they do not influence the spread of the neurotoxin. Xeomin is the only botulinum toxin that is free from complexing proteins and is stable at room temperature for a period of 4 years. When injected directly into muscles, Xeomin inhibits local neuromuscular cholinergic transmission, causing focal weakness. It binds to motor nerve terminal pre-synaptic receptors, is internalized via receptor-mediated endocytosis and then selectively cleaves a protein called SNAP-25. This is one of several so-called 'SNARE' proteins involved in exocytosis. Cleavage of SNAP-25 inhibits the secretion of acetylcholine causing the paralysis of the muscle. The clinical effects begin 24-72 h after injection, peak at approximately 4-6 weeks and are sustained for several months.

An integrated approach to structural genomics.

Structural genomics aims at determining a set of protein structures that will represent all domain folds present in the biosphere. These structures can be used as the basis for the homology modelling of the majority of all remaining protein domains or, indeed, proteins. Structural genomics therefore promises to provide a comprehensive structural description of the protein universe. To achieve this, a broad scientific effort is required. The Berlin-based "Protein Structure Factory" (PSF) plans to contribute to this effort by setting up a local infrastructure for the low-cost, high-throughput analysis of soluble human proteins. In close collaboration with the German Human Genome Project (DHGP) protein-coding genes will be expressed in Escherichia coli or yeast. Affinity-tagged proteins will be purified semi-automatically for biophysical characterization and structure analysis by X-ray diffraction methods and NMR spectroscopy. In all steps of the structure analysis process, possibilities for automation, parallelization and standardization will be explored. Major new facilities that are created for the PSF include a robotic station for large-scale protein crystallization, an NMR center and an experimental station for protein crystallography at the synchrotron storage ring BESSY II in Berlin.

New trichothecenes isolated from Holarrhena floribunda.

Bioassay-guided fractionation of an extract of Holarrhena floribunda stem, has led to the isolation of the new trichothecenes, 8-dihydrotrichothecinol A (1), loukacinol A (2), and loukacinol B (3), and the known compounds, trichothecolone (4), trichothecin (5), trichothecinol A (6), rosenonolactone (7), 6beta-hydroxyrosenonolactone (8), and rosololactone (9). The structures were determined by spectral and chemical methods, and absolute configurations were established by a modified Horeau's method using HPLC. Compounds 1 and 6 exhibited significant cytotoxicity against several human tumor cell lines, whereas compound 8 showed moderate and weak antileishmanial activity toward extracellular and intracellular Leishmania donovani, respectively.

Antimicrobial, antitumor and antileishmania screening of medicinal plants from Guinea-Bissau.

Following an ethnobotanical search carried out in Guinea-Bissau, eighteen extracts derived from sixteen medicinal species were screened for antimicrobial, antitumor and antileishmania activity. Significant antitumor activity was found for Holarrhena floribunda against KB (squamous carcinoma), SK-Mel 28 (melanoma), A 549 (lung carcinoma) and MDA-MB 231 (mamma carcinoma) cell lines, with corresponding IC50 values of 7.9, 9.0, 3.4 and 9.9 micrograms/ml. Khaya senegalensis and Anthostema senegalense exhibited a significant activity against Leishmania donovani with IC50 values of 9.8 and 9.1 micrograms/ml, respectively. Most of the extracts showed week or moderate antibacterial and antifungal activity, with MIC values in the range 0.25-1.0 mg/ml. Active extracts were submitted to bioassay-guided fractionation, and the IC50 and MIC of the active fractions were determined.

Botulinum A toxin therapy: neutralizing and nonneutralizing antibodies--therapeutic consequences.

Although muscle-relaxant doses of botulinum A toxin (BoNT/A) are generally lower than doses stimulating the immune system, specific antibodies are raised in a substantial number of patients. As a rule, this necessitates the termination of treatment. Therefore, a reliable determination of specific anti-BoNT/A antibodies is helpful and we introduced, for this purpose, a novel in vitro toxin-neutralizing assay based on a nerve-muscle preparation. We measured the antibody titers in four groups of subjects: Group 1 comprised 75 randomly selected patients of a total of 295 who responded to treatment with Dysport in our local clinic. Five patients, in group 2, were nonresponders. Group 3 consisted of 32 untreated volunteers and group 4 of 8 subjects immunized with a toxoid more than 10 years ago. Two of the responders had marginal titers of neutralizing antibodies, while they were present in all nonresponders. The sera of all responders were also tested for nonneutralizing antibodies by ELISA. Their occurrence, however, was of no consequence to the therapeutic success. The blood samples of volunteers were free from specific antibodies, whereas antibodies persisted in the immunized subjects for longer than a decade. Patients from various clinics who had been treated unsuccessfully with the toxin-14 patients had received BOTOX, 7 had been treated with Dysport, and 7 with both products-all had neutralizing antibodies. Whether there was an antibody response depended on the amount of toxin administered. We believe, however, the effective toxin dose can be reduced by so much as to make antibody production highly improbable.

