A new toxin, Lqh alpha IT, which caused a unique mode of paralysis of blowfly larvae, was purified from the venom of the scorpion Leiurus quinquestriatus hebraeus, and its structural and pharmacological properties were compared to those of three other groups of neurotoxins found in Buthinae scorpion venoms. Like the excitatory and depressant insect-selective neurotoxins, Lqh alpha IT was highly toxic to insects, but it differed from these toxins in two important characteristics: (a) Lqh alpha IT lacked strict selectivity for insects; it was highly toxic to crustaceans and had a measurable but low toxicity to mice. (b) It did not displace an excitatory insect toxin, 125I-AaIT, from its binding sites in the insect neuronal membrane; this indicates that the binding sites for Lqh alpha IT are different from those shared by the excitatory and depressant toxins. However, in its primary structure and its effect on excitable tissues, Lqh alpha IT strongly resembled the well-characterized alpha scorpion toxins, which affect mammals. The amino acid sequence was identical with alpha toxin sequences in 55%-75% of positions. This degree of similarity is comparable to that seen among the alpha toxins themselves. Voltage- and current-clamp studies showed that Lqh alpha IT caused an extreme prolongation of the action potential in both cockroach giant axon and rat skeletal muscle preparations as a result of the slowing and incomplete inactivation of the sodium currents. These observations indicate that Lqh alpha IT is an alpha toxin which acts on insect sodium channels.(ABSTRACT TRUNCATED AT 250 WORDS)

The human immunodeficiency virus (HIV) genome codes for a trans-activating regulatory protein, tat. Using chemically synthesized tat, it was found that 125I-tat and 12s51_ t384;6 specifically bound to rat brain synaptosomal membranes with moderate affinity (K0.5 = 3 ,uM). Interaction of tat with nerve cells was also revealed by flow cytometry, which showed its binding to rat glioma and murine neuroblastoma cells, using both direct fluorescence with fluorescein isothiocyanate-labeled tat and indirect immunofluorescence assays. This interaction was investigated with electrophysiology using isolated excitable frog muscle fibers and cockroach giant interneuron synapses. tat acted on the cell membrane and induced a large depolarization, accompanied by a decrease in membrane resistance, thereby modifying cell permeability. It is now established that infection with human immunodeficiency virus type 1 (HIV-1) is often complicated by neurological syndromes that include dementia, subacute encephalitis, and vacuolar degeneration of the spinal cord (9,19,28,32). The identification and isolation of HIV-1 from the brain suggests that the retrovirus itself is responsible for the neurological disorders observed in HIV-infected patients. Other lentiviruses, including visna virus (15) and simian immunodeficiency virus (22), are also associated with brain infections. Among central nervous system (CNS) cells, monocyte and macrophage lines are preferentially infected by HIV, but infection of other neural cell types has also been discussed (20,35).The pathogenic mechanism by which the virus causes encephalopathy remains unknown. It was reported recently that the HIV envelope glycoprotein manifests neurotoxic activity by increasing free Ca2+ in rat neurons, thus causing cellular damage (4, 7). This effect can be prevented by Ca2+ channel antagonists.As an approach to another possible cause of neurological dysfunction, we investigated whether other HIV proteins could be implicated in this pathology. For this study, numerous peptides were chemically synthesized on an Applied Biosystems peptide synthesizer (model 430A) with the stepwise solid-phase method (25,29).By testing the neurotoxicity of synthetic fragments of various lengths, derived from gp160, p25, nef, and tat proteins, we discovered that the intracerebroventricular injection of tat or some tat fragments caused toxic and lethal effects in mice. The 86-residue tat protein from HIV-1 has been previously reported to be critical for virus replication through its role in viral trans activation (1,11,12,14,18,33 We have further investigated tat neurotoxicity by structure-activity relationships, using binding experiments and electrophysiology. We first investigated the capacity of radiolabeled tat3886 from HIV-1, LAVBru isolate, to bind to rat brain synaptic nerve ending particles (synaptosomal membranes) prepared by the method of Gray and Whittaker (13). Protein was measured by a modified Lowry method (24). 125I-tat38 86 (>10-8 M), the most active peptide in vivo (Table 1), bound to...

