Botulin

Ann Otol Rhinol Laryngol, 1989 Feb, 98(2), 130 - 4
Implantation of a recurrent laryngeal nerve stimulator for the treatment of spastic dysphonia; Friedman M et al.; Spastic dysphonia, a rare speech disorder, is characterized by strained phonation with excessively adducted vocal cords . Recurrent laryngeal nerve section, botulinum toxin injection into the vocalis-thyroarytenoid muscle complex, and other techniques have been used to treat this disorder . We have used percutaneous electrical stimulation of the recurrent laryngeal nerve with good results . Previous dog studies demonstrated the relative safety of an implantable recurrent laryngeal nerve stimulator . In this study, we directly stimulated the recurrent laryngeal nerve and vagus nerve in a dog without change in cardiorespiratory status . A Medtronic peripheral nerve stimulator was implanted in a patient with abductor spastic dysphonia . The cuff electrode was positioned around the recurrent laryngeal nerve and stimulation resulted in improvement in her voice . Extensive cardiopulmonary monitoring did not reveal any adverse response to stimulation and there was no discomfort to the patient . On the basis of the good results of this preliminary study, further study with long-term follow-up is under way.

Dev Biol, 1989 Feb, 131(2), 401 - 14
Lack of fiber type selectivity during reinnervation of neonatal rabbit soleus muscle; Soha JM et al.; Fast and slow contracting fibers in neonatal mammalian skeletal muscle are each innervated in a highly specific manner by motor neurons of the corresponding type, even at an age when polyinnervation is widespread . Chemospecific recognition is a possible mechanism by which this pattern of innervation could be established . We have investigated this possibility by studying the degree of specificity during reinnervation of rabbit soleus muscle following nerve crush on Postnatal Day 1 or 4 . We assayed fiber type composition by measuring the twitch rise times of motor units within 2 days of the onset of functional reinnervation (5-6 days after nerve crush) . In contrast to the broad, bimodal distribution of single motor unit twitch rise times seen in normal muscles, motor units in reinnervated muscles yielded a narrower, unimodal distribution of rise times . Rise times of reinnervated units were intermediate to those of normal fast and slow units, suggesting that reinnervated units were composed of a mixture of fast and slow contracting fibers . An alternative possibility, that specific reinnervation was masked by contractile dedifferentiation of muscle fibers, was examined by maintaining a transmission blockade induced by botulinum toxin poisoning for an equivalent interval . Twitch rise times of treated motor units exhibited the distinctly bimodal distribution characteristic of normal muscles, suggesting that muscle fibers can retain contractile diversity during a transient period of denervation . We carried out computer simulations to estimate the amount of rise time diversity induced by varying degrees of specificity during reinnervation . Based on this analysis, we conclude that there is little if any selective reinnervation of muscle fiber types at the ages studied.

J Neurochem, 1989 Feb, 52(2), 370 - 6
Actin involvement in exocytosis from PC12 cells: studies on the influence of botulinum C2 toxin on stimulated noradrenaline release; Matter K et al.; Botulinum C2 toxin is known to ADP-ribosylate actin . The toxin effect was studied on {3H}noradrenaline secretion of PC12 cells . {3H}Noradrenaline release was stimulated five- to 15-fold by carbachol (100 microM) or K+ (50 mM) and 10-30-fold by the ionophore A23187 (5 microM) . Pretreatment of PC12 cells with botulinum C2 toxin for 4-8 h at 20 degrees C, increased carbachol-, K+-, and A23187-induced, but not basal, {3H}noradrenaline release maximally 1.5-to three-fold, whereas approximately 75% of the cellular actin pool was ADP-ribosylated . Treatment of PC12 cells with botulinum C2 toxin for up to 1 h at 37 degrees C also increased stimulated {3H}noradrenaline secretion, whereas toxin treatment for greater than 1 h decreased the enhanced {3H}noradrenaline release stimulated by carbachol and K+ but not by A23187 . Concomitantly with toxin-induced stimulation of secretion, 20-50% of the cellular actin was ADP-ribosylated, whereas greater than 60% of actin was modified when exocytosis was attenuated . The data indicate that ADP-ribosylation of actin by botulinum C2 toxin largely modulates stimulation of {3H}noradrenaline release . Moreover, the biphasic toxin effects suggest that distinct mechanisms are involved in the role of actin in secretion.

Mol Cell Biochem, 1989 Jan 23, 85(1), 67 - 73
Structure of heavy and light chain subunits of type A botulinum neurotoxin analyzed by circular dichroism and fluorescence measurements; Singh BR et al.; The secondary and tertiary structural features of botulinum neurotoxin (NT) serotype A, a dichain protein (Mr 145,000), and its two subunits, the heavy (H) and light (L) chains (Mr 97,000 and 53,000, respectively) were examined using circular dichroism and fluorescence spectorscopy . Nearly 70% of the amino acid residues in each of the three polypeptide preparations were found in ordered structure (sum of alpha helix, beta sheet and beta turns) . Also, the alpha helix, beta sheet, beta turns and random coil contents of the dichain NT were nearly equal to the weighted mean of each of these secondary structure parameters of the L and H chains; e.g., sum of alpha helix of L chain (22%) and H chain (18.7%), as weighted mean, 19.8% was similar to that of NT (20%) . These agreements suggested that the secondary structures of the subunits of the dichain NT do not significantly change when they are separated as isolated L and H chains . Fluorescence emission maximum of L chain, 4 nm less (blue shift) than that of H chain, suggested relatively more hydrophobic environment of fluorescent tryptophan residue(s) of L chain . Tryptophan fluorescence quantum yields of L chain, H chain and the NT, 0.072, 0.174 and 0.197, respectively, suggested that a) an alteration in the micro-environment of the tryptophan residues was possibly caused by interactions of L and H chain subunits of the NT and b) quantum yields for L and H chains were altered when they are together as subunits of the NT . Possible implications of structural features of the L and H chains, their interactions and the molecular mechanism of action of botulinum NT are assessed.

Neurosci Lett, 1989 Jan 16, 96(2), 127 - 32
Sprouting of frog motor nerve terminals after long-term paralysis by botulinum type A toxin; Diaz J et al.; A single sublethal injection of botulinum type A toxin (BoTx-A) to winter frogs induced a general and complete paralysis of skeletal muscles, which lasted several months . Quantitative analysis of 483 end-plates from 8 BoTx-A poisoned muscles and 495 endplates from 8 control muscles revealed a higher and significant incidence of terminal and ultraterminal sprouts in poisoned junctions when taking into account the normal remodelling of motor innervation . We conclude that prolonged neuromuscular blockade by BoTx-A results in the extension of the nerve terminal arborization.

FEBS Lett, 1989 Jan 16, 243(1), 70 - 6
Purification of the 22 kDa protein substrate of botulinum ADP-ribosyltransferase C3 from porcine brain cytosol and its characterization as a GTP-binding protein highly homologous to the rho gene product; Braun U et al.; The 22 kDa protein substrate of botulinum ADP-ribosyltransferase C3 was purified from porcine brain cytosol by acetone precipitation, CM-Sephadex, octyl-Sepharose and TSK phenyl-5PW HPLC chromatography to apparent homogeneity . ADP-ribosylation of the protein was increased by guanine nucleotides (GTP, GDP, GTP gamma S, each 100 microM) but not by GMP, ATP or ATP gamma S . The purified 22 kDa protein bound maximally 0.9 mol {35S}GTP gamma S and hydrolyzed GTP with the rate 0.007 mol per mol protein . Amino acid sequences were obtained from two tryptic peptides, selected from an in situ digestion of Immobilon electrotransferred, gel purified ADP-ribosylated substrate . The two sequences obtained, cover 23 residues from the corresponding sequences in human rho.

Biochem Biophys Res Commun, 1989 Jan 16, 158(1), 209 - 13
The rho gene product expressed in E . coli is a substrate of botulinum ADP-ribosyltransferase C3; Aktories K et al.; The ras-related rho A protein expressed in E . coli, was ADP-ribosylated by botulinum ADP-ribosyltransferase C3 . C3 also modified the valine-14 mutant rho protein but not the products of H-ras, R-ras, ral, ypt, and rap 1 genes . A ras-rho chimaera consisting of 60 amino acids from the amino terminus of ras fused to 133 amino acids from the carboxy terminus of rho was not modified by C3 . Antibodies raised against the porcine brain cytosolic substrate of C3 cross reacted with the rho, valine-14 rho and ras-rho proteins, but not with the gene products of H-ras, R-ras, ral or rap 1 . Polyclonal anti-H-ras antibodies cross reacted with H-ras but not with ral, rho, or the C3 substrate purified from porcine brain.

Infect Immun, 1989 Jan, 57(1), 18 - 22
Sequence homology between tetanus and botulinum toxins detected by an antipeptide antibody; Halpern JL et al.; The extent of immunological similarity between tetanus toxin and botulinum toxins A, B, C1, and E was studied by using 10 antibodies produced against synthetic peptides representing different sequences of tetanus toxin, mouse antitetanus serum, and human Tetanus Immune Globulin . Antibodies produced against the synthetic peptides recognized tetanus toxin in an enzyme-linked immunosorbent assay and on Western blots (immunoblots) but did not appear to recognize the native protein . One of the antitetanus peptide antibodies, which was produced against a peptide from the amino terminal, cross-reacted with three of the four botulinum toxins on immunoblots . This antibody, 1, reacted strongly with botulinum toxins B and C1 and weakly with E but did not recognize type A toxin . None of the other peptide antibodies cross-reacted with the botulinum toxins . Mouse antitetanus serum and human Tetanus Immune Globulin did not recognize any of the botulinum toxins on immunoblots . The amino-terminal region of the light chain of tetanus toxin and botulinum toxin types A, B, C1, and E are known to have sequence homology . Our data demonstrate that for tetanus toxin and botulinum toxin types B, C1, and E this region also has immunological homology . Type A, which has the least amount of homology with tetanus toxin in this region, does not share this immunological homology . These data also suggest that although the native structures of tetanus and botulinum toxins have relatively few common immunological determinants, the two toxins may contain short stretches of identical or very similar amino acid sequences.

Mov Disord, 1989, 4(4), 287 - 96
Adductor laryngeal dystonia (spastic dysphonia): treatment with local injections of botulinum toxin (Botox); Brin MF et al.; Adductor spastic dysphonia (SD) is a laryngeal dystonia characterized by a choked, constrained voice pattern with breaks in vocal flow . Treatment with a variety of therapies including speech and pharmacotherapy have minimal benefit; only one-third of patients undergoing recurrent laryngeal nerve section have benefitted at 3 years . We have used local injections of botulinum toxin (Botox) bilaterally into vocalis muscles in 42 patients with SD . Injections were through a teflon-coated hollow electromyography (EMG) recording needle . Unilateral small doses (2.5-3.75 U) were of no clinical benefit . Bilateral small doses resulted in sustained improvement lasting 84.4 +/- 9.3 days . The degree of improvement was 61.1 +/- 4.6% . Common side-effects included a brief period of breathy hypophonia (8.5 +/- 2.5 days) and a mild sensation of choking/aspiration of fluids (1.7 +/- 0.6 days); there were no serious adverse effects . Vocal cord paralysis was not necessary for benefit . Follow-up vocalis muscle EMGs revealed denervation . All patients responded to retreatment (longest follow-up 3.5 years) . Patients with prior recurrent laryngeal nerve surgery and residual uncomplicated dysphonia had similar results . Our results indicate that local injection of low-dose Botox is the treatment of choice for SD.

Neuroscience, 1989, 31(2), 521 - 7
Increase in reactive cholesterol in the presynaptic membrane of depolarized Torpedo synaptosomes: blockade by botulinum toxin type A; Egea G et al.; We have investigated the redistribution of filipin-cholesterol complexes at freeze-fractured presynaptic membrane of pure cholinergic synaptosomes isolated from Torpedo electric organ during acetylcholine release . After chemical depolarization, filipin-induced lesions increase at the presynaptic membrane . These changes do not take place when synaptosomes are stimulated in a calcium-free medium . Botulinum neurotoxin type A blocks both acetylcholine release and the rearrangement of filipin-induced lesions induced by depolarization . Since botulinum neurotoxin type A does not block either membrane depolarization or calcium entry into the nerve terminal, our results suggest that the redistribution of filipin-cholesterol complexes is linked to the acetylcholine release process.

Toxicon, 1989, 27(8), 849 - 59
Gangliosides mediate inhibitory effects of tetanus and botulinum A neurotoxins on exocytosis in chromaffin cells; Marxen P et al.; Bovine chromaffin cells in monolayer culture were preloaded with 3H-NA (noradrenaline) and subsequently stimulated with carbachol . Botulinum A neurotoxin partially inhibited the evoked release of 3H-NA and the basal efflux of the hormone . The inhibition of evoked release did not exceed 40%, although the cells were exposed to 10 micrograms/ml of toxin for 6 days . The inhibitory effect of botulinum A neurotoxin was neutralized by its antibodies . In contrast to botulinum A neurotoxin, tetanus toxin at even higher concentrations did not influence evoked release . This difference in sensitivity could be explained by the ganglioside pattern of chromaffin cells . Ganglioside GD1a, a putative receptor for botulinum A neurotoxin, could be identified in lipophilic extracts, whereas the tetanus toxin binding gangliosides GT1b and GD1b could not be detected by means of thin-layer chromatography . Treatment of the cells with neuraminidase abolished both, GD1a and the inhibitory effect of botulinum A neurotoxin . Incubation of chromaffin cells with a mixture of gangliosides (21% GM1, 44% GD1a, 15% GD1b, 20% GT1b) not only increased the efficacy of botulinum A neurotoxin but also made the cells sensitive towards tetanus toxin . The concentration-response curve of botulinum A neurotoxin was shifted to the left about five-fold and the maximum inhibition of evoked release was increased up to 60%, even though the cells were exposed to the toxin for 3 days only . In contrast, the maximum inhibition that could be achieved by tetanus toxin was 40% . The results indicate that polysialogangliosides are important for the intracellular accumulation of these clostridial neurotoxins.

J Neurol Neurosurg Psychiatry, 1989 Jan, 52(1), 57 - 62
Vestibulo-ocular abnormalities in spasmodic torticollis before and after botulinum toxin injections; Stell R et al.; In order to establish whether vestibular abnormalities often found in spasmodic torticollis are secondary to the abnormal head posture, the vestibulo-ocular reflex (VOR) was studied in eight patients before and after correction of head posture with botulinum toxin . Eye movements were recorded in the dark during sinusoidal and velocity step rotation . Four patients showed a significantly asymmetric response, with the slow phase of the VOR more active ipsilateral to the torticollis (chin) . Despite significant improvement of the head posture in all patients for up to 10 weeks following treatment, no correction of the vestibular asymmetry occurred . This suggests that the VOR abnormalities are not caused by the head posture itself . We interpret the findings as evidence of primary involvement of the vestibular system in torticollis and we postulate a widespread derangement of the sensory-motor mechanisms controlling head posture in this disease.

Prog Brain Res, 1989, 80, 183 - 96; discussion 171-2
Open-loop and closed-loop optokinetic nystagmus in squirrel monkeys (Saimiri sciureus) and in man; Behrens F et al.; Horizontal optokinetic nystagmus (OKN) was measured in 3 normal Squirrel monkeys by means of the electromagnetic search coil technique . Binocular and monocular stimulation of each eye to the left and right by moving vertical stripe patterns of 2.37 or 15 degree period were applied at angular velocities of 0.5 to 400 deg/s . After measurement of horizontal OKN under normal conditions, open-loop OKN gain was determined by monocular stimulation of an eyeball immobilized by means of retrobulbar injections of 11 units botulinum toxin (BoTx type A) and compared with the pre-injection data or with monocular stimulation of the other eye, which remained mobile . In normal Squirrel monkeys gain of optokinetic nystagmus reached values between 0.8 and 0.97 at angular velocities below 1.5 deg/s . Gain under these conditions was related to stimulus angular velocities Vs, i.e . Ge = Ve/Vs . A slightly higher gain was found for binocular than for monocular stimulation . No significant differences were found in OKN when monocular stimulation in the naso-temporal and in the temporo-nasal direction was applied . The upper cut-off angular velocity (-3 dB-point) reached values of 180-230 deg/s, significantly above those observed in man under similar stimulus conditions . Monocular optokinetic stimulation of an immobilized eye led to vigorous optokinetic nystagmus and OKAN of the other eye, whereby maximum gain (Gi = Ve/Vr) was found to be between 20 and 30 at lower retinal stimulus velocities (2-5 deg/s) . Gain was related to retinal stimulus velocity Vr . Increase in Vr above 10 deg/s led to a decrease in gain with a slope of about 20 dB per decade . Measurement of gain of closed-loop OKN related to retinal stimulus velocity Vr (which was determined by the difference between Vs and Ve) led to a similar dependence of OKN as in closed-loop stimulus conditions . Differences in sensitivity between the temporal and the nasal visual hemifield stimulation evoking horizontal open-loop OKN are described . Directional selectivity appeared in these experiments . Open-loop OKN data from a human subject are reported . With highly attentive horizontal optokinetic gaze nystagmus, Ve depended on the duration of the pursuit phases of OKN . Ve accelerated with the duration of the individual slow phase of OKN and was reset by each backward saccade (of the covered mobile eye) . OKN gain was considerably smaller when the subject intentionally pursued as many stripes as possible of the 1.15 degree period stripe pattern (gain related to Vr about 1.5-3).(ABSTRACT TRUNCATED AT 400 WORDS)

Neurology, 1989 Jan, 39(1), 80 - 4
Controlled trial of botulinum toxin injections in the treatment of spasmodic torticollis; Gelb DJ et al.; We administered local injections of botulinum toxin to 20 patients with torticollis in a blinded, placebo-controlled study . Each patient received four sets of injections: three different doses of botulinum toxin and one placebo . The order of the sessions was random and unknown to the patients . Sixteen of the patients (80%) reported subjective improvement to at least one dose of botulinum toxin; 11 (55%) reported substantial improvement . No objective benefit was documented . Side effects were minor and transient, although dysphagia occurred in four . Some patients reported that the effect waned despite persistent relaxation or even flaccidity of previously overactive muscles, suggesting a change in the pattern of muscle activity after botulinum toxin injections.

Eye, 1989, 3 ( Pt 4), 409 - 14
Doppler ultrasound velocity mapping of extra-ocular muscles: a preliminary report; Canning CR et al.; Colour doppler imaging and conventional spectral doppler/B-mode techniques were used to detect and quantify rectus muscle movement during voluntary saccades . A velocity gradient was evident in all muscles studied--low velocities near the muscle origin at the back of the orbit increasing linearly to a maximum near the muscle insertion on the globe . A consistent reduction in velocity occurred along muscles injected with botulinum toxin . Doppler ultrasound complements current clinical eye movement recording techniques--electronystagmography, infrared light reflection methods and magnetic induction methods--insofar as it detects movement in parts of the muscle itself rather than movement of the globe secondary to muscle contraction . Future developments in colour doppler imaging will make the technique clinically more applicable.

Eye, 1989, 3 ( Pt 4), 391 - 400
The role of botulinum toxin in the management of sixth nerve palsy; Fitzsimons R et al.; Fifty-five patients with sixth nerve palsy have been treated with Botulinum toxin injection to the antagonist medial rectus, either in isolation or in combination with rectus muscle surgery . Forty of these patients (72 per cent) obtained significant benefit from injection at some stage in their management . Botulinum toxin has a major role in conjunction with transposition muscle surgery for the treatment of complete unrecovered sixth nerve palsy . In less severe paresis, a functional cure may be obtained in a significant number of cases with the use of Botulinum toxin alone, 37 per cent in this series . No serious complications were observed . In view of the safety of this procedure it is reasonable to consider an injection of Botulinum toxin in any adult patient with a persisting sixth nerve palsy.

J Neural Transm Park Dis Dement Sect, 1989, 1(3), 229 - 42
Effects of botulinum neurotoxin and Lambert-Eaton myasthenic syndrome IgG at mouse nerve terminals; Lande S et al.; The interaction between two presynaptically acting agents, Lambert-Eaton myasthenic syndrome (LEMS) immunoglobulin G (IgG) and purified botulinum neurotoxin (BoNT) type A, was studied . Intracellular microelectrode recordings were carried out on mouse muscles after injection with LEMS IgG . BoNT was either injected before recordings were made or applied in vitro . The time course of the in vitro actions of BoNT on miniature end-plate potential and end-plate potential parameters were not affected by pretreatment with LEMS IgG . After in vivo injection of BoNT, end-plate potential quantal content was reduced to less than 2% of control values, whether or not LEMS IgG had also been previously given . Quantitative electron-microscope autoradiographical analysis showed that neither the binding of 125I-BoNT to acceptors on the nerve terminal membrane nor the pattern of its internalisation were affected by pretreatment with LEMS IgG . We conclude that the effects of BoNT are not affected by LEMS IgG, suggesting different presynaptic binding sites for the two agents.

Toxicon, 1989, 27(10), 1143 - 50
Modification of carboxyl groups in botulinum neurotoxin types A and E; Woody MA et al.; Effects of chemical modification of carboxyl groups of botulinum neurotoxin serotypes A and E were studied by using a water soluble carbodiimide-nucleophile reaction that is highly specific for modifying carboxyl groups of proteins . In both types A and E, increasing levels of the reagents, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and norleucine methyl ester or glycine methyl ester, at pH 4.8 caused increased loss of toxicity . More glycine could be incorporated than norleucine . Amino acid analysis did not reveal modification of any amino acid residue other than carboxyl groups (possible reaction of sulfhydryl groups was not studied) . Loss of one carboxyl group did not severely affect toxicity, but modification of three carboxyl groups caused greater than 95% detoxification in both types . Complete detoxification could not be achieved with any amount of the reagents . Modification of three to five carboxyl groups did not affect serological activity.

Toxicon, 1989, 27(9), 989 - 93
Botulinum C2 toxin treatment increases the G-actin pool in intact chicken cells: a model for the cytopathic action of actin-ADP-ribosylating toxins; Aktories K et al.; Botulinum C2 toxin ADP-ribosylates actin in intact chicken embryo cells in a concentration-dependent manner . This effect correlates with an enhancement in the inhibitory potency of the respective cell lysates on DNAse I activity, indicating an increase in the cellular G-actin content of toxin-treated cells . The data support our view, that ADP-ribosylation of cellular actin with subsequent depolymerization of cytoskeleton-associated F-actin to monomeric G-actin is involved in the cytotoxic effects of botulinum C2 toxin . A model of the cytopathic action of actin-ADP-ribosylating toxins is presented.

Acta Neuropathol (Berl), 1989, 78(1), 72 - 85
Dithiobiuret neurotoxicity: an ultrastructural investigation of the lesion in preterminal axons and motor endplates in the rat lumbrical muscle; Jones HB; 2,4-Dithiobiuret was given i.p . to rats for 4 days at a daily dosage of 1 mg/kg and the development of the lesion associated with neuromuscular dysfunction studied in hindlimb lumbrical muscles . The first morphological indication of neurointoxication was the appearance in some motor endplates of masses of branching tubular smooth endoplasmic reticulum (SER) on day 2 which correlated with the initial functional disturbances . By the 3rd day, most motor endplates were distended by accumulations of dense-cored, lucent and synaptic vesicles, abnormally swollen mitochondria, intermediate filaments and branching, tubular SER . Evidence of collateral axonal sprouting was seen first at this time . On days 4 and 5, many motor endplates were markedly enlarged and showed axoplasmic organelle congestion . A significant increase in synaptic vesicle size was noted at these times in some terminals . Interposition of Schwann cell processes between the pre- and postsynaptic membranes and terminal retraction was now evident . Some intramuscular nerves showed hydropic Schwann cell cytoplasm with separation of the outermost myelin lamellae, mitochondrial swelling and adaxonal vacuoles as early as the 1st day . Proliferation and segregation of SER around central cores of neurofilaments was seen in myelinated nerve fibres and preterminals on the 3rd day . At this and later times accumulations of SER and swollen mitochondria were found at sites of axonal varicosities and at the paranodal constrictions at nodes of Ranvier . These ultrastructural data are discussed with regard to reduced terminal Ca2+ content (demonstrated by oxalate-pyroantimonate cytochemistry) and compared with the sequelae of botulinum intoxication.

Proc Natl Acad Sci U S A, 1989 Jan, 86(1), 372 - 6
Botulinum toxin type A blocks the morphological changes induced by chemical stimulation on the presynaptic membrane of Torpedo synaptosomes; Marsal J et al.; The action of botulinum neurotoxin on acetylcholine release, and on the structural changes at the presynaptic membrane associated with the transmitter release, was studied by using a subcellular fraction of cholinergic nerve terminals (synaptosomes) isolated from the Torpedo electric organ . Acetylcholine and ATP release were continuously monitored by chemiluminescent methods . To catch the membrane morphological changes, the quick-freezing method was applied . Our results show that botulinum neurotoxin inhibits the release of acetylcholine from these isolated nerve terminals in a dose-dependent manner, whereas ATP release is not affected . The maximal inhibition (70%) is achieved at neurotoxin concentrations as low as 125 pM with an incubation time of 6 min . This effect is not linked to an alteration of the integrity of the synaptosomes since, after poisoning by botulinum neurotoxin type A, they show a nonmodified occluded lactate dehydrogenase activity . Moreover, membrane potential is not altered by the toxin with respect to the control, either in resting condition or after potassium depolarization . In addition to acetylcholine release inhibition, botulinum neurotoxin blocks the rearrangement of the presynaptic intramembrane particles induced by potassium stimulation . The action of botulinum neurotoxin suggests that the intramembrane particle rearrangement is related to the acetylcholine secretion induced by potassium stimulation in synaptosomes isolated from the electric organ of Torpedo marmorata.

J Physiol, 1988 Dec, 407, 263 - 74
Neural regulation of {3H}saxitoxin binding site numbers in rat neonatal muscle; Bambrick LL et al.; 1 . Neural regulation of the density of sodium (Na+) channels in rat muscle was studied by measuring specific binding of tritiated saxitoxin ({3H}STX) to muscles from rat hindlimbs during normal development and in rats in which neuromuscular function was interrupted by sciatic nerve section or neuromuscular blockade with botulinum toxin (BoTX) . 2 . The normal developmental increase in {3H}STX binding site numbers followed a simple exponential with a time constant of 12 days . The most rapid incorporation of channels coincided with the onset of accelerated muscle growth and increased neuromuscular activity at 2 weeks of age . 3 . Elimination of neuromuscular activity retarded muscle growth and inhibited the normal incorporation of Na+ channels into neonatal muscle . Muscles denervation was more effective than BoTX paralysis: denervation at 2 weeks of age prevented the normal 3-fold increase in the binding site density between 2 and 3 weeks of age while age-matched BoTX-treated muscles incorporated an average of 66% of the normal Na+ channel incorporation . 4 . Denervation and BoTX treatment were equally effective in reducing the numbers of {3H}STX binding sites in adult muscle . A reduction of 30% in binding sites brought the numbers to levels which corresponded with levels normally seen in muscles at 3 weeks of neonatal development . 5 . It was concluded that the neural influence on incorporation of Na+ channels into membranes of neonatal muscle is, at least in part, mediated by neuromuscular activity.

Eur J Biochem, 1988 Nov 15, 177(3), 683 - 91
Involvement of the constituent chains of botulinum neurotoxins A and B in the blockade of neurotransmitter release; Maisey EA et al.; 1 . The abilities of botulinum neurotoxins, types A and B (single and two-chain forms) to inactivate an intraneuronal component required for transmitter release were quantified in a phrenic-nerve-diaphragm preparation, cerebrocortical synaptosomes or the buccal ganglion of Aplysia californica and compared with the mouse toxicity assay . 2 . Homogeneous preparations of the individually renatured polypeptide chains of both toxin types showed low residual toxicity in the whole animal and had no effect on neurotransmission in all three systems, when tested singly . 3 . Mixtures of individually renatured heavy chain, from type A or B, and either light chain proved very effective in blocking the evoked release of acetylcholine when bath-applied to the buccal ganglion of Aplysia whilst they were relatively inactive on mammalian nerve terminals, indicating a less efficient uptake of the polypeptides in the latter . 4 . When renatured together, the homologous, but not the heterologous, chains of each toxin type yielded toxic, disulphide-linked two-chain species . 5 . A role for the heavy chain alone in acceptor recognition and membrane translocation was implicated by the blockade of acetylcholine release produced when light chain was applied to a ganglion of Aplysia previously bathed in heavy chain and washed extensively . No blockade was observed when the order of application of the two chains was reversed . 6 . These findings are discussed in the context of the intracellular requirement for both the constituent toxin chains for toxicity, and in the apparent need for these chains to be linked via a disulphide bond for uptake in rodents but not in Aplysia.

Am J Ophthalmol, 1988 Nov 15, 106(5), 584 - 6
Treatment of acquired nystagmus with botulinum A toxin; Helveston EM et al.; We injected botulinum A toxin into the retrobulbar space of one eye each in two patients who had acquired nystagmus with oscillopsia and decreased vision . After injection of 25 units of botulinum A toxin, visual acuity improved from 20/80 to 20/30 in one patient, and both patients were able to read and watch television . Improved vision lasted from five to 13 weeks . No adverse side effects were observed after a total of five injections in each patient.

J Biol Chem, 1988 Nov 15, 263(32), 16744 - 9
Purification and characterization of the 22,000-dalton GTP-binding protein substrate for ADP-ribosylation by botulinum toxin, G22K; Bokoch GM et al.; GTP-binding proteins were purified from human neutrophils, including a 40,000-Da pertussis toxin substrate (Gn) and 22,000-, 24,000-, and 26,000-Da proteins, termed G22K, G24K, and G26K, respectively . The latter proteins were shown to be immunologically unrelated to Gn . G22K cross-reacted with anti-ras monoclonal antibody 142-24EO5, but not with monoclonal antibody Y13-259 . A single 22,000-Da substrate for botulinum toxin-catalyzed ADP-ribosylation present in neutrophil membranes co-migrated upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis with G22K . In the presence of a cytosolic factor, G22K could serve as a specific botulinum toxin substrate . The 22,000-Da botulinum toxin substrate in neutrophil membranes could be immunoprecipitated by antibody 142-24EO5, but not by antibody Y13-259 . G22K appears to be a unique GTP-binding protein which serves as a substrate for ADP-ribosylation by a component of botulinum toxin and which may be involved in exocytotic secretion or cellular differentiation.

J Biol Chem, 1988 Nov 5, 263(31), 16303 - 8
ADP-ribosylation of the bovine brain rho protein by botulinum toxin type C1; Kikuchi A et al.; We have separated at least six GTP-binding proteins (G proteins) with Mr values between 20,000 and 25,000 from bovine brain crude membranes (Kikuchi, A., Yamashita, T., Kawata, M., Yamamoto, K., Ideda, K., Tanimoto, T., and Takai, Y . (1988) J . Biol . Chem . 263, 2897-2904) . Three of these G proteins were copurified with the proteins ADP-ribosylated by botulinum toxin type C1 . One G protein ADP-ribosylated by this toxin was identified to be the bovine brain rho protein (rho p20) which was purified to near homogeneity (Yamamoto, K., Kondo, J., Hishida, T., Teranishi, Y., and Takai, Y . (1988) J . Biol . Chem . 263, 9926-9932) . rho p20 was ADP-ribosylated by botulinum toxin type C1 in time- and dose-dependent manners . About 0.4 mol of ADP-ribose was maximally incorporated into 1 mol of rho p20 . The ADP-ribosylation of rho p20 was dependent on the presence of Mg2+ . GTP enhanced the ADP-ribosylation in the presence of a low concentration (50 nM) of Mg2+ but not in the presence of a high concentration (0.5 mM) of Mg2+ . The high concentration of Mg2+ fully stimulated the ADP-ribosylation even in the absence of GTP . The ADP-ribosylation of rho p20 did not affect its GTP gamma S-binding and GTPase activities . These results indicate that there are at least three G proteins ADP-ribosylated by botulinum toxin type C1 in bovine brain crude membranes and that one of them is rho p20 . Two other G proteins have not yet been identified, but neither the c-ras protein, ADP-ribosylation factor for Gs, nor a G protein with a Mr of 24,000 was ADP-ribosylated by this toxin.