Botulinum A toxins: units versus units.

We investigated the efficacies and potencies of two commercial preparations of botulinum neurotoxin type A (BoNt/A) reputed to differ in potency. Tests were conducted in vitro using the mouse phrenic nerve-hemidiaphragm which is an approved tool for measuring clostridial toxicity. In addition, in a double-blind trial on volunteers, varying amounts of one product were injected into the Musculus extensor digitorum brevis of the left foot, while equal amounts, i.e. units, of the other preparation were injected into the same muscle of the right foot. Compound muscle action potentials (CMAPs) were recorded before and at various points in time after the injections. As opposed to wide-spread anecdotal reports, no difference in effectiveness was found. The dose-response curves obtained from the mouse organ preparation with both commercial products equalled one another in potency (number of units) and corresponded to previous toxicity tests in mice conducted elsewhere. Dose-response curves from volunteers were also identical for both commercial preparations. The time course of paralysis and recovery of muscle function did not differ either. At lower concentrations of toxin, however, restoration of muscle function was more rapid than at higher concentrations. Since the results obtained from man and the animal organ preparation are in excellent accord, we conclude that 1 unit of Botox corresponds to 1 unit of Dysport.

Processing of tetanus and botulinum A neurotoxins in isolated chromaffin cells.

Tetanus and botulinum A neurotoxins were introduced into the cytosol of chromaffin cells by means of an electric field in which the plasma membrane is forced to form pores of approximately 1 micron at the sites facing the electrodes. As demonstrated by electron microscopy, both [125I] and gold-labelled tetanus toxin (TeTx) diffuse through these transient openings. Dichain-TeTx, with its light chain linked to the heavy chain by means of a disulfide bond, causes the block of exocytosis to develop more slowly than does the purified light chain. The disulfide bonds, which in both toxins hold the subunits together, were cleaved by the intrinsic thioredoxin-reductase system. Single chain TeTx, in which the heavy and light chains are interconnected by an additional peptide bond, was far less effective than dichain-TeTx at blocking exocytosis, which indicates that proteolysis is the rate-limiting step. The toxins were degraded further to low-molecular weight fragments which, together with intact toxins and subunits, were released by the cells. The intracellular half-life of [125I] dichain-TeTx was approximately three days. The number of light-chain molecules required to maintain exocytosis block in a single cell, as calculated by two different methods, was less than 10. The long duration of tetanus poisoning may result from the persistence of intracellular toxin due to scarcity of free cytosolic proteases. This may also hold for the slow recovery from botulism.

Specific antibodies against the Zn(2+)-binding domain of clostridial neurotoxins restore exocytosis in chromaffin cells treated with tetanus or botulinum A neurotoxin.

Although tetanus and botulinum A neurotoxins are ineffective in cultured chromaffin cells, they will inhibit carbachol-induced release of noradrenaline provided they gain access to the cytosol either through artificial pores generated in the plasma membrane or by binding to incorporated exogenous gangliosides. The block of exocytosis persists for weeks followed by a slow recovery of cell function. When specific anti-botulinum A toxin antibodies are introduced into cells through pores after manifestation of the block by botulinum A neurotoxin, restoration of exocytotic function is accelerated and fully reestablished within 4 days. The same time course of restoration is seen with anti-tetanus toxin antibodies in cells poisoned by tetanus toxin. Since the light chains of the toxins are enzymatically active, we have introduced polyclonal and monoclonal anti-light chain antibodies into the cytosol. Of all light chain antibodies tested, only those directed against the peptide homologous to the zinc-binding sequence, which is present in both neurotoxins, restored exocytosis regardless of which toxin caused the block. These results indicate that the zinc-binding domain is directly involved in the interaction of the light chains with their substrates and that the toxins have to be present continuously to maintain the block.

Alteration in Ca2+ homeostasis by a trauma peptide.

