THE MARINE DINOFLAGELLATE ALEXANDRIUM OSTENFELDII: PARALYTIC SHELLFISH TOXIN CONCENTRATION,COMPOSITION, AND TOXICITY TO A TINTINNID CILIATE1

The composition of paralytic shellfish toxins in the marine dinoflagellate Alexandrium ostenfeldii (Paulsen) Balech et Tangen grown in unialgal culture was determined by high-performance liquid chromatography. The toxin profile revealed that the low-potency sulfamate toxin B₂ was dominant (90 molar % of total toxins), but small amounts of the weakly toxic 21-N-sulfocarbamoyl derivatives C₁₊₂ and trace amounts of the carbamate toxins GTX₂ and GTX₃ were also present. The mammalian toxicity was confirmed by a modification of the conventional AOAC mouse bioassay (0.6–1.4 pg STX_eq· cell^-1). The acute toxicity to a potential predator, the tintinnid ciliate Favella ehrenbergi (Clap, et Lach.) Jörg., was also investigated. The ciliate was able to graze on A. ostenfeldii when the cell concentration of the dinoflagellate was low (<2000 cells · mL^-1). At higher concentrations the ciliate was affected by exudates (presumably PSP toxins) that induced backward swimming followed by swelling and lysis of the cell. Fluorescence microscopy of calcofluor-stained cells was employed as an easy and rapid method to identify this and other thecate dinoflagellates.     

Characterization of high affinity GTPase activity correlated to β-adrenergic receptor stimulation of adenylyl cyclase in rat parotid membranes

β-Adrenergic receptor stimulation of adenylyl cyclase involves the activation of a GTP-binding regulatory protein (G-protein, termed here Gs). Inactivation of this G-protein is associated with the hydrolysis of bound GTP by an intrinsic high affinity GTPase activity. In the present study, we have characterized the GTPase activity in a Gs-enriched rat parotid gland membrane fraction. Two GTPase activities were resolved; a high affinity GTPase activity displaying Michaelis-Menten kinetics with increasing concentrations of GTP, and a low affinity GTPase activity which increased linearly with GTP concentrations up to 10 mM. The β-adrenergic agonist isoproterenol (10 μM) increased the V_max of the high affinity GTPase component approx. 50% from 90 to 140 pmol/mg protein per min, but did not change its K_m value (≈ 450 nM). Isoproterenol also stimulated adenylyl cyclase activity in parotid membranes both in the absence or presence of GTP. In the presence of a non-hydrolyzable GTP analogue, guanosine 5′-(3-O-thio)triphosphate (GTPγS), isoproterenol increased cAMP formation to the same extent as that observed with AlF₄^?. Cholera toxin treatment of parotid membranes led to the ADP-ribosylation of two proteins (≈ 45 and 51 kDa). Cholera toxin also specifically decreased the high affinity GTPase activity in membranes and increased cAMP formation induced by GTP in the absence or the presence of isoproterenol. These data demonstrate that the high affinity GTPase characterized here is the ‘turn-off’ step for the adenylyl cyclase activation seen following β-adrenergic stimulation of rat parotid glands.     

First chemical synthesis of a scorpion alpha-toxin affecting sodium channels: the Aah I toxin of Androctonus australis hector.

Aah I is a 63-residue alpha-toxin isolated from the venom of the Buthidae scorpion Androctonus australis hector, which is considered to be the most dangerous species. We report here the first chemical synthesis of Aah I by the solid-phase method, using a Fmoc strategy. The synthetic toxin I (sAah I) was renatured in DMSO-Tris buffer, purified and subjected to thorough analysis and comparison with the natural toxin. The sAah I showed physico-chemical (CD spectrum, molecular mass, HPLC elution), biochemical (amino-acid composition, sequence), immunochemical and pharmacological properties similar to those of the natural toxin. The synthetic toxin was recognized by a conformation-dependent monoclonal anti-Aah I antibody, with an IC50 value close to that for the natural toxin. Following intracerebroventricular injection, the synthetic and the natural toxins were similarly lethal to mice. In voltage-clamp experiments, Na(v) 1.2 sodium channel inactivation was inhibited by the application of sAah I or of the natural toxin in a similar way. This work describes a simple protocol for the chemical synthesis of a scorpion alpha-toxin, making it possible to produce structural analogues in time.     