Riv Neurol, 1988 Nov-Dec, 58(6), 245 - 8
Use of botulinum toxin in Meige's disease; Maurri S et al.; Four patients with severe Meige's disease (blepharospasm-oromandibular dystonia) have been treated, after having given an informed consent, by local injections of purified botulinum toxin type "A" . Previous systemic therapy with anticholinergics, dopamine antagonists and other drugs had been unsuccessful in all these subjects . Each patient was treated by saline solution injected with the same method as botulinum toxin, just once . The self-evaluation of patients and the clinical evaluation that some of us- unaware of the kind of therapy which had been performed- gave to the symptoms on the basis of videotapes, for each session of injection, showed that the injections of botulinum toxin are effective in the treatment of such disorder . The duration of the beneficial effect was slightly shorter in these patients than in patients with blepharospasm treated by the same method.

J Clin Microbiol, 1988 Nov, 26(11), 2351 - 6
Human immune response to botulinum pentavalent (ABCDE) toxoid determined by a neutralization test and by an enzyme-linked immunosorbent assay; Siegel LS; To determine the immune status of persons receiving botulinum pentavalent (ABCDE) toxoid and to evaluate the effectiveness of the vaccine, we surveyed immunized individuals for neutralizing antibodies to type A and to type B botulinum toxins . After the primary series of three immunizations administered at 0, 2, and 12 weeks, 21 of 23 persons tested (91%) had a titer for type A that was greater than or equal to 0.08 international units (IU)/ml, and 18 (78%) had a titer for type B of greater than or equal to 0.02 IU/ml . (One international unit is defined as the amount of antibody neutralizing 10,000 mouse 50% lethal doses of type A or B botulinum toxin) . Just before the first annual booster, 10 of 21 (48%) and 14 of 21 (67%) people lacked a detectable titer for type A and for type B, respectively . After the first booster, all individuals tested had a demonstrable titer to both types A and B . Of 77 persons who had previously received from one to eight boosts of the toxoid, 74 (96%) had an A titer of greater than or equal to 0.25 IU/ml and would not require an additional booster, according to the recommendations of the Centers for disease Control . However, only 44 of 77 (57%) had a B titer of greater than or equal to 0.25 IU/ml . In each group by booster number, even the group having had eight boosts, at least one person would require reimmunization on the basis of B titer . There was a wide range of antibody levels among individuals at the same point in the immunization scheme . Results from an enzyme linked immunosorbent assay, with purified type A or type B neurotoxin as the capture antigen, were compared with neutralization test results on 186 serum samples for type A and 168 samples for type B . Statistically, the correlation coefficients for results from the two assays were high (r = 0.69, P < 0.0001, for type A and r = 0.77, P < 0.0001, for type B) . However, due to the wide dispersion of values obtained, using enzyme-linked immunosorbent assay results to predict neutralizing antibody levels is unwarranted.

Ophthalmology, 1988 Nov, 95(11), 1535 - 42
Treatment of sixth nerve palsy in adults with combined botulinum toxin chemodenervation and surgery; Fitzsimons R et al.; This study reports the results of treating unrecovered sixth nerve palsy in adults with a combination of botulinum toxin and surgery . Twenty-two adults, 11 with unilateral and 11 with bilateral unrecovered sixth nerve palsy, were treated with injections of botulinum toxin to the contracted medial rectus followed by transposition surgery to the vertical rectus muscles . This management technique produced a satisfactory reduction in esotropia, averaging 46 prism diopters (PD), comparable with that achieved by conventional surgery in other series . The advantages of this form of treatment are discussed.

Ophthalmology, 1988 Nov, 95(11), 1529 - 34
Long-term results and complications of botulinum A toxin in the treatment of blepharospasm; Dutton JJ et al.; The authors review their long-term results and complications with the use of botulinum A toxin in the treatment of facial dystonias . Two hundred thirty-two patients in three diagnostic groups--essential blepharospasm, hemifacial spasm, and Meige's syndrome--were treated with botulinum A toxin . A total of 1044 treatments were given over a 4-year period . A reduction in orbicularis spasm intensity was noted in 1012 (96.9%) treatments (mean duration, 13.3 weeks) . There was no clear relationship between toxin dose and the amount of spasm reduction or duration of response, and average duration of beneficial effect remained constant from the first through the twelfth injections . Complications occurred in 236 (22.6%) treatments . In most cases, these were local and transient . Symptomatic dry eye was the most common side effect, noted in 7.5% of cases . Ptosis was reported in 7.3% of treatments and photophobia in 2.5% . Diplopia involving the inferior oblique or lateral rectus muscles was seen in less than 1% of cases . There were no differences in degree of response or in complications among the three diagnostic groups, although there was a slight difference in duration of effect . Patients who had undergone previous eyelid surgery for blepharospasm did not respond differently from those without prior surgery.

Brain Res, 1988 Nov 1, 463(2), 218 - 22
Motor nerve outgrowth: reduced capacity for sprouting in the terminals of longer axons; Pestronk A et al.; The ability of axons to grow or sprout can vary considerably . In this study we have examined the relation between axon length and the abundance of outgrowth from motor nerve terminals in vivo . Outgrowth from nerve terminals was evoked using botulinum toxin . Terminal axons, sprouts and neuromuscular junctions were visualized using a cholinesterase-silver stain . The amount of axonal outgrowth was compared in several proximal (e.g . rhomboid and paraspinous), intermediate, and distal (e.g . soleus and foot) muscles . Our results show that sprouting is generally more abundant in proximal than in distal muscles . There is a significant inverse correlation between nerve length and the abundance of sprouting from terminal axons . Thus, terminals of short axons appear to have more ability or potential to sprout than those of long axons.

J Neurol Sci, 1988 Nov, 87(2-3), 175 - 85
Axonal sprouting after botulinum toxin does not elicit a histological axon reaction; Pamphlett R; In an attempt to determine which elements of the axon reaction are essential for early axonal outgrowth, axonal sprouting was induced with botulinum toxin (BoTx) and the nerve cell body changes compared with those accompanying axonal growth after nerve trauma . Anterior horn cells of mice were examined histologically at times ranging from 3 days to 3 weeks after either BoTx hindlimb injection or sciatic nerve crush . After sciatic nerve crush there was dispersion of Nissl substance, increase in cell body size, and an increase in neurofilament protein staining . None of these changes were found after BoTx-induced terminal axonal sprouting, suggesting that these morphological features of the axon reaction are not essential for early axonal outgrowth.

Aust N Z J Ophthalmol, 1988 Nov, 16(4), 333 - 5
Botulinum toxin--the rationale of its use; Dunlop C; Treatment with Oculinum, (Botulinum toxin, Type A), may induce three possible effects . These are a pharmacologic inhibitory effect, a mechanical effect and a sensory effect.

CMAJ, 1988 Nov 1, 139(9), 837 - 44
Cranial dystonia, blepharospasm and hemifacial spasm: clinical features and treatment, including the use of botulinum toxin; Kraft SP et al.; Blepharospasm, the most frequent feature of cranial dystonia, and hemifacial spasm are two involuntary movement disorders that affect facial muscles . The cause of blepharospasm and other forms of cranial dystonia is not known . Hemifacial spasm is usually due to compression of the seventh cranial nerve at its exit from the brain stem . Cranial dystonia may result in severe disability . Hemifacial spasm tends to be much less disabling but may cause considerable distress and embarrassment . Patients affected with these disorders are often mistakenly considered to have psychiatric problems . Although the two disorders are quite distinct pathophysiologically, therapy with botulinum toxin has proven very effective in both . We review the clinical features, proposed pathophysiologic features, differential diagnosis and treatment, including the use of botulinum toxin, of cranial dystonia and hemifacial spasm.

Neurology, 1988 Nov, 38(11), 1780 - 3
Neuromuscular effects distant from the site of botulinum neurotoxin injection; Olney RK et al.; We assessed the severity and temporal profile of distant neuromuscular effects from a single dose (280 units) of botulinum neurotoxin injected into neck muscles for torticollis . We performed single-fiber EMG studies on the biceps brachii of six patients to measure jitter (20 pairs) and fiber density on the initial treatment day and then again, at least once more, after 2 to 12 weeks . No patient developed weakness beyond the neck muscles or decrement of the biceps response to repetitive 3-Hz nerve stimulation . Between the baseline and the last follow-up study, the average of mean MCD increased from 29 microseconds to 38 microseconds (31%) . Mean fiber density increased concurrently or earlier from 1.35 to 1.79 (33%) . There were no electrophysiologic signs of presynaptic blockade, even at 2 and 4 weeks . The effects we observed are compatible with stimulation of terminal sprouting by the neurotoxin, without significant presynaptic inhibition of acetylcholine release . We therefore believe that higher dosages of the neurotoxin may be used if clinically indicated.

Biochem Biophys Res Commun, 1988 Oct 14, 156(1), 361 - 7
Different types of ADP-ribose protein bonds formed by botulinum C2 toxin, botulinum ADP-ribosyltransferase C3 and pertussis toxin; Aktories K et al.; We attempted to characterize ADP-ribose-amino acid bonds formed by various bacterial toxins . The ADP-ribose-arginine bond formed by botulinum C2 toxin in actin was cleaved with a half-life of about 2 h by treatment with hydroxylamine (0.5 M) . In contrast, the ADP-ribose-cysteine bond formed by pertussis toxin in transducin and the ADP-ribose-amino acid linkage formed by botulinum ADP-ribosyltransferase C3 in platelet cytosolic proteins were not affected by hydroxylamine . HgCl2 cleaved the ADP-ribose-amino acid bond formed by pertussis toxin in transducin but not those formed by botulinum C2 toxin or botulinum ADP-ribosyltransferase C3 in actin and platelet cytosolic proteins, respectively . NaOH (0.5 M) cleaved the ADP-ribose-amino acid bonds formed by botulinum C2 toxin and pertussis toxin but not the one formed by botulinum ADP-ribosyltransferase C3 . The data indicate that the ADP-ribose bond formed by botulinum ADP-ribosyltransferase C3 differs from those formed by the known bacterial ADP-ribosylating toxins.

FEBS Lett, 1988 Oct 10, 238(2), 277 - 80
Botulinum toxin-induced ADP-ribosylation and inhibition of exocytosis are unrelated events; Adam-Vizi V et al.; The hypothesis that inhibition of secretion by botulinum neurotoxin type D occurs by an intracellular process involving ADP-ribosylation has been directly tested by measuring both the extent of inhibition of secretion and of ADP-ribosylation in the same cells . Although the inhibitory effect of unpurified toxin closely parallels intracellular ribosylation, the two events are clearly unrelated, as using purified D and C3 toxins together with their antibodies, each of these events can be either stimulated or inhibited independently of each other.

J Neurosci, 1988 Oct, 8(10), 3909 - 19
Motor nerve terminal sprouting in formamide-treated inactive amphibian skeletal muscle; Wines MM et al.; Motor axons can form sprouts from their terminal arborizations in response to partial denervation, and when exposed to pharmacological blocking agents like TTX, botulinum toxins alpha-bungarotoxin, or curare . Each of these experimental procedures has cessation of muscle contractile activity as a common feature . We tested the specific role of muscle fiber inactivity in regulating nerve terminal sprouting by chronically treating adult frog (Rana pipiens) cutaneous pectoris muscles with formamide . Exposure to formamide, unlike the other compounds used to study sprouting, selectively inhibits muscle contractions without blocking pre- or postsynaptic transmission or muscle fiber action potentials . Repeated formamide applications were used to achieve chronic block of muscle contractile activity in vivo for up to 6 weeks . Motor axons in formamide-treated inactive muscle sprouted only from their terminal arborizations, but not from nodes of Ranvier . The onset of this sprouting was protracted compared with that seen in pharmacologically blocked mammalian muscles, and sprouts in formamide-treated muscles were more complex and ornate . The frequency of sprouting terminals was less in these formamide-treated muscles than that seen after alternate methods of contractile block, and this suggests that contractile inactivity alone serves as only a moderate cue for sprouting . The possibility is discussed that the prolific sprouting seen following neurotoxin administration may, in fact, be due to perturbations in synaptic transmission or muscle electrical activity rather than muscle fiber inactivity.

J Clin Invest, 1988 Oct, 82(4), 1376 - 82
Botulinum C2 toxin ADP-ribosylates actin and enhances O2- production and secretion but inhibits migration of activated human neutrophils; Norgauer J et al.; The binary botulinum C2 toxin ADP-ribosylated the actin of human neutrophils . Treatment of human neutrophils with botulinum C2 toxin for 45 min increased FMLP-stimulated superoxide anion (O2-) production 1.5-5-fold, whereas only a minor fraction of the cellular actin pool (approximately 20%) was ADP-ribosylated . Effects of botulinum C2 toxin depended on toxin concentrations, presence of both components of the toxin, and incubation time . Cytochalasin B similarly enhanced O2- production . The effects of botulinum C2 toxin and cytochalasin B were additive at submaximally, but not maximally effective concentrations and incubation time of either toxin . Botulinum C2 toxin also enhanced stimulation of O2- production by Con A and platelet-activating factor, but not by phorbol 12-myristate 13-acetate (PMA) . Botulinum C2 toxin increased FMLP-induced release of N-acetyl-glucosaminidase by 100-250%; release of vitamin B12-binding protein induced by FMLP and PMA was enhanced by approximately 150 and 50%, respectively . Botulinum C2 toxin blocked both random migration of neutrophils and migration induced by FMLP, complement C5a, leukotriene B4, and a novel monocyte-derived chemotactic agent . The data suggest that botulinum C2 toxin-catalyzed ADP-ribosylation of a minor actin pool has a pronounced effect on the activation of human neutrophils by various stimulants.

J Biol Chem, 1988 Sep 25, 263(27), 13739 - 42
ADP-ribosylated actin caps the barbed ends of actin filaments; Wegner A et al.; The mode of action on actin polymerization of skeletal muscle actin ADP-ribosylated on arginine 177 by perfringens iota toxin was investigated . ADP-ribosylated actin decreased the rate of nucleated actin polymerization at substoichiometric ratios of ADP-ribosylated actin to monomeric actin . ADP-ribosylated actin did not tend to copolymerize with actin . Actin filaments were depolymerized by the addition of ADP-ribosylated actin . The maximal monomer concentration reached by addition of ADP-ribosylated actin was similar to the critical concentration of the pointed ends of actin filaments . ADP-ribosylated actin had no effect on the rate of polymerization of gelsolin-capped actin filaments which polymerize at the pointed ends . The results suggest that ADP-ribosylated actin acts as a capping protein which binds to the barbed ends of actin filaments to inhibit polymerization . Based on an analysis of the depolymerizing effect of ADP-ribosylated actin, the equilibrium constant for binding of ADP-ribosylated actin to the barbed ends of actin filaments was determined to be about 10(8) M-1 . As actin is ADP-ribosylated by perfringens iota toxin and by botulinum C2 toxin, it appears that conversion of actin into a capping protein by ADP-ribosylation is a pathophysiological reaction catalyzed by bacterial toxins which ultimately leads to inhibition of actin assembly.

FEBS Lett, 1988 Sep 12, 237(1-2), 168 - 72
Gn-proteins are distinct from ras p21 and other known low molecular mass GTP-binding proteins in the platelet; Bhullar RP et al.; The 27 kDa platelet membrane protein (Gn27) that binds {alpha-32P}GTP on nitrocellulose blots of SDS-polyacrylamide gels {(1987) Biochem . J . 245, 617-620} was compared with other low molecular mass GTP-binding proteins . Platelet membranes also contained 21 kDa proteins that bound anti-ras p21 antibody and 22-23 kDa proteins that could be ADP-ribosylated by botulinum neurotoxin type D . These groups of proteins were resolved electrophoretically from each other and from Gn27 . A low molecular mass GTP-binding protein from bovine brain {(1987) Biochem . J . 246, 431-439} was also resolved from Gn27 . At the levels normally present in cell membranes, only Gn-proteins bound significant amounts of {32P}GTP after transfer of protein from SDS-polyacrylamide gels to nitrocellulose.

J Chromatogr, 1988 Sep 9, 430(2), 279 - 89
Fast protein liquid chromatography of botulinum neurotoxin types A, B and E; Woody MA et al.; Three antigenically different botulinum neurotoxins (NTs, relative molecular mass approximately 150,000), classically distinguished only by specific antisera, were for the first time chromatographically resolved . Mixed NTs eluted from a Mono-Q column in order of types E, A and B, and from Mono-S as B, E and A . Type A and B NTs were successfully chromatographed on the cation-exchange Mono-S column above their isoelectric points . Purification of type A and B NTs by automated liquid chromatography was also accomplished for the first time . Type A, B and E NTs were purified by application on an anion-exchange Mono-Q column, followed by use of a cation-exchange Mono-S column.

Acta Otolaryngol, 1988 Sep-Oct, 106(3-4), 178 - 85
Experimental paralysis of tensor veli palatini muscle; Casselbrant ML et al.; In an effort to study the effects of experimental paralysis of tensor veli palatini (TVP) muscle on Eustachian tube (ET) function and middle-ear (ME) status, botulinum toxin A (Oculinum) was injected into the TVP muscles of 8 Rhesus monkeys . Tubal function was tested longitudinally in 2 animals with tympanostomy tubes using the forced-response test, while in the remaining 6 animals; ME condition was documented daily using tympanometry . The postinjection tubal function was characterized by abolished active muscular function and decreased closing pressure . Activity associated with tubal dilations gradually reappeared by the fifth week . The lack of lumen constrictions following injection suggested that the TVP muscle is the cause of constriction as well as normal dilation . In 6 animals with intact tympanic membranes, 10 of the 12 ears developed flat tympanograms associated with otitis media with effusion (OME) within 8-30 days of injection and serous effusions were recovered by tympanocentesis in seven ears . These results show that a non-traumatic reversible functional obstruction of the ET was created by injecting botulinum toxin A into the TVP muscle . This functional obstruction was associated with the development of high negative ME pressure and serous effusion.

Mol Cell Biochem, 1988 Sep, 83(1), 65 - 72
Reductive methylation of lysine residues of botulinum neurotoxin types A and B; Sathyamoorthy V et al.; Reductive methylation of botulinum neurotoxin (NT) serotypes A and B at various ratios of protein to reagent modified up to 75% of the lysine residues . Amino acid analysis of the modified proteins (HCl hydrolysed) confirmed selective modifications of lysine . The derivative N,N-dimethyl lysine was more abundant than monomethyl lysine; trimethyl lysine was not detected . Distribution of modified lysine residues among the heavy and light chains (Mr approximately 100,000 and approximately 50,000, respectively) of the dichain type A NT (Mr approximately 150,000) was approximately proportional to the lysine contents of the two subunit chains of the NT . Toxicity (mouse lethality) and serological reactivity (polyclonal antibody) of serotype A NT were not (or insignificantly) damaged following methylation of up to 72 lysine residues . Modification of 3 additional residues caused precipitous loss in toxicity . Toxicity of serotype B NT, unlike type A, appeared more sensitive to lysine modification . The large number of lysine residues that can be methylated without damaging toxicity of type A NT can be exploited to a) radiolabel the dichain protein exclusively in one chain keeping the other chain unlabelled, b) restrict the number of tryptic cleavage sites of the NT, and c) tag the protein with various markers or reactive ligands.

Neurotoxicol Teratol, 1988 Sep-Oct, 10(5), 393 - 416
Effects of neurotoxicants on synaptic transmission: lessons learned from electrophysiological studies; Atchison WD; A number of environmentally-important neurotoxicants affect chemical synaptic transmission in the peripheral and central nervous system . These include heavy metals such as lead, mercury, cadmium and tin; organophosphates; pyrethroid insecticides, and 2,5-hexanedione . Electrophysiological techniques including intracellular microelectrode recording of nerve-evoked and spontaneously occurring synaptic potentials, iontophoresis of neurotransmitter, and voltage clamp of presynaptic and postsynaptic membrane ionic current have proven to be especially useful in analyzing the cellular mechanisms by which these toxicants affect neurotransmission . The process of synaptic transmission can be broadly subdivided into those processes associated with transmitter synthesis, storage and release and sometimes termination of transmitter action (presynaptic processes), and those processes associated with binding of transmitter to its receptors on the receiving cell, activation of the receptor-associated ionic channel and degradation of chemical transmitter (postsynaptic processes) . The processes associated with release of neurotransmitter are the target of a number of naturally-occurring toxins and environmentally important toxicants . General mechanisms by which these agents disrupt presynaptic processes associated with transmission include: prevention or disruption of axonal excitability (pyrethroid insecticides); disruption of calcium-dependent neurotransmitter release (heavy metals, antibiotics, certain snake and spider venom toxins, botulinum toxin); and disruption of intracellular buffering of calcium (heavy metals), Mechanisms by which these agents may disrupt postsynaptic processes include effects on transmitter degradation (organophosphates) or effects on the postsynaptic membrane receptors or associated ionic channels (organophosphates, antibiotics, and perhaps pyrethroids) . Microelectrode studies have shown that cadmium, lead and mercury (organic and inorganic forms) suppress release of neurotransmitter by presynaptic mechanisms and increase spontaneous discharge of transmitter quanta from the presynaptic nerve terminal . This has led to the suggestion that a component of synaptic toxicity of these agents entails block of Ca entry into and buffering by the presynaptic nerve terminals . Conventional and patch voltage clamp studies have been used to measure effects of neurotoxicants on ionic currents carried through voltage-sensitive and receptor-operated ionic channels.(ABSTRACT TRUNCATED AT 400 WORDS)

Biochem Biophys Res Commun, 1988 Aug 30, 155(1), 263 - 9
Botulinum toxin D ADP-ribosylates a 22-24 KDa membrane protein in platelets and HL-60 cells that is distinct from p21N-ras; Banga HS et al.; Botulinum toxin D ADP-ribosylates a 22-24 KDa protein in platelets, GH3 and HL-60 cells, and a mouse T-cell line CTLL . In platelet homogenates the protein is localized to the membrane fraction, and ADP-ribosylation can also be produced in saponin-permeabilized and intact cells . In the latter, the toxin also potentiates secretion caused by a variety of agonists . In platelets and HL-60 cells the toxin substrate is shown, by use of anti-ras monoclonal antibody, to be distinct from the ras family of proteins . This toxin substrate may represent an additional class of proteins involved in stimulus-response coupling.

Neurology, 1988 Aug, 38(8), 1220 - 5
Effects of botulinum toxin injections on speech in adductor spasmodic dysphonia; Ludlow CL et al.; Adductor spasmodic dysphonia involves an overadduction of the vocal folds during speech causing uncontrolled voice and pitch breaks and slow, effortful speech . The disorder is resistant to speech therapy and often recurs following initial benefit from unilateral recurrent laryngeal nerve resection . Botulinum toxin injections into multiple sites of the thyroarytenoid muscle on one side were performed in 16 patients . Speech was recorded prior to injection and three times post-injection . Symptoms were measured by two examiners from speech spectrograms without knowledge of speaker identity or recording session . Significant (p less than or equal to 0.03) reductions in pitch and voice breaks, phonatory aperiodicity, and sentence time occurred only when injections resulted in unilateral vocal fold paralysis . Symptoms returned with the restoration of vocal fold movement, 3 months later . Reduction in speed of swallowing without aspiration was reported in 80% of cases . Although speech volume was reduced, there were no instances of aphonia.

J Neurochem, 1988 Aug, 51(2), 522 - 7
Tetanus toxin and botulinum A and C neurotoxins inhibit noradrenaline release from cultured mouse brain; Habermann E et al.; Primary nerve cell cultures from the brainstem of embryonic mice take up {3H}noradrenaline . Release can be evoked by high K+ or sea anemone toxin II and depends on Ca2+ . The cultures allow neurochemical studies on the long-term actions of clostridial neurotoxins . Tetanus and botulinum A and C neurotoxins partially inhibit the absolute and fractional release evoked by high K+, as well as the fractional basal release . The detection limit for the toxins is below 5 pM . Total radioactivity is higher in the poisoned cultures, although the initial velocity of uptake is not measurably influenced by tetanus or botulinum A toxin . Pretreatment with neuraminidase prevents the effects of botulinum A toxin and diminishes those of botulinum C and tetanus toxins . Within 6 days, the cultures partially recover from tetanus toxin poisoning . Antitoxin prevents the actions of the toxin, but only slightly promotes recovery . The data indicate close pharmacological analogies between the clostridial neurotoxins.

Ophthalmology, 1988 Aug, 95(8), 1042 - 5
Hemifacial spasm due to intracranial tumor . An international survey of botulinum toxin investigators; Sprik C et al.; Hemifacial spasm (HFS) due to intracranial mass lesions is rare . Most cases are thought to be due to compression of the facial nerve by small vessels near the root of the facial nerve . A survey was undertaken of all botulinum toxin investigators to determine the incidence of imaged mass lesions causing HFS . Responders contributed information on 1676 patients with HFS . Of this group, nine tumors were reported for an incidence of 0.54% of patients . However, of this group only 52.5% underwent computed tomography (CT) or magnetic resonance (MR) scanning so the incidence of tumor causing HFS could be as high as 1.0% . No one tumor type was predominant, and most patients were women older than 50 years of age . The incidence compares with another large series of HFS patients in which one tumor was found in 367 patients . The authors also report as an illustrative case a 26-year-old man with HFS due to a presumed lipoma of the cerebellopontine angle . This diagnosis can be made with increased certainty with MR scanning . If the incidence of unsuspected diagnostically significant mass lesions is 1 in 200 patients with HFS referred for botulinum toxin injection, the cost of detecting one such lesion would be $100,000 at an average imaging cost of $500 per MR imaging or CT examination . Although mass lesions are uncommon, any patient with HFS whose general clinical course could justify intervention should be considered for imaging studies to rule out treatable conditions other than vascular compression.

Ital J Neurol Sci, 1988 Aug, 9(4), 337 - 44
Beneficial effect of botulinum A toxin in blepharospasm: 16 months' experience with 16 cases; Maurri S et al.; After introducing the problem of blepharospasm, we report our experience on treatment with purified botulinum A toxin in 16 cases of blepharospasm, symptomatic in two and essential in 14, than had not responded to drugs . The changes in intensity and frequency of spasm after treatment were evaluated on a clinical scale and by review of videotapes . The beneficial effect appeared within a week in most patients, lasting from 6 to 28 weeks (mean 13), and reached the maximum at the third-seventh week . Mild spasms and female patients responded better . Repeated injections were followed by better response to the drug . Complications, exclusively local, were represented by transient corneal exposure, ptosis, lacrimation or diplopia.

Can J Neurol Sci, 1988 Aug, 15(3), 276 - 80
Botulinum toxin injections in the treatment of blepharospasm, hemifacial spasm, and eyelid fasciculations; Kraft SP et al.; Seventy-six patients with blepharospasm (mean age 56.9 years) received 248 injection treatments with botulinum A exotoxin (mean 3.1 treatments per patient): 87.0% of treatments led to total relief of spasms for a mean interval of 14.1 weeks . The average duration of response remained fairly constant over the first six injection series, although patients with the most severe spasms had shorter intervals than patients with less severe symptoms . Twenty patients with hemifacial spasm (mean age 56.9 years) received 44 treatments (mean 1.9 treatments per patient): In 93.1% of cases there was total relief of periocular and perioral spasms, with a mean interval of 17.4 weeks . The average duration of response for the third series of treatments was much shorter than the mean durations for the first two treatments . Side effects were always transient and included ptosis (23.3%), dry eyes (18.1%), tearing (5.5%), and strabismus (1.4%) . No patient had a systemic reaction to the drug . Chronic benign eyelid fasciculations were also successfully treated in 3 patients with single treatments.

Am J Ophthalmol, 1988 Jul 15, 106(1), 45 - 7
The effect of omitting botulinum toxin from the lower eyelid in blepharospasm treatment; Frueh BR et al.; We randomly selected 26 patients with essential blepharospasm to receive either botulinum toxin or saline injection in their lower eyelids to evaluate the necessity of lower eyelid injection to relieve blepharospasm . As diplopia may occur from botulinum toxin injections for blepharospasm, most commonly from injection of the lower eyelid, and surgical relief of blepharospasm is often achieved by excision of only the upper eyelid protractors, omission of toxin from the lower eyelid seemed both desirable and possible . All patients received botulinum toxin in the upper eyelids, above the eyebrows, across the glabella, and near the lateral canthus . Thirteen of 15 patients who received saline in their lower eyelids had relief of spasm, with the same spasm-free interval as those who received toxin . We recommend avoiding injection of toxin in the medial two thirds of the lower eyelid in order to diminish the likelihood of diplopia from inferior oblique muscle paresis.

J Neurol Neurosurg Psychiatry, 1988 Jul, 51(7), 920 - 3
Botulinum toxin in spasmodic torticollis; Stell R et al.; Ten patients with spasmodic torticollis were treated by injection of a total dose of 30 ng of botulinum toxin type A into the affected sternomastoid and posterior cervical muscles . Nine patients reported improvement in head position and control, which was confirmed in seven cases by clinical assessment and "blind" videotape ratings before and 6 weeks after injection . Five patients who had pain reported relief . Seven patients had mild transient dysphagia after injection; two who were given a more concentrated solution of the toxin developed more severe dysphagia, but this also recovered . Other minor transient side effects included weakness of the voice and local pain . The beneficial effects of botulinum toxin injections lasted some 2 to 3 months . A slight reduction in the total dose of toxin injected avoided the main side effects, and this method of treatment appears to offer successful control of head position and pain in the majority of patients with torticollis.

J Neurol Neurosurg Psychiatry, 1988 Jun, 51(6), 767 - 72
Blepharospasm: a review of 264 patients; Grandas F et al.; The natural history and response to different treatments have been evaluated in 264 patients with blepharospasm . The mean age of onset was 55.8 years and there was a female preponderance of 1.8 to 1 . Dystonia elsewhere was found in 78% of patients, usually in the cranial-cervical region, and appeared to follow a somatotopic progression . A family history of blepharospasm or dystonia elsewhere was found in 9.5% of cases, which suggests a genetic predisposition . Ocular lesions preceded the onset of blepharospasm in 12.1% of cases . The response to drugs was inconsistent, although initial improvement was experienced by one fifth of patients treated with anticholinergics . Twenty-nine bilateral facial nerve avulsion operations were performed with benefit in 27 cases; but recurrences appeared in 22, on average one year after surgery . Botulinum toxin injections were performed in 151 patients . Significant improvement was achieved in 118 cases . Mean duration of benefit was 9.2 weeks . Transient ptosis and diplopia were the commonest side effects.

Laryngoscope, 1988 Jun, 98(6 Pt 1), 636 - 40
Clinical and laboratory characteristics of focal laryngeal dystonia: study of 110 cases; Blitzer A et al.; Spastic dysphonia is a syndrome often producing a strain-strangle voice . We have previously classified most of these patients as having focal laryngeal dystonia, a disorder of central motor processing . In a study of 1,280 cases of dystonia registered at the Dystonia Clinical Research Center at the Columbia-Presbyterian Medical Center, we found 110 patients who had vocal cord involvement . These patients had historical information evaluated for age of onset (mean 34.6 years), duration of symptoms (mean 13.8 years), sex (1.4:1 female to male) family history (positive in 23%), and primary (66%) and secondary (34%) etiology; neurological evaluation for other dystonic involvement (25% with segmental cranial involvement, 23% with generalized dystonia) or tremor (irregular 23%, regular 6% on EMG) . Treatment options were evaluated and included speech therapy, psychotherapy, biofeedback (with limited success), systemic medication (limited success except in abductor cases), nerve section (with late failure rate), and the use of botulinum toxin (improvement in all 34 injected patients).

J Neurochem, 1988 Jun, 50(6), 1808 - 16
Characterization of the inhibitory action of botulinum neurotoxin type A on the release of several transmitters from rat cerebrocortical synaptosomes; Ashton AC et al.; Under optimised conditions for intoxication, botulinum neurotoxin type A was shown to inhibit approximately 90% of Ca2+-dependent K+-evoked release of {3H}acetylcholine, {3H}noradrenaline, and {3H}dopamine from rat cerebrocortical synaptosomes; cholinergic terminals were most susceptible . In each case, the dose-response curve for the neurotoxin was extended, with about 50% of evoked release being inhibited at approximately 10 nM whereas 200 nM was required for the maximal blockade . This may suggest some heterogeneity in the release process . The action of the toxin was time and temperature dependent and appeared to involve binding and sequestration steps prior to blockade of release . The neurotoxin failed to exert any effect on synaptosomal integrity or on Ca2+-independent release of the transmitters tested; it produced only minimal changes in neurotransmitter uptake although small secondary effects were detected with cholinergic terminals . Blockade by the neurotoxin of Ca2+-dependent resting release of transmitter was apparent; Sr2+, Ba2+, or high concentrations of Ca2+ restored the resting release of 3H-catecholamine but not {3H}acetylcholine . Interestingly, none of the latter conditions or 4-aminopyridine could reverse the toxin-induced blockade of evoked release . This lack of specificity in its action on synaptosomes, and other published findings, lead to the conclusion that toxin-sensitive component(s) exist in all nerve terminals that are concerned with transmitter release.