Postinjury tissue inflammation with PMN elastase proteolysis generates immunosuppressive fibronectin peptides (FNDP) impairing chemotaxis, T-cell activation, and proliferation. Excess intracellular Ca2+ ([Ca2+]i) impairs T-cell activation. This study quantifies the changes in [Ca2+]i following exposure to a degradation peptide of fibronectin to determine the mechanism of action of these peptides on calcium homeostasis. Isolated human PBLs were exposed to immunosuppressive concentrations of FNDP after loading with the [Ca2+]i probe FURA-2AM. Resting and sustained [Ca2+]i concentrations were calculated and compared to buffer control. The mechanism of action was determined by pretreatment with: (1) EDTA binding extra cellular Ca2+: [Ca2+]e, (2) the Ca2+ channel blockers verapamil and nifedipine, and (3) inhibition of [Ca2+]i released by dantrolene. Inositol triphosphate (IP3) essential for [Ca2+]i release was measured following T-cell stimulation as well. FNDP caused 200-400% increases in [Ca2+]i concentration relative to buffer control at known suppressive doses. Verapamil and nifedipine partially block [Ca2+]i influx by as much as 50% suggesting the slow Ca2+ (voltage independent) channels are partially responsible for the increased [Ca2+]i seen following FNDP. EDTA completely suppressed [Ca2+]e influx but did not completely inhibit the release of [Ca2+]i although IP3 was 80% suppressed. The increase in [Ca2+]i following FNDP stimulation is due to release of intracellular stores.

The complete sequence of botulinum neurotoxin type A and comparison with other clostridial neurotoxins.

The seven serologically different botulinum neurotoxins are highly potent protein toxins that inhibit neurotransmitter release from peripheral cholinergic synapses. The activated toxins consist of the toxifying A-subunits (Mr approximately 50,000) linked by a disulfide bond to the receptor-binding BC-subunits (Mr approximately 100,000). We have established the complete sequence of botulinum neurotoxin type A (BoNT/A; 1,296 amino acid residues, Mr = 149,425) and a partial sequence of botulinum neurotoxin type E (273 amino acid residues) as deduced from the corresponding nucleotide sequences of the chromosomally located structural genes. The promoter of the BoNT/A gene is inactive in Escherichia coli. Primer extension experiments indicated that initiation of transcription of the BoNT/A gene occurred 118 nucleotides upstream from the ATG codon. A comparison of the protein sequence revealed an overall identity of 33.8% to that of tetanus toxin. No significant similarity to other known proteins including ADP-ribosylating toxins could be detected. Three of the six histidine residues of the A-subunit of BoNT/A were found in the peptide sequence H223ELIHXXH230 within a domain of predicted alpha-helical secondary structure. This motif is also found in similar positions of the A-subunits of tetanus toxin and BoNT/E.

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.

ADP-ribosylation of a 21-24 kDa eukaryotic protein(s) by C3, a novel botulinum ADP-ribosyltransferase, is regulated by guanine nucleotide.

Besides botulinum C2 toxin, Clostridium botulinum type C produces another ADP-ribosyltransferase, which we termed 'C3'. ADP-ribosyltransferase C3 has a molecular mass of 25 kDa and modifies 21-24 kDa protein(s) in platelet and brain membranes. C3 was about 1000 times more potent than botulinum C1 toxin in ADP-ribosylation of membrane proteins. C3-catalysed ADP-ribosylation of the 21-24 kDa protein(s) was decreased by stable guanosine triphosphates, with the potency order GTP[S] much greater than p[NH]ppG greater than p[CH2]ppG. GTP[S] inhibited the ADP-ribosylation caused by C3 by maximally 70-80%, with half-maximal and maximal effects occurring at 0.3 and 10 microM-GTP[S] respectively. The concomitant addition of GTP decreased the inhibitory effect of GTP[S]. GTP[S]-induced inhibition of ADP-ribosylation was resistant to washing of pretreated platelet membranes. The data suggest that the novel botulinum ADP-ribosyltransferase C3 modifies eukaryotic 21-24 kDa guanine nucleotide-binding protein(s).

Saccharomyces cerevisiae structural cell wall mannoprotein.

A novel mannoprotein fraction with an average molecular weight of 180 000 has been isolated from Saccharomyces cerevisiae mnn9 mutant cell wall that was solubilized by beta-glucanase digestion. The same material could be extracted from purified wall fragments with 1% sodium dodecyl sulfate. The protein component, 12% by weight, is rich in proline, whereas the carbohydrate, mainly mannose, is about evenly distributed between asparagine and hydroxyamino acids. Endoglucosaminidase H digestion of the isolated mannoprotein reduced its average molecular weight to 150 000, but the mannoprotein, while still embedded in the cell wall, was inaccessible to the enzyme. Biosynthesis and translocation of the mannoprotein were investigated by following incorporation of [3H]proline into this fraction. In the presence of tunicamycin, both mnn9 and wild-type X2180 cells made a mannoprotein fraction with an average molecular weight of 140 000, whereas in the absence of the glycosylation inhibitor, the mnn9 mutant made material with a molecular weight of 180 000 and the mannoprotein made by wild-type cells was too large to penetrate the polyacrylamide gel. Although the cell wall mannoprotein was resistant to heat and proteolytic enzymes, attempts to isolate the carbohydrate-free component failed to yield any characteristic peptide material.

