Plasmid distribution among unicellular and filamentous toxic cyanobacteria

Plasmid content of 5 hepatotoxin and 2 neurotoxin producing cyanobacterial strains were analyzed. Among the hepatotoxin-producing strains, Microcystis aeruginosa PCC7820, M. aeruginosa M228 and M. aeruginosa UV027 were found to carry plasmids, whereas other hepatotoxin and neurotoxin producing strains did not harbor any plasmids. Correlations were sought between toxicity and the presence of plasmids in toxic cyanobacteria as a function of age. Aged cultures of M. aeruginosa PCC7820 exhibited toxicity and harbored plasmids. In other cyanobacterial strains, plasmids were not detected. The data add to and support the current understanding that plasmids are probably not involved in toxin production in cyanobacteria.Author for Correspondence     

Presynaptic regulation of dopaminergic transmission in the striatum

1. In vitro studies have indicated that several transmitters present in the striatum can regulate presynaptically the release of dopamine (DA) from nerve terminals of the nigrostriatal DA neurons. 2. The receptors involved in these local regulatory processes are located or not located on DA nerve terminals. 3. Recent in vivo investigations have demonstrated that the corticostriatal glutamatergic neurons facilitate presynaptically the release of DA and have allowed the analysis of the respective roles of presynaptic events and nerve activity in the control of DA transmission.     

Role of the N-terminal region of the A chain in beta 1-bungarotoxin from the venom of Bungarus multicinctus (Taiwan-banded krait)

The N-terminal -amino groups of ₁-bungarotoxin (₁-Bgt) fromBungarus multicinctus venom were modified with trinitrobenzene sulfonic acid and the modified derivative was separated by high performance liquid chromatography. The trinitrophenylated (TNP) derivative contained two TNP groups at the -amino groups of A chain and B chain and showed a marked decrease in enzymatic activity. Methionine residues at positions 6 and 8 of the A chain were oxidized with chloramine T or cleaved with cyanogen bromide to remove the N-terminal octapeptide. Oxidation of methionine residues and removal of the N-terminal octapeptide caused a precipitous decrease in enzymatic activity, whereas antigenicity remained unchanged. The presence of dihexanoyllecithin influenced the interaction between ₁-Bgt and 8-antilinonaphthalene sulfonate (ANS) and revealed that ₁-Bgt consists of two types of ANS-binding sites, one at the substrate binding site of the A chain and the other might be at the B chain. The modified derivatives still retained their affinity for Ca²⁺ and ANS, indicating that the N-terminal region is not involved in Ca²⁺ and substrate binding. A fluorescence study revealed that the -amino group of the A chain was in the vicinity of substrate binding site and that the TNP -amino groups were in proximity to Trp-19 of the A chain. In addition, the study showed that the N-terminal region is important for stabilizing the architectural environment of Trp-19. The results, together with the proposal that Trp-19 of the A chain is involved in substrate binding, suggest that the N-terminal region of the A chain plays a crucial role in maintaining a functional active site for ₁-Bgt.     

Presynaptic Nicotinic Cholinergic Receptors Labeled by [3H]Acetylcholine on Catecholamine and Serotonin Axons in Brain

Nicotinic cholinergic receptor binding sites labeled by [3H]acetylcholine were measured in the cerebral cortices, thalami, striata, and hypothalami of rats lesioned by intraventricular injection of either 6-hydroxydopamine or 5, 7-dihydroxytryptamine. In addition, [3H]acetylcholine binding sites were measured in the cerebral cortices of rats lesioned by injection of ibotenic acid into the nucleus basalis magnocellularis. [3H]Acetylcholine binding was significantly decreased in the striata and hypothalami of both 6-hydroxydopamine- and 5,7-dihydroxytryptamine-lesioned rats. There was no change in binding in the cortex or thalamus by either lesion. Ibotenic acid lesions of the nucleus basalis magnocellularis, which projects cholinergic axons to the cortex, did not alter [3H]acetylcholine binding. These results provide evidence for a presynaptic location of nicotinic cholinergic binding sites on catecholamine and serotonin axons in the striatum and hypothalamus.     

Two Components of Glutamate Exocytosis Differentially Affected by Presynaptic Modulation

Abstract: The total Ca²⁺-dependent release of glutamate induced by depolarization of cerebrocortical nerve terminals with KCl was analyzed into a fast and a slow component. The fast component exhibited a decay time of <1 s and accounted for 0.95 ± 0.10 nmol of glutamate, whereas the slow component, which exhibited a decay time of 52 ± 7 s, accounted for the release of 2.48 ± 0.19 nmol of glutamate. These two components were differentially affected by the Ca²⁺ chelator BAPTA, the divalent cation Sr²⁺, or the botulinum neurotoxin A. The adenosine A₁ receptor agonist N ⁶-cyclohexyladenosine strongly reduced the fast component without altering the slow component. In contrast, the inhibitory effect of arachidonic acid and the facilitatory action of the metabotropic glutamate receptor agonist (1 S ,3 R )-1-aminocyclopentane-1,3-dicarboxylic acid were observed as a decrease and an increase, respectively, in the two components. It is concluded, first, that the fast and slow components correspond to the release of docked and mobilized vesicles, respectively, and second, that presynaptic modulation more significantly alters the fast component of release.     