Poplar (Populus nigra L.) plants transformed with aBacillus thuringiensis toxin gene: insecticidal activity and genomic analysis

Insect-resistant poplar (Populus nigra L.) plants have been produced by infecting leaves withAgrobacterium tumefaciens strains carrying a binary vector containing different truncated forms of aBacillus thuringiensis (B.t.) toxin gene under a duplicated CaMV 35S promoter. Putative transgenic plants were propagated by cuttings at two experimental farms (in Beijing and Xinjiang, China). At 2–3 years after transformation, 17 of them were selected on the bases of insect-tolerance and good silvicultural traits, and evaluated for insect resistance, for the presence of theB.t. toxin DNA fragment (Southern blots and PCR) and for the expression of the transgene (western and northern blots). Somaclonal variation, as suggested by the appearance of permanent changes in the shape of the leaves, was also investigated with molecular tools (RFLP (restriction fragment length polymorphism), RAPD (random amplified polymorphic DNA) and microsatellite DNA).Bioassays withApochemia cineraius andLymantria dispar on the leaves of the selected clones showed different and, in some cases, high levels of insecticidal activity. The molecular analysis demonstrated integration and expression of the foreign gene. Somatic changes were correlated to extensive genomic changes and were quantified in dendrograms, in terms of genomic similarity. The analysis of control plants suggested that genomic changes were correlated to thein vitro culture step necessary forA. tumefaciens-mediated gene transfer, rather than to the integration of the foreign genes.Three transgenic clones (12, 153 and 192), selected for insect resistance, reduced morphological changes and promising silvicultural traits, are now under large-scale field evaluation in six different provinces in China.     

Ca2+-activated K+ channels of human and rabbit erythrocytes display distinctive patterns of inhibition by venom peptide toxins

Despite recent progress in the molecular characterization of high-conductance Ca²⁺-activated K⁺ (maxi-K) channels, the molecular identities of intermediate conductance Ca²⁺-activated K⁺ channels, including that of mature erythrocytes, remains unknown. We have used various peptide toxins to characterize the intermediate conductance Ca²⁺-activated K⁺ channels (Gardos pathway) of human and rabbit red cells. With studies on K⁺ transport and on binding of ¹²⁵I-charybdotoxin (ChTX) and ¹²⁵I-kaliotoxin (KTX) binding in red cells, we provide evidence for the distinct nature of the red cell Gardos channel among described Ca²⁺-activated K⁺ channels based on (i) the characteristic inhibition and binding patterns produced by ChTX analogues, iberiotoxin (IbTX) and IbTX-like ChTX mutants, and KTX (1–37 and 1–38 variants); (ii) the presence of some properties heretofore attributed only to voltage-gated channels, including inhibition of K transport by margatoxin (MgTX) and by stichodactyla toxin (StK); (iii) and the ability of scyllatoxin (ScyTX) and apamin to displace bound ¹²⁵I-charybdotoxin, a novel property for K⁺ channels. These unusual pharmacological characteristics suggest a unique structure for the red cell Gardos channel.We thank Dr. Chris Miller of Brandeis University for generously providing recombinant ChTX mutants, Dr. Maria Garcia of Merck Research Laboratories for MgTX and Dr. Regine Romi of Laboratoire d'Ingenierie des Proteines (Marseille, France) for synthetic KTX,1–37 and KTX,1–38. This research was supported by grant HL-15157 from the National Institutes of Health.     

Crystal structure of a non-toxic mutant of heat-labile enterotoxin, which is a potent mucosal adjuvant.