J Pharmacol Exp Ther, 1988 Jun, 245(3), 867 - 72
Use of pharmacologic antagonists to deduce commonalities of biologic activity among clostridial neurotoxins; Simpson LL; The pharmacologic activity of several clostridial neurotoxins was assayed on the mouse phrenic nerve-hemidiaphragm preparation . The substances that were assayed included botulinum neurotoxin types A, B, C and E and tetanus toxin . Experiments were done in the presence or absence of antagonists that inhibit either the internalization of toxins or intracellular expression of toxicity . Ammonium chloride and methylamine hydrochloride, agents that inhibit toxins that enter cells by receptor-mediated endocytosis, antagonized botulinum and tetanus neurotoxins . The magnitude of antagonism was substantial for all toxins . Calcium, 3,4-diaminopyridine and guanidine, agents that alter the intracellular expression of toxicity, produced a variable result . They were effective antagonists of botulinum neurotoxin type A, but they were less effective or inactive against the other neurotoxins . The ability of 3,4-diaminopyridine and guanidine to antagonize botulinum neurotoxin type A was highly calcium dependent . When ambient levels of the cation were reduced from 1.8 to 1.0 mM, the activity of the drugs was substantially reduced . The ability of these drugs to produce antagonism was also time dependent . When added simultaneously with toxin, they were maximally active; when added at later times, activity was diminished . A host of agents that alter intracellular levels of cyclic AMP, including theophylline, forskolin, isobutylmethylxanthine and cholera toxin, were evaluated as potential neurotoxin antagonists . Theophylline and isobutylmethylxanthine produced a transient increase in nerve-evoked muscle twitch . None of the drugs that alter tissue levels of cyclic AMP had a universal effect in antagonizing clostridial toxins . The data here have been compared with published data on drugs that antagonize binding of botulinum toxin and tetanus toxin.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochem Biophys Res Commun, 1988 May 16, 152(3), 957 - 61
Separation of the 24 kDa substrate for botulinum C3 ADP-ribosyltransferase and the cholera toxin ADP-ribosylation factor; Tsai SC et al.; Botulinum C3 ADP-ribosyltransferase modifies a approximately 24 kDa membrane protein believed to bind guanine nucleotides . Cholera toxin ADP-ribosylation factors are approximately 19 kDa GTP-binding proteins that directly activate the toxin . To evaluate a possible relationship between C3 ADP-ribosyltransferase substrate and ADP-ribosylation factor, they were partially purified from bovine brain . ADP-ribosylation factor, but not C3 ADP-ribosyltransferase substrate, stimulated auto-ADP-ribosylation of the choleragen A1 subunit whereas C3 ADP-ribosyltransferase substrate, but not ADP-ribosylation factor, was ADP-ribosylated by C3 ADP-ribosyltransferase . Thus, although both may be GTP-binding proteins, no functional similarity between ADP-ribosylation factor and C3 ADP-ribosyltransferase substrate was found.

FEBS Lett, 1988 May 9, 232(1), 217 - 20
Escherichia coli hemolysin permeabilizes small unilamellar vesicles loaded with calcein by a single-hit mechanism; Menestrina G; Escherichia coli hemolysin produces small unilamellar lipid vesicles permeable to the fluorescent dye calcein by forming pores through their membrane . The process of permeabilization proceeds as a pseudo first-order reaction, indicating that the toxin is active as a monomer; consistently no evidence for cooperativity has been found in a dose-response titration . The rate of interaction increases on lowering the pH of the solution and by introducing negatively charged lipids into the vesicles . The overall pore formation mechanism resembles that of other toxins of bacterial origin such as colicins, diphtheria, tetanus and botulinum toxin.

Arch Int Pharmacodyn Ther, 1988 May-Jun, 293, 69 - 83
Cardiac effects of botulinal toxin; Lamanna C et al.; Crystalline type A botulinal toxin rapidly caused temporary bradycardia and electrocardiographic (ECG) changes in mice, rats, rabbits and dogs . In addition, in the dog the force of contraction was measured and found to be depressed . The ECG changes were indicative of conduction defects . The hemagglutinin present in the toxin played no role in the effects on the heart, since a derivative toxin without hemagglutinin also caused these phenomena . The cardiac effects were spontaneously reversible in the intact animal without removal of the toxin . On the other hand, in the in vitro isolated heart of the rat, recovery from the cardiac effects occurred only after the toxin was washed out of the preparation . The findings are consistent with, but do not prove, a physical rather than a chemical mechanism for the effects of toxin on the heart.

Br J Ophthalmol, 1988 May, 72(5), 361 - 2
Treatment of senile entropion with botulinum toxin; Clarke JR et al.; Botulinum toxin was used to treat senile entropion in 12 patients by injection into the preseptal orbicularis muscle of the lower lids . It produced relief of symptoms in 10 patients but this was transient, lasting for an average of 14.8 weeks before orbicularis function returned . The technique is simple and easy to perform, and may be useful in selected patients, but surgery offers a more permanent result.

Muscle Nerve, 1988 May, 11(5), 493 - 501
Spinal irradiation does not inhibit distal axonal sprouting; Pamphlett RS; In an attempt to determine the relative importance of the nerve cell body and of the axon in initiating and controlling axonal regeneration, nerve cell bodies were irradiated and the ability of the distal axon to sprout was examined . Mice were subjected to either 25 or 50 Gray (Gy) of x-irradiation localized to the lumbar spinal cord . After times varying from 1 day to 6 months after irradiation, a sublethal dose of botulinum toxin (BoTx) was injected into the calf muscles of one leg . The soleus muscle was examined histologically after times varying from 1 week to 6 months after injection, and BoTx-induced ultraterminal axonal sprouting was assessed by the number of motor endplates showing sprouts, the length of the sprouts, and the long term endplate morphology . Apart from some irradiated subgroups having slightly shorter sprout lengths, no significant differences were found between irradiated and nonirradiated groups . The results suggest either that the processes in the nerve cell body responsible for initiating and supporting axonal growth are resistant to large doses of irradiation, or that growth regulatory mechanisms in the distal axon are under local control.

J Urol, 1988 May, 139(5), 919 - 22
Effects of botulinum A toxin on detrusor-sphincter dyssynergia in spinal cord injury patients; Dykstra DD et al.; We evaluated the ability of low doses of botulinum A toxin, an inhibitor of acetylcholine release at the neuromuscular junction, to denervate and relax the spastic rhabdosphincter in 11 men with spinal cord injury and detrusor-sphincter dyssynergia . Toxin concentration, injection volume, percutaneous versus cystoscopic injection of the sphincter and number of injections were evaluated in 3 treatment protocols . All 10 patients evaluated by electromyography after injection showed signs of sphincter denervation . Bulbosphincteric reflexes in the 10 patients evaluated after injection were more difficult to obtain, and they showed a decreased amplitude and normal latency . The urethral pressure profile in the 7 patients in whom it was measured before and after treatment decreased an average of 27 cm . water after toxin injections . Post-void residual urine volume decreased by an average of 146 cc after the toxin injections in 8 patients . In the 8 patients for whom it could be determined toxin effects lasted an average of 50 days . The toxin also decreased autonomic dysreflexia in 5 patients.

Biochem Biophys Res Commun, 1988 Apr 29, 152(2), 926 - 32
Effect of botulinum D toxin on neutrophils; Mege JL et al.; Activated botulinum D toxin ADP-ribosylates a 22 kDa molecular weight protein in homogenates obtained by sonication of a suspension of rabbit peritoneal neutrophils . The ADP-ribosylation catalyzed by activated botulinum D toxin is inhibited in homogenates obtained from cells pretreated with the toxin, suggesting that it is able to enter into these cells and be activated by them . The rise in intracellular concentration of free calcium in toxin treated cells stimulated by fMet-Leu-Phe is similar to that found in control cells . The basal concentration of intracellular free calcium is significantly elevated in neutrophils treated with the intact but not with the activated form of the botulinum D toxin . Superoxide generation in control and native toxin treated cells stimulated with fMet-leu-Phe, phorbol 12-myristate 13-acetate or opsonized zymosan is the same . The release of beta-glucosaminidase produced by fMet-Leu-Phe or Concanavalin A in botulinum D toxin treated neutrophils was slightly higher than the corresponding release in control cells . Furthermore, the fMet-Leu-Phe-induced increase in the amount of actin associated with the cytoskeleton is not inhibited by botulinum D toxin . These results suggest that the 22 kDa protein which can be ADP-ribosylated by botulinum D toxin is not involved in these stimulated neutrophil responses.

Ophthalmology, 1988 Apr, 95(4), 473 - 80
Botulinum toxin A-induced protective ptosis in corneal disease; Kirkness CM et al.; Botulinum toxin A produces a temporary, flaccid ptosis when injected into the levator palpebrae superioris muscle . The resulting protective ptosis was used to aid healing in 21 cases of indolent ulceration, and, prophylactically, in 4 cases of neuroparalytic keratitis . Of the indolent ulcers, 90% healed completely . In all but one case, the cornea was covered completely by the lid and complete ptosis was produced in 75% of cases in an average of 3.6 days, lasting for 16 days on average before recovery began . Recovery of levator function was complete in 8.5 weeks on average . Superior rectus underaction was seen in 68% of cases but this recovered completely in all cases in an average of 6 weeks . Impression cytology showed a trend toward normal conjunctival morphology as healing progressed.

Experientia, 1988 Mar 15, 44(3), 224 - 6
Inhibition by tetanus and botulinum A toxin of the release of {3H}noradrenaline and {3H}GABA from rat brain homogenate; Habermann E; Rat brain homogenate was preloaded with {3H}noradrenaline or {3H}GABA and stimulated with high K+ . Tetanus toxin and botulinum A neurotoxin partially prevent the evoked {3H}noradrenaline release in the same range of toxin concentrations starting below 10(-10) M . In contrast, release of gamma-amino butyric acid (GABA) is much more sensitive to tetanus than to botulinum A toxin.

Am J Ophthalmol, 1988 Mar 15, 105(3), 313 - 5
Negative antibody response to long-term treatment of facial spasm with botulinum toxin; Gonnering RS; Over a period of 163 weeks, 223 injections of botulinum A toxin were administered to 38 patients with facial spasm syndromes . The maximum cumulative toxin dose was 553 units, the maximum number of injections in any given patient was 16, and the maximum dosage of any given injection was 52.5 units . Sera from these patients showed no antibody production when measured with a standard mouse lethality bioassay.

Neurology, 1988 Mar, 38(3), 452 - 8
Spinal cord TRH deficiency is associated with incomplete recovery of denervated muscle in the rat; Van den Bergh P et al.; The raphe-spinal pathway, which contains co-localized serotonin (5-HT), thyrotropin-releasing hormone (TRH), and several TRH-prohormone-derived non-TRH peptides, projects to the ventral horn of the spinal cord . Pharmacologic ablation of this pathway with the 5-HT neurotoxin, 5,7-dihydroxytryptamine, in neonatal rats resulted in deficient recovery of plantar foot muscles, functionally denervated with botulinum toxin type A . Failure of reinnervation was suggested by slower and incomplete recovery of the plantar foot compound muscle action potential amplitude and by a reduced mean diameter of plantar foot muscle fibers in ablated rats . These findings indicate that deprivation of alpha motor neurons from descending raphe-spinal input interferes with their ability to respond to muscle-derived signals for reinnervation.

Aust N Z J Ophthalmol, 1988 Feb, 16(1), 15 - 20
Botulinum toxin in ophthalmology; Dunlop D et al.; Alan B . Scott selected, researched and developed Type A Botulinum toxin for clinical use in ophthalmology . This unique drug has proved invaluable for treatment of a number of conditions which are difficult to treat in ophthalmology and in a variety of other disciplines . The indications, methods and problems of its use are described and the results of treatment of 133 patients are discussed . Up to December 1986 over 13,000 patients have been treated in a multicentre international trial without significant complications.

Laryngoscope, 1988 Feb, 98(2), 193 - 7
Localized injections of botulinum toxin for the treatment of focal laryngeal dystonia (spastic dysphonia); Blitzer A et al.; Spastic dysphonia is a condition producing a strain-strangle phonation . We have previously classified most of these patients as having focal laryngeal dystonia, a disorder of central motor processing . The initial success of recurrent nerve section in many of these patients has been followed by recurrence of symptoms in months to years . Bilateral involvement of the vocal cords with hyperfunction of the nonparalyzed vocal cord could explain these failures . Injection of botulinum toxin (BOTOX) has been effective treatment for many focal dystonias . We have treated more than 100 patients with dystonia including five with laryngeal dystonia . All of the patients laryngeal had dramatic improvement after 48 to 72 hours; benefit lasted 3 to 9 months for each injection period . BOTOX injection can be performed on awake, ambulatory patients . Bilateral treatment and titration of dose can achieve the desired degree of weakness.

J Neurochem, 1988 Feb, 50(2), 431 - 9
Energy metabolism and quantal acetylcholine release: effects of botulinum toxin, 1-fluoro-2,4-dinitrobenzene, and diamide in the Torpedo electric organ; Dunant Y et al.; In the Torpedo electric organ, a modified nerve-muscle system, type A botulinum toxin blocked the release of acetylcholine (ACh) quanta, both neurally evoked and spontaneous . At the same time, the toxin increased the release of a class of small miniature potentials (the subminiature potentials), reduced the ATP and more the creatine phosphate content of the tissue, and impaired the activity of creatine kinase (CK) . Thus, we compared this pattern of changes with those provoked by 1-fluoro-2,4-dinitrobenzene (FDNB), an efficient inhibitor of CK . As expected, FDNB rapidly inactivated CK, which resulted in a profound depletion of ATP whereas the stores of creatine phosphate were preserved . In addition, FDNB caused conspicuous morphological alterations of nerve endings and ACh depletion . This agent also suppressed evoked and spontaneous quantal release whereas the occurrence of subminature potentials was markedly increased . Diamide, a penetrating thiol oxidizing substance, provoked first a transient rise in quantal ACh release and then blockade of transmission with, again, production of a large number of subminiature potentials . Creatine phosphate was depleted in the tissue by diamide, the ATP content reduced, and CK activity partly inhibited . The morphology of nerve terminals did not show obvious changes with either diamide or botulinum toxin at the stage of transmission failure . Although the three poisons acted by different mechanisms, this resulted in a rather similar pattern of physiological changes: failure of quantal release and enhancement of subquantal release . These results and experiments on synaptosomes indicated that CK inhibition was probably a crucial mechanism for FDNB but not for diamide or botulinum intoxication.(ABSTRACT TRUNCATED AT 250 WORDS)

Experientia, 1988 Jan 15, 44(1), 18 - 20
Zinc antagonizes the effect of botulinum type A toxin at the mouse neuromuscular junction; Nishimura M et al.; Zn2+ (10-100 microM) elevated the frequency of miniature end-plate potentials (MEPPs) in the mouse diaphragm . The effect did not depend on external Ca2+ . Botulinum type A toxin (BTXA, 50 ng/ml) abolished MEPPs almost completely within 30 min . Zn2+ (100 microM) restored MEPPs and increased their frequency after they had been abolished by BTXA in Ca2+ -free solutions . The antagonistic effect of Zn2+ in the Ca2+ -free solution was reduced by exposing the diaphragm to the toxin in the Ca2+ -free solutions containing high K+ . Thus, the action of BTXA is probably enhanced by depolarization of the motor nerve terminals.

J Biol Chem, 1988 Jan 15, 263(2), 696 - 700
Botulinum C2 toxin ADP-ribosylates cytoplasmic beta/gamma-actin in arginine 177; Vandekerckhove J et al.; Isolated cytoplasmic actin of human platelet and pig liver actin, but not rabbit skeletal muscle actin, was ADP-ribosylated by botulinum C2 toxin in the presence of {32P}NAD . Tryptic digestion of the {32P}ADP-ribosylated platelet actin generated two labeled peptides: a soluble peptide covering residues 174-183 and an insoluble fragment containing residues 148-183 . Further digestion of these two peptides with thermolysin yielded the same radioactive peptide, which was in both cases peptide 175-177 . Amino acid sequence analysis of peptides 174-183 and 175-177 located the ADP-ribosylation on Arg177.

J Fr Ophtalmol, 1988, 11(3), 237 - 40
{Treatment of blepharospasm and facial hemispasm by injection of botulinum toxin}; Saraux H; 13 patients with hemifacial spasm and 19 patients with blepharospasm (essential blepharospasm or Meige syndrome) were treated with injection of botulinum A toxin in the eyelids . The effectiveness of this therapy is constant and the treatment offers relief to almost all patients . The response times for repeated treatment was 6 months for blepharospasm and 7 months for hemifacial spasm . Repeated injections were performed in several patients and gave the same improvement . Transient ptosis or diplopia were observed in 11 patients . Possible systemic side effects (respiratory trouble) were documented in two patients . Botulinum toxin injection is an effective treatment but the relief is usually temporary and repeated treatments are necessary.

Eye, 1988, 2 ( Pt 1), 24 - 8
Botulinum toxin therapy for squint; Lee J et al.; Four hundred and five patients have been treated with injections of Botulinum neurotoxin A to extraocular muscles in the Botulinum Toxin Clinic at Moorfields Eye Hospital from November 1982 until the present . The indications and outcome of therapy are described and discussed.

Toxicon, 1988, 26(4), 329 - 36
Tetanus and botulinum toxins block the release of acetylcholine from slices of rat striatum and from the isolated electric organ of Torpedo at different concentrations; Rabasseda X et al.; Tetanus toxin, like botulinum toxin type A, blocks cholinergic synaptic transmission at the central and peripheral nervous systems . Nevertheless, the diseases induced by the two toxins are different since tetanus toxin induces a spastic paralysis and botulinum toxin elicits a flaccid paralysis . Thus, we have investigated the sensitivity of a central and a peripheral cholinergic synapse to these two toxins . We have studied the action of both poison on the release of acetylcholine from slices of the rat striatum and from the isolated electric organ of Torpedo, which is homologous to the neuromuscular junction . Acetylcholine release from the rat striatum was continuously monitored by a chemiluminescent method . The secretion of acetylcholine from the electric organ was estimated both by measuring the amplitude of the evoked electrical discharge from stacks of electroplaques, and by continuously monitoring the neurotransmitter release from isolated nerve terminals . Tetanus toxin blocks the electrical discharge of electric organ prisms, and also impairs the release of acetylcholine from the Torpedo electric organ nerve endings . Our results on acetylcholine release show that tetanus toxin is more potent than botulinum toxin type A at the central cholinergic synapse (tetanus/botulinum toxins potency ratio about 100-200) whereas botulinum toxin is the most potent at the peripheral cholinergic synapse (botulinum/tetanus toxins potency ratio about 100).

Graefes Arch Clin Exp Ophthalmol, 1988, 226(2), 141 - 4
Botulinum toxin treatment of acute sixth and third nerve palsy; Metz HS et al.; Thirty-four patients with acute sixth nerve palsy and nine patients with acute third nerve palsy were treated with botulinum toxin injection to the antagonist, nonparalytic horizontal rectus muscle . In a control group of 52 patients with acute sixth nerve palsy not treated with botulinum in the acute stage, only 16 (31%) recovered spontaneously and did not require surgery . Twenty-two of the 31 surviving patients who could be followed with acute sixth nerve palsy had lateral rectus recovery and surgery was avoided . Four required prisms in their glasses to obtain fusion . Nine patients developed chronic sixth nerve palsy and required surgery . In this group of acute sixth nerve palsy patients, eleven were bilateral . Seven of these eleven developed chronic sixth nerve paralysis, and required strabismus surgery . This suggests the prognosis for recovery following botulinum treatment in cases of acute bilateral sixth nerve palsy is not as good as in the unilateral cases . Botulinum toxin treatment does not appear to be effective in chronic sixth nerve palsy, as judged by results of treatment in one patient known to have a chronic palsy . Nine of nine patients with acute third nerve palsy had medial rectus recovery with fusion horizontally in primary gaze . None have required surgery . Only four of nine showed improvement in vertical rotations . The remaining five patients avoid vertical diplopia by a compensatory chin position . Botulinum toxin treatment of patients with acute sixth and third nerve palsy appears beneficial . However, since some in this group of patients may recover spontaneously, a randomized, double-blind study may be necessary to more definitively determine the effectiveness of this therapy.

Ann Clin Lab Sci, 1988 Jan-Feb, 18(1), 58 - 71
Bacterial toxins; Lubran MM; Many bacterial toxins are proteins, encoded by the bacterial chromosomal genes, plasmids or phages . Lysogenic phages form part of the chromosome . The toxins are usually liberated from the organism by lysis, but some are shed with outer membrane proteins in outer membrane vesicles . An important non-protein toxin is lipopolysaccharide or endotoxin, which is a constituent of the cell wall of gram negative bacteria . Toxins may damage the eukaryotic cell membrane by combining with some structural component, or otherwise alter its function . Many toxins combine with specific receptors on the surface membrane, frequently glycoproteins or gangliosides, and penetrate the cell to reach their intracellular target . A common mechanism of entry is absorptive endocytosis . Many protein toxins have an A-B structure, B being a polypeptide which binds to the receptor and A being an enzyme . Many toxins are activated, either when produced by the bacterium or when bound to the membrane receptor, by proteases (nicking) . An enzymatic process common to many toxins is adenosine diphosphate (ADP)-ribosylation of the adenylate cyclase regulatory proteins, leading to an increase in intracellular cyclic adenosine monophosphate (cAMP) . This is the mechanism of action of cholera toxin . Diphtheria toxin catalyzes the transfer of ADP-ribose to elongation factor-2, inhibiting protein synthesis . Most toxins act on the target cells to which they bind, but tetanus toxin, and, to a lesser degree, botulinum toxin, ascend axons and affect more distant structures . Although many toxin effects caused by bacteria have been described, only a few toxins have been identified, characterized, and their mode of action determined at the molecular level . The best known of these are discussed.

Mov Disord, 1988, 3(4), 333 - 5
Systemic toxicity of botulinum toxin by intramuscular injection in the monkey; Scott AB et al.; Botulinum toxin (Oculinum) was injected intramuscularly into eight monkeys . The LD50 dose is estimated to be approximately 39 U/kg body weight . The lowest dose that caused systemic toxicity, 33 U/kg, was close to the fatal dose range, 38-42 U/kg.

Eye, 1988, 2 ( Pt 5), 506 - 16
Monitoring ocular disease by impression cytology; Adams GG et al.; The technique for obtaining and processing conjunctival impression cytology is described . The method of grading the stained samples is outlined . Our findings in various ocular conditions with particular emphasis on; alkali burns, uveitis, hypopyon ulcer and Botulinum toxin protective ptosis are shown . Cytology from severely damaged eyes is highly abnormal, improving as the ocular disease settles . In alkali burns a return to normal cytology does not correlate with return of corneal clarity . Loss of goblet cells is a common finding in the ocular diseases demonstrated, and appears to be a non-specific indicator of ocular problems.

Toxicon, 1988, 26(11), 1089 - 94
Mosquito inoculation: an alternative bioassay for toxins; Turell MJ et al.; Mosquitoes were evaluated as a bioassay host for several classes of biological toxins . Mosquitoes were sensitive to snake toxic or neurotoxic phospholipase A2 enzymes (but not to nontoxic phospholipase A2 enzymes), cobrotoxin, saxitoxin, microcystin and the scorpion insect sodium channel toxin . Mosquitoes were not sensitive to ricin, diphtheria toxin, anthrax toxin, botulinum toxin, tetanus toxin, conotoxin G or a scorpion sodium channel toxin toxic to mammals . Specific antisera neutralization tests with mosquitoes gave comparable results to those of a mouse assay . The mosquito is a suitable bioassay animal for many, but not all biological toxins, and offers a safer, more efficient and economical assay than mice.

C R Acad Sci III, 1988, 306(15), 483 - 8
{Ganglionic synapses of Aplysia as a model for the study of the mechanism of action of botulinum neurotoxins}; Poulain B et al.; The action of type A and type B botulinum neurotoxin on neurotransmitter release was studied on identified ganglionic synapses of Aplysia . Using this model, we have shown that botulinum neurotoxins at concentrations used in vertebrate preparations had the same specificity of action and that both heavy and light chains of these toxins are intracellularly required to inhibit neurotransmitter release.

Zentralbl Bakteriol Mikrobiol Hyg {B}, 1988 Jan, 185(4-5), 368 - 78
{Microbiological research methods of drinking water regulation in West Germany from 1986 . Suitability of the specifications of DIN 38411, Part 7, for the detection of sulfite-reducing, spore-forming anaerobes (Clostridia)}; Schneider J et al.; The drinking-water regulations of the Federal Republic of Germany, from 22.05.1986, contains in paragraph 1 the instructions: "Drinking-water must be free of pathogens", and further in paragraph 11, "Responsibilities of the employer or other owner of a water supplying facility", include that: "The official authority may direct, that the employer...of a water supplying facility has to extend or has to cause to extend the microbiological examinations in order to determine, that...sulfite-reducing, spore-forming anaerobes (Clostridia) can not be detected in 20 ml of water..." The drinking-water regulations do not prescribe a bacteriological examination method in detail . Appendix 1 rules only that the examination for sulfite-reducing, spore-forming anaerobes (Clostridia) has to be performed after heating the sample to 75 degrees C (+/- 5 degrees C) for 10 min, by either the multiple-tube or membrane filtration method and cultivation in DRCM1-medium . If growth occurs, the presence of Clostridia must be confirmed by anaerobic and aerobic subcultivation . Furthermore, a DIN-instruction (DIN 38411, part 7) exists, which prescribes a detailed procedure for multiple-tube and membrane filtration methods, but does not provide for strict anaerobiosis . We were, however, unable to detect Clostridia in a multitude of water samples with the methods of the DIN-regulation . In order to examine if neglect of strict anaerobiosis was the reason for these failures, we checked the suitability of the DIN-regulation for the isolation of Clostridia from drinking water . In preliminary tests we examined up to four strains of the species C . botulinum, C . cadaveris, C . cochlearium, C . difficile, C . innocuum, C . perfringens and C . tertium for their ability to form heat-resistent spores in four sporulation media . It was, however, not possible to find a medium, in which all strains could sporulate within one week . In order to characterize the detection of these anaerobes in water, one particularly well-sporulating strain of each of the following, C . cadaveris, C . difficile and C . perfringens, was selected and the multiple-tube and membrane-filtration methods were compared . Counts of C . difficile and C . perfringens detected by the multiple-tube method were identical with counts of test-suspensions determinded by the most probable number (MPN) method . It was found to be decisive that only freshly prepared DRCM-medium be used and that, disagreeing with the DIN-instruction, cultivation at 37 degrees C is continued for at least four days.(ABSTRACT TRUNCATED AT 400 WORDS)

FEBS Lett, 1987 Dec 21, 226(1), 115 - 20
The N-terminal half of the heavy chain of botulinum type A neurotoxin forms channels in planar phospholipid bilayers; Blaustein RO et al.; The heavy chain of botulinum type A neurotoxin forms channels in planar phospholipid bilayer membranes . Channel activity is confined to the N-terminal half of this chain; the C-terminal half is inactive . Channel activity is stimulated by low pH (4.5-5.5) on the cis side (the side to which protein is added), neutral pH on the opposite (trans) side, and cis positive voltages . These findings are strikingly similar to those previously reported for analogous fragments of diphtheria and tetanus toxins.

Arch Ophthalmol, 1987 Dec, 105(12), 1703 - 11
Botulinum toxin paralysis of adult monkey extraocular muscle . Structural alterations in orbital, singly innervated muscle fibers; Spencer RF et al.; Botulinum toxin was injected into the medial rectus muscles of adult rhesus monkeys, with postinjection survival periods of three to 56 days . Light and electron microscopic changes were most apparent in the orbital, singly innervated muscle fibers, which during the acute stage (seven to 28 days), exhibited denervationlike hypertrophy with dispersion of the central mitochondria toward the periphery of the fibers . Withdrawal of the capillary network on which this fiber type normally is dependent for oxidative function was a secondary adaptive response to the disuse resulting from botulinum toxin paralysis of neuromuscular transmission . Neuromuscular junctions still were present on all muscle fiber types, although evidence of sprouting was apparent . In the long term (following return of function at 42 to 56 days), the muscle fibers appeared normal and the vasculature recovered in proportion to the decreased cross-sectional area of the muscle fibers.

J Biochem (Tokyo), 1987 Dec, 102(6), 1355 - 64
Action of botulinum neurotoxin on acetylcholine release from rat brain synaptosomes: putative internalization of the toxin into synaptosomes; Murayama S et al.; The action of botulinum neurotoxin type C1 on the release of acetylcholine from rat brain synaptosomes was studied by using anti-toxin heavy chain Fab and anti-toxin light chain Fab . The toxin was bound to synaptosomes at 0 degrees C for 10 min, in which {14C}acetylcholine had been accumulated previously . The toxin-binding synaptosomes were pre-incubated at 37 degrees C, and the release of acetylcholine was determined after the synaptosomes had been incubated in 25 mM KCl-incubation medium for 20 min at 37 degrees C . Inhibition of {14C}acetylcholine release from the synaptosomes was observed with increasing pre-incubation time and toxin concentration, and the maximum inhibition was seen after pre-incubation for at least 15 min, which was called the "lag time." The toxin-binding synaptosomes were reacted with anti-toxin heavy chain and anti-toxin light chain Fabs at 0 degrees C for 1.5 min before pre-incubation of the synaptosomes at 37 degrees C . Both Fabs reversed the acetylcholine release inhibition by the toxin . However, when the Fabs were added during the pre-incubation time at 37 degrees C, they showed less restoration with increasing pre-incubation time . The restoration was completely abolished if the Fabs were added to the synaptosomes after the first half of the "lag time." On the other hand, when 125I-labeled toxin-binding synaptosomes were reacted with the Fabs at 0 degrees C for 1.5 min before pre-incubation of the synaptosomes at 37 degrees C, anti-heavy chain Fab removed 125I-toxin from the synaptosomes, but anti-light chain Fab did not . However, if the Fabs were added to toxin-binding synaptosomes during the pre-incubation time at 37 degrees C, the Fabs could not remove 125I-toxin from the synaptosomes, and the synaptosomes retained more labeled toxin with increasing pre-incubation time . These results suggest that there are three distinct steps in the inhibition of acetylcholine release from synaptosomes by botulinum neurotoxin . The first is binding, which is reversible, temperature-independent, and mediated by the heavy chain of the toxin . The second is temperature-dependent internalization, that takes place in the first half of the "lag time," in which both the chains are internalized into synaptosomes . The third is the development of toxicity, which requires the latter half of the "lag time."

Scand J Immunol, 1987 Dec, 26(6), 737 - 43
Persistence and selectivity of the immune phagocytosis inhibition by major histocompatibility complex antibodies; Neppert J; Major histocompatibility complex (MHC) antibodies induce immune phagocytosis inhibition (IPI) which lasts for at least 7 days . IPI-inducing antibodies do not inhibit the phagocytosis mediated by the beta-glucan receptor . This corresponds well to recent findings that these antibodies do not interfere with the phagocytosis of deactivated saccharomyces, mediated by the mannose-fucose receptor, or polyacrylic acid particles, also mediated by non-Fc receptors . Substances that interact with certain MHC antigens or with Fc receptors, certain toxins that inhibit surface molecule mobility, and ciclosporin do not cause IPI and do not suppress the induction of IPI by MHC antibodies . These substances are: opioid peptides, insulin, penicillin G, immune complexes, aggregated IgG, Fc fragments, ciclosporin, botulinum C2 toxin, sodium azide . Some lectins and EDTA are inhibitory in a non-selective fashion, since the Fc receptor independent phagocytosis is also abrogated.

FEBS Lett, 1987 Nov 16, 224(1), 38 - 42
Botulinum ADP-ribosyltransferase C3 but not botulinum neurotoxins C1 and D ADP-ribosylates low molecular mass GTP-binding proteins; Rosener S et al.; Botulinum ADP-ribosyltransferase C3 modified 21-24 kDa proteins in a guanine nucleotide-dependent manner similar to that described for botulinum neurotoxin C1 and D . Whereas GTP and GTP gamma S stimulated C3-catalyzed ADP-ribosylation in the absence of Mg2+, in the presence of added Mg2+ ADP-ribosylation was impaired by GTP gamma S . C3 was about 1000-fold more potent than botulinum C1 neurotoxin in ADP-ribosylation of the 21-24 kDa protein(s) in human platelet membranes . Antibodies raised against C3 blocked ADP-ribosylation of the 21-24 kDa protein by C3 and neurotoxin C1 but neither cross reacted with neurotoxin C1 immunoblots nor neutralized the toxicity of neurotoxin C1 in mice . The data indicate that the ADP-ribosylation of low molecular mass GTP-binding proteins in various eukaryotic cells is not caused by botulinum neurotoxins but is due to the action of botulinum ADP-ribosyltransferase C3 . The weak enzymatic activities described for botulinum neurotoxins appear to be due to the contamination of C1 and D preparations with ADP-ribosyltransferase C3.

