Botulin

Otolaryngol Clin North Am, 1991 Oct, 24(5), 1227 - 37
Treatment options in spasmodic dysphonia; Miller RH et al.; Options in the treatment of spasmodic dysphonia include surgical interruption of the recurrent laryngeal nerve, anterior laryngoplasty, voice therapy, and Botulinum toxin injection . Although none of these treatments is ideal, Botulinum toxin injection appears to have the greatest potential to benefit the greatest number of patients.

Jpn Circ J, 1991 Oct, 55(10), 1036 - 43
Small GTP-binding proteins in bovine aortic smooth muscle; Kawahara Y et al.; In bovine aortic smooth muscle, GTP-binding activity was equally distributed in the membrane and cytosol fractions . The most abundant GTP-binding