Two closely related bacterial toxins, heat-labile enterotoxin (LT-I) and cholera toxin (CT), not only invoke a toxic activity that affects many victims worldwide but also contain a beneficial mucosal adjuvant activity that significantly enhances the potency of vaccines in general. For the purpose of vaccine design it is most interesting that the undesirable toxic activity of these toxins can be eliminated by the single-site mutation Ser63Lys in the A subunit while the mucosal adjuvant activity is still present. The crystal structure of the Ser63Lys mutant of LT-I is determined at 2.0 A resolution. Its structure appears to be essentially the same as the wild-type LT-I structure. The substitution Ser63Lys was designed, based on the wild-type LT-I crystal structure, to decrease toxicity by interfering with NAD binding and/or catalysis. In the mutant crystal structure, the newly introduced lysine side chain is indeed positioned such that it could potentially obstruct the productive binding mode of the substrate NAD while at the same time its positive charge could possibly interfere with the critical function of nearby charged groups in the active site of LT-I. The fact that the Ser63Lys mutant of LT-I does not disrupt the wild-type LT-I structure makes the non-toxic mutant potentially suitable, from a structural point of view, to be used as a vaccine to prevent enterotoxigenic E. coli infections. The structural similarity of mutant and wild-type toxin might also be the reason why the inactive Ser63Lys variant retains its adjuvant activity.     

Structural studies of receptor binding by cholera toxin mutants.

The wide range of receptor binding affinities reported to result from mutations at residue Gly 33 of the cholera toxin B-pentamer (CTB) has been most puzzling. For instance, introduction of an aspartate at this position abolishes receptor binding, whereas substitution by arginine retains receptor affinity despite the larger side chain. We now report the structure determination and 2.3-A refinement of the CTB mutant Gly 33-->Arg complexed with the GM1 oligosaccharide, as well as the 2.2-A refinement of a Gly 33-->Asp mutant of the closely related Escherichia coli heat-labile enterotoxin B-pentamer (LTB). Two of the five receptor binding sites in the Gly 33-->Arg CTB mutant are occupied by bound GM1 oligosaccharide; two other sites are involved in a reciprocal toxin:toxin interaction; one site is unoccupied. We further report a higher resolution (2.0 A) determination and refinement of the wild-type CTB:GM1 oligosaccharide complex in which all five oligosaccharides are seen to be bound in essentially identical conformations. Saccharide conformation and binding interactions are very similar in both the CTB wild-type and Gly 33-->Arg mutant complexes. The protein conformation observed for the binding-deficient Gly 33-->Asp mutant of LTB does not differ substantially from that seen in the toxin:saccharide complexes. The critical nature of the side chain of residue 33 is apparently due to a limited range of subtle rearrangements available to both the toxin and the saccharide to accommodate receptor binding. The intermolecular interactions seen in the CTB (Gly 33-->Arg) complex with oligosaccharide suggest that the affinity of this mutant for the receptor is close to the self-affinity corresponding to the toxin:toxin binding interaction that has now been observed in crystal structures of three CTB mutants.     

Rapid Identification by Polymerase Chain Reaction of Staphylococcal Exfoliative Toxin Serotype A and B Genes

A new system was designed to detect staphylococcal exfoliative toxin A (ETA) and B (ETB) genes by the polymerase chain reaction (PCR). The primer pairs for the ETA gene (eta) were 20 and 20-mer, and its PCR product was a 741-bp eta fragment, while the primer pairs for the ETB gene (etb) were also 20 and 20-mer, and its PCR product was a 629-bp etb fragment. When these primers were simultaneously used in the PCR, the two types of ET were clearly detected as two bands in an ETA and ETB double-producer using only one colony within 3 hr. We examined 66 strains of Staphylococcus aureus isolated from patients with staphylococcal scalded skin syndrome (SSSS) and compared the results obtained by ELISA and PCR. The same results were obtained for 56 of the strains, i.e., 30 strains were ETA producers, 20 strains were ETB producers, and 6 strains were double-producers. However, positive results were obtained for 5 of the 10 non-ET-producing strains. Two of these strains were judged by PCR as ETA producers and three as ETB producers. Thus, PCR is very sensitive and rapid in detecting ETA and ETB gene fragments in colonies isolated from patients with SSSS.