Neurosci Lett, 1987 Nov 10, 82(1), 83 - 8
The levator auris longus muscle of the mouse: a convenient preparation for studies of short- and long-term presynaptic effects of drugs or toxins; Angaut-Petit D et al.; We describe here the levator auris longus muscle of the mouse as a convenient neuromuscular preparation for the in vitro study of presynaptic effects of drugs and toxins applied in vivo in young or adult mice . The good visibility of its motor axons and terminals using Nomarski optics allows accurate electrophysiological studies of presynaptic signals . In addition, the levator auris longus muscle is sufficiently thin to be stained as a whole mount preparation . Preliminary results indicate that some correlation can be established between changes in time course of the presynaptic signal and the morphology of motor endings after poisoning the levator auris longus muscle with botulinum type A toxin.

Ann Ophthalmol, 1987 Nov, 19(11), 416 - 8, 422
Abducens paralysis repaired with muscle transposition and intraoperative botulinum toxin; Armenia JV et al.; Surgical management of abducens paralysis has been modified frequently since the turn of the century in a continuing effort to produce not only orthotropia in the primary position but good or excellent abduction . We report a bilateral case repaired surgically by a single, whole-muscle, superior rectus transposition . Secondary medial rectus contracture, a common problem, is usually managed by surgical weakening procedures which it is difficult, if not impossible, to grade . We attempted to avoid the problem by weakening the antagonist medial rectus by intraoperative injection of botulinum toxin which might allow a self-adjusting mechanism . Using this approach, fusion in down-gaze without correction and in the primary position with a small amount of vertical and horizontal prism correction was achieved . Good to excellent abduction was restored.

Neuroscience, 1987 Nov, 23(2), 767 - 79
Selective location of acceptors for botulinum neurotoxin A in the central and peripheral nervous systems; Black JD et al.; The main site of action for botulinum neurotoxin is cholinergic motor nerve terminals where specific acceptors concentrate the toxin on the cell surface, thereby facilitating its internalization and inactivation of a component essential for transmitter release . In this study, the interaction in vitro of {125I}botulinum neurotoxin type A with central and peripheral nerve terminals of different types was investigated using Ultrofilm and electron-microscope autoradiography . It was found that: (i) The neurotoxin binds to synapse-rich areas of rat brain, particularly in the hippocampus and cerebellum; identity of the neuron types labelled is unclear although cholinergic nerves seem to be labelled, perhaps not exclusively, in many areas . (ii) Toxin uptake at central nerve terminals appears to be minimal and its penetration into intact brain slices is restricted; this may account for the toxin's lower central toxicity . (iii) Selective labelling of cholinergic nerves but not purinergic, peptidergic or adrenergic nerve terminals in mouse ileum suggests that the toxin may be a specific marker for cholinergic nerves in the periphery . Based on these localization studies and published pharmacological observations, it is concluded that efficient toxin-induced blockade of neurotransmission depends on the presence of specific acceptors of high affinity for the toxin and of an effective neuronal uptake mechanism . Inhibition of the release of numerous transmitters from different kinds of nerve terminals lacking one of these features can be produced by high toxin concentrations when uptake occurs via low affinity acceptors or by non-specific means . Notably, this widespread action of the toxin indicates the occurrence of a common intracellular target in several, possibly all, nerve types.

Br J Ophthalmol, 1987 Sep, 71(9), 664 - 8
Long-term results of treatment of idiopathic blepharospasm with botulinum toxin injections; Elston JS; One hundred and one patients with idiopathic blepharospasm have been treated with injections of botulinum toxin A into the orbicularis oculi . Ten had previously had facial nerve avulsions and responded well, normal visual function being restored in the majority (7/10) for an average of 14 weeks . Without prior surgical treatment the response was more variable, but 71/91 regained normal or near normal vision . Older patients, those with a family history of the condition, and those without oromandibular dystonia responded slightly better . The severity of the blepharospasm, the length of the history, and spontaneous resolution of an episode of focal dystonia in the past had no influence on the outcome . Results were poor in the presence of an associated neurological disorder . Side effects, particularly a temporary partial ptosis, were common but were well tolerated . The average duration of improvement was eight weeks in men, nine in women, and there was no evidence of any increase in duration after multiple injections . Eighty nine patients continued with injections, 11 opted for surgical treatment, and one resumed drugs.

Arch Ophthalmol, 1987 Sep, 105(9), 1265 - 8
Experimental doxorubicin myopathy . A permanent treatment for eyelid spasms?
Baker L, Wirtschafter JD.
Doxorubicin may effect a more permanent chemomyectomy and chemodenervation (by retrograde "suicide transport") of the orbicularis oculi muscle than the chemodenervation produced by botulinum A toxin used for the treatment of blepharospasm . Graded doses (0.4 mg and 1.0 mg) of doxorubicin hydrochloride (2 mg/mL) were injected into the eyelids and antecubital and popliteal skin of rabbits in an attempt to evaluate the drug's efficacy and safety . Histologic changes in the orbicularis oculi muscle included necrosis and decreased mass . The results are promising because the eyelids appear to be more resistant than other skin regions to the development of tissue necrosis . Moreover, doxorubicin might not be excessively toxic in the treatment of eyelid cancer.

Ann Otol Rhinol Laryngol, 1987 Sep-Oct, 96(5), 534 - 41
Use of botulinum toxin to lateralize true vocal cords: a biochemical method to relieve bilateral abductor vocal cord paralysis; Cohen SR et al.; Using the mongrel dog as an animal model, we studied the effectiveness of botulinum toxin (Oculinum) to lateralize the true vocal cord . This experiment was undertaken in order to determine whether the toxin can improve the airway in subjects with bilateral abductor vocal cord paralysis . The toxin was injected into the cricothyroid muscle to block neuromuscular transmission at the motor end-plate . Paralysis of the cricothyroid muscle was achieved and documented by electromyography and videotaped endoscopy . Paralysis of the cricothyroid muscle decreases the tension of the true vocal cord and allows the cord to take a more lateral position . In this preliminary report, the literature is reviewed, the effect of and action of the toxin are discussed, and the results of the experimental protocol, establishment of dose-response curves, and techniques of injection are presented . Preliminary data suggest that there is an increase in the airway by lateralizing the true vocal cord with this biologic substance, and that this method may have many applications in clinical medicine for the otolaryngologist-head and neck surgeon.

Can J Neurol Sci, 1987 Aug, 14(3 Suppl), 533 - 5
Local treatment of spasmodic torticollis with botulinum toxin; Tsui JK et al.; Fifty-six patients with spasmodic torticollis were treated with local injections of botulinum toxin . The drop out rate was 21% . The remaining 44 patients were followed for a period of 3 to 21 months . Thirty-two patients (76%) had pain relief out of 42 presenting with pain; 37 (66%) improved in the amount of sustained movements of torticollis . The efficacy was reproducible after repeated injections.

Ophthalmology, 1987 Aug, 94(8), 976 - 9
Use of botulinum toxin in the treatment of one hundred patients with facial dyskinesias; Mauriello JA Jr et al.; One hundred patients with involuntary eyelid and facial spasms resulting from essential blepharospasm, Meige syndrome, and hemifacial spasm continued to respond to repeated injections of botulinum toxin at expected time intervals during a 30-month period . This series of more than 372 injections done by one of us (JAM) supports the finding of earlier, smaller series that tolerance to the toxin does not develop . The drug had a greater duration of effect in patients with hemifacial spasm than in patients with blepharospasm; the duration of effect was 17.25 weeks in the former group and 12.6 weeks in the latter . Eighteen percent of patients required a higher than standard dose for a therapeutic response . Fourteen of 15 patients with lower facial spasms and one patient with torticollis had significant relief . Dry eye may be managed by titrating the lower lid dose.

Ophthalmology, 1987 Aug, 94(8), 971 - 5
Effectiveness of botulinum toxin therapy for essential blepharospasm; Engstrom PF et al.; The effectiveness of Botulinum toxin (Oculinum) therapy in 76 patients with the diagnosis of essential blepharospasm was analyzed . Botulinum offers relief to almost all patients suffering from essential blepharospasm, however, this relief is usually temporary . The response time for repeated treatments tended to be longer than the first treatment . Patients with mild blepharospasm responded significantly longer to Botulinum injection, than those with severe spasms . The response to Botulinum was not significantly different in patients with Meige syndrome than in patients with only essential blepharospasm . Patients previously treated surgically for essential blepharospasm did not respond differently than those patients with no previous surgical therapy . The authors believe that Botulinum toxin injection is an effective, although temporary, mode of therapy for the signs and symptoms of this focal dystonia . The authors recognize that there may be psychologic factors affecting the response.

J Am Optom Assoc, 1987 Aug, 58(8), 646 - 51
Benign essential blepharospasm; Malinovsky V; A 57-year-old female had vague visual complaints of pressure behind her right eye during an ocular examination . One month later the patient was visually impaired due to uncontrollable spasms of both eyelids . A diagnosis of benign essential blepharospasm (BEB) was made, and the patient was eventually treated with botulinum toxin to temporarily relieve her symptoms . I have reviewed and summarized the literature relating to the characteristics and management of this often misdiagnosed and misunderstood ocular muscular condition.

Pflugers Arch, 1987 Aug, 409(4-5), 533 - 9
Distinct sites of action of clostridial neurotoxins revealed by double-poisoning of mouse motor nerve terminals; Gansel M et al.; (1) We investigated the effects of single- and double-poisoning with tetanus toxin (TeTx), botulinum neurotoxin type A (BoTx A) and botulinum neurotoxin type B (BoTx B) on spontaneous and nerve-evoked quantal transmitter release at motor endplates of the triangularis sterni preparation of the mouse . (2) Inhibitory effects of TeTx and BoTx B on spontaneous and nerve-evoked transmitter release were very similar, except that the action of BoTx B required 500-fold lower concentrations and was less dependent on temperature . BoTx A caused stronger inhibition of quantal release than TeTx or BoTx B, but was comparatively much easier counteracted by 4-aminopyridine (4-AP) . (3) In contrast to BoTx A, with TeTx or BoTx B the increase of transmitter release following onset of 50 Hz nerve stimulation was delayed for a few seconds and synaptic latencies of quanta showed large variations . This release pattern was also evident in all double-poisoning experiments, regardless of intoxication sequence . (4) Inhibition of evoked release was found to be slightly stronger with TeTx than with BoTx B, so the amount of nerve-evoked quanta released after double-poisoning with any sequence of these toxins always approached that of TeTx . In no case supra-additive actions were observed . (5) A strong reduction of evoked quanta was observed when BoTx A was applied in addition to either of the two other toxins . With reversed poisoning sequences (BoTx A - TeTx or BoTx A - BoTx B) the resulting values remained at the extremely low level of BoTx A . (6) In the presence of 4-AP double-poisoning with any combination between BoTx A and TeTx or BoTx B (regardless of intoxication sequence) revealed supra-additive effects, since the number of quanta released was considerably lower than that obtained with any of the toxins alone (in the presence of 4-AP) . (7) Our results indicate that tetanus toxin and botulinum toxin type B have a common site of action which is different and independent from that of botulinum toxin type A.

FEBS Lett, 1987 Jul 13, 219(1), 40 - 4
Botulinum C2 toxin potentiates activation of the neutrophil oxidase . Further evidence of a role for actin polymerization; Al-Mohanna FA et al.; Botulinum C2 toxin was employed as a specific inhibitor of actin polymerization in rat neutrophils to determine its role in oxidase activation . This toxin was shown to inhibit actin polymerization and the microfilament-dependent function, phagocytosis . Oxidase activation in response to the chemotactic peptide, f-Met-Leu-Phe (FMLP) was enhanced approx . 3-fold . The enhancement by C2 toxin did not occur in cells pretreated with cytochalasin B . C2 toxin had no significant effect on the FMLP-induced intracellular Ca2+ rise . These data are consistent with an inhibitory role for actin polymerization in oxidase activation.

FEBS Lett, 1987 Jul 13, 219(1), 219 - 23
Botulinum neurotoxin inhibits depolarization-stimulated protein phosphorylation in pure cholinergic synaptosomes; Guitart X et al.; Botulinum neurotoxin, a strong blocker of acetylcholine release at peripheral cholinergic synapses, inhibits depolarization-stimulated protein phosphorylation in pure cholinergic synaptosomes isolated from the electric organ of Torpedo marmorata . Moreover, tetrodotoxin has the same effect on protein phosphorylation when cholinergic synaptosomes are depolarized by veratridine . Correlation between presynaptic protein phosphorylation and acetylcholine release is suggested by the fact that botulinum neurotoxin blocks specifically neurotransmitter release without affecting membrane depolarization and calcium fluxes in our synaptosomal preparation.

Muscle Nerve, 1987 Jul-Aug, 10(6), 552 - 5
Distant effects of local injection of botulinum toxin; Lange DJ et al.; We studied five patients who received local injections of botulinum toxin for dystonic disorders to determine if there had been any distant effects on neuromuscular transmission . No patient developed weakness or abnormalities on routine electrophysiologic testing . In all patients who received more than 245 U of toxin, SFEMG in the extensor digitorum communis muscle, a muscle distant to all those injected, was abnormal . Fiber density was increased in the two patients who received the largest cumulative dose . Jitter was maximally increased at slow firing rates, confirming its presynaptic basis . Our results reveal that there is an effect on neuromuscular transmission in muscles distant to those injected with botulinum toxin for dystonia.

J Neurosci, 1987 Jul, 7(7), 2019 - 24
Mechanisms of postsynaptic plasticity: remodeling of the junctional acetylcholine receptor cluster induced by motor nerve terminal outgrowth; Yee WC et al.; Motor nerve terminal outgrowth (NTO) at neuromuscular junctions (NMJs) occurs rapidly in response to denervation changes in muscle . We have previously found that NTO can produce an elongation of the synaptic area of the NMJ as defined by cholinesterase-silver staining . In the present study, we examined the effects of NTO on a postsynaptic muscle membrane component, the usually stable cluster of acetylcholine receptors (AChRs) at the NMJ . NTO was evoked in rat soleus muscles using botulinum toxin . AChRs were demonstrated using immunocytochemistry or autoradiography of alpha-bungarotoxin binding . Our results show that NTO induces rapid elongation of the cluster of AChRs at the NMJ within 7 d of treatment with botulinum toxin . The growth in the size of the AChR clusters was accompanied by an increase in the number of AChRs/NMJ . No elongation of AChR clusters was seen following surgical denervation, suggesting that cluster growth is related to NTO and not to denervation changes in muscle per se . Growth of NMJ-AChR clusters appeared to result primarily from 2 processes: insertion of new AChRs into the NMJ membrane and, surprisingly, redistribution of preexisting NMJ-AChRs . These results show that NTO can cause rapid changes in the normally stable cluster of AChRs at the NMJ . Motor nerve terminals provide a strong and anatomically precise control of AChRs at the NMJ.

Am J Ophthalmol, 1987 Jun 15, 103(6), 741 - 4
Persistent binocular diplopia after cataract surgery; Hamed LM et al.; We reviewed the records of 38 consecutive patients who had persistent binocular diplopia after cataract surgery . The patients were divided into ten categories based on the suspected conditions underlying the diplopia . Of 16 patients who underwent strabismus surgery, five achieved the therapeutic goal of single binocular vision in the primary and reading position, and four attained this with prismatic or botulinum toxin therapy in addition to surgery . Seven patients continued to have diplopia despite strabismus surgery and adjunctive therapy . Even small residual deviations were often intolerable, because of highly diminished fusional amplitudes.

Eur J Biochem, 1987 Jun 15, 165(3), 675 - 81
Botulinum toxin A blocks glutamate exocytosis from guinea-pig cerebral cortical synaptosomes; Sanchez-Prieto J et al.; The exocytotic release of L-glutamate from guinea-pig cerebral cortical synaptosomes can be extensively inhibited by preincubation with botulinum neurotoxin type A at 37 degrees C for 1-2 h . The toxin has no effect on synaptosomal respiratory control, respiratory capacity, ATP synthesis, plasma-membrane 86Rb+ permeability or plasma-membrane potential, does not inhibit the entry of 45Ca2+ into the synaptosome upon depolarization and does not alter the ability of intrasynaptosomal mitochondria to sequester Ca2+ . The blockade of Ca2+-dependent glutamate release may be totally reversed by the Ca2+/2 H+-exchange ionophore ionomycin, but not by increasing extracellular Ca2+ concentration . It is suggested (a) that exocytosis is triggered by the penetration of Ca2+ into an intracellular hydrophobic milieu; (b) that this stage is blocked by the toxin and (c) that ionomycin is able to bypass this block and deliver Ca2+ to the exocytotic apparatus.

Acta Ophthalmol (Copenh), 1987 Jun, 65(3), 313 - 9
Use of botulinum toxin in blepharospasm and other facial spasms; Ruusuvaara P et al.; Essential blepharospasm is an involuntary spasmodic closure of the eyelids . It frequently makes normal social human life such as reading, driving and even walking almost impossible . Botulinum toxin has been approved for investigational use for treatment of blepharospasm . We report the results and side-effects of botulinum A toxin treatment of the first 13 patients in Finland . Temporary control of spasms was achieved in all patients varying from 1 to 38 weeks with a mean of 11 weeks after the first injection . Most patients needed re-treatment after varying intervals . All complications were transient.

Can J Ophthalmol, 1987 Jun, 22(4), 212 - 7
Injection of type A botulinum toxin into extraocular muscles for correction of strabismus; Flanders M et al.; Between July 1984 and March 1985, 51 injections of type A botulinum toxin were given to 39 patients aged 11 to 81 years with various types of strabismus, including paralytic strabismus, sensory exotropia, consecutive and residual postsurgical deviations and partially accommodative esotropia . All deviations were documented by orthoptic assessment and photography . After treatment the patients were followed for up to 3 years . The beneficial effects of a single injection were modest, and a second injection was often necessary to achieve satisfactory ocular alignment . Thirteen of the 26 patients (50%) with nonparalytic strabismus and 10 of the 13 patients (77%) with paralytic strabismus had a good outcome (final deviation 12 prism dioptres or less) . Adverse side effects included transient ptosis and diplopia and inadvertent vertical deviation . Twenty-one consecutive cases of nonparalytic horizontal strabismus treated with adjustable sutures were also reviewed . The results in these patients were more predictable and longer lasting than those in the patients who received botulinum toxin.

Arch Otolaryngol Head Neck Surg, 1987 Jun, 113(6), 603 - 5
Botulinum toxin injection of the vocal fold for spasmodic dysphonia . A preliminary report; Miller RH et al.; A number of different therapies have been used in the management of spasmodic dysphonia . None are curative, and the results of the most widely used surgical procedure (resection of the recurrent laryngeal nerve) are not universally good, with a high recurrence rate in some series . Furthermore, the procedure is not reversible . Using a special electromyographic hypodermic needle, we injected botulinum A toxin into one of the vocal folds of two patients with severe spasmodic dysphonia . Both had significant improvement without any complications . Although the resultant paresis is not permanent, the injection can be repeated as needed as an outpatient procedure.

Muscle Nerve, 1987 Jun, 10(5), 464 - 70
Aminoglycosides and 3,4-diaminopyridine on neuromuscular block caused by botulinum type A toxin; Molgo J et al.; Impulse-evoked transmitter release was greatly reduced at frog neuromuscular junctions 3-20 days after botulinum type A toxin (BoTx) poisoning . The reduction in transmitter release was accompanied by an increased variability in the latency between the presynaptic spike and the release of transmitter . The aminoglycoside antibiotics amikacin, gentamycin, and bekanamycin, when applied at concentrations within their therapeutic levels, markedly enhanced the blockade of transmitter release in BoTx-poisoned junctions . 3,4-diaminopyridine strongly antagonized the effects of BoTx at early stages of poisoning, and the combined presynaptic effects of BoTx and aminoglycoside antibiotics provided that transmitter release was not completely blocked by the toxin . The antagonism was apparent at all frequencies of stimulation . Since the aminoglycoside antibiotics enhanced the neuromuscular block caused by BoTx, these drugs should be avoided in patients suspected of poisoning by this toxin.

Brain Res, 1987 May 5, 410(2), 385 - 9
In botulinum type A-poisoned frog motor endings ouabain induces phasic transmitter release through Na+-Ca2+ exchange; Molgo J et al.; Ouabain (100 microM) applied for 60 min to botulinum A (BoTx) poisoned motor junctions increases, in a time-dependent manner, the mean number of acetylcholine quanta released by nerve stimulation and enhances the delayed transmitter release . The drug does not affect spontaneous quantal release . The observed effects on evoked transmitter release cannot be explained by changes in the configuration of presynaptic currents recorded from motor terminals . They suggest that in BoTx-poisoned motor endings the level of intraterminal Ca2+, lower than that required for the activation of quantal transmitter release, can be effectively increased through the reversed operation of an Na+-Ca2+ exchange system that normally uses the Na+ gradient to extrude Ca2+.

J Physiol, 1987 May, 386, 475 - 84
The effects of in vitro application of purified botulinum neurotoxin at mouse motor nerve terminals; Dolly JO et al.; 1 . Purified botulinum neurotoxin type A (10 nM) was applied in vitro to mouse diaphragm muscles . Intracellular micro-electrode recordings were made continuously in single fibres . 2 . This treatment reduced end-plate potential (e.p.p.) amplitudes with a time to half-maximal effect of about 75 min at 22-25 degrees C . E.p.p . rise-times remained fast and unaffected by the toxin . 3 . Miniature end-plate potential (m.e.p.p.) frequency was reduced by the toxin to less than 5% of control frequency, and followed a similar time course to the block of e.p.p . amplitudes . The m.e.p.p . rise-time and coefficient of variation (c.v.) of m.e.p.p . amplitude distributions both increased, but the time course of these increases lagged significantly behind the change in frequency . 4 . A population of slow rise-time m.e.p.p.s was present in controls at low frequency . This population was found to be unaffected by the toxin . 5 . The above-detailed in vitro changes could be explained by the toxin acting by a single common mechanism to inhibit the release process underlying both fast rise-time m.e.p.p.s and e.p.p.s . A distinct release process, which leads to slow rise-time m.e.p.p.s, was unaffected by the toxin.

J Pediatr, 1987 May, 110(5), 719 - 22
Botulinum toxin chemodenervation in infants and children: an alternative to incisional strabismus surgery; Magoon E et al.; Eighty-two children aged 13 years or younger were given injections of botulinum toxin for horizontal strabismus . Improvement was achieved in all but one patient . Children younger than 1 year or older than 6 years of age received only topical drop anesthesia and no sedation . Young children generally required low-dose ketamine sedation . The technique typically undercorrects, so reinjection was necessary in 85% of the patients . There were no systemic complications . Side effects, lasting up to a few weeks, included transient ptosis and hypertropia caused by involvement of other extraocular muscles.

Biosci Rep, 1987 May, 7(5), 355 - 67
Calcium and diacylglycerol control of secretion; Knight DE; Measurements of intracellular Ca2+ in adrenal medullary cells suggest that a transient rise in Ca2+ leads to a transient secretory response, the rise in Ca2+ being brought about by an influx through voltage-sensitive Ca channels which subsequently inactivate . The level of Ca2+ observed is much smaller than the Ca2+ needed to trigger secretion when introduced directly into the cell . The discrepancy is removed by the presence of diacylglycerol, which increases the sensitivity of the secretory process to Ca2+ . The site of action of Ca2+ and diacylglycerol is probably protein kinase C, and the different secretory responses to increases of Ca2+ and diacylglycerol can be modelled in terms of a preferential order of binding of these two substrates to the enzyme . ATP is needed for secretion: one role is possibly to confer stability to the secretory apparatus; another may involve phosphorylation of some key protein . The kinetics of secretion suggest that if Ca2+ regulates phosphorylation or dephosphorylation, then it is the rate of change of phosphorylation that controls secretion rather than the extent of phosphorylation or dephosphorylation . Guanine nucleotide-binding proteins may play a role not only at the level of signal transduction coupling, but also at or near the site of exocytosis, and the mechanism by which some Botulinum toxins inhibit secretion may be associated with these proteins.

J Physiol, 1987 Apr, 385, 677 - 92
Botulinum toxin inhibits quantal acetylcholine release and energy metabolism in the Torpedo electric organ; Dunant Y et al.; 1 . Type A Botulinum toxin (BoTX) blocked nerve-electroplaque transmission in small fragments of Torpedo marmorata electric organ incubated in vitro . The effect was observed either with the crystalline toxin complex (associated with haemagglutinin) or with the purified neurotoxin (molecular weight approximately 150,000) . 2 . The quantal content of the evoked post-synaptic response was reduced by BoTX but the quantum size remained unchanged till complete blockade of the evoked response . 3 . Spontaneous electroplaque potentials were composed of two populations: one with a bell-shaped amplitude distribution (miniature potentials or quanta) and a population of small events with a skewed distribution (subminiatures) . In BoTX-poisoned tissue, the bell-distributed miniatures progressively disappeared, but the subminiatures kept on occurring . Occasionally, larger spontaneous potentials with a slow time course were recorded; they were also BoTX resistant . 4 . A biochemical assay showed that evoked acetylcholine (ACh) release was impaired by BoTX . During the period when evoked transmission was blocked, spontaneous ACh release transiently increased . 5 . At the time of transmission blockade, there was no significant change of ACh content, of ACh turnover, of ACh repartition in the vesicle-bound and free compartments, or of the number of synaptic vesicles . 6 . The amount of ATP was reduced to 50% by BoTX, and that of creatine phosphate (CrP) to less than 20% . The ATP-CrP-converting enzyme, creatine kinase, was inhibited in BoTX-poisoned tissue . 7 . Thus, the electrophysiological effects of BoTX are very similar at the nerve-electroplaque and the neuromuscular junctions . The present work suggests in addition that suppression of quantal release by BoTX is related to marked alterations of the energy metabolism in the tissue.

J Neurosci Methods, 1987 Apr, 19(4), 285 - 95
The plantar nerves-lumbrical muscles: a useful nerve-muscle preparation for assaying the effects of botulinum neurotoxin; Clark AW et al.; The plantar nerves-lumbrical muscles (PL) of the hindpaw of the mouse is a preparation that is particularly useful for assaying the effects of botulinum neurotoxin (NT) on the mammalian neuromuscular junction . Each mouse provides a minimum of 4 and a maximum of 8 such preparations . The muscles are thin enough so that neuromuscular junctions can be easily located with Nomarski interference contrast optics for impaling with microelectrodes . The preparations are small enough so they can be sectioned transversely or longitudinally, in their entirety, for light and electron microscopy, or just the regions containing end-plates can be used . An examination of the responses of this preparation to types A and E NT reveal its exceptional suitability for experiments with NT . The small number of fibers and their arrangement permit assays of low concentrations of NT to proceed to complete and total paralysis in a convenient period of time.

Neurology, 1987 Apr, 37(4), 616 - 23
Botulinum A toxin for cranial-cervical dystonia: a double-blind, placebo-controlled study; Jankovic J et al.; We studied the effects of botulinum A toxin in 12 patients with blepharospasm and 10 patients with oromandibular-cervical dystonia received in a double-blind manner . All blepharospasm patients improved, 71.6% on a clinical rating score, 60.7% by self-assessment, and 38.9% by video-rating; there was no improvement with placebo . The beneficial effects lasted a mean of 12.5 weeks (range, 5 to 28) . Only 37.5% of the patients with oromandibular-cervical dystonia improved . Patients with pharyngeal dystonia and spasmodic dysphonia also improved.

Biochem Biophys Res Commun, 1987 Feb 13, 142(3), 1032 - 8
ADP-ribosylation by type C1 and D botulinum neurotoxins: stimulation by guanine nucleotides and inhibition by guanidino-containing compounds; Ohashi Y et al.; We recently reported that type D botulinum neurotoxin ADP-ribosylates a specific protein of Mr 21,000 in membrane fractions of various tissues (Ohashi, Y . and Narumiya, S . (1987) J . Biol . Chem . in press) . We examined similar enzyme activities in other types (types A, B, C1 and E) of botulinum neurotoxins . Of these, only type C1 toxin showed the activity similar to type D toxin and ADP-ribosylated the same Mr 21,000 protein in membranes of mouse brain . No enzyme activities were detected in type A, B and E toxins under the present experimental conditions . GTP stimulated ADP-ribosylation by the two toxins in a concentration dependent manner from 10 nM to 100 microM . The maximum stimulation was about 6 fold . GDP was 10 times less potent than GTP and achieved similar maximum at 1 mM, while GMP, ADP and ATP had little effect . Several guanidino-containing compounds dose-dependently inhibited the activities of both toxins . The IC50 values were 8.5, 14.5 and 45 mM for agmatine, L-arginine methyl ester and guanidine, respectively.

J Biol Chem, 1987 Feb 5, 262(4), 1430 - 3
ADP-ribosylation of a Mr 21,000 membrane protein by type D botulinum toxin; Ohashi Y et al.; When crude membrane fraction from bovine adrenal gland was incubated with type D botulinum toxin in the presence of NAD, a membrane protein with a molecular weight of 21,000 was specifically ADP-ribosylated . This ADP-ribosylation occurred dependent on the dose of the toxin and was abolished by prior boiling ADP-ribose transfer to the membrane protein was significantly suppressed when agmatine and L-arginine methyl ester were included in the reaction mixture . Dithiothreitol stimulated this ADP-ribosylation about 3-fold . Incubation of membrane fractions from mouse brain and pancreas with this toxin also resulted in ADP-ribosylation of a protein of the same molecular weight . These results suggested that type D botulinum toxin catalyzed transfer of an ADP-ribose moiety of NAD to the specific membrane protein common to secretory cells.

Am J Optom Physiol Opt, 1987 Feb, 64(2), 79 - 82
Botulinum toxin and its uses in the treatment of ocular disorders; Alpar AJ; The recent utilization of type A botulinum toxin in clinical ophthalmology makes it important for all health professionals involved in eye care to understand the action of this "non-surgical" tool . This paper summarizes the present knowledge concerning the chemistry, toxicology, and pathophysiology of the toxin . It also discusses the indications, contraindications, safety, and efficacy of this use of botulinum toxin.

Can J Neurol Sci, 1987 Feb, 14(1), 42 - 5
Botulinum toxin for benign essential blepharospasm, hemifacial spasm and age-related lower eyelid entropion; Carruthers J et al.; Purified botulinum A exotoxin was used in the treatment of forty seven patients with benign essential blepharospasm, 11 patients with hemifacial spasm and 2 patients with age-related entropion . The treatment was effective in all three groups for an average of 3-4 months when symptoms recurred and repeated chemodenervation with toxin was needed . The commonest complication was transient ptosis with an overall frequency of 7.8% . This incidence increased to 11.1% with toxin doses higher than 25 units per orbicularis . The treatment was well accepted by the patients, who were subsequently able to return to pre-blepharospasm lifestyles.

Can J Ophthalmol, 1987 Feb, 22(1), 24 - 8
Treatment of blepharospasm with medication, surgery and type A botulinum toxin; Arthurs B et al.; A total of 39 patients with essential blepharospasm and 2 patients with hemifacial spasm were treated with one or more forms of therapy . All patients underwent neurologic and ophthalmic assessment to rule out ocular causes of blepharospasm . Thirty-six patients were given a trial of various medications . Only one patient was successfully treated: her condition was markedly improved with pimozide after benztropine mesylate, clonazepam and amantadine hydrochloride had failed to help . Patients who did not respond to drug therapy were offered the option of undergoing eyebrow-eyelid muscle stripping surgery . The six patients who underwent surgery showed considerable improvement; however, side effects such as frontal anesthesia, exposure keratitis, lagophthalmus, scarring and eyelid malposition occurred, and three of the six had residual spasm . At this point type A botulinum toxin became available . A total of 27 patients (26 who did not respond to drug therapy, including the 3 with residual spasm after surgery, and 1 previously untreated patient) received type A botulinum toxin injections . Most experienced rapid relief from their spasms . The beneficial effects lasted weeks to months, and there were no major side effects . Treatment with type A botulinum toxin appears to be a safe and effective means of temporarily relieving blepharospasm . The long-term results with repeated injections are yet to be determined.