Sequential1H-NMR assignments of neurotoxin III from the sea anemoneHeteractis macrodactylus and structural comparison with related toxins

The complete sequence-specific assignment of resonances in the¹H-NMR spectrum of the polypeptide neurotoxin III (Hm III) from the sea anemoneHeteractis macrodactylus is described. Comparison of the chemical shifts and pattern of NOEs for Hm III with those for the related toxin Hp III fromHeteractis paumotensis, which differs only in the substitution of Asn for Tyr at position 11, shows that the overall secondary and tertiary structures are conserved. The largest differences in chemical shift caused by the substitution at position 11 are observed for the NH resonances of Arg-13, Thr-14, Ala-15, Leu-17, and Cys-26. The CH resonances influenced most are those of ASP-6, Gly-9, Leu-17, and Glu-42, while the most affected CH resonances are from Leu-17, Glu-28, and Lys-32. The absence of long-range NOEs to the aromatic ring of Tyr-11 as well as the lack of significant chemical shift effects on residues outside the loop comprising residues 7–16 confirm that this part of the loop makes no long-lived contacts with the rest of the molecule. The deviations from random coil shifts of Hm III are compared with those of the related anemone toxins Hp II, Hp III, and toxin I fromStichodactyla helianthus (Sh I). The similarity in deviations in chemical shift as a function of sequence position for these four toxins emphasizes the overall structural homology among these polypeptides.     

Algae in mosquito breeding sites and the effectiveness of the mosquito larvicide Bacillus thuringiensis H-14

The presence of cyanobacteria generally decreased the effectiveness of Bacillus thuringiensis H-14 (BTI) as a mosquito larvicide. The effect was more pronounced when the mosquito larvae were exposed to BTI in the presence of several cyanobacterial strains. No synergistic or antagonistic effect between the -endotoxin from BTI and the hepatotoxin from cyanobacteria was seen. Neurotoxic cyanobacterial strains caused very fast paralysis in mosquito larvae; the decreases in the effectiveness of BTI when tested in combination with a neurotoxic strain might be due to the effect of this paralytic action on the feeding rate of the mosquito larvae.     

Modeling of the H-reflex facilitation during ramp and hold contractions

Healthy subjects were asked to make a voluntary ramp and hold contraction. The duration of the ramp stage was 500 ms, and the torque increment in this period was set to 15 Nm. The contraction was made from a relaxed and from a 5 Nm background torque situation. Hoffmann (H-) reflexes were elicited during the voluntary contraction, mostly with 100 ms intervals. These experiments showed an increase (facilitation) in the H-reflex before the torque or the EMG started to increase. This facilitation of the H-reflex remained during all the stages of the voluntary movement and declined to normal levels again only at the very end of the hold phase, which lasted for one second. This specific pattern of facilitation during a voluntary contraction was modeled using a modeling language, that is specifically designed to calculate neuronal systems with a high degree of reality (Ekeberg et al., 1991). Our model consisted of a motoneuron pool with 200 neurons connected to an EMG-model of the human soleus muscle and an extra group of higher-level neurons for controlling the amount of decrease of presynaptic inhibition. The model was used to simulate the observed modulation of the H-reflex with both a presynaptic and a postsynaptic mechanism. Simulations showed that a continuous change in the descending control signals is needed to make the model based on postsynaptic mechanism fit with the experimental data, whereas no extra control from the CNS over the excitatory drive to the motoneuron pool is needed when the decrease of presynaptic inhibition mechanism is applied.     

Non-crystallographic symmetry of crystal of neutral phospholipase A2 from Agkistrodon halys Pallas

Agkistrodotoxin, a neutral phospholipase A₂ with high presynaptic neurotoxicity from the venom of Agkistrodon halys Pallas, has been crystallized by hanging drop vapor diffusion method. The crystal belongs to P2₁ space group with the cell dimensions a = 10.836 nm, b=8.486nm, c = 7.082nm, β=109.87$ showing C2 pseu-do-symmetry. Diffraction data to 0. 26 nm resolution have been collected on a Siemens X-200B area detector. C2 pseu-do-symmetry suggests that there exists a non-crystallographic two-fold axis parallel to crystallographic b axis. Self-rotation function calculation with different integrated radius and resolution ranges using the program POLARRFN yields four stable high peaks corresponding to three more non-crystallographic two-fold axis and one special non-crystal-lographic symmetry. The molecules in the asymmetric unit are suggested to be arranged in a manner of "dimer of dimers" by inference.