Carbohydrate structure of Saccharomyces cerevisiae mnn9 mannoprotein.

The neutral oligosaccharides from Saccharomyces cerevisiae mnn1 mnn9, mnn2 mnn9, and mnn9 mutant mannoproteins, and from mnn1 and wild type carboxypeptidase Y, have been characterized. The major oligosaccharide from the mnn1 mnn9 mutant, Man10GlcNAc, has the structure (formula; see text) whereas the largest oligosaccharide from the mnn9 mutant, Man13GlcNAc, has the structure (formula; see text) the differences being due to the mnn1 mutation. The smaller mnn9 homologs had lesser amounts of terminal alpha 1----3-linked mannose and may be precursors of the mature oligosaccharide. The mnn2 mutation had no effect on the mnn9 oligosaccharide structures. Carboxypeptidase Y and mnn9 oligosaccharides were identical, which suggests that the mnn9 mutation eliminates the differences in carbohydrate structure that distinguish intra- from extracellular mannoproteins. One mnn1 mnn9 oligosaccharide, Man11GlcNAc, retained the terminal alpha 1----2-linked mannose of the lipid-linked core precursor, which suggests that processing to give the larger oligosaccharides can occur without removal of this unit. A smaller mnn1 mnn9 oligosaccharide, Man9GlcNAc, was a mixture of isomers that must, in part, have arisen by action of an alpha 1----2-mannosidase.

Yeast invertase polymorphism is correlated with variable states of oligosaccharide chain phosphorylation.

Saccharomyces cerevisiae invertase (EC 3.2.1.26) isolated from wild-type strain X2180 can be resolved by isoelectric focusing into at least seven bands revealed by an activity stain. Most of this polymorphism is eliminated in mutants that are defective in phosphorylation of the mannoprotein carbohydrate chains (mnn4 and mnn6). In contrast to strain X2180, invertase from the mnn9 mutant, which makes mannoprotein lacking the outer portion of the polymannose chains, shows only two major bands on isoelectric focusing. Although mnn2 mannoprotein is though not to have any branches in its outer chain, the invertase of this mutant shows at least six bands on isoelectric focusing, and digestion of this invertase with an endo-alph aI leads to 6-mannanase that removes the unbranched outer chain produces an invertase with two bands that are similar to those from the mnn9 mutant. The invertase from mnn2 cells, grown with [32P]orthophosphate and precipitated with specific antiserum, gives at least five radioactive bands on isoelectric focusing, and after digestion with the endomannanase the radioactivity no longer migrates with the residual invertase. Mutants with shortened and unbranched outer chains (mnn2 mnn7, mnn2 mnn8, and mnn2 mnn10) give invertase patterns similar to mnn2. The results suggest that multiple states of outer chain phosphorylation lead to isoelectric polymorphism of S. cerevisiae external invertase and, because invertase has nine carbohydrate chains, no more than one phosphate group per chain would be required to account for this property.

Occurrence and biosynthesis of glyoxysomal enzymes in ripening cucumber seeds.

Glyoxysomal enzymes, being necessary during seed germination, are already synthesized at the stage of seed maturation. Two stages of embryogenesis of cucumber seeds (Cucumis sativus) were investigated. One was characterized by the presence of microbodies showing catalase and enoyl-CoA hydratase activities. Microbodies at a later stage contained, in addition, malate synthase and isocitrate lyase. The biosynthesis of three microbody components was followed in a pulse chase-labelling experiment which demonstrated that the biosynthesis of cytosolic species of malate synthase, isocitrate lyase and enoyl-CoA hydratase preceded the appearance of these proteins in microbodies.

Purification of glyoxysomal acetyl-CoA acyltransferase.

Acetyl-CoA acyltransferase was extracted from cotyledons of germinating cucumber seeds and purified to apparent heomogeneity. The purification based primarily on cation exchange chromatography and separation on an affinity gel. The enzyme is a dimer consisting of two subunits of molecular weight 45 000. Acetyl-CoA acyltransferase was localized in glyoxysomes, the matrix was found to be the site of thiolase function within the organelle.