Ophthalmic Surg, 1987 Feb, 18(2), 118 - 9
The effect of intravitreal botulinum toxin on rabbit visual evoked potential; Hoffman RO et al.; Intravitreal injections of botulinum A toxin in two doses, 1.25 and 25 units, were performed in two rabbit eyes . The fellow eyes were injected with an equal volume of saline to serve as controls . The visual evoked potential was unchanged at one and two weeks post-injection when compared to pre-injection recordings in both botulinum and saline injected eyes.

Eur J Cell Biol, 1987 Feb, 43(1), 134 - 40
Botulinum C2 toxin ADP-ribosylates actin and disorganizes the microfilament network in intact cells; Reuner KH et al.; Botulinum C2 toxin ADP-ribosylates actin in {32P}orthophosphate-labelled intact chick embryo cells (CEC) . The toxin-induced rounding up of CEC is correlated with ADP-ribosylation of actin in intact cells in a time and concentration-dependent manner . Both, rounding up of cells and actin ADP-ribosylation, depend on the presence of both components of botulinum C2 toxin (components I and II) and are independent of the ability of CEC to divide . Treatment of CEC with botulinum C2 toxin induced a time-dependent disorganization of the typical architecture of the microfilament network as shown by fluorescein-phalloidin staining . Botulinum C2 toxin decreased the amount of Triton X-100 insoluble actin, while the fraction of Triton soluble actin was increased . Actin, which was 32P-labelled by botulinum C2 toxin in intact CEC, was recovered in the Triton soluble but not in the Triton insoluble actin fraction . It is suggested that in intact CEC botulinum C2 toxin causes ADP-ribosylation of G-actin but not of F-actin thereby leading to an accumulation in the pool of monomeric actin.

Mov Disord, 1987, 2(4), 237 - 54
Localized injections of botulinum toxin for the treatment of focal dystonia and hemifacial spasm; Brin MF et al.; Medical treatment of dystonia usually results in an incomplete response and is frequently unsuccessful . Peripheral surgical therapy is available for some focal dystonias, but may only offer temporary relief and may have unacceptable complications . We have used local injections of botulinum toxin into the appropriate muscles for treatment of disabling focal or segmental dystonia in 93 patients with torticollis, blepharospasm, oromandibular dystonia (OMD), limb dystonia, lingual dystonia, and dystonia adductor dysphonia, in addition to four patients with hemifacial spasm . Significant relief of motor symptoms was seen in 69% of the patients with blepharospasm and 64% of patients with torticollis; 74% of the latter group with pain experience relief . Relief of symptoms was noted in most patients with OMD and limb dystonia, and all with lingual dystonia, dystonic adductor spastic dysphonia, and those with hemifacial spasm . Benefit averaged 2 1/2-3 months initially; however some patients experienced longer relief with subsequent injections . Adverse effects were transient, although 2 patients developed antibodies against the toxin, and we documented evidence for distant effects in others . This approach of chemically weakening contracting muscles in focal dystonia offers many advantages over pharmacotherapy and surgical therapy . Additional experience is needed to explore the proper doses, and potential for long term adverse effects.

Eye, 1987, 1 ( Pt 5), 603 - 8
Botulinum toxin A induced protective ptosis; Adams GG et al.; Botulinum Toxin A injected into the levator palpebrae superioris produces a flaccid ptosis of the upper lid and provides a safe and effective protection for the cornea to aid healing in indolent ulceration or as prophylaxis when there is fifth or seventh cranial nerve damage . Fifteen patients have received this treatment . Levator paresis, producing ptosis for a mean of 2-3 weeks and recovering in a mean of 8.1 weeks was successfully produced in all patients and complete corneal healing was produced in 80% of patients . The major side effect was weakness of the superior rectus muscle which occurred in 80% of cases and lasted a mean of 6 weeks.

Ophthal Plast Reconstr Surg, 1987, 3(1), 25 - 7
Treatment of blepharospasm with high dose brow injection of botulinum toxin; Kristan RW et al.; We treated 12 patients who had essential blepharospasm with brow and eyelid injections of botulinum toxin . The eyelid injections were kept constant while varying doses were used in the brow . The duration of the effect of the toxin was longer and the degree of improvement greater in those patients receiving more than 20 U in the brow than in those receiving less than 20 U . However, the degree of improvement in spasm intensity in those patients receiving greater than 20 U was similar . Ptosis occurred in seven patients . No systemic side effects were noted.

Naunyn Schmiedebergs Arch Pharmacol, 1987 Jan, 335(1), 1 - 7
Differential effects of various secretagogues on quantal transmitter release from mouse motor nerve terminals treated with botulinum A and tetanus toxin; Dreyer F et al.; Electrophysiological and electron microscopic techniques were used to investigate the actions of potassium depolarization, black widow venom (BWSV), Ca2+-ionophore A 23187 and hyperosmotic solution on mouse hemidiaphragms poisoned in vitro with botulinum A toxin (BoTx) and tetanus toxin (TeTx) . These neurotoxins reduced the frequency of miniature endplate potentials (m.e.p.ps) from 5/s of the control to 2/min and 21/min, respectively . High potassium (25 mmol/l) increased the m.e.p.p.-frequency at BoTx- and TeTx-poisoned endplates to 30/min and 50/s, respectively . The ultrastructure of endplates showed no obvious changes . BWSV (0.04 glands/ml) was just as effective in promoting transmitter release from BoTx-treated endplates as in control preparations . Electron micrographs revealed depletion of vesicles as well as swollen and disrupted mitochondria . When preparations were pretreated with TeTx, BWSV only moderately increased transmitter release and no alterations of the ultrastructure could be observed . At TeTx- or BoTx-poisoned endplates Ca2+-ionophore A 23187 usually produced an extreme reduction of m.e.p.p.-frequency (0.005/s), sometimes preceded by a short burst-like release . The ultrastructure of these endplates was not obviously affected . Application of hyperosmotic solution to BoTx- or TeTx-poisoned preparations further reduced the already low m.e.p.p.-frequency . These results further demonstrate that TeTx and BoTx act at different sites in the transmitter releasing process.

Int Arch Allergy Appl Immunol, 1987, 84(4), 380 - 4
Inhibition of histamine release from rat mast cells by botulinum C2 toxin; Bottinger H et al.; The effects of botulinum C2 toxin, which ADP-ribosylates actin, were studied on the stimulated histamine release of rat peritoneal mast cells . Treatment of mast cells with botulinum C2 toxin for 4 h inhibited histamine release stimulated by compound 48/80 or MCD-peptide maximally by about 50% . A half-maximal and maximal inhibition occurred at about 30 and 450 ng/ml botulinum C2 toxin . At similar concentrations the toxin ADP-ribosylated actin in intact mast cells . Botulinum C2 toxin largely impaired the histamine release stimulated by 12-O-tetradecanoylphorbol-13-acetate but not by the ionophore A23187 . The data indicate that botulinum C2 toxin partially inhibits the stimulated histamine release and suggest that actin is involved in the stimulus-secretion coupling in mast cells.

J Physiol, 1987 Jan, 382, 69 - 86
Acetylcholine receptors and sodium channels in denervated and botulinum-toxin-treated adult rat muscle; Bambrick L et al.; 1 . The number of acetylcholine (ACh) receptors and Na channels was measured in adult rat hind-limb muscles after denervation or injection of botulinum toxin type A (BoTX), using specific binding of radiolabelled neurotoxins . 2 . Denervation by sciatic nerve section increased the number of {125I}iodo-alpha-bungarotoxin ({125I}BTX) binding sites from low, unmeasurable levels to 39 +/- 3 fmol of toxin bound per milligram muscle protein at 21 days . 3 . Subcutaneous injection of BoTX produced complete neuromuscular blockade for 11-14 days over which time the number of {125I}BTX binding sites increased with the same time course and to the same extent as following denervation . 4 . Neither denervation nor BoTX treatment significantly altered the number of tritiated saxitoxin ({3H}STX) binding sites from normal values of 7.8 fmol/mg muscle weight or 57 +/- 3 fmol/mg homogenate protein . This may, however, correspond to a lower density of {3H}STX sites in the muscle membrane . 5 . It was concluded that neuromuscular blockade with BoTX is equivalent to denervation in its effects on synthesis of ACh receptors . Numbers of Na channels are more stable than ACh receptors but may also be modulated by neuromuscular activity.

Ophthalmology, 1986 Dec, 93(12), 1621 - 7
Ptosis associated with botulinum toxin treatment of strabismus and blepharospasm; Burns CL et al.; The incidence, severity, and duration of ptosis after botulinum neurotoxin type A (BAT) injections into extraocular or orbicularis muscles were reviewed retrospectively . Even though lid droop frequently complicated botulinum toxin treatment in this series, no loss of vision or permanent ptosis was encountered . Sixty-seven injections were completed in 44 patients between September 1982 and December 1983 . The records were reviewed to determine the incidence of ptosis after these 67 injections . Information was not adequate to ascertain the presence or absence of ptosis after 10 injections . Some degree of ptosis developed after 30 (53%) of the 57 injections with adequate follow-up history . Marked ptosis occurred after 12 (21%) of the 57 injections . The posttreatment lid droop in these 12 cases was potentially visually significant . All cases of ptosis, which impinged on the visual axis, resolved to a level permitting undisturbed vision by eight weeks after toxin injection . The incidence of ptosis was similar in patients who received injections into their medial rectus muscles and in patients who received injections into their lateral rectus muscles . However, an increased incidence of marked ptosis may be associated with the injection of previously operated muscles . Toxin dosage, patient age, and quality of the electromyogram used to monitor the injection showed no statistically significant relationship to the ptosis.

J Physiol, 1986 Dec, 381, 607 - 18
The nature and origin of calcium-insensitive miniature end-plate potentials at rodent neuromuscular junctions; Lupa MT et al.; 1 . To study the nature and origin of slow-rising, Ca2+-insensitive miniature end-plate potentials (m.e.p.p.s) in mammalian muscle we used intracellular recording techniques and drugs which block acetylcholine (ACh) synthesis or the uptake of ACh into synaptic vesicles . Slow m.e.p.p.s were induced in vivo by paralysing the extensor digitorum longus muscle of the rat with botulinum toxin type A or in vitro by the application of 4-aminoquinoline to the mouse diaphragm nerve-muscle preparation . 2 . Hemicholinium-3, which blocks ACh synthesis, reduced the amplitude of all synaptic potentials including slow m.e.p.p.s, but only if the nerve was stimulated . 3 . 2(4-phenylpiperidino)cyclohexanol (AH-5183), which blocks the active uptake of ACh into synaptic vesicles, reduced both the frequency and the amplitude of slow m.e.p.p.s and did so without requiring nerve stimulation . 4 . No correlation was observed between the molecular leakage of ACh from the motor nerve and the frequency and amplitude of slow m.e.p.p.s . 5 . We conclude that slow m.e.p.p.s are caused by the release of ACh from the nerve terminal, possibly from a small pool of synaptic vesicle-like structures.

Eur J Biochem, 1986 Nov 17, 161(1), 155 - 62
ADP-ribosylation of platelet actin by botulinum C2 toxin; Aktories K et al.; Botulinum C2 toxin is a microbial toxin which possesses ADP-ribosyltransferase activity . In human platelet cytosol a 43-kDa protein was ADP-ribosylated by botulinum C2 toxin . Labelling of the 43-kDa protein using {32P}NAD as substrate was reduced by unlabelled NAD and nicotinamide . The label was removed by treatment with snake venom phosphodiesterase . Half-maximal and maximal ADP-ribosylation occurred at 0.1 microgram/ml and 3 micrograms/ml botulinum C2 toxin, respectively . The Km value of the ADP-ribosylation reaction for NAD was about 1 microM . The peptide map of the ADP-ribosylated 43-kDa protein was almost identical with platelet actin . The ADP-ribosylated 43-kDa substrate protein bound to and was eluted from immobilized DNase I in a manner similar to G-actin . Trypsin treatment of platelet cytosol decreased subsequent ADP-ribosylation of the 43-kDa protein without occurrence of smaller labelled polypeptides . Purified platelet actin was also ADP-ribosylated by botulinum C2 toxin with similar characteristics found with actin in platelet cytosol . Phalloidin decreased the ADP-ribosylation of actin in platelet cytosol and of isolated platelet actin . Half-maximal and maximal, about 90%, reduction of actin ADP-ribosylation was observed at 0.4 microM and 10 microM phalloidin, respectively . ADP-ribosylation of purified actin, induced by botulinum C2I toxin, abolished the formation of the typical microfilament network . The data indicate that platelet G-actin but not F-actin is a substrate of botulinum C2 toxin and that this covalent modification largely affects the functional properties of actin.

Nature, 1986 Nov 6-12, 324(6092), 76 - 8
Intracellularly injected tetanus toxin inhibits exocytosis in bovine adrenal chromaffin cells; Penner R et al.; The clostridial neurotoxins tetanus and botulinum toxin type A are known to block transmitter release from nerve terminals, probably by interfering with some essential process controlling exocytosis after the entry of Ca2+ ions . Although exocytosis occurs in many secretory cells, these toxins show a high specificity for neurones and the secretory response of cultured bovine adrenal medullary cells is not inhibited by exposure to medium containing tetanus or botulinum toxin type A (although it is by botulinum toxin type D) . Here we report that when tetanus toxin and botulinum neurotoxin type A are injected intracellularly into chromaffin cells they strongly inhibit secretion, as revealed by the measurement of cell capacitance . These results indicate that these toxins are normally ineffective in chromaffin cells because they are not bound and internalized, so do not reach their site of action . Furthermore, we have localized the secretion-blocking effects of the toxin to a fragment comprising the light chain covalently linked to part of the heavy chain, suggesting that this part of the molecule contains the active site.

Ophthalmic Surg, 1986 Nov, 17(11), 747 - 53
Botulinum toxin for the treatment of essential blepharospasm; Shore JW et al.; Twenty-six patients with essential blepharospasm were treated with botulinum toxin by injection . The onset of protractor weakness in all patients ranged from one to five days following treatment . Maximal weakness developed within 12 days . There was a variable and gradual return of protractor strength over eight to 29 weeks in most patients and, with it, a return of spasm . Twenty-five patients received some degree of functional relief following initial injection . In most patients, however, the post-injection result could not be stabilized and repeat injections have been necessary to control recurrent spasms . There was one treatment failure . Three patients treated by injection following previous neurectomy and myectomy appeared to have a reduced requirement for subsequent injections . Complications included transient ptosis in six patients and mild exposure symptoms in four patients . Extraocular muscle paresis did not occur . There were no systemic side effects from the botulinum toxin injections.

Acta Physiol Scand, 1986 Nov, 128(3), 429 - 36
Pharmacological characterization of the calcium-insensitive, intermittent acetylcholine release at the rat neuromuscular junction; Tabti N et al.; A variety of pharmacologically active compounds was surveyed for effects on the Ca2+-insensitive miniature end-plate potentials (slow mepps) induced by botulinum toxin type A (Botx) poisoning in rat muscle . The purpose was to gain insight into the release process responsible for this type of acetylcholine secretion . It was found that caffeine and dibutyryl cyclic adenosine 3',5'-monophosphate increased significantly the frequency of slow mepps in Botx-poisoned muscles, but had no effect on slow mepps in control muscles . Vinblastine and cytochalasin B significantly increased the slow mepp frequency in Botx-poisoned as well as in normal control muscles . Inhibitors of oxidative metabolism reduced the frequency of slow mepps by 90%, indicating a high energy requirement for this type of release . No agent was found to augment the slow mepp frequency above 1-2 Hz, suggesting that an upper limit exists for this type of packaging and release of acetylcholine.

Pflugers Arch, 1986 Nov, 407(5), 476 - 81
A comparison of miniature end-plate potentials at normal, denervated, and long-term botulinum toxin type A poisoned frog neuromuscular junctions; Lupa MT et al.; The effects of denervation and long-term botulinum toxin type A (BoTx) poisoning on miniature end-plate potentials (m.e.p.p.s) in the frog were studied with intracellular microelectrode recording . BoTx reduced the frequency of m.e.p.p.s to less than 1% of the level seen in untreated frogs, leaving a large percentage of tiny m.e.p.p.s and slow-rising m.e.p.p.s (slow m.e.p.p.s) . Unlike what is observed in the rat, the frequency of slow m.e.p.p.s never increased above the low rate measured in the untreated controls, and in fact slightly but significantly decreased after BoTx . A comparison of the m.e.p.p.s seen after BoTx poisoning (BoTx m.e.p.p.s) and m.e.p.p.s seen after denervation (Schwann m.e.p.p.s) revealed many similarities between the two including amplitude and time-to-peak distributions, temperature Q10 values and responses to several drugs and procedures . However, it was concluded that BoTx m.e.p.p.s do not originate from the Schwann cells because denervation of BoTx-paralysed frogs abolishes all m.e.p.p.s and the drug 4-aminoquinoline affects BoTx m.e.p.p.s and Schwann m.e.p.p.s in opposite ways, increasing the frequency of the former while almost eliminating the latter . BoTx m.e.p.p.s and Schwann m.e.p.p.s probably represent similar processes of secretion which are non-specific in nature, having a lower energy barrier than for normal release and not originating from specialized areas of transmitter release.

FEBS Lett, 1986 Oct 27, 207(2), 222 - 6
Botulinum toxin types A, B and D inhibit catecholamine secretion from bovine adrenal medullary cells; Knight DE; Evoked catecholamine secretion from cultured bovine adrenal medullary cells is inhibited by commercially available botulinum toxins - types A, B and D (10(4)-10(6) MLD/ml of culture medium) . Basal secretion is also inhibited . The catecholamine content of such toxin-treated cells is larger than that of control cells and may in part be a result of the inhibition of basal release . The onset of action of botulinum toxin types A and D can be neutralised by their respective antisera . Concentrations of botulinum toxins A, B or D that inhibit secretion leave unaffected the 45Ca2+ influxes normally associated with secretion . These data provide further evidence to support the idea {(1985) Nature 317, 719-721} that botulinum toxins block secretion by acting downstream of the Ca2+ transient at or near the site of exocytosis.

Klin Monatsbl Augenheilkd, 1986 Oct, 189(4), 283 - 5
{Blepharospasm treatment with botulinum toxin (follow-up)}; Roggenkamper P; Within a period of 14 months 73 patients with idiopathic blepharospasm which could not be treated satisfactorily by any other form of therapy were treated by injections of botulin toxin . In 55 cases the follow-up period was sufficiently long to permit an evaluation . Thirty-five patients were temporarily completely free of complaints; in one patient there was no improvement . In the remaining cases there was a slight to distinct reduction in symptoms, which often cause severe suffering . The full effect of the drug lasted between 1 and 27 weeks (average 7.5 weeks) . No systemic side-effects were observed; local side-effects (subcutaneous bleeding during injection, slight ptosis, intermittent diplopia) seen in 6 cases were of very minor significance.

J Pediatr Ophthalmol Strabismus, 1986 Sep-Oct, 23(5), 216 - 21
Treatment of facial spasm with Oculinum (C . botulinum toxin); Biglan AW et al.; Forty-six consecutive adult patients with facial spasm were treated with one or more Botulinum toxin (Oculinum) injections to control muscle spasm . During the follow-up period of 6.6 months (one week-12 months), 27 patients required retreatment with a mean time interval of 3.4 months between treatments . Control of facial spasm was achieved in all patients . Complications included occasional bruising around the injection site and a transient blepharoptosis in one patient . No systemic effect was observed . This preliminary report suggests that treatment with Botulinum toxin (Oculinum) is an acceptable alternative to surgical management in select patients.

Pflugers Arch, 1986 Sep, 407(3), 275 - 8
Effects of botulinum toxin induced muscle paralysis on endocytosis and lysosomal enzyme activities in mouse skeletal muscle; Tagerud S et al.; The effects of botulinum toxin (type A) induced muscle paralysis on endocytosis and lysosomal enzyme activities in skeletal muscle were compared with the effects of surgical denervation . Muscle atrophy, measured as decrease in total muscle protein content, was as large or larger after botulinum toxin treatment as after denervation . Endocytic activity, measured as the in vitro uptake of horseradish peroxidase, and the specific activities of the lysosomal enzymes N-acetyl-beta-D-glucosaminidase and cathepsin D were all increased six days after denervation . Only the specific activity of cathepsin D was increased six days after botulinum toxin poisoning . The uptake of horseradish peroxidase and the specific activity of N-acetyl-beta-D-glucosaminidase were also increased eleven days after poisoning . Transverse sections of eleven days botulinum poisoned muscles from animals injected with horseradish peroxidase showed fibres with dense peroxidase staining similar to those seen in denervated muscle although they seemed to occur less frequently . The results show that increases in endocytic activity and lysosomal enzyme activities may occur in skeletal muscle without the presence of degenerating axons . The differences in effects of surgical denervation and botulinum toxin induced paralysis are discussed in terms of what is known about the mechanism of action of botulinum toxin and the possible functional roles of the two lysosomal enzymes studied.

Lancet, 1986 Aug 2, 2(8501), 245 - 7
Double-blind study of botulinum toxin in spasmodic torticollis; Tsui JK et al.; In a double-blind trial in 21 patients with spasmodic torticollis botulinum-A toxin produced both subjective and objective improvement, including significant pain relief in 14 of the 16 patients presenting with pain . Side-effects were more frequently reported during placebo administration and no significant systemic adverse reactions were noted.

Can J Ophthalmol, 1986 Aug, 21(5), 167 - 9
The orbicularis stripping operation for intractable blepharospasm: surgical results in eighteen patients; Hurwitz JJ et al.; The orbicularis stripping operation may be the only method of relieving the symptoms of essential blepharospasm when medical means and botulinum toxin injections fail.

J Cell Biol, 1986 Aug, 103(2), 521 - 34
Interaction of 125I-labeled botulinum neurotoxins with nerve terminals . I . Ultrastructural autoradiographic localization and quantitation of distinct membrane acceptors for types A and B on motor nerves; Black JD et al.; The labeling patterns produced by radioiodinated botulinum neurotoxin (125I-BoNT) types A and B at the vertebrate neuromuscular junction were investigated using electron microscopic autoradiography . The data obtained allow the following conclusions to be made . 125I-BoNT type A, applied in vivo or in vitro to mouse diaphragm or frog cutaneous pectoris muscle, interacts saturably with the motor nerve terminal only; silver grains occur on the plasma membrane, within the synaptic bouton, and in the axoplasm of the nerve trunk, suggesting internalization and retrograde intra-axonal transport of toxin or fragments thereof . 125I-BoNT type B, applied in vitro to the murine neuromuscular junction, interacts likewise with the motor nerve terminal except that a lower proportion of internalized radioactivity is seen . This result is reconcilable with the similar, but not identical, pharmacological action of these toxin types . The saturability of labeling in each case suggested the involvement of acceptors; on preventing the internalization step with metabolic inhibitors, their precise location became apparent . They were found on all unmyelinated areas of the nerve terminal membrane, including the preterminal axon and the synaptic bouton . Although 125I-BoNT type A interacts specifically with developing terminals of newborn rats, the unmyelinated plasma membrane of the nerve trunk is not labeled, indicating that the acceptors are unique components restricted to the nerve terminal area . BoNT types A and B have distinct acceptors on the terminal membrane . Having optimized the conditions for saturation of these binding sites and calibrated the autoradiographic procedure, we found the densities of the acceptors for types A and B to be approximately 150 and 630/micron 2 of membrane, respectively . It is proposed that these membrane acceptors target BoNT to the nerve terminal and mediate its delivery to an intracellular site, thus contributing to the toxin's selective inhibitory action on neurotransmitter release.

J Cell Biol, 1986 Aug, 103(2), 535 - 44
Interaction of 125I-labeled botulinum neurotoxins with nerve terminals . II . Autoradiographic evidence for its uptake into motor nerves by acceptor-mediated endocytosis; Black JD et al.; Using pharmacological (Simpson, L.L., 1980, J . Pharmacol . Exp . Ther . 212:16-21) and autoradiographic techniques (Black, J.D., and J.O . Dolly, 1986, J . Cell Biol., 103:521-534), it has been shown that botulinum neurotoxin (BoNT) is translocated across the motor nerve terminal membrane to reach a postulated intraterminal target . In the present study, the nature of this uptake process was investigated using electron microscopic autoradiography . It was found that internalization is acceptor-mediated and that binding to specific cell surface acceptors involves the heavier chain of the toxin . In addition, uptake was shown to be energy and temperature-dependent and to be accelerated by nerve stimulation, a treatment which also shortens the time course of the toxin-induced neuroparalysis . These results, together with the observation that silver grains were often associated with endocytic structures within the nerve terminal, suggested that acceptor-mediated endocytosis is responsible for toxin uptake . This proposal is supported further by the fact that lysosomotropic agents, which are known to interfere with the endocytic pathway, retard the onset of BoNT-induced neuroparalysis and also affect the distribution of silver grains at nerve terminals treated with 125I-BoNT . Possible recycling of BoNT acceptors (an important aspect of acceptor-mediated endocytosis of toxins) at motor nerve terminals was indicated by comparing the extent of labeling in the presence and absence of metabolic inhibitors . On the basis of these collective results, it is concluded that BoNT is internalized by acceptor-mediated endocytosis and, hence, the data support the proposal that this toxin inhibits release of acetylcholine by interaction with an intracellular target.

Ophthalmology, 1986 Jul, 93(7), 917 - 23
Treatment of facial spasm with botulinum toxin . An interim report; Frueh BR et al.; Forty-eight patients were given serial injections of botulinum toxin in their eyelids for treatment of eyelid spasm during a two-year interval . Ninety-four percent obtained relief of spasm from botulinum toxin injection . The duration of the spasm-free interval as well as the incidence of ptosis and of diplopia was dose dependent . The marked increase in the incidence of these side effects with only a small increase in the duration of the spasm-free interval, when a dose of 25 units per lid was used, leads the authors to conclude that this dose is too high and should not be used . Since diplopia was most commonly caused by paresis of the inferior oblique muscle, and since blepharospasm usually can be controlled by excising the upper lid protractors, further studies are required to determine whether lower lid injection is necessary and, if it is found to be so, whether injecting only the lateral portion of the lid would be adequate.

J Neurol Neurosurg Psychiatry, 1986 Jul, 49(7), 827 - 9
Botulinum toxin treatment of hemifacial spasm; Elston JS; Six patients with hemifacial spasm were treated with injections of botulinum toxin A into the orbicularis oculi; the abnormal movements around the eye were relieved for an average of 15 weeks . There were no systemic or significant local side effects, and in view of the risks involved in neurosurgical treatment, a trial of botulinum toxin injections is recommended in the first instance in this condition.

J Neurol Neurosurg Psychiatry, 1986 Jul, 49(7), 817 - 9
Sera from patients with motor neuron disease and associated paraproteinaemia fail to inhibit experimentally induced sprouting of motor nerve terminals; Donaghy M et al.; IgG kappa paraproteinaemia was discovered in one patient with amyotrophic lateral sclerosis and one with chronic spinal muscular atrophy . Serum from these patients was injected into mouse muscles paralysed by botulinum toxin which is known to induce sprouting from motor nerve terminals . Daily injection of serum for 7 days failed to inhibit terminal sprouting . It is concluded that the paraproteins did not recognise a growth factor postulated as being implicated in the pathogenesis of motor neuron disease.

Anal Biochem, 1986 Jul, 156(1), 213 - 9
Purification of type E botulinum neurotoxin by high-performance ion exchange chromatography; Schmidt JJ et al.; A purification procedure for type E botulinum neurotoxin has been developed, based solely on high-performance ion exchange chromatography . The method exploits the differential chromatographic behavior of the free neurotoxin versus the neurotoxin-protein 12S complex . The high purity of the product was demonstrated with sodium dodecyl sulfate-gel electrophoresis and amino acid sequencing . Beginning with dialyzed crude extract, at least 4 mg of pure neurotoxin could be obtained in two working days . The method has been adapted to user-prepared columns for processing large volumes of crude neurotoxin in one batch.

Neuroscience, 1986 Jun, 18(2), 499 - 505
Nerve dependent regulation of neural cell adhesion molecule expression in skeletal muscle; Moore SE et al.; The expression of neural cell adhesion molecule was analysed by indirect immunofluorescence on adult mouse skeletal muscle subjected to a variety of experimental lesions . Adult mouse muscle does not express neural cell adhesion molecule at the sarcolemma . However, following denervation there is a rapid rise in neural cell adhesion molecule levels; this is initially in the cytoplasm of the myofibres but by 18 days there is intense reactivity at the sarcolemma . A nerve crush lesion was used to show that the increase in neural cell adhesion molecule levels following denervation is accompanied by a switch-off of neural cell adhesion molecule expression following reinnervation . Paralysis of skeletal muscle by botulinum toxin injection is sufficient to activate neural cell adhesion molecule expression . As paralysis of skeletal muscle by botulinum toxin is not accompanied by activation of muscle satellite cells or degeneration products of nerve or myelin, it suggests that the observed levels of neural cell adhesion molecule are synthesised by myofibres . As the expression of neural cell adhesion molecule in these lesions parallels the ability of skeletal muscle to accept innervation it is possible that neural cell adhesion molecule acts as a molecular cue at the sarcolemma in regulating synaptogenesis.

Pflugers Arch, 1986 Jun, 406(6), 636 - 40
Facilitation, augmentation and potentiation of transmitter release at frog neuromuscular junctions poisoned with botulinum toxin; Lupa MT et al.; Botulinum toxin type A (Botx) is a potent neurotoxin which inhibits specifically cholinergic synaptic transmission by an unknown mechanism . In order to gain further insight into the mode of action of this toxin, the effect of conditioning nerve stimuli on neuromuscular transmission was studied at endplates of Botx-poisoned and unpoisoned control cutaneous pectoris muscles in the frog . Effects of single conditioning stimuli (facilitation) and multiple high-frequency stimuli (augmentation and potentiation) on epp amplitude and mepp frequency were studied . The main results were that initial facilitation was significantly increased and its decay time constant significantly decreased in Botx-poisoned muscles, while augmentation was unchanged and potentiation was abolished . These changes could be detected before the muscle became completely paralysed, suggesting that they reflect a primary disturbance in the Ca2+-dependent release process.

Biochemistry, 1986 May 20, 25(10), 2872 - 6
Ion-conducting channels produced by botulinum toxin in planar lipid membranes; Donovan JJ et al.; The interaction of botulinum neurotoxin (Botx) with planar lipid membranes was studied by measuring the ability of the toxin to form ion-conducting channels . Channel formation was pH dependent . At physiological pH, Botx formed no channels, whereas at pH 6.6, the toxin formed channels with a unit conductance of 12 pS in 0.1 M NaCl . The rate of channel formation increased with decreasing pH, reaching a maximum at pH 6.1, and then decreased at lower values of pH . The channels, once formed, were permanent entities in the membrane throughout the course of an experiment and fluctuated between an open and a closed state . The rate of channel formation depended upon the square of the toxin concentration, suggesting an aggregation step is involved in channel formation . The data were consistent with the hypothesis that Botx enters cells through endocytosis, followed by its release into the cytoplasm at low pH.

Neurology, 1986 Apr, 36(4), 545 - 7
Botulinum toxin for blepharospasm: single-fiber EMG studies; Sanders DB et al.; In four patients who received periocular injections of botulinum toxin for blepharospasm, abnormal neuromuscular transmission was demonstrated by single-fiber EMG in arm muscles . The time course with which the abnormalities developed and cleared, as well as the inverse relationship between the neuromuscular jitter and the firing rate in the abnormal muscles, indicated that the toxin caused the abnormalities in arm muscles . No weakness was detected clinically in muscles distant from the face, but the abnormal neuromuscular transmission indicates that the toxin spread remotely from the site of injection.

Arch Neurol, 1986 Apr, 43(4), 380 - 2
Botulinum toxin in the management of blepharospasm; Dutton JJ et al.; Seventy-seven patients with blepharospasm unresponsive to other forms of therapy were treated with botulinum A toxin . The drug was injected into the orbicularis oculi muscle of both upper and lower eyelids and, in some patients, into the brows and upper part of the face as well . A total of 192 treatment sessions were given, with follow-up ranging from four weeks to 18 months . Orbicularis muscle spasms decreased markedly over the first five days following injection, both in patients with essential blepharospasm and in those with hemifacial spasm . Beneficial effect was temporary, however, and repeated injections were required for sustained relief . Fifty-two patients received multiple treatments, ranging from two to eight injections each, with a mean duration of benefit of 11.8 weeks . Results of subsequent injections were similar to the first in rapidity of onset, degree of benefit, and duration of effect . Complications were local, mild, and transient, and no systemic side effects were encountered.

