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.     

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.     

Experimental comparison of selection methods to improve a non-linear trait in Tribolium

Summary Five lines of Tribolium castaneum (flour beetle) were selected in each of three, replicates for biomass, using quadratic (Q) and linear (L) selection indices, direct selection for biomass (B) and indirect selection for each component trait, i.e. pupal weight (P) and family size (F), respectively, for five generations. The highest response in biomass was obtained in the Q line using an exact quadratic index in which the genetic value for each trait was based on information from both traits. The expected results were that Q selection would not exceed L selection while the L method would be superior with respect to direct selection for biomass (B line); P selection would be better and F selection worse than B selection. Selection was effective for pupal weight in the Q, L, B and P lines, but was not effective for family size, although the Q line increased slightly. The lines had no linear response for family size. Selection for biomass was influenced more by family size than by pupal weight. It is advantageous to include terms such as squares and products in the selection index, even though the response to selection may have been non-linear because of the low heritability of family size and an intermediate optimum for this component trait.     

Fluoride can activate the respiratory burst independently of Ca2+, stimulation of phosphoinositide turnover and protein kinase C translocation in primed human neutrophils

Evidences have been provided in our laboratory that in neutrophils different signal transduction sequences for the activation of O2(-)-forming NADPH oxidase can be triggered by the same stimulus (Biochem. Biophys. Res. Commun. 1986, 135, 556-565; 1986, 135, 785-794; 1986, 140, 1-11). The results presented here show that the transduction sequence triggered by fluoride via dissociation of G-proteins and involving messengers produced by stimulation of phosphoinositide turnover, Ca2+ changes and translocation of protein kinase C from the cytosol to the plasmamembrane, can be bypassed when a primed state of neutrophils is previously induced. In fact: i) fluoride causes a pertussis toxin insensitive and H-7 sensitive respiratory burst in human neutrophils, which is linked to the activation of hydrolysis of PIP2, rise in [Ca2+]1 and translocation of PKC. In Ca2+-depleted neutrophils these responses to fluoride do not occur and are restored by addition of CaCl2. ii) The pretreatment of Ca2+-depleted unresponsive neutrophils with non stimulatory doses of PMA restores the activation of the NADPH oxidase by fluoride but not the turnover of phosphoinositides and PKC translocation. The nature of the alternative transduction sequence, the reactions different from phospholipase C activated by G-protein for the alternative sequence and the role of these discrete pathways for NADPH oxidase activation are discussed.     

Novel 1,4-Dihydropyridine (Bay K 8644) Facilitates Calcium-Dependent [3H]Noradrenaline Release from PC 12 Cells

The effects of the novel 1,4-dihydropyridine Bay K 8644 [methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)-pyridine- 5-carboxylate] on the release of [3H]noradrenaline in cultured PC 12 cells were investigated. K+ in a concentration-dependent manner evoked 3H-transmitter release with an EC50 of 50-56 mM. Bay K 8644 at 30 nM potentiated the K+-evoked [3H]noradrenaline release; however, in the absence of calcium neither K+ evoked nor Bay K 8644 enhanced [3H]noradrenaline release. At a K+ concentration of 25 mM, Bay K 8644 stimulated [3H]noradrenaline release fivefold, with an EC50 of 10 nM, and 100 nM of the calcium channel blocker nitrendipine shifted the concentration response curve of Bay K 8644 to the right in an apparently competitive fashion. Nitrendipine blocked the Bay K 8644-potentiated release with an EC50 of 700 nM in the presence of 500 nM Bay K 8644. [3H]Nitrendipine bound to a saturable population of binding sites on PC 12 cell membranes with a Bmax of 180 fmol X mg-1 of membrane protein and a KD of 0.9 nM. Bay K 8644 inhibited [3H]nitrendipine binding with a Ki of 16 nM. It is concluded that Bay K 8644 binds to, and stabilizes, the open state of calcium channels and thus acts as a "calcium agonist" to mediate calcium-dependent cellular events such as catecholamine release from PC 12 cells.     

Interleukin-1β-Mediated Regulation of μ-Opioid Receptor mRNA in Primary Astrocyte-Enriched Cultures