The human immunodeficiency virus (HIV) genome codes for a trans-activating regulatory protein, tat. Using chemically synthesized tat, it was found that 125I-tat and 12s51_ t384;6 specifically bound to rat brain synaptosomal membranes with moderate affinity (K0.5 = 3 ,uM). Interaction of tat with nerve cells was also revealed by flow cytometry, which showed its binding to rat glioma and murine neuroblastoma cells, using both direct fluorescence with fluorescein isothiocyanate-labeled tat and indirect immunofluorescence assays. This interaction was investigated with electrophysiology using isolated excitable frog muscle fibers and cockroach giant interneuron synapses. tat acted on the cell membrane and induced a large depolarization, accompanied by a decrease in membrane resistance, thereby modifying cell permeability. The neurotoxicity of tat was further demonstrated in vitro, on glioma and neuroblastoma cell growth, as well as by a 51Cr release assay in both tumor cell lines. Interestingly, no hemolytic activity of tat for human erythrocytes was found even when tat was tested at its highly neurotoxic concentration. Experiments in vivo showed that synthetic tat is a potent and lethal neurotoxic agent in mice. The use of tat peptide derivatives showed that basic region from 49 to 57 is necessary and sufficient for binding to cell membranes and toxicity.

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The present study shows that most of variables obtained using 3DGA in hip OA patients are reliable. Moreover, for most variables, 5-10 trials are needed to obtain good reliability and to overcome intrinsic variability, rather than 30 or more, thus improving the feasibility of measurement.

The effects of synthetic poneratoxin (PoTX), a new toxin isolated from the venom of the ant Paraponera clavata, were studied under current- and voltage-clamp conditions in frog skeletal muscle fibres. PoTX induces a concentration-dependent (10(-9) M-5 x 10(-6) M) prolongation of the action potentials and, at saturating concentration, a slow repetitive activity developing at negative potentials. PoTX specifically acts on voltage-dependent Na channels by decreasing the peak Na current (INa) and by simultaneously inducing a slow INa which starts to activate at -85 mV and inactivates very slowly. Both the fast and the slow components of INa are suppressed by tetrodotoxin and reverse at the same potential corresponding to the equilibrium potential for Na ions. The fast component of INa has voltage dependence, activation and steady-state inactivation almost similar to those of the control INa. The voltage dependence of the slow Na conductance is 40 mV more negative than that of the fast one. The results suggest that PoTX affects all the Na channels and that the fast and the slow INa components originate from a possible PoTX-induced interconversion between a fast and a slow operating mode of the Na channels.

SUMMARY1. The double sucrose-gap technique has been applied to rat skeletal muscle fibres to study the ionic currents under voltage-clamp conditions.2. The iliacus muscle was found to be of 'fast' type according to the characteristics of the twitch generated by an action potential.3. Micro-electrode measurements have shown that the intracellular potential is under good control even when an inward current develops.4. The components of an equivalent circuit with two time constants have been estimated from the records of the capacitive current.5. In rat muscle, between 15 and 21 TC, inward and outward currents are similar to sodium and potassium currents found in frog muscle at lower temperature (1-3 0C).6. The inward current which depends on [Na]o and is abolished by tetrodotoxin is carried by sodium ions. Related to the mean value for the holding potential (-90 5 mV) this current reaches its maximum amplitude at + 40 and + 50 mV, reverses between + 130 and + 150 mV and its half inactivation occurs between +14 and +22 mV. The effect of low doses of tetrodotoxin suggests that two components participate in the sodium current.7. The delayed outward current which shows inactivation is divided in two components: (i) the fast has a linear instantaneous current-voltage relation and differs from the fast component of frog muscle in that its equilibrium potential is more negative than the resting potential; (ii) the slow has a linear instantaneous currentvoltage relation and the mean value for its equilibrium potential is 26 mV less negative than the resting potential.8. Inward-going rectification is present in rat muscle.

SUMMARY1. A comparison of the delayed outward current of isolated fibres from rat soleus and iliacus muscle has been made using a double sucrose-gap voltage-clamp method.2. The fast and slow components of the outward current were separated using time constants of the tail currents. The results indicate that in both iliacus and soleus fibres there is a shift in reversal potential which depends on the quantity of current that flows during depolarization.3. The shift is larger in iliacus than in soleus; it is absent in glycerol-treated muscles.4. The results obtained in normal and in detubulated fibres show that the shift is due to an accumulation process of potassium ions in the lumen of the T-tubules.5. In detubulated soleus fibres the outward current is composed of a fast and a slow component, each with the same reversal potential; in detubulated iliacus the slow component is absent. 6. In both types of muscles TEA produces a dose-dependent block of the total outward current. 4-aminopyridine has different effects; it inhibits the total outward current in iliacus fibres and only the fast component in soleus fibres.7. These results show that in soleus fibres a fast and a slow component participate in the potassium outward current, while only a fast component is present in iliacus muscle.