Ophthalmology, 1986 Apr, 93(4), 470 - 5
Botulinum toxin for the treatment of dysthyroid ocular myopathy; Dunn WJ et al.; Eight consecutive patients with acquired deviations due to dysthyroid ocular myopathy were injected with botulinum A toxin for relief of their diplopia . Seven patients were acute in the onset of symptoms and one was chronic . All showed improvement in motility and experienced a reduction if not total relief of their symptoms . Six patients required reinjection . Complications were limited to transient ptosis, transient involvement of adjacent muscles and transient but prolonged paralysis that eventually resolved . No systemic complications were noted . We conclude that chemodenervation with botulinum A toxin may have a role in the management of dysthyroid ocular myopathy not amenable to prism treatment and may act as an adjunct to or eliminate the need for surgical correction in some patients.

J Physiol, 1986 Mar, 372, 395 - 404
Botulinum toxin prevents stimulus-induced backfiring produced by neostigmine in the mouse phrenic nerve-diaphragm; Aizenman E et al.; The origin of motor nerve antidromic activity (backfiring) induced by anticholinesterase treatment was examined in the mouse phrenic nerve-hemidiaphragm preparation . Botulinum toxin was used to determine whether backfiring is due to (a) a direct effect of the cholinesterase inhibitor on the nerve terminal, or (b) an indirect effect via the prolongation of the action of acetylcholine . In previously untreated control preparations, neostigmine produced spontaneous and stimulus-induced antidromic activity in the phrenic nerve when rapidly introduced into the diaphragm via its vasculature . This activity could be reversibly blocked by d-tubocurarine and decamethonium, but not by atropine . Neostigmine-induced backfiring did not occur in preparations in which transmitter release was blocked with botulinum toxin . Infusion of a small bolus of a high concentration of acetylcholine following neostigmine treatment resulted in a short-term increase in the incidence of antidromic activity, followed by block, in both controls and botulinum toxin-treated preparations . It is concluded that transmitter release is necessary for the production of backfiring following cholinesterase inhibition since neostigmine alone does not elicit antidromic activity in botulinum toxin-treated preparations at concentrations which are effective in controls . Our results support the hypothesis that the effects of neostigmine on the motoneurone terminal are mediated by the prolonged action of acetylcholine that occurs with inhibition of acetylcholinesterase.

Am J Ophthalmol, 1986 Feb 15, 101(2), 232 - 5
Absence of antibody production in patients treated with botulinum A toxin; Biglan AW et al.; To test the possibility of the formation of an antibody to botulinum A toxin after multiple injections of this potent neurotoxin, we collected serum samples from 28 patients who received 57 doses . These injections over a nine-month period with as much as 50 units per injection formed no detectable antibody.

Klin Monatsbl Augenheilkd, 1986 Feb, 188(2), 89 - 94
{Use of botulinum toxin in ophthalmology}; Huber A et al.; Botulin toxin A was introduced as a treatment in ophthalmology by Dr . Scott of San Francisco . One important application is in cases of blepharospasm, where the toxin is injected into the lateral parts of the lower and upper lid and, if necessary, over the eyebrows in a single dose of 1-2 nanograms, preferably using a needle under electromyographic control . The effect on the blepharospasm is visible after a few days and lasts for several months . The procedure can be repeated several times . The second application is in cases of strabismus . In paralytic strabismus, contracture of the antagonist of the paralyzed muscle can be weakened by local injection of botulin toxin with a coaxial electrode under electromyographic control . Good results were observed in cases of eye muscle disorders in endocrine ophthalmopathy . In concomitant strabismus (exotropia or esotropia) administration of botulin toxin is also possible although a certain paresis of the injected muscle has to be taken into account . The doses for strabismus vary between 1/2 and 2 nanograms of the toxin . The administration of botulin toxin either in blepharospasm or strabismus has no systemic side effects and is a safe procedure if performed under careful electromyographic control . First personal experiences in the treatment both of blepharospasmus as well paralytic strabismus and concomitant strabismus are reported.

Ophthalmology, 1986 Jan, 93(1), 1 - 3
The use of botulinum toxin in the medical management of benign essential blepharospasm; Perman KI et al.; Twenty-eight patients with mild or moderate cases of benign essential blepharospasm were treated with botulinum toxin Type A . Average follow-up was six months . The injection technique used on these patients is illustrated . The treatment was effective in virtually all patients treated, although transient; the mean interval of relief of spasm was approximately two and one-half months . Potential side effects include ptosis and epiphora . Botulinum toxin as an initial treatment or as an adjunct in postsurgical residual blepharospasm shows promise in this preliminary study.

Toxicon, 1986, 24(11-12), 1065 - 74
Botulinum A neurotoxin unlike tetanus toxin acts via a neuraminidase sensitive structure; Bigalke H et al.; The binding and effects of tetanus and botulinum A neurotoxins were studied on mouse spinal cord cultures treated with neuraminidase . In untreated cultures both neurotoxins blocked synaptic transmission . Treatment of the cell cultures with neuraminidase, 25 mU/ml for 24 hr, decreased the potency of botulinum A neurotoxin . At 7 X 10(-11) M no toxin effect on inhibitory or excitatory synapses was observed, whereas at higher concentrations of the toxin the concentration-response curve was shifted to the right by a factor of about 30 . Surprisingly, the action of tetanus toxin over a large concentration range was unaffected by pretreatment of the neurones with the enzyme . Accordingly, neurones treated with neuraminidase failed to bind 125I-botulinum A neurotoxin, whereas labelled tetanus toxin was still fixed by cell bodies, as well as by neurites, as shown by histoautoradiography . Chromatographic extraction of gangliosides from cultures prelabelled with 14C-glucosamine showed a dramatic loss in the contents of polysialogangliosides following treatment with neuraminidase . Our results indicate that neuraminidase-sensitive structures might be important for the action of botulinum A neurotoxin . The effect of tetanus toxin appears to be mediated by a different site which is insensitive to neuraminidase.

Ophthal Plast Reconstr Surg, 1986, 2(3), 143 - 6
Treatment of hemifacial spasm with botulinum A toxin . Results and rationale; Gonnering RS; Hemifacial spasm is characterized by unilateral, periodic, tonic contractions of facial muscles, thought to be caused by mechanical compression at the root-exit zone of the facial nerve . Electrophysiologic abnormalities such as ectopic excitation and synkinesis are typical . Although posterior fossa microsurgical nerve decompression is successful in bringing about relief of the spasm in most cases, it carries a risk to hearing . As an alternative treatment, 15 patients with hemifacial spasm were given a total of 41 sets of injections with botulinum A toxin, with a mean follow-up of 14.3 +/- 1.1 months . Relief of symptoms lasted a mean of 108.3 +/- 4.2 days . Mild transient lagophthalmos and ptosis were the only complications . Although the exact mechanism of its action and beneficial effect is speculative at this time, botulinum A toxin appears to offer an effective, safe alternative to more radical intracranial surgery for patients with hemifacial spasm.

Ophthal Plast Reconstr Surg, 1986, 2(2), 71 - 4
Orbicularis muscle pathology after botulinum toxin injection; Wojno T et al.; A 49-year-old woman underwent 1 year (six injections) of botulinum toxin treatments for essential blepharospasm . When she became dissatisfied with the need for repeat injections, she elected to undergo an orbicularis muscle stripping procedure for control of spasms . Pathologic examination of the orbicularis muscles showed only minimal degenerative changes . Botulinum toxin injection into the orbicularis is known to provide only temporary relief of essential blepharospasm . This may be because the resulting orbicularis atrophy is insufficient to maintain permanent control of these spasms.

Toxicon, 1986, 24(2), 123 - 30
Actions of beta-bungarotoxin on spontaneous release of transmitter at muscle end-plates treated with botulinum toxin; Tse CK et al.; Rat leg muscles were injected subcutaneously with sublethal doses of type A botulinum neurotoxin, and the extensor digitorum longus muscle removed three days later . Intracellular microelectrode recordings were then made of miniature end-plate potentials (mepps) . The mepp frequency was reduced by botulinum toxin, while mepp rise times were slowed . Mepp amplitude distributions became characteristically skew . beta-Bungarotoxin (140 nM) was applied to normal muscles in vitro and recordings were made 10-30 min later . The main effect was an increase in mepp frequency during this period . Mepp rise times were unaffected . When beta-bungarotoxin was applied in vitro to muscles treated with botulinum toxin there was also an increase in mepp frequency, although to a value less than in normal muscles . The mepp rise times were speeded up to normal values . The mepp amplitude and rise time distributions showed no obvious evidence for the addition of a second component to the distribution . The data appear to support the hypothesis that the sites for spontaneous release in botulinised muscle may be located at or near the usual release sites at the active zones.

Brain Res, 1985 Dec 23, 360(1-2), 318 - 24
Botulinum A neurotoxin inhibits non-cholinergic synaptic transmission in mouse spinal cord neurons in culture; Bigalke H et al.; The effects of botulinum A neurotoxin and tetanus toxin were studied in cultured mouse spinal cord neurons . In approximately 60% of the neurons (n = 150), botulinum A neurotoxin caused paroxysmal depolarizing events . In two cells hyperpolarizing shifts were observed . The pattern of the burst-like activity varied in shape and frequency in individual cells . Between the paroxysmal events, ongoing synaptic activity could be recorded . The other 40% of the treated neurons did not develop a characteristic pattern of bursts, but there was a decrease in frequency of synaptically generated events . In contrast to botulinum A neurotoxin, tetanus toxin invariably produced well organized paroxysmal events without any synaptic activity between them . At later stages botulinum A neurotoxin and tetanus toxin blocked inhibitory and excitatory postsynaptic potentials in all neurons studied . These results have demonstrated, for the first time using electrophysiological techniques, that botulinum A neurotoxin blocks both excitatory and inhibitory synaptic transmission in the mammalian central nervous system . There are however differences between these effects of botulinum A neurotoxin and the actions of tetanus toxin on these cells . It is suggested that at the femtomolar range tetanus toxin blocks selectively central inhibitory systems and botulinum A neurotoxin the motor endplate . At the picomolar range both toxins affect many if not all, transmitter systems.

Br J Ophthalmol, 1985 Dec, 69(12), 891 - 6
Paralytic strabismus: the role of botulinum toxin; Elston JS et al.; Five adults with acute extraocular muscle palsies followed by limited recovery of muscle function were treated with injections of botulinum toxin A to the ipsilateral antagonist of the affected muscle . Three were cases of unilateral sixth nerve palsy, one of bilateral sixth nerve palsy, and one of third nerve palsy . After a period of paralysis, during which the strabismus was overcorrected, the injected muscle recovered and binocular sensory and motor functions were restored and have been maintained for periods of up to 21 months . The treatment is a safe and effective alternative to surgery, particularly in cases in which a general anaesthetic is not advisable.

Can J Neurol Sci, 1985 Nov, 12(4), 314 - 6
A pilot study on the use of botulinum toxin in spasmodic torticollis; Tsui JK et al.; Dystonic torticollis has been treated with local injections of botulinum toxin in a single blind study of 12 patients . A significant decrease of abnormal movements was recorded, and pain improved . Further studies are desirable to define the optimum dosage and site for injections, and the long term effects of repeated injections.

Nature, 1985 Oct 24-30, 317(6039), 719 - 21
Inhibition of exocytosis in bovine adrenal medullary cells by botulinum toxin type D; Knight DE et al.; Botulinum toxins are known to block transmitter release at peripheral cholinergic synapses, producing muscular weakness and paralysis . The toxins may also block adrenergic transmission, although this effect is less well understood . The mechanisms by which toxins act are unclear . They are proteins of relative molecular mass approximately 150,000 and are structurally similar to tetanus toxin . It is generally accepted that a rise in intracellular calcium concentration is sufficient to trigger secretion by exocytosis, but it is not known whether the toxins block secretion by preventing this Ca transient or whether they act downstream from Ca entry by interfering with the process of exocytosis itself . We have attempted to resolve these questions in the case of the adrenergic system by studying the effects of botulinum toxins (types A, B, D and E) on the secretory response of isolated bovine adrenal medullary cells maintained in culture . The cells were either challenged with various secretagogues or rendered leaky and challenged directly with Ca buffers . We report here that botulinum toxin type D inhibits secretion in a time- and dose-dependent manner, the results being entirely consistent with the idea that the toxin acts at or near the site of exocytosis rather than at the sites controlling the rise in free Ca.

Br J Ophthalmol, 1985 Oct, 69(10), 718 - 24
Treatment of strabismus in adults with botulinum toxin A; Elston JS et al.; Eighty-five adults with horizontal concomitant strabismus were treated with an injection of a low dose of botulinum toxin A (BTXA) into the lateral or medial rectus muscle . The ocular deviation was reduced by an average of 60% independently of its size and whether or not surgery had previously been performed . The change was temporary, however, except in those cases with binocular functions, when fusion was re-established . Repeated low dose injections can maintain the improvement, but at higher doses, although larger reductions are produced, temporary local side effects--ptosis and vertical strabismus--are common . The technique is simple, well tolerated, and has no systemic side effects.

Neurology, 1985 Oct, 35(10), 1499 - 500
Blepharospasm, Meige syndrome, and hemifacial spasm: treatment with botulinum toxin; Mauriello JA Jr; In 34 patients with idiopathic blepharospasm, 5 with Meige syndrome, and 11 with hemifacial spasm, local injection of botulinum toxin gave temporary relief from the involuntary movements of the eyelids . Local ophthalmologic side effects were minimal . There are no known systemic side effects . The procedure had to be repeated every 12 weeks . Tolerance to the toxin did not appear.

Arch Ophthalmol, 1985 Sep, 103(9), 1305 - 6
Hemifacial spasm treated with botulinum A toxin injection; Savino PJ et al.; Fifteen patients with hemifacial spasm were treated with botulinum A toxin injections . All patients experienced relief from spasm, with the effect lasting an average of 12.2 weeks . Complications were tearing in four patients, inability to close the involved eye in three patients, corneal exposure in one patient, and ectropion in one patient . All complications were transient and deemed minor by the patients.

Am J Ophthalmol, 1985 Aug 15, 100(2), 305 - 7
Sequelae of botulinum toxin injection; Lingua RW; A total of 136 injections was given to 83 patients for strabismus (99 injections), blepharospasm (29 injections), and spastic entropion (eight injections) . All four patients with entropion experienced temporary benefits and early recurrence; one injection resulted in temporary paralytic ectropion . Two of 13 patients treated for blepharospasm developed transient bilateral blepharoptosis . Temporary and related sequelae of extraocular muscle injection included one periocular hemorrhage, one total ophthalmoplegia, and a 44% incidence (29 of 66 patients) of blepharoptosis, which in two patients lasted more than six months . Within three days of injection one patient developed homolateral acute herpes simplex keratitis and a second died of an acute myocardial infarction . No causal relationship for these events has been established.

J Neurol Sci, 1985 Jul, 69(3), 171 - 82
Neurogenic origin of an experimental myopathy induced by the ionophore A23187; Llados FT; An experimental myopathy was induced in frog skeletal muscle incubated in vitro with the calcium ionophore A23187 . This myopathy could be prevented if Ca2+ ions in the incubating medium were replaced by either Mg2+ or Co2+ ions . Similarly, preincubation of the preparation with acetylcholine (ACh) receptor blockers such as d-tubocurarine (d-TC) or alpha-bungarotoxin (alpha-BuTX) prevented the development of muscle damage . In muscles that had been previously denervated or treated with botulinum toxin (BTX) the ionophore failed to induce morphological alterations . These results suggest that spontaneous transmitter release, greatly increased by the ionophore A23187, triggers Ca2+ influx into the muscle fibres at the endplate region . Elevated levels of intracellular Ca2+ presumably activate proteolytic systems leading to myofilament disassembly.

Toxicol Appl Pharmacol, 1985 Jun 30, 79(2), 211 - 7
Comparison of the action of types A and F botulinum toxin at the rat neuromuscular junction; Kauffman JA et al.; Blockade of neuromuscular transmission was produced in the lower hind limb of the rat by local injection of either type A or type F botulinum toxin (BoTx) . At 1, 3, 7, and 10 days after injection, the extensor digitorum longus (edl) nerve-muscle preparation was excised and analyzed for alterations in muscle mechanical properties or spontaneous and nerve stimulus-evoked quantal transmitter release . Muscles receiving type A toxin were paralyzed up to and including 7 days after injection . Muscles treated with type F toxin, although completely paralyzed at 1 and 3 days after injection, twitched in response to nerve stimulation by 7 days . Both toxins induced a marked decrease in the frequency of miniature end-plate potentials, but type A did so to a greater extent . Between 1 and 3 days after toxin injection nerve impulse-evoked transmitter release was reduced in both type A- and type F-treated muscles . Evoked release was temperature sensitive in type A-treated muscles but not in those treated with type F . 3,4-Diaminopyridine (3,4-DAP), a compound which increases nerve-evoked transmitter release by increasing Ca2+ influx, was more effective in reversing the paralysis in type A than in type F-treated muscles . 3,4-DAP induced asynchronous end-plate potentials in response to nerve stimulation in type F-paralyzed muscles, but not in muscles treated with type A . Amidination of the amino groups (presumably lysine) on the toxin by treatment with ethylacetimidate increased the potency and efficacy of only type F BoTx . The results show that type F BoTx differs from type A, mainly by its lower potency, efficacy, shorter duration of action, and by being less effectively antagonized by 3,4-DAP.

Br Med J (Clin Res Ed), 1985 Jun 22, 290(6485), 1857 - 9
Effect of treatment with botulinum toxin on neurogenic blepharospasm; Elston JS et al.; Botulinum toxin type A creates temporary localised flaccid paralysis after injection into skeletal muscle . Thirty four patients with blepharospasm, of whom 28 also had the oromandibular dystonia syndrome, were treated with injections of botulinum toxin type A into the orbicularis oculi, and 28 showed functional improvement after the treatment . A high incidence of local side effects occurred, especially partial ptosis, which was well tolerated . There were no systemic side effects . The average period of relief was 2.5 months, increasing to 2.8 months after a second injection . Functional improvement was limited in patients with severe associated dystonia.

Can J Ophthalmol, 1985 Jun, 20(4), 135 - 41
Ophthalmologic use of botulinum A exotoxin; Carruthers JD; Purified botulinum A exotoxin was used to treat 9 adults with strabismus, 22 adults with incapacitating essential blepharospasm and 1 adult with "senile" spastic lower-eyelid entropion . Eight of the strabismus patients received one injection each into one horizontal extraocular muscle under electromyographic control in the outpatient clinic; the ninth patient received two injections . One week after the injection there was an 81% change on average in the angle of deviation . In the three patients followed up for 4 to 9 months the average change was 66% . For the patients with blepharospasm the toxin was injected into the orbicularis oculi . Relief of spasm lasted an average of 12 weeks after the first treatment and 15 weeks after the second . In the patient with spastic entropion the symptoms resolved with repeated injection of the lower-lid orbicularis . In all three groups the injections were well tolerated . The main complication was transient ptosis, which occurred in about 30% of the first two groups.

Klin Monatsbl Augenheilkd, 1985 Jun, 186(6), 453 - 4
{The treatment of eye muscle diseases with botulinum toxin}; Lang J; The author discusses the possible methods of treatment with botulin (Scott) in cases of blepharospasm, paralytic strabismus, and concomitant strabismus.

Am J Ophthalmol, 1985 May 15, 99(5), 542 - 6
The use of botulinum toxin in blepharospasm; Shorr N et al.; Twenty-two patients, 17 with benign essential blepharospasm and five with hemifacial spasm, received botulinum toxin by subcutaneous injections in the eyelids and eyebrows . All 22 patients received a standard treatment of 12.5 units of botulinum toxin per side at each injection session . The orbicularis oculi muscle showed pronounced weakness after injection and the spasms decreased . Spasms often recurred despite persistent orbicularis oculi muscle weakness . Side effects were minimal . All patients received some relief, which generally lasted six to 12 weeks.

Brain Res, 1985 May 13, 334(1), 139 - 46
The use of antibody Fab fragments specifically directed to two different complementary parts of the tetanus toxin molecule for studying the mode of action of the toxin; Gawade S et al.; The injection of 500 minimal lethal doses (MLDs) of tetanus toxin into mice routinely causes a flaccid-type paralysis and death within 8 h . Non-precipitating antibody fragments (Fab) directed against each of two papain cleavage products of tetanus toxin (Ibc and IIc) were used to study this botulinum toxin-type effect of tetanus toxin . Ibc (100,000 daltons) is a toxic fragment which does not bind to gangliosides but will produce a flaccid type paralysis when injected into mice . Treatment of intact tetanus toxin (500 MLDs) with Fab-Ibc prevents the flaccid type paralysis and such mice will die from a spastic paralysis after about 24 h . IIc (50,000 daltons) is an atoxic fragment of tetanus toxin which binds tightly to gangliosides . Treatment of tetanus toxin with Fab-IIc prior to intracerebral injection converts the characteristic spastic paralysis to a flaccid paralysis . It is proposed that the botulinum toxin-type effect of tetanus toxin complexed to Fab-IIc results from the inability of such complexes to be transported to the central nervous system . Moreover, the ability of Fab-Ibc to prevent flaccid paralysis, but not spastic paralysis, suggests that both types of paralysis may be mediated by the same portion of the tetanus toxin molecule.

Ophthalmology, 1985 May, 92(5), 676 - 83
Botulinum toxin injection in the management of lateral rectus paresis; Scott AB et al.; Seventeen patients with lateral rectus paresis (3 bilateral) were treated by injection of botulinum toxin to the antagonist medial rectus to eliminate its unopposed action or to eliminate its contracture . This allowed maintenance of single binocular vision in most patients while waiting for the palsy to heal, especially important in two children . It allowed avoidance of surgery in some cases, and a reduction or elimination of medial rectus surgery when later intervention was needed for persistent paralysis . The release of medial rectus shortening and stiffness (contracture) after just a few days of denervation was unexpected . This implies an internal muscular mechanism of contracture, perhaps sarcomere overlap, different from the fibrotic changes found in muscles after inflammation or trauma . These results also provide a rationale for experimental denervation treatment of contracture in limb muscle disorders.

Biochem Biophys Res Commun, 1985 Mar 29, 127(3), 768 - 72
Partial amino acid sequences of the heavy and light chains of botulinum neurotoxin type E; Sathyamoorthy V et al.; The dichain type E botulinum neurotoxin, a product of nicking the single chain protein by trypsin, is composed of a heavy and light chains . Sequence of the first 13 and 20 N-terminal residues of these two chains were determined . Also, proof is provided here that (i) the light chain of the nicked (dichain) is derived from the N-terminal one-third of the parent single chain neurotoxin, and (ii) molecular events leading to the activation, of the single chain neurotoxin cannot involve tryptic cleavage at or very close to the N-terminal of the single chain protein . The partial amino acid sequence of the light chain of botulinum type E and tetanus neurotoxins show significant similarity between the two clostridial neurotoxins.

Arch Ophthalmol, 1985 Mar, 103(3), 347 - 50
Botulinum A toxin injection as a treatment for blepharospasm; Scott AB et al.; Thirty-nine patients with blepharospasm were treated with injections of botulinum A toxin into the lid and brow . The maximum number of injections in one patient was 16 over a period of 24 months . A reduction of abnormal movement occurred in all patients, lasting up to 170 days . Both the amount and the duration of effect were dose dependent . Reinjection for recurrence had effects similar to the original injection . Tearing, dry-eye symptoms, or transient ptosis occurred in 20% of injections, especially in patients who had had previous eyelid surgery.

J Neurosci, 1985 Mar, 5(3), 751 - 8
Motor nerve terminal outgrowth and acetylcholine receptors: inhibition of terminal outgrowth by alpha-bungarotoxin and anti-acetylcholine receptor antibody; Pestronk A et al.; Motor nerves undergo extensive terminal outgrowth when the muscles they supply are "functionally denervated." In this study, we have investigated the role of the acetylcholine receptors (AChRs), newly appearing in such muscles, in promoting nerve terminal outgrowth . The amount of outgrowth was determined by morphometric measurement of nerve terminal branching, endplate length, and ultraterminal sprouts, in cholinesterase-silver-stained neuromuscular junctions . Presynaptic neuromuscular blockade with botulinum toxin induced pronounced nerve terminal outgrowth in both the rat and mouse soleus muscles, although ultraterminal sprouts did not occur in the rat soleus . By contrast, postsynaptic neuromuscular blockade with alpha-bungarotoxin (alpha-BuTx) induced little or no terminal outgrowth, although it caused "functional denervation." Moreover, alpha-BuTx and anti-AChR antibody inhibited the terminal outgrowth otherwise induced by botulinum toxin . Other types of motor nerve growth, such as nerve regeneration, were unaffected by these agents . Our results are consistent with the concept that extrajunctional AChRs in skeletal muscle play an important role in the control of motor nerve terminal outgrowth at neuromuscular junctions.

Proc Natl Acad Sci U S A, 1985 Mar, 82(6), 1692 - 6
Channels formed by botulinum, tetanus, and diphtheria toxins in planar lipid bilayers: relevance to translocation of proteins across membranes; Hoch DH et al.; The heavy chains of both botulinum neurotoxin type B and tetanus toxin form channels in planar bilayer membranes . These channels have pH-dependent and voltage-dependent properties that are remarkably similar to those previously described for diphtheria toxin . Selectivity experiments with anions and cations show that the channels formed by the heavy chains of all three toxins are large; thus, these channels could serve as "tunnel proteins" for translocation of active peptide fragments . These findings support the hypothesis that the active fragments of botulinum neurotoxin and tetanus toxin, like that of diphtheria toxin, are translocated across the membranes of acidic vesicles.

Am J Ophthalmol, 1985 Feb 15, 99(2), 176 - 9
Treatment of blepharospasm with botulinum toxin; Tsoy EA et al.; In 43 patients (81 eyes) with blepharospasm resistant to other forms of therapy, 149 outpatient injections of botulinum A toxin were given into the orbicularis oculi muscle . Subsequent follow-up periods ranged from ten to 210 days . Orbicularis oculi spasm, eyelid forced closure, and eyebrow spasm all decreased substantially over the first ten days following the initial injection . Patients with essential blepharospasm, hemifacial spasm, or previous surgery all responded in a similar fashion . This beneficial effect was transient, however, and additional treatment was required for sustained relief . Twenty-five patients received multiple injections with a mean interval of 65 days . Results of second and third injections were similar to the first in rapidity of onset and duration of effect . Complications were local, mild, and transient, and no systemic side effects were noted.

Q J Exp Physiol, 1985 Jan, 70(1), 63 - 73
Effects of botulinum toxin on synapse formation and acetylcholine sensitivity in skeletal muscle in the newt; Sayers H et al.; A sublethal dose of botulinum toxin (type E) was injected into the biceps muscle of the forelimb of adult newts, Triturus cristatus, causing local paralysis for up to 3 weeks and impaired transmission for about 3 months . The paralysed muscles became supersensitive to the acetylcholine analogue, carbachol, and also accepted innervation from an implanted foreign nerve . These results suggest that in the newt activity is important in the control of muscle sensitivity to acetylcholine and synapse formation . During the first 2 months after botulinum toxin, stimulation of the implanted nerve caused contraction in every muscle examined (n = 13) . In muscles examined between 3 and 14 months after toxin injection there was no contraction in response to stimulation of the implanted nerve in eight out of nineteen cases, suggesting that regression of the foreign innervation might have occurred . Further evidence for regression of foreign innervation was obtained by intracellular recordings from those muscles which contracted in response to stimulation of both nerves . The proportion of muscle fibres responding to stimulation of the implanted nerve fell significantly (P less than 0.05) from 65.7 +/- 7.1% (+/- S.E.M., n = 239) in thirteen muscles examined during the first 4 months to 39.5 +/- 5.5% (+/- S.E.M., n = 188) in nine muscles examined between 9 and 14 months after toxin injection . There was a strong negative correlation (r = 0.81; P less than 0.001) between the proportion of muscle fibres responding to stimulation of the original and implanted nerves in individual muscles.

Ophthal Plast Reconstr Surg, 1985, 1(4), 253 - 61
Management of blepharospasm; Kennedy RH et al.; From 1950 through 1984, 123 patients underwent surgical treatment of blepharospasm at the Mayo Clinic . During this period, four different operations (proximal and distal neurectomy and two types of myectomy) were used . Significant recurrent or residual blepharospasm was observed more frequently (p less than 0.01) and need for subsequent operations was greater (p less than 0.01) among patients who had undergone distal neurectomy than among those who had had myectomy . These data support the view that myectomy is a more effective procedure than neurectomy . Initial results with botulinum toxin injection seem to indicate that it is an effective short-term treatment for blepharospasm . However, its long-term efficacy and safety need further study, as does the role it should play in combination with myectomy.

Trans Ophthalmol Soc U K, 1985, 104 ( Pt 2), 208 - 10
The use of botulinum toxin A in the treatment of strabismus; Elston JS; Alteration of the position of the eye within the orbit is conventionally achieved by surgery on the extraocular muscles; a new method, selective weakening of one of the muscles with Botulinum Toxin A is described . Results in the first 85 cases treated show that it is a safe, and effective treatment, particularly suited to certain cases of strabismus.

Trans Am Ophthalmol Soc, 1985, 83, 367 - 86
Management of blepharospasm; Waller RR et al.; From 1950 through 1984, 123 patients underwent surgical treatment of blepharospasm at the Mayo Clinic . During this period, four different operations (proximal and distal neurectomy and two types of myectomy) were used . Significant recurrent or residual blepharospasm was observed more frequently (P less than 0.01), and need for subsequent operations was greater (P less than 0.01), among patients who had undergone distal neurectomy than among those who had had myectomy . These data support the view that myectomy is a more effective procedure than neurectomy . Initial results with botulinum toxin injection seem to indicate that it is an effective short-term treatment for blepharospasm . However, its long-term efficacy and safety need further study, as does the role it should play in combination with myectomy.

Zh Nevropatol Psikhiatr Im S S Korsakova, 1985, 85(11), 1634 - 9
{Myasthenic syndromes linked to mediator secretion disorders}; Sanadze AG et al.; On the basis of clinical and electrophysiological examinations of 23 patients with an impaired secretion of the mediator, the following 5 groups of patients with myasthenic syndromes (MS) characterized by disturbances of the neuromuscular transision were identified: patients with bronchogenic carcinoma of the lung; males with a clinical picture closely resembling the one described in bronchogenic cancer of the lung; young males and females with an abnormal thyroid gland; patients with a mild subcortical syndrome and with ataxia; patients with chronic botulinic intoxication . Correlation of the results of electrophysiological examination did not reveal any specificity for any of the identified groups of patients.

Vopr Med Khim, 1985 Jan-Feb, 31(1), 65 - 8
{Effect of botulinum toxin on the activity of transport ATPases in biological membranes}; Chesnokova NP et al.; Activity of transport ATPases was studied in erythrocyte membranes and synaptosomal fraction of cervical department of spinal cord obtained from rats in dynamics of botulinic C intoxication Na+, K+-ATPase was inhibited by the competitive type in the synaptosomal brain fraction at the preclinical period of intoxication and by the noncompetitive type at the step of skeletal muscle paresis . In erythrocyte membranes activity of Na+, K+-ATPase was inhibited by the mixed type at the preclinical period of intoxication and the enzymatic activity was inhibited by the noncompetitive type at the step of skeletal muscle paresis . The Na+, K+-ATPase from biological membranes was reactivated by unithiol and nicotinamide in dynamics of intoxication . The toxin was shown to activate Mg2+-ATPase in brain synaptosomal fraction.