Abstract: Opioids have been found to modulate the immune system by regulating the function of immunocompetent cells. Several studies suggest that the interaction between immune and opioid systems is not unidirectional, but rather reciprocal, in nature. In the CNS, one cellular target of immune system activation is the astrocytes. These glial cells have been shown to produce the opioid peptide, proenkephalin, to express the μ-, δ-, and κ-opioid receptors, and to respond to the immune factor interleukin-1β (IL1β) with an increased proenkephalin synthesis. To characterize more completely the astrocytic opioid response to immune factor stimulation, we examined the effect of IL1β (1 ng/ml) on the μ-receptor mRNA expression in primary astrocyte-enriched cultures derived from rat (postnatal day 1–2) cortex, striatum, cerebellum, hippocampus, and hypothalamus. A 24-h treatment with IL1β produced a 70–80% increase in the μ-receptor mRNA expression in the striatal, cerebellar, and hippocampal cultures but had no effect on this expression in the cortical and hypothalamic cultures. This observation represents one of the few demonstrated increases in levels of the μ-receptor mRNA in vitro or in vivo, since the cloning of the receptor. The enhanced μ-receptor mRNA expression, together with the previous observation that IL1β stimulates proenkephalin synthesis in astrocytes, supports the IL1β-mediated regulation of an astroglial opioid peptide and receptor in vitro, a phenomenon that may be significant in the modulation of the gliotic response to neuronal damage. Therefore, the astroglial opioid "system" may be important in the IL1β-initiated, coordinated response to CNS infection, trauma, or injury.     

Molecular Modeling Studies on Novel Open-chain and Cyclic Thia Compounds and their Ag(I) and Hg(II) Complexes

Molecular modeling techniques were used to generate structures of several HLA-DQ proteins associated with insulin-dependent diabetes mellitus (IDDM). A peptide fragment from glutamic acid decarboxylase (GAD), a known IDDM autoantigen, binds to certain HLA-DQ molecules positively associated with IDDM. Modeling studies were used to explore possible binding interactions between this GAD peptide and several HLA-DQ molecules. Based on the characterization of anchor pockets in the HLA-DQ binding groove and of peptide side chains, a novel binding mode was proposed. This binding mode predicts the GAD peptide is positioned in the binding groove in the direction opposite the orientation observed for class I proteins and the class II DR1, DR3, and I-E^k proteins. Peptide docking exercises were performed to construct models of the HLA-DQ/peptide complexes, and the resulting models have been used to design peptide binding experiments to test this reverse-orientation binding mode. A variety of experimental results are consistent with the proposed model and suggest that some peptide ligands of class II molecules may bind in a reversed orientation within the binding groove.Supplementary material to this paper is available in electronic form at http://dx.doi.org/10.1007/s0089460020205     

Limited proteolysis of tetanus toxin. Relation to activity and identification of cleavage sites

Tetanus toxin is synthesized by Clostridium tetani as a 151-kDa peptide chain. The primary gene product is processed post-translationally by removal of the initiating methionine residue, formation of disulfide bridges and limited proteolysis by bacterial or exogenous proteinases. The mature toxins consist of a 52-kDa light chain and a 98-kDa heavy chain, linked together by a disulfide bond. Proteolytic nicking is accompanied by increased pharmacological potency. To identify the structural alterations involved, single-chain toxin has been subjected to limited proteolysis with various enzymes. The new N-termini have been determined by Edman degradation and the C-termini by isolation of short C-terminal peptide fragments and subsequent analysis of the sequence and composition. All two-chain toxins result from proteolytic nicking within the 17-residue segment of residues 445-461. Thus, the protease(s) of the culture broth cleave on the C-terminal side of Glu449 and partially Ala456, giving rise to two heavy chain N-termini. Trypsin and clostripain first attack the C-terminal of Arg454 and later Arg448, whereas endoproteinase Arg-C cleaves the former bond only. Chymotrypsin and endoproteinase Glu-C each split a single peptide bond, i.e. that located after Tyr452 and Glu449, respectively. Papain gives rise to a large number of cleavages within the 17-residue segment, the new C-terminus being Thr445 or Asn446 and the new N-terminus being Asp460 or Leu461. Further papain digestion leads to an additional cleavage within the heavy chain between Ser863 and Lys864. The original N-terminal Pro1 and C-terminal Asp1314, predicted from the nucleotide sequence, are conserved in all proteolytic digests. The pharmacological activity of the various two-chain toxins was 5-11 times that of the single-chain toxin, as estimated from the inhibition of [3H]noradrenaline release from rat-brain homogenate. The present data on the processing and activation by limited proteolysis prove the existence of several active tetanus isotoxins. These data, together with our previous data on the localization of disulfide bridges and sulfhydryl groups (Krieglstein, K., Henschen, A., Weller, U. & Habermann, E. (1990) Eur. J. Biochem. 188, 39-45), provide the detailed protein chemical characterization of the tetanus isotoxins.     

Heterologous gene expression in continuous cultures of budding yeast

Summary In a previous paper we have studied the expression of -galactosidase from Escherichia coli, driven from the inducible GAL1-10/CYC1 hybrid promoter, in batch cultures of budding Saccharomyces cerevisiae and have described operating conditions for maximal productivity. In this paper we show that the plasmid instability in continuous cultures can be overcome by utilizing appropriate selection markers and a high copy number vector. The maximal level of expression is influenced by the dilution rate. Moreover, enzyme accumulation appears to depend also upon the degree of oxygenation. A possible explanation of these modulations is discussed, taking into account the interactions of the UAS-GAL and TATA-CYC1 elements. Offprint requests to: D. Porro     

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)