J Physiol, 1984 Nov, 356, 587 - 99
Miniature end-plate potentials in rat skeletal muscle poisoned with botulinum toxin; Kim YI et al.; Spontaneous transmitter release, recorded as miniature end-plate potentials (m.e.p.p.s), was studied in rat extensor digitorum longus (e.d.l.) and soleus muscles partially or completely paralysed by botulinum toxin type A (BoTx) . Normal unpoisoned muscles were examined for comparison . Analysis of m.e.p.p.s in both normal and BoTx-poisoned muscles confirmed the presence of two populations of potentials . One population, which comprised about 96% of the m.e.p.p.s recorded at non-poisoned end-plates, was characterized by a uniform time course and a mean time-to-peak of 0.5-0.7 ms . These potentials had a shape and time-to-peak similar to that of quantal end-plate potentials (e.p.p.s) evoked by nerve stimuli . These were designated 'fast m.e.p.p.s' . The other population of m.e.p.p.s was characterized by a slower, more variable rise-time, the time-to-peak exceeding 1.1 ms, and generally a larger amplitude . These were designated 'slow m.e.p.p.s' . In both partial and complete paralysis by BoTx the frequency of fast m.e.p.p.s was reduced by more than 90% and the reduction lasted several weeks . After 6-10 days of poisoning the frequency of slow m.e.p.p.s gradually increased . The highest frequency of slow m.e.p.p.s (0.4 Hz) was recorded in the partially paralysed soleus muscle, the frequency being about ten times that at unpoisoned end-plates . In both partially paralysed muscles slow m.e.p.p . frequency returned towards normal 28 days after poisoning . A significant correlation (r = 0.67) was observed between the quantal content of e.p.p.s and the frequency of fast m.e.p.p.s in partially paralysed e.d.l . muscles . No significant correlation was observed between quantal content of e.p.p.s and the frequency of slow m.e.p.p.s . To further study if muscle activity influenced the appearance of slow m.e.p.p.s, partially paralysed soleus muscles were directly stimulated in vivo during the first 11-13 days following BoTx poisoning, using a stimulation pattern which inhibits nerve terminal sprouting and the appearance of denervation changes . This procedure did not alter the frequency of slow m.e.p.p.s as compared to unstimulated poisoned controls . It is concluded that enhancement of slow m.e.p.p . frequency in muscles poisoned with BoTx is related to the blockade of evoked Ca2+-dependent quantal transmitter release . However, additional factors influence this type of spontaneous and Ca2+-insensitive release of acetylcholine since there is a great variability between fibres and a time lag between the disappearance of fast m.e.p.p.s and the activation of slow m.e.p.p . frequency.(ABSTRACT TRUNCATED AT 400 WORDS)

Exp Neurol, 1984 Nov, 86(2), 405 - 9
Axon growth in tendon organs of botulinum-paralyzed neonatal mouse muscles; Hopkins WG; Paralysis of neonatal mouse gluteus muscles with botulinum toxin stimulated the growth not only of motor nerve terminals but also of the terminals of the sensory axons innervating the tendon organs . However, the adult mouse tendon organ axons, unlike motor nerve terminals, did not sprout in response to paralysis.

N Engl J Med, 1984 Oct 11, 311(15), 933 - 9
Inhibition of terminal axonal sprouting by serum from patients with amyotrophic lateral sclerosis; Gurney ME et al.; To investigate the pathogenesis of amyotrophic lateral sclerosis, we compared the effect of serum from patients with this disease on the regenerative sprouting of terminal axons in botulinum-treated mouse gluteus muscle with the effects of serum from controls and from patients with diabetic peripheral neuropathy . Serum from 9 of 19 patients with the sporadic form of amyotrophic lateral sclerosis and from 2 of 6 patients with the familial form caused a reduction in the proportion of sprouting terminal axons, as compared with that found in muscles treated with serum from controls or diabetic patients . Immunoglobulin from patients with amyotrophic lateral sclerosis, when tested on immunoblots, recognized a 56-kilodalton protein secreted by denervated rat diaphragm muscle; rabbit antiserum raised against this protein also suppressed terminal axonal sprouting . Thus, we have detected an antibody in the serum of patients with amyotrophic lateral sclerosis that inhibits sprouting of neurons and subsequent reinnervation of skeletal muscle . Whether this antibody is of primary pathogenic importance or represents a secondary response to neuromuscular destruction is not known . In either case, serum from patients with amyotrophic lateral sclerosis may provide reagents for studies of the trophic communications between muscle and motor neurons.

Arch Ophthalmol, 1984 Oct, 102(10), 1464 - 8
Treatment of blepharospasm with botulinum toxin . A preliminary report; Frueh BR et al.; The effects of botulinum A toxin injections for the treatment of facial spasm were analyzed for 22 patients . Sixteen patients had unoperated on essential blepharospasm, three had essential blepharospasm with residual spasm following previous surgical treatment, and three had unoperated on hemifacial spasm . Treatment was effective for most patients, but transient, with the mean interval of relief of spasm after the first injections being ten weeks . The injection of botulinum toxin reduced the maximum lid force by about 10% . While side effects were common, they were generally mild and well tolerated . No cumulative effect of botulinum toxin was evident in those receiving three series of injections . There is no significant difference in either the interval free of spasm or the rate of lid-force recovery following any of three sequential injections of increasing doses of botulinum toxin.

Doc Ophthalmol, 1984 Aug 15, 58(1), 141 - 5
Injection treatment of endocrine orbital myopathy; Scott AB; Eight patients with endocrine orbital myopathy received botulinum toxin injection of extraocular muscles for strabismus or injections of the levator for lid retraction . Strabismus of 25 prism diopters or less, especially during early stages of eye muscle involvement, responded well to injection with realignment and, probably, with avoidance of fixed muscle shortening . Long-standing strabismus, large angles, and lid retraction responded less well.

Brain Res, 1984 Jul 2, 305(1), 177 - 80
Botulinum toxin and tetanus toxin recognize similar membrane determinants; Simpson LL; The binding fragment of tetanus toxin (50,000 dalton carboxy-terminus of heavy chain) does not block neuromuscular transmission, but it does antagonize the ability of native tetanus toxin to block neuromuscular transmission . The binding fragment of tetanus toxin also antagonizes certain botulinum toxins, including types C and E . Antagonism is at the cell surface, suggesting that the various molecules compete for a similar membrane binding site . The data indicate that the binding site is specific for botulinum toxin rather than tetanus toxin.

Arch Biochem Biophys, 1984 Jul, 232(1), 172 - 8
Effect of diethylpyrocarbonate on the biological activities of botulinum neurotoxin types A and E; Dasgupta BR et al.; The single chain (unnicked) type-E and the dichain (nicked) type-A botulinum neurotoxins were modified with diethylpyrocarbonate (ethoxyformic anhydride), a reagent highly specific for histidine residues . The type-E neurotoxin could be completely detoxified without causing detectable damage to its serological reactivity . Under identical modification reaction conditions, the type A was incompletely detoxified with some alteration in its serological reactivity . Modification of histidine residues was evident from the increase in absorbance at 240 nm, and reactivation of the detoxified proteins by reversing the modification with hydroxylamine . The completely detoxified type-E neurotoxin, used as toxoid, elicited antibodies in rabbits . The antiserum precipitated and neutralized the neurotoxin . This toxoid is homogeneous as tested by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, whereas the traditional toxoid produced with formaldehyde is heterogeneous.

J Pediatr Ophthalmol Strabismus, 1984 May-Jun, 21(3), 110 - 3
Botulinum toxin chemo-denervation for strabismus in infants and children; Magoon EH; Fifteen children and five infants with short follow-up show chemo-denervation with botulinum toxin to be a safe and effective alternative to surgery.

Brain Res, 1984 Apr 16, 297(2), 309 - 16
Studies on the mode of action of botulinum toxin type A at the frog neuromuscular junction; Molgo J et al.; In frogs poisoned with botulinum toxin type A the quantal content of endplate potentials is greatly reduced . Lowering the temperature of the preparation increases quantum content; between 14 and 4 degrees C the mean Q10 for this effect is 6.3 . Facilitation of synaptic transmission is marked with pairs of stimuli and cooling further enhances facilitation . The time constant of decay of facilitation is 34 ms at 20 degrees C and 116 ms at 4 degrees C . The increase in facilitation and in its time constant of decay at low temperature are presumably not a result of a prolongation of the duration of the nerve terminal action potential since such changes are not seen in the presence of K+-channel blockade by 3,4-diaminopyridine . Electrotonic depolarization of nerve terminals in the presence of tetrodotoxin and 3,4-diaminopyridine induces all-or-none endplate currents . Such endplate currents, at a holding potential of -50 mV, show that the amount of charge entry is about 1/3 of that in unpoisoned junctions but still corresponds to 5-10 X 10(3) transmitter quanta . Transmitter release at this level is maintained during repetitive stimulation even in the presence of 82 mM Ca2+ in the extracellular solution . We speculate that the blockade of transmitter release in BoTx -poisoned muscles results from a stimulatory effect of the toxin on metabolic systems of Ca2+ disposal in the nerve terminal.

Ophthalmic Surg, 1984 Apr, 15(4), 310 - 4
Botulinum toxin injection into rabbit vitreous; Wienkers K et al.; Botulinum A toxin in a dose of 5.0 X 10(-4) micrograms was injected into the vitreous of eight rabbit eyes . An equal volume of normal saline was injected into the fellow eye as a control . External examination, intraocular pressure, ophthalmoscopy, electroretinography, and light microscopy showed no significant difference between toxin injected and control eyes over a two-month period.

J Pharmacol Exp Ther, 1984 Apr, 229(1), 182 - 7
The binding fragment from tetanus toxin antagonizes the neuromuscular blocking actions of botulinum toxin; Simpson LL; A chromatographically homogenous preparation of the binding fragment from tetanus toxin was tested for its ability to antagonize the neuromuscular blocking properties of native tetanus toxin . At a concentration of 1 X 10(-6) M, the binding fragment antagonized the paralytic effects of native toxin (1 X 10(-9) M) on mouse phrenic nerve-hemidiaphragms . The binding fragment of tetanus toxin (1 X 10(-6) M) also was tested for its ability to antagonize types A to G botulinum neurotoxin . The fragment did not produce statistically significant antagonism of types A, B, D, F and G neurotoxins, but it did produce highly significant antagonism of types C and E neurotoxin . A series of experiments involving column chromatography, dialysis and high-performance liquid chromatography confirmed that the binding fragment rather than a contaminant was responsible for antagonism . Experiments with type C neurotoxin showed that antagonism between the binding fragment and the toxin occurred extracellularly at the level of the cell membrane . The fragment did not act directly on the toxin to produce inactivation, but instead competed with the toxin for a binding site on the membrane . The fact that the binding moiety of tetanus toxin and the binding moiety of botulinum toxin compete for a similar membrane site suggests that the molecules have areas of structural homology . Such homologies, if confirmed, would have important therapeutic implications.

Biochem Biophys Res Commun, 1984 Mar 30, 119(3), 900 - 4
Partial amino acid sequence of the heavy and light chains of botulinum neurotoxin type A; Schmidt JJ et al.; The dichain (nicked) type A botulinum neurotoxin is a protein (mol . wt . 145,000) composed of a heavy and a light chain (mol . wt . 97,000 and 53,000, respectively) that are held together by disulfide bond(s) . We report here the sequence of the first 17 amino acid residues of the light chain, and the first 10 residues of the heavy chain . The heavy chain was isolated from the neurotoxin by two different methods, while the light chain was isolated by the only available method . The identical amino acid sequence was found in both preparations of heavy chain . Two samples of the light chain isolated from two separately prepared batches of the neurotoxin also had identical sequences.

Brain Res, 1984 Mar 19, 295(2), 368 - 71
Stimulation of nodal and terminal sprouting of mouse motor nerves by gangliosides; Robb GA et al.; Treatment of mice with daily intraperitoneal gangliosides (50 mg/kg) enhanced both the number and mean length of nodal sprouts 4 days after partial denervation of the gluteus maximus muscle . Terminal sprouting in the soleus muscle 14 days after botulinum toxin paralysis was also enhanced by gangliosides, as assessed by zinc iodide-osmium tetroxide staining . Functional recovery from paralysis, assessed electrophysiologically, was simultaneously accelerated; this may have resulted from the enlarged terminal area, rather than reflecting an additional action of gangliosides upon synaptogenesis.

Klin Monatsbl Augenheilkd, 1984 Mar, 184(3), 216 - 7
{Treatment of nystagmus using injections of botulinum toxins into the eye muscles}; Crone RA et al.; Two patients with severe nystagmus were treated by injection of botulinum toxin into the eye muscles . The first patient had a vertical nystagmus of large amplitude with oscillopsia; the second, monocular patient had a congenital horizontal nystagmus.

J Physiol, 1984 Mar, 348, 1 - 17
A study of the action of tetanus toxin at rat soleus neuromuscular junctions; Bevan S et al.; Tetanus toxin (TeTX) inhibits the evoked release of acetylcholine (ACh) at rat soleus end-plates . The effects of various procedures which evoke ACh release by raising the level of free intracellular calcium have been investigated at various stages of tetanus intoxication . At all stages studied TeTX has little or no effect on either the frequency or the amplitude of spontaneous miniature end-plate potentials (m.e.p.p.s) . After TeTX poisoning, e.p.p . latency is more variable than normal and the slope of the relationship between ln m (quantal content) and ln {Ca}o is reduced from the control value of about 4 . Plots of m-1/n against 1/{Ca}o, for n = 1-4, suggest that mmax, the maximum number of quanta releasable by nerve stimulation, is reduced at intoxicated end-plates . Blocking delayed rectification with 3-aminopyridine (1-5 mM) increases m, but has little or no effect on either the slope of ln m-ln {Ca}o plots or estimates of mmax . Several treatments which raise m.e.p.p . rate (high {Ca}o, hyperosmotic medium, addition of lanthanum) are less effective after TeTX poisoning . Some of the tested agents increase m.e.p.p . frequency by a mechanism which is thought to involve a mobilization of calcium from intracellular stores . The decline in m.e.p.p . rate after a period of high-frequency nerve stimulation is different at normal and TeTX-treated end-plates . At tetanus-intoxicated end-plates, the decline differs from that expected if TeTX acted simply to block calcium entry into the terminal . In addition, the increase in m.e.p.p . frequency observed with a high rate of nerve stimulation suggests that considerable amounts of calcium can enter the terminal with each action potential . It is concluded that TeTX blocks transmitter release by acting at a step between calcium influx to the terminal and transmitter release such that the mechanism for ACh release shows a reduced sensitivity to intracellular calcium . The possibility of an additional effect on the presynaptic calcium conductance cannot be excluded . Some differences between the properties of end-plates poisoned with TeTX and botulinum toxin are discussed.

J Pharmacol Exp Ther, 1984 Mar, 228(3), 600 - 4
Fragment C of tetanus toxin antagonizes the neuromuscular blocking properties of native tetanus toxin; Simpson LL; Tetanus toxin, fragment B and fragment C were assayed for toxicity on the mouse phrenic nerve-hemidiaphragm preparation . The native toxin was a potent blocker of neuromuscular transmission; fragment B possessed little toxicity and fragment C was atoxic . Pretreatment of tissues with fragment C antagonized the neuromuscular blocking properties of tetanus toxin, but not those of type A botulinum toxin or beta-bungarotoxin . Agents that antagonize cholera toxin (B subunit) and diphtheria toxin (CRM197) did not antagonize tetanus toxin and did not alter the ability of fragment C to antagonize tetanus toxin . Fragment C exerted its effect by competing with unbound toxin for receptor sites on the nerve membrane . The fragment did not: 1) displace bound toxin; 2) inhibit internalization of toxin; or 3) inhibit intracellular expression of toxicity . In assays on intact cells, under conditions in which toxin binding was not dissociable, fragment C binding to phrenic nerves had an apparent KD of approximately 1.4 X 10(-7) M . Homogenates of mouse cerebral cortex adsorbed tetanus toxin and these homogenates competed with phrenic nerves for unbound toxin . Homogenized cortex did not displace or promote desorption of toxin already bound to phrenic nerves . Homogenates of eel and torpedo electric organ were not very effective in adsorbing toxin.

Biokhimiia, 1984 Mar, 49(3), 426 - 31
{Characterization of the subunits of botulinum neurotoxin type A}; Vinogradova ID et al.; A comparative amino acid analysis of botulinum neurotoxin type A and its subunits has been carried out . The heavy and light chains of neurotoxin have the same ratios of polar and non-polar amino acids (1.3:1), the amount of tryptophan residues in the heavy chain is 4 times as much as that in the light chain, and the number of SH-groups exceeds that in the light chains 2-fold . In neurotoxin, two N-terminal amino acid residues--alanine and leucine--were identified . Alanine was found to be the N-terminus of the heavy chain . The fluorescence spectra of neurotoxin subunits indicate differences in the conformational state of the polypeptide chains . The antigenic non-identity of botulinum neurotoxin A subunits suggests the presence in the neurotoxin molecule of at least two antigenic determinants, corresponding to the heavy and light chains.

Science, 1984 Feb 17, 223(4637), 714 - 6
Susceptibility of skeletal muscle to Coxsackie A2 virus infection: effects of botulinum toxin and denervation; Andrew CG et al.; Coxsackie A viruses can infect denervated but not innervated mature skeletal muscles . The role of synaptic transmission in preventing susceptibility to Coxsackievirus infection was studied by surgically denervating leg muscles of mice or injecting the muscles with botulinum toxin to block quantal release of acetylcholine . Control muscles were injected with heat-inactivated toxin . Subsequent injection of Coxsackie A2 virus resulted in extensive virus replication and tissue destruction in the denervated and botulinum toxin-treated muscles, while the control muscles showed only minimal changes . This suggests that the susceptibility of skeletal muscle to Coxsackievirus infection is regulated by synaptic transmission.

Nature, 1984 Feb 9-15, 307(5951), 546 - 8
Suppression of sprouting at the neuromuscular junction by immune sera; Gurney ME; Injury of afferent motor axons or pathological loss of motoneurones from the spinal cord causes the remaining axons within a muscle to sprout and to reinnervate the denervated muscle fibres . Sprouting occurs at two sites along intramuscular axons, at nodes of Ranvier (nodal sprouting) and at the neuromuscular junction (terminal sprouting) . Terminal sprouting is also produced by treatment with botulinum toxin and by other agents that render muscle inactive . The muscle probably provides a signal for terminal sprouting as restoration of muscle activity by direct electrical stimulation prevents sprouting . Such a signal might be a local change on the muscle fibre surface or a 'soluble' sprouting factor, although the failure to induce terminal sprouting in one muscle by denervating adjacent muscles argues against the latter hypothesis . I now report that rabbit antisera against a 56,000 (56K)-molecular weight protein secreted by denervated rat muscle suppress botulinum toxin-induced terminal sprouting in the mouse gluteus muscle . An immune response against this protein has also been detected in serum of patients with amyotrophic lateral sclerosis (ALS), a disease in which loss of motoneurones from the spinal cord is not accompanied by the degree of sprouting and reinnervation seen in other motoneurone diseases.

Muscle Nerve, 1984 Feb, 7(2), 101 - 9
Effects of acrylamide and some other sulfhydryl reagents on spontaneous and pathologically induced terminal sprouting from motor end-plates; Kemplay S et al.; The innervation of the normal rat sternocostalis muscle exhibits a constant low level of short spontaneous terminal sprouts visible in zinc iodide-osmium (ZIO) and in methylene blue-stained preparations . Acrylamide inhibits these spontaneous sprouts in a dose-dependent manner . This inhibition is mimicked by N-ethylmaleimide (a sulfhydryl group blocking agent) and can be nullified when acrylamide is given after the sulfhydryl group protecting agent, dithiothreitol (DTT) . This could not be reversed by giving DTT 3 hours after acrylamide . Furthermore, when given alone DTT increases the level of spontaneous terminal sprouting seen in ZIO and in methylene blue-stained preparations . These findings suggest that the binding of acrylamide to sulfhydryl groups is involved in the inhibitory process . Acrylamide also reduces the number and length of the reactionary terminal sprouts, seen in ZIO and in methylene blue-stained preparations, that follow partial denervation or local injection of botulinum toxin . These inhibitory effects are long-lasting; recovery still has not fully occurred 4 weeks after a single dose of acrylamide (50 mg/kg) . The roles of glutathione and other sulfhydryl components of axons are discussed in relation to the mechanism of acrylamide neurotoxicity.

Soc Appl Bacteriol Symp Ser, 1984, (12), 241 - 56
Detection and enumeration of injured bacterial spores in processed foods; Johnson KM et al.; Injury of bacterial spores is manifested in ways similar to those for vegetative cells, i.e . altered nutritional requirements, increased sensitivity to inhibitors and modified optimum temperatures . However, injury in spores may involve activation, germination and/or outgrowth systems, in addition to the vegetative growth processes . Food processes in the form of heat, acid, inhibitors, preservatives, low water activity and combinations of these factors can stress spores . Consequently, detection and enumeration methods designed to recover bacterial spores from processed foods must always consider the requirements of injured spores . Injury to germination systems has been circumvented through the addition of non-nutritive agents, such as lysozyme, to recovery media, or by incubation at an alternative temperature which favours a secondary germination system . A shift in temperature has also permitted repair of damaged outgrowth systems . Addition of materials such as starch and charcoal can reduce the sensitivity of injured spores to some inhibitors . Our current research on hypochlorite injury of Cl . botulinum, differing injury responses for B . cereus in rice and TSB, and injury of B . cereus earlier in the heating process at 85 degrees C than at 90 degrees C indicate a need for more information on the injury of spores in processed foods.

Toxicon, 1984, 22(3), 415 - 24
Purification and amino acid composition of type A botulinum neurotoxin; DasGupta BR et al.; A method to purify type A botulinum neurotoxin from a 64 liter bacterial culture is reported . The procedure includes cation exchange chromatography at pH 7.0 . The final product, essentially homogeneous (according to polyacrylamide gel-sodium dodecylsulfate electrophoresis), is a mixture of two forms of the neurotoxin (mol . wt 145,000); the dichain or nicked form (over 95%) and its precursor the single chain or unnicked form . Two batches of the neurotoxin purified by the method described here and one batch purified according to the method of Sugii and Sakaguchi were similar in purity and amino acid composition . The best estimate of the number of amino acid residues per neurotoxin molecule (mol . wt 145,000) is: Asp200Thr75Ser79Glu114Pro44Gly64Ala53Val70CyS10Met22Ile111Leu104Tyr71 Phe68Lys100His14Arg43Trp17.

J Physiol (Paris), 1984, 79(4), 246 - 51
Electrophysiological and neurochemical investigations of the action of presynaptic neurotoxins; Gundersen CB et al.; Previous experiments have suggested that hemicholinium-3 might directly antagonize certain actions of beta-bungarotoxin at the neuromuscular junction . Data presented here show that, on the contrary, hemicholinium-3 neither inhibits the phospholipase activity of beta-bungarotoxin nor does it affect the characteristic pattern of transmitter release observed at end plates exposed to the toxin . Lanthanum ions were found to promote the release of acetylcholine from sartorius nerve-muscle preparations that had been paralyzed by botulinum toxin . However, the acceleration of transmitter release by lanthanum was not nearly as great as in control preparations as monitored either electrophysiologically or by chemical measurement of ACh.

Acta Neuropathol (Berl), 1984, 63(2), 144 - 9
The effects of chronic isoniazid intoxication on motor end plate sprouting in rat sternocostalis muscle and on responses to partial denervation and local botulinum toxin; Kemplay S et al.; Chronic dosing of rats with isoniazid (INH) leads to an increase in the incidence of short "spontaneous" sprouts on motor end plates in the rat sternocostalis muscle . After partial denervation there is a slight increase in terminal sprouting after 1 week of dosing: this changes to a significant decrease from 2 to 6 weeks of dosing . The same is noted after local botulinum toxin injection, and in both conditions sprout length is significantly reduced . In vitro studies show that glutathione, cysteine and cystathionine all increase the incidence of short, "spontaneous" sprouts from end plates, while homocysteine and cystine have no effect . These findings are interpreted in the light of the hypothesis that in INH intoxication there may be a reduction of available axonal glutathione and cysteine due to inhibition of the pyridoxal phosphate-dependent enzymes cystathionine synthetase and cystathioninase .

J Physiol (Paris), 1984, 79(4), 280 - 303
Botulinum neurotoxin and dendrotoxin as probes for studies on transmitter release; Dolly JO et al.; Acceptors for BoNT have been detected autoradiographically on the terminal membrane of motor nerves at a density of approximately 150/micron2 and shown to mediate toxin internalization, a process deemed essential for its inhibition of transmitter release . DTX, a protein with pronounced central neurotoxicity, was shown to induce convulsive states in hippocampal slices from guinea-pig . Synaptic transmission was facilitated and spontaneous epileptiform activity produced in intact cell populations . Voltage clamp analysis of hippocampal neurones revealed that DTX specifically attenuated a transient voltage-dependent K+ conductance (A-current) and this could account for the excitatory effects observed . Proteinaceous acceptors with high affinity for DTX were identified on brain synaptosomal membranes and found to contain a 65 000 Mr polypeptide . Their location in rat brain regions was established and contrasted with that of the binding sites for beta-bungarotoxin . These findings indicate the usefulness of DTX as a probe for a protein associated with one variety of K+ channel while the larger subunit of BoNT was found to interact with a membraneous component that resides at cholinergic nerve terminals and, hence, is likely to have a unique role.

J Physiol (Paris), 1984, 79(4), 252 - 8
Action of botulinum A toxin and tetanus toxin on synaptic transmission; Dreyer F et al.; Intracellular recordings of the spontaneous activity from mammalian spinal cord neurons in culture demonstrated different sensitivities of excitatory and inhibitory synaptic transmission for the action of tetanus toxin (Tetx) and botulinum toxin type A (Botx) . The effects of Tetx and Botx on spontaneous and nerve-evoked transmitter release were compared under identical experimental conditions in experiments on in vitro poisoned mouse diaphragms . At 37 degrees C completely paralyzed endplates are characterized by a very low frequency of spontaneous miniature endplate potentials (m.e.p.p.s) and by a 100% failure to evoke endplate potentials (e.p.p.s) in response to single nerve stimuli . Striking differences in the action of both toxins have been observed when the very low transmitter release probabilities of paralyzed nerve-muscle preparations were increased by tetanic nerve stimulation and/or application of potent K+-channel blockers and/or by reduction of temperature to 25 degrees C . While Botx did not change the short latency between nerve impulse and postsynaptic response, Tetx produced a temporal dispersion of the quantal release suggesting that the toxins act at different sites in the chain of events that result in transmitter release . To find further evidence to support the different actions of the toxins the spontaneous transmitter release was studied in more detail . Tetx blocked preferentially the release of so-called large mode m.e.p.p.s without affecting the frequency of the small mode ones . In contrast, Botx strongly inhibited both the small and large mode m.e.p.p.s.(ABSTRACT TRUNCATED AT 250 WORDS)

J Physiol (Paris), 1984, 79(4), 192 - 5
Transmitter release in botulinum-poisoned muscles; Thesleff S; Examination of miniature end-plate potentials (m.e.p.ps) in rat skeletal muscle poisoned in vivo by botulinum toxin type A reveals the presence of two populations of potentials . One population which corresponds to m.e.p.ps in unpoisoned muscles and to quantal end-plate potentials . The frequency of these m.e.p.ps is greatly reduced by botulinum toxin . The second population of m.e.p.ps has quite different characteristics . These m.e.p.ps have a more variable, but generally much larger amplitude, and their time to peak is longer than normal m.e.p.ps . The frequency of these m.e.p.ps increases during poisoning and reaches 0.3-1 Hz after 10-14 days . In addition to the variability in amplitude and time-to-peak these m.e.p.ps differ from those at unpoisoned junctions by being unaffected by procedures which alter extra- or intracellular Ca2+ concentrations . The appearance of this Ca2+-insensitive spontaneous quantal secretion of acetylcholine is apparently not a direct effect of the toxin but secondary to blockade of impulse transmission since it also appears at unpoisoned end-plates when transmission is impaired for other reasons . Procedures which increase the intracellular Ca2+ concentration in nerve terminals restore transmitter release from botulinum toxin poisoned nerves . Furthermore, the block caused by the toxin is very temperature-dependent, a reduction in temperature relieving the block . Since presynaptic Ca2+ currents are unaltered by the toxin it is proposed that the block of transmission is due to a reduction in the calcium content of the nerve terminal to a level where the amount of Ca2+, which normally enters, is insufficient to activate transmitter release.

Farmakol Toksikol, 1983 Nov-Dec, 46(6), 9 - 12
{Mechanism of the effect of methylcobalamin on the recovery of neuromuscular functions in mechanical and toxin denervation}; Mikhailov VV et al.; It has been shown in experiments on rats that daily administration of methylcobalamine in a dose of 50 micrograms/100 g bw produces marked activation of the regeneration of mechanically damaged axons of motoneurons . Systematic administration of the drug has a protective action on the development of neuromuscular transmission blockade induced by botulinum toxoid.

Biokhimiia, 1983 Nov, 48(11), 1825 - 30
{Role of arginine and histidine residues in the biological activity of botulinic neurotoxin A}; Shibaeva IV et al.; Treatment of botulinic neurotoxin A with cyclohexanedione demonstrated that modification of 5 to 10 arginine residues does not change the neurotoxin toxicity, while after modification of 15-20 arginine residues the toxicity is decreased by 40-50% of the original value . Butanedione exerts a stronger detoxicating effect on neurotoxin than cyclohexanedione . The molecular conformation of the modified toxin derivatives and their precipitability upon interaction with antisera against toxin and toxin fragments does not change thereby . The non-toxic derivatives of toxin containing 40 modified arginine residues possess a partial serological affinity for the original toxin in a reaction with antiserum against toxin but do not interact with the antifragment sera . The molecular conformation of these preparations is changed considerably . It is assumed that one or two arginine residues are located near the toxic site of the neurotoxin molecule and are also components of its antigenic determinants . Modification of histidine residues in the neurotoxin molecule by diethylpyrocarbonate is accompanied by a decrease of its toxicity . An additional 10% toxicity is revealed upon modification of 11-13 histidine residues . The molecular conformation of the modified derivatives of neurotoxin and their precipitability do not change thereby . It is probable that 1 or 2 histidine residues are located at or near the toxic site . The data obtained suggest that histidine residues are not localized in antigenic determinants of the neurotoxin molecule.

Pflugers Arch, 1983 Nov, 399(3), 228 - 34
Transmitter release in tetanus and botulinum A toxin-poisoned mammalian motor endplates and its dependence on nerve stimulation and temperature; Dreyer F et al.; The effects of tetanus toxin (TeTx) and botulinum A toxin (BoTx) on spontaneous and nerve-evoked transmitter release have been compared in mouse hemidiaphragms poisoned in vitro . At 37 degrees C endplates poisoned with either of these agents were characterized by (1) a decrease of miniature endplate potential (m.e.p.p.)-frequency to less than 30/min for TeTx and 3/min for BoTx, (2) reduced mean m.e.p.p.-amplitude and (3) 100% failure to show endplate potentials (e.p.p.s) in response to single nerve stimuli . In addition (4) tetanic nerve stimulation and/or reduction of temperature to about 20 degrees C caused a remarkable increase in the nerve-evoked transmitter release, but did not affect the low frequency of spontaneous m.e.p.p.s . However, several important differences exist between the effects of both toxins . (1) At room temperature even single nerve stimuli could elicit e.p.p.s in BoTx-muscles the failure rate being about 80% . For TeTx the failure was 100% . However, if the nerve was stimulated with higher frequencies (greater than 5 Hz), the probability of quantal release increased, the delay for release from the onset of stimulation being several seconds and similar to that observed at 37 degrees C . (2) TeTx distorted the synchronous release of quanta increasing the distribution of their synaptic delays . BoTx did not influence the time course of the phasic secretion process in response to nerve action potentials . (3) TeTx preferentially blocked the release of spontaneous m.e.p.p.s of large amplitude without affecting the frequency of the small amplitude ones, while BoTx inhibited both the small and large amplitude m.e.p.p.s . The distribution of the amplitudes of the nerve-evoked m.e.p.p.s were similar to those of spontaneous m.e.p.p.s before the blockade with the toxins.(ABSTRACT TRUNCATED AT 250 WORDS)

J Anat, 1983 Oct, 137 (Pt 3), 477 - 82
Effects of acrylamide and botulinum toxin on horseradish peroxidase labelling of trigeminal motor neurons in the rat; Kemplay S et al.; The extent of neuronal labelling with horseradish peroxidase (HRP) was examined in rat trigeminal motor neurons at various stages of acrylamide intoxication, following HRP uptake by motor nerve endings in the anterior digastric muscle . After 7 days of acrylamide (5 daily injections of 30 mg acrylamide/kg body weight), the pattern of HRP labelling was altered from normal, and this changed pattern persisted without further alteration when survival time and dosage were increased to 14 and 21 days (equivalent to 10 X 30 mg/kg and 15 X 30 mg/kg respectively) . Six hours after HRP injection, the number of labelled cells in the ipsilateral trigeminal motor nucleus was reduced in treated animals compared to controls . By 18 hours, cells containing label were present in similar numbers to controls; but by 24 hours, the number in treated animals had fallen again, unlike controls in which labelling remained constant between 6 and 24 hours . At longer intervals, this reduction in labelling continued, but more slowly, so that by 96 hours after HRP injection, numbers of labelled cells were again similar in poisoned and control animals . One and three days after a single intramuscular injection of 0.05 microgram botulinum toxin type A, HRP labelling in the trigeminal motor neurons was unaffected, although at three days after toxin, mild chromatolytic changes could be seen in a few of the neurons.

Vet Pathol, 1983 Sep, 20(5), 553 - 62
Cytology of equine cerebrospinal fluid; Beech J; The cytology of cerebrospinal fluid samples from horses is described . The samples were obtained from 24 normal horses, 35 horses with axonal degeneration and/or spinal cord compression, 29 horses with encephalomyelitis, 14 horses with other lesions of the nervous system, and eight horses with signs of neurologic dysfunction of undetermined origin . (Three of the latter were suspected botulinum intoxications.) Fluid was aspirated from the atlanto-occipital space following general anesthesia or immediately after a lethal dose of barbiturate . In two horses, fluid also was aspirated from the lumbosacral space . Small mononuclear cells were predominant in normal horses, and in most horses with axonal degeneration and encephalomyelitis . Several horses with encephalomyelitis also had neutrophils, eosinophils, and some mitotic figures . Although the cytologic findings were abnormal in many of the horses with disease of the central nervous system, in most horses the cytologic findings were normal.

Ann Otol Rhinol Laryngol, 1983 Sep-Oct, 92(5 Pt 1), 424 - 9
Three-dimensional computer reconstruction of the distribution of neuromuscular junctions in the thyroarytenoid muscle; Rosen M et al.; Microinjections of botulinum toxin have recently been shown to be effective in the treatment of strabismus, and it has also been suggested that microinjections of this myoneural blocking agent might be of value in the treatment of spastic dysphonia . The success of such a microinjection technique would rely on a precise knowledge of the distribution of myoneural junctions in the thyroarytenoid muscle . In view of this potential application as well as the need for such information in reinnervation procedures, we have used computer graphics to reconstruct the three-dimensional distribution of motor end-plates in the thyroarytenoid muscle . Three cat and one human (fresh autopsy specimen) larynges were frozen and sectioned on a cryostat . Serial sections were then processed for the histochemical localization of acetylcholinesterase activity to demarcate the neuromuscular junctions . An X-Y digitizer was used to reference the position of the motor end-plates in each serial section, and the three-dimensional distribution of the neuromuscular junctions was reconstructed on a computer graphics terminal . The results are discussed in regard to their applicability to clinical treatment of spastic dysphonia and other disorders of phonation.

J Neurochem, 1983 Aug, 41(2), 395 - 402
Tetanus and botulinum toxins inhibit, and black widow spider venom stimulates the release of methionine-enkephalin-like material in vitro; Janicki PK et al.; The actions of tetanus toxin, botulinum A toxin, and black widow spider venom on the release of methionine-enkephalin-like immunoreactivity have been studied; a particulate fraction prepared from rat striata was used . Depending on the duration of preincubation, tetanus toxin diminished the release evoked by veratridine (50 microM final concentration), and abolished it at final concentrations between 0.1 and 1 micrograms/ml . Botulinum A toxin was about 10 to 20 times less potent . Heating or pretreatment with antitoxin inactivated the clostridial toxins . The particulate fraction pretreated with V . cholerae neuraminidase retained its toxin sensitivity . Tetanus toxin also depressed the release due to sea anemone toxin II and high K+ . Spider venom stimulated the release in a concentration-dependent manner and required the presence of Ca2+; its effects were depressed by tetanus toxin . These results support the view that both clostridial toxins and spider venom act as broad-range presynaptic neurotoxins on peptidergic transmitter systems.

Brain Res, 1983 Jul 4, 270(2), 373 - 5
Botulinum A toxin and tetanus toxin do not affect presynaptic membrane currents in mammalian motor nerve endings; Dreyer F et al.; The hypothesis according to which BoTx and TeTx block neuromuscular transmission by impairing Ca2+ influx was tested by recording presynaptic membrane currents at motor endplates of the mouse by means of external electrodes . The use of K-channel blockers allowed us to observe inward Ca current which was unaffected by bath application of BoTx or TeTx at doses which block neuromuscular transmission.

Pflugers Arch, 1983 Jun 1, 397(4), 319 - 22
Decrease of the spontaneous non-quantal release of acetylcholine from the phrenic nerve in botulinum-poisoned rat diaphragm; Dolezal V et al.; Botulinum type A toxin (BoTx) has been found to diminish by 40% the spontaneous release of acetylcholine (ACh) from normal (acutely denervated) rat diaphragms incubated in the presence of 5 mM K+, while the release of ACh from chronically (4 days) denervated diaphragms was not affected during 2 h incubations . The toxin has been found to rapidly remove (within 10 min) the local depolarization of about 8 mV which developed in the end-plate zones of the diaphragms after the inhibition of cholinesterases; after the administration of BoTx, tubocurarine lost its ability to increase the resting membrane potential (H-response, Katz and Miledi 1977) in the end-plate area of anticholinesterase-treated muscles . It is concluded that BoTx inhibits the non-quantal release of ACh from the motor nerve fibres and that it probably acts directly on the nerve terminal surface membrane (without internalization) . The H-response in the rat diaphragm reflects the non-quantal release of ACh from the nerve terminals and not from the muscle fibres.

J Pharmacol Exp Ther, 1983 Jun, 225(3), 546 - 52
Ammonium chloride and methylamine hydrochloride antagonize clostridial neurotoxins; Simpson LL; Ammonium chloride (1-8 mM) and methylamine hydrochloride (1-16 mM) produce concentration-dependent antagonism of the onset of neuromuscular blockade caused by botulinum toxin types A, B and C (all at 1 X 10(-11) M) and by tetanus toxin (3 X 10(-10) M) . Neither drug antagonizes the onset of paralysis caused by beta-bungarotoxin (1 X 10(-7) M) or by taipoxin (1 X 10(-8) M) . At concentrations that produce antagonism of clostridial neurotoxins, ammonium chloride and methylamine hydrochloride (8-10 mM) do not inactivate toxin molecules, nor do they produce irreversible changes in tissue function . When studied under conditions that impose partial synchrony on the mechanism of clostridial neurotoxin action, ammonium chloride and methylamine hydrochloride do not inhibit ligand binding and do not reverse neuromuscular blockade . The drugs act solely to antagonize internalization of toxins by cholinergic nerve endings . As a result of inhibiting the process of internalization, the drugs trap the toxins at an antitoxin sensitive site.

Neuroscience, 1983 May, 9(1), 1 - 8
A new type of transmitter release at the neuromuscular junction; Thesleff S et al.; Examination of spontaneous miniature endplate potentials (MEPPs) in murine skeletal muscle has revealed that in conditions such as botulinum poisoning, during nerve terminal regeneration or in the presence of the drug 4-aminoquinoline, two types of acetylcholine release are responsible for the MEPPs . In addition to the MEPPs which correspond to the quantal component of a nerve impulse-evoked endplate potential a second type of acetylcholine release occurs . The latter type of transmitter release gives rise to MEPPs with a more prolonged time-to-peak and frequently a larger than normal amplitude . It is unaffected by nerve terminal depolarization and transmembrane Ca2+ fluxes . The relationship between MEPP frequency and temperature has a Q10 of about 12 compared to 2-3 for normal MEPPs . In botulinum-poisoned muscles this secretory type of transmitter release dominates, being exclusively present in muscles where nerve stimulation fails to release transmitter . In normal muscle such a release is induced by 4-aminoquinoline which may cause up to 45% of all the spontaneous MEPPs to be of that kind . It is suggested that the described spontaneous secretion of acetylcholine serves in inductory and neurotrophic function.

Infect Immun, 1983 Apr, 40(1), 336 - 9
Lethal and vascular permeability activities of botulinum C2 toxin induced by separate injections of the two toxin components; Ohishi I; Two components, designated I and II, of botulinum C2 toxin were injected separately into the same animal . The intravenous injection of one component at different time intervals after intravenous injection of the other component, irrespective of the sequence, was lethal to mice . When components I and II were injected intradermally into separate sites, vascular permeability increased only at the site where component II was injected . The sizes of blued areas were smaller with increased distance between the injection sites of components I and II . When one component was injected intravenously and the other intradermally, an increase in vascular permeability was induced at the intradermal site of injection of component II but not at that of component I . These results indicate that the simultaneous injection of components I and II is not always required to elicit the biological activity of C2 toxin . The vascular permeability response induced by separate injections of the two toxin components suggests that the activity of C2 toxin results from component II binding to the tissue around its injection site and component I recognizing the altered tissue.

Med Biol, 1983 Apr, 61(2), 120 - 5
Comparison of the effects of botulinum neurotoxin types A and E at the rat neuromuscular junction; Sellin LC et al.; Local blockade of transmitter release was produced by s.c . injection of purified botulinum neurotoxin (NT) types A or E above the tibialis anterior muscle of adult male rats . Extensor digitorum longus nerve-muscle preparation was examined for toxin-induced alterations in single twitch and tetanic tension (in situ) or transmitter release (in vitro) . For both single twitch and tetanic tension, muscles treated with type E NT recovered from an initial partial paralysis (induced with 56 mouse LD50) or full paralysis (induced with 565 mouse LD50) by 7 days after NT injection, while those treated with only 5 mouse LD50 of type A remained either fully or partially paralysed through 10 days . Also, miniature end-plate potential frequency and mean quantal content were reduced for a longer period of time and/or to a greater extent for muscles treated with type A NT than for those treated with type E . The present results are consistent with the observed higher specific toxicity (i.p . injections in mice) for type A NT than for type E, although these differences may be exaggerated after s.c . injections . The differences in the paralytic effect between types A and E may be determined by differences in amino acid sequence, which causes type E to dissociate more easily from its site of action and/or be detoxified more rapidly . The clinical implications of these findings are discussed.

Brain Res, 1983 Mar 28, 264(1), 89 - 97
Botulinum toxin and 4-aminoquinoline induce a similar abnormal type of spontaneous quantal transmitter release at the rat neuromuscular junction; Thesleff S et al.; Intracellular recordings from botulinum toxin type A (BoTx)-poisoned extensor digitorum longus muscles from adult rats have shown that the toxin initially reduced the frequency of miniature endplate potentials (m.e.p.ps) to about 1/200 of normal . After a few days the m.e.p.p . frequency rose and was subsequently maintained at a level of about 1/3 of that at normal endplates . Depolarization of the nerve terminals with 20-30 mM KCl-Ringer initially failed to affect the frequency of m.e.p.ps and later caused only a 2-3--fold increase in their frequency . The temperature dependence of m.e.p.p . frequency at BoTx-poisoned endplates had a Q10 of about 12 compared to 2-3 for normal junctions . The time to peak of a population of m.e.p.ps at Botx-poisoned junctions was prolonged as compared to normal and fast- and slow-rising m.e.p.ps originated within the same post-synaptic membrane field area . M.e.p.ps in BoTx-poisoned muscles resembled the m.e.p.ps which 4-aminoquinoline (4-AQ) has been shown to induce in normal muscle, and we therefore examined and compared these two release processes for acetylcholine . Procedures known to markedly affect m.e.p.p . frequency at normal junctions, such as nerve terminal depolarization or changes in extra- and intracellular Ca2+ concentrations, failed to affect m.e.p.p . frequency in BoTx-poisoned muscles and similarly the frequency of m.e.p.ps induced by 4-AQ in normal muscle . Tonicity changes in the extracellular medium altered m.e.p.p . frequency in both the experimental conditions, but in a direction opposite to that at normal junctions . The temperature dependence of the frequency of 4-AQ-induced m.e.p.ps was similar to that of m.e.p.ps at BoTx-poisoned junctions . It is concluded that BoTx poisoning induces an abnormal type of spontaneous quantal transmitter release, characterized by being insensitive to nerve terminal depolarization and to transmembrane Ca2+ fluxes . This transmitter release has characteristics similar to that previously described for the release induced, at normal junctions, by 4-AQ.

Mol Biol (Mosk), 1983 Mar-Apr, 17(2), 286 - 92
{Effect of urea on botulinum neurotoxin type A}; Uvarova RN et al.; Kinetic and concentration-mediated dependences of urea effects on toxicity and upon values of negative extremes (279, 287, 292 nm), shifts of lambda max fluorescence from 332 nm to the red region, decrease of fluorescence intensity, changes in accessibility of SH-groups were found . These data point to direct correlation between conformational changes in neurotoxin in urea solutions and decrease of toxicity . An investigation was carried out on renaturation of conformational changed neurotoxin in 8 M urea solution . In no cases was the toxicity restored, though there was a shift of lambda max fluorescence in protein from 340 to 333 nm, that was the evidence for reverse transition of the neurotoxin molecule from a disordered structure to the globule shape . The data lead to the conclusion that the toxicity region is probably a conformationally depending determinant.

Brain Res, 1983 Feb 14, 261(1), 172 - 5
Botulinum toxin blocks quantal but not non-quantal release of ACh at the neuromuscular junction; Stanley EF et al.; Botulinum (BOT) toxin is known to block quantal acetylcholine (ACh) release at the neuromuscular junction but little is known about its effect on non-quantal ACh release . We have examined the effect of BOT on non-quantal ACh release directly using a variant of the electrophysiological technique described by Katz and Miledi . This method is based on the observation that non-quantally released ACh results in a small, continual depolarization of the postsynaptic membrane, after inhibition of cholinesterase . This depolarization can be revealed by suddenly blocking ACh receptors with a pulse of curare, resulting in an abrupt hyperpolarization, the amplitude of which is presumed to be proportional to the rate of non-quantal ACh release . BOT treatment resulted in a marked decrease in quantal ACh release as shown by miniature endplate potential (m.e.p.p.) frequencies (control 0.65 +/- 0.33 m.e.p.p.s/s; BOT 0.03 +/- 0.03 m.e.p.p.s/s) . However, non-quantal ACh release measured by the curare induced hyperpolarization, was not significantly different in control and BOT treated diaphragms (control 1.01 +/- 0.09 mV: BOT 1.03 +/- 0.11 mV) . Our results show that BOT does not block non-quantal ACh release at a time when it has a profound effect on spontaneous quantal ACh release . This suggests that quantal and non-quantal ACh release take place through different release mechanisms.

Arch Ophthalmol, 1983 Feb, 101(2), 211 - 3
Botulin ophthalmoplegia . Clinical and oculographic observations; Hedges TR 3rd et al.; Two patients with botulin ophthalmoplegia had rapid quivering eye motions during attempts to refixate laterally placed objects; these abnormal eye movements occurred in conjunction with a disjunctive limitation of range of eye movements . Oculographic examination showed that the quivering motions were composed of multiple hypometric saccades, many of which had subnormal and stuttering velocities . By blocking acetylcholine release into myoneural junctions, botulin toxin seems to limit the duration of saccadic burst innervation reaching ocular muscle.

J Pharmacol Exp Ther, 1983 Feb, 224(2), 265 - 8
Spontaneous output of acetylcholine from rat diaphragm preparations declines after treatment with botulinum toxin; Gundersen CB et al.; A gas chromatographic spectrometric assay was used to measure tissue and released acetylcholine and choline in diaphragm preparations of rats previously injected with botulinum toxin type A . Botulinum intoxication was found not to alter the acetylcholine content of rat diaphragms in vivo or in fully paralyzed muscles in vitro . This result provides direct support for the hypothesis that botulinum toxin blocks transmitter release without affecting acetylcholine synthesis . However, in diaphragm preparations in vitro, this toxin was found to inhibit not only the evoked release of acetylcholine but also the spontaneous "leakage" of acetylcholine that is measured at rest . Additional experiments were performed to characterize this action of the toxin . The magnitude of the decline in resting acetylcholine output appears to be too large to be accounted for solely by the known effect of botulinum toxin to reduce the frequency of miniature endplate potentials . The mechanism of this action of botulinum toxin remains an enigma.

Cell Tissue Res, 1983, 228(1), 1 - 12
Neural control of embryonic acetylcholine receptor and skeletal muscle; Creazzo TL et al.; The manner by which motor neurons exert control over the distribution and number of acetylcholine receptors, and muscle development was investigated in the superior oblique muscle of white Peking duck embryos . Clusters of receptors in the normally developing muscle first appeared on day 10 of incubation as determined with I125 alpha-bungarotoxin autoradiography . The initial appearance of receptor clusters coincided with the arrival of motor nerve fibers in the muscle . Clusters of receptors also appeared in normal fashion in muscles made aneural by destruction of motor neurons on day 7 . But after day 14 these clusters had disappeared and no new clusters were seen thereafter in the aneural muscle . Receptor clusters persisted throughout development in muscle in which neuromuscular transmission was blocked with either curare or botulinum toxin and in muscles denervated on day 10.5, i.e., shortly after the initial nerve-muscle contact but prior to the onset of muscle activity . A progressive increase in the total number of receptors and in the total amount of protein occurred during the course of normal development . However, the specific activity of the receptor protein declined sharply following innervation on day 10 . The total number of receptors and the specific activity of the receptor was affected depending on whether the motor neurons were destroyed before or after innervation and following chronic blockade of neuromuscular transmission . The half-life of the receptor protein was similar in normal, aneural, and paralyzed muscles (26, 25, 26 h, respectively) . Measurements of total protein indicated that essentially no muscle growth occurred in the complete absence of innervation . Paralyzed muscles continued to develop but at a slower pace.

Biokhimiia, 1983 Jan, 48(1), 33 - 9
{Effect of photooxidation on biological properties of botulinum neurotoxin A}; Kolesnikova VA et al.; Photooxidation of botulinic neurotoxin A in the presence of methylene blue is associated with a decrease in toxicity down to complete detoxication . During neurotoxin photooxidation, when the toxicity makes up to 1 to 3% of the original one, the conformation of the neurotoxin molecule and its antigenic properties remain unchanged . Under these conditions, using diethylpyrocarbonate, a specific reagent for histidine, the photooxidized neurotoxin was found to contain 5-6 oxidized histidine residues per molecule of neurotoxin; this was accompanied by changes in the UV absorbance spectrum around 280 nm . It was assumed that the main decrease in neurotoxin toxicity during photooxidation is probably due to oxidation of tryptophane, since the differential UV spectra suggest that the higher the extremum around 280 nm, the greater the decrease of toxicity; chemical modification of histidine residues alone causes no noticeable detoxication.

Toxicon, 1983, 21(4), 535 - 45
Purification and amino acid composition of type E botulinum neurotoxin; DasGupta BR et al.; The procedure we have adopted to purify type E botulinum neurotoxin (mol . wt . approximately 147,000) from bacterial cultures consistently yields a pure protein (a single band in polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate) . Our procedure is a modification of one of the five published procedures . Other procedures have failed to yield pure neurotoxin . To develop reliable data on the amino acid composition, three batches of the neurotoxin were analyzed, each batch isolated from a separate neurotoxin producing culture . The best estimate of the number of amino acid residues per neurotoxin molecule was: Asp240 Thr75 Ser98 Glu118 Pro45 Gly58 Ala40 Val62 CyS7 Met17 Ile123 Leu107 Tyr70 Phe62 Lys97 His15 Arg34 Trp16.

Jpn J Physiol, 1983, 33(4), 677 - 80
Anti-botulismic effect of toosendanin and its facilitatory action on miniature end-plate potentials; Shih YL et al.; The time for blocking neuromuscular transmission by botulinum toxin is lengthened in phrenic nerve-diaphragm preparation of rats sacrificed at various times after subcutaneous injection of toosendanin (7 mg/kg) . This effect of toosendanin manifests itself 15 min after injection and lasts for 3 days . During this period the frequency of miniature end-plate potentials is increased.

Vopr Med Khim, 1983 Jan-Feb, 29(1), 107 - 10
{Alterations in activity of the ATPase systems in subcellular fractions of brain and spinal cord in the preclinical period of botulinum poisoning}; Chesnokova NP et al.; Under conditions of the C type botulinic intoxication of rats blockade of cholinergic synaptic transmission and development of paralytic syndrome followed the distinct alteration in activity of the ATPase systems in various subcellular fractions of brain and spinal cord . This suggests that the toxin affects the various intracellular metabolic reactions related directly or indirectly to active transmembrane transport of ions.

Acta Physiol Scand, 1983, 119(2), 127 - 33
Different effects of types A and B botulinum toxin on transmitter release at the rat neuromuscular junction; Sellin LC et al.; Blockade of neuromuscular transmission was produced in the lower hind limb of the rat by local injection of either crystalline type A botulinum toxin or purified type B botulinum neurotoxin . At 1, 3, 5 and 7 days after injection, the extensor digitorum longus nerve-muscle preparation was excised and analyzed in vitro for alterations in spontaneous and nerve stimulus-evoked quantal transmitter release . Muscles receiving type A toxin were paralyzed up to and including 7 days after injection . Muscles treated with type B toxin, although completely paralyzed at 1 and 3 days, twitched in response to nerve stimulation at 5 and 7 days after injection . Both toxins induced a marked decrease in the frequency of miniature endplate potentials but type A did so to a greater extent . The remaining population of miniature endplate potentials contained a greater frequency of potentials with small or large amplitudes and prolonged rise times compared to normal muscle . These changes were more pronounced with type A toxin than with type B toxin . In the presence of alpha-dinitrophenol (1 mM), high frequency, fast-rising miniature endplate potentials of uniform size reappeared . High K+ (20 mM) was less effective in this respect . At 3 days after toxin injection nerve impulse evoked transmitter release was reduced more for type A treated muscles than for type B . However, 3,4-diaminopyridine, an agent which increases nerve-evoked transmitter release by increasing Ca2+ influx, was more effective in reversing the paralysis in type A than in type B-treated muscles.(ABSTRACT TRUNCATED AT 250 WORDS)

Brain Res, 1982 Dec 16, 253(1-2), 13 - 8
The effects of pyronin on sprouting and regeneration of mouse motor nerves; Keynes RJ; Administration to mice of a 0.1% solution of pyronin G in their drinking water caused an acceleration both of axonal sprouting from nodes of Ranvier in partly denervated gluteus maximus muscles, and of motor nerve regeneration following a crush to the soleus nerve . Sprouting from soleus motor nerve terminals in response to botulinum toxin-induced paralysis was, however, unaffected . Removal of degenerating axons following nerve section was also accelerated by pyronin treatment . Pyronin is therefore likely to act upon the process of Wallerian degeneration, rather than upon intact motor nerves directly.

Proc R Soc Lond B Biol Sci, 1982 Nov 22, 216(1205), 497 - 507
An endplate potential due to potassium released by the motor nerve impulse; Katz B et al.; A small endplate potential can be recorded in frog muscle fibres, after all acetylcholine-mediated transmission has been eliminated by pre- or postsynaptic blocking agents (botulinum toxin, calcium lack, manganese, curare, alpha-bungarotoxin) . It is usually necessary to hyperpolarize the muscle membrane to detect this 'non-cholinergic' endplate potential . Below--100 mV little or no response is seen; a maximum is reached at about--140 mV, when the amplitude can be as large as 100 microV (endplate current up to about 1 nA) . Other characteristic features are: the response shows no quantal fluctuations; its amplitude is not facilitated by repetitive impulses; its size and time course are not noticeably affected by prostigmine, curare or alpha-bungarotoxin; the half-time of decline of the endplate current is approximately 1.7 ms at 20 degrees C, and is lengthened by lowering the temperature with a Q10 of about 1.3; the response is abolished by barium . When iontophoretic pulses of potassium are applied to the endplate, local depolarization is recorded whose amplitude varies with membrane potential similarly to that of the nerve-evoked response . These observations strongly indicate that this 'non-cholinergic', 'non-quantal' endplate potential arises from a rapid synaptic transfer of potassium ions, released by the active nerve terminal into the synaptic cleft and entering the muscle fibre through 'anomalous rectifier' channels in the endplate membrane.

Eur J Biochem, 1982 Nov, 128(1), 267 - 76
Preparation of neurotoxic 3H-beta-bungarotoxin: demonstration of saturable binding to brain synapses and its inhibition by toxin I; Othman IB et al.; 1 . Homogeneous beta-bungarotoxin, isolated from the venom of Bungarus multicinctus was radiolabelled with N-succinimidyl-{2.3-(3) H}propionate . Stable, di-propionylated material was obtained which was tritiated on both subunits and had a specific radioactivity of 102 Ci/mmol . 2 . After separation from unlabelled toxin by isoelectric focussing, it was shown to exhibit significant biological activity in both the peripheral and central nervous systems but had negligible phospholipase A2 activity towards lecithin or cerebrocortical synaptosomes . 3 . The labeled neurotoxin binds specifically to a single class of non-interacting sites of high affinity (Kd = 0.6 nM) on rat cerebral cortex synaptosomes; the content of sites is about 150 fmol/mg protein . This binding was inhibited by unlabelled beta-bungarotoxin with a potency which indicates that tritiation does not alter the affinity significantly . 4 . The association of toxin with its binding component and its dissociation were monophasic; rate constants observed were 7.8 x 10(5) M-1 s-1 and 5.6 x 10(-4) s-1 at 37 C, respectively . 5 . beta-Bungarotoxin whose phospholipase activity had been inactivated with p-bromophenacyl bromide inhibited to some extent the binding of tritiated toxin but with low efficacy . Taipoxin and phospholipase A2 from bee venom, but not Naja melanoleuca, inhibited the synaptosomal binding of toxin with low potencies in the presence, but not the absence, of Ca2+ . 6 . Toxin I, a single-chain protein from Dendroaspis polylepis known to potentiate transmitter release at chick neuromuscular junction, completely inhibited the binding of 3H-beta-bungarotoxin with a Ki of 0.07 nM; this explains its ability to antagonise the neuroparalytic action of beta-bungarotoxin . Other pure presynaptic neurotoxins, alpha-latrotoxin and botulinum neurotoxin failed to antagonise the observed binding; likewise tityustoxin, which is known to affect sodium channels, had no effect on 3H-beta-bungarotoxin binding . 7 . Trypsinization of synaptosomes completely destroyed the binding activity, suggesting that the binding component is a protein; the functional role of the latter is discussed in relation to the specificity of toxin binding.

Proc R Soc Lond B Biol Sci, 1982 Oct 22, 216(1204), 369 - 76
The antagonism between botulinum toxin and calcium in motor nerve terminals; Gundersen CB et al.; The effects of tetraethylammonium and manganese, which modify calcium entry into motor nerve terminals, have been studied during advanced stages of botulinum paralysis . Evidence has been obtained that the voltage-activated calcium current in the nerve endings is not significantly reduced by botulinum toxin . The depression of transmitter release that the toxin produces must arise at a later stage, at an intracellular site of the release mechanism.

Zhonghua Min Guo Wei Sheng Wu Ji Mian Yi Xue Za Zhi, 1982 Aug, 15(3), 221 - 6
Effects of type A botulinus toxin on primary chick embryo cell culture; Liu YT et al.; Type A botulinus toxin was studied for its ability to inhibit the DNA, RNA, and protein synthesis of a chick embryo primary cell culture . 3H-thymidine, 3H-uridine, and 14C-leucine were employed as tracers for measuring the synthesis of DNA, RNA, and protein, respectively . In the presence of large doses (2 x 10(6) MLD/ml) of type A botulinus toxin, RNA synthesis was inhibited to a lesser extent than DNA synthesis, and a very weak inhibition of protein synthesis was observed . The effect of type A botulinus toxin on DNA synthesis of a chick embryo brain cell suspension culture was also tested . The DNA synthesis of the brain cells was strongly inhibited by this toxin.

Brain Res, 1982 Jul 15, 243(2), 345 - 9
Importance of pathway formation for nodal sprout production in partly denervated muscles; Brown MC et al.; Experiments were carried out to investigate possible factors controlling nodal sprout growth in partly denervated mouse gluteal muscles . Pretreatment of the muscle with botulinum toxin for up to 20 days, which produces denervation-like change and elicits terminal and nodal sprouting, increased reinnervation by terminal sprouting after partial denervation but did not alter the rate of reinnervation by nodal sprouts . This implies that nodal sprout growth is not limited by the development of an adequate growth stimulus from denervated muscle . A disto-proximal gradient of degeneration was observed in denervated intramuscular nerves in the electron microscope, suggesting that nodal sprout growth may be modulated by the availability of endoneurial pathways sufficiently degenerated to permit reinnervation by nodal sprouts, although the initial outgrowths from nodes of Ranvier may appear in response to a growth stimulus from denervated muscle.

Q J Exp Physiol, 1982 Jul, 67(3), 495 - 506
The effects of black widow spider venom on the innervation of muscles paralysed by botulinum toxin; Gomez S et al.; Botulinum toxin (BoTx) was injected into the muscles of one leg in mice causing local paralysis . Black widow spider venom (b.w.s.v.) was then injected into the paralysed muscles 3 or 15 d later . In both groups b.w.s.v . destroyed the nerve terminals poisoned by BoTx . In the 15 d group axonal sprouts, which had former due to the block of neuromuscular transmission by BoTx, were also destroyed . Within a few days the motor nerve terminal regenerated and the muscles recovered from paralysis at a faster rate than after BoTx alone . Recovery seemed to begin earlier in muscles where axonal sprouting was already advanced when b.w.s.v . was injected . The normal pattern of innervation was re-established in both groups, which was in marked contrast with muscles after BoTx alone where numerous sprouts and many ectopic end-plates had formed.

J Pharmacol Exp Ther, 1982 Jul, 222(1), 43 - 8
The interaction between aminoquinolines and presynaptically acting neurotoxins; Simpson LL; Chloroquine and hydroxychloroquine block neuromuscular transmission in isolated tissues from mouse, rat, guinea pig and chick . Blockade is associated with depressed muscle responses to potassium and abolished muscle responses to nicotinic cholinergic agonists . Within certain time and concentration limits, the blocking effects of chloroquine and hydroxychloroquine are reversible . Both drugs antagonize the onset of paralysis caused by botulinum neurotoxin types A and B, but neither drug antagonizes tetanus toxin or beta-bungarotoxin . The ability of chloroquine and hydroxychloroquine to antagonize botulinum toxin is not due to blockade of nicotinic cholinergic receptors . At concentrations that produce neuromuscular blockade, d-tubocurarine does not antagonize botulinum toxin types A and B . Chloroquine causes botulinum toxin to remain at an antitoxin sensitive site . These data could mean that chloroquine acts at the cell membrane to inhibit toxin binding or internalization, or that it acts in the cell interior to inhibit lysosomal processing of toxin . Whatever its action, chloroquine is the most effective antagonist of botulinum toxin yet described.

Ophthalmology, 1982 May, 89(5), 489 - 91
Diagnostic injection of Xylocaine into extraocular muscles; Magoon E et al.; In situations where it is unclear which extraocular muscle is causing anomalous eye movement or to what extent one of two muscles is responsible, temporary paralysis of the muscle with Xylocaine may provide the answer . The procedure is to inject 0.2 to 0.5 cc 2% Xylocaine directly into the muscle using electromyography (EMG) for precise localization, a technique similar to that of therapeutic injections of botulinum toxin (Oculinum) . The procedure is especially useful for Duane's syndrome, superior oblique palsy, and other situations where abnormal muscle insertions or innervations make diagnosis of the muscle responsible for an eye movement anomaly difficult and surgery unpredictable.

Proc R Soc Lond B Biol Sci, 1982 Apr 22, 215(1198), 63 - 74
Discrepancies between spontaneous and evoked synaptic potentials at normal, regenerating and botulinum toxin poisoned mammalian neuromuscular junctions; Colmeus C et al.; Amplitudes and times to peak of spontaneous miniature endplate potentials (m.e.p.ps) and evoked quantal endplate potentials (e.p.ps) were compared at normal, regenerating and botulinum toxin poisoned neuromuscular junctions of the extensor digitorum longus muscle of the rat . At normal junctions the mean time to peak of m.e.p.ps was longer and more variable than that of similar-sized e.p.ps . At endplates where nerve regeneration was induced by mechanical crushing of the motor nerve the frequency of m.e.p.ps was reduced and their amplitude distribution was broader than normal . The distribution of times to peak of m.e.p.ps was considerably broader than that of quantal e.p.ps recorded at the same endplates . At neuromuscular junctions poisoned with botulinum toxin type A, spontaneous and evoked transmitter release were greatly reduced . The amplitude distribution of m.e.p.ps was wider than that of e.p.ps and the time to peak of e.p.ps was about twice as fast as and less variable than that of m.e.p.ps . To explain the observed differences in time to peak among m.e.p.ps and between m.e.p.ps and quantal e.p.ps we suggest that some m.e.p.ps, but not e.p.ps, originate from transmitter quanta released from sites at a greater distance from postsynaptic receptors or that the release or diffusion process for acetylcholine is more prolonged when producing some of the m.e.p.ps . Such mechanisms produce at normal junctions a small population of m.e.p.ps with prolonged times to peak, at regenerating junctions a greater proportion of such m.e.p.ps and in botulinum toxin poisoning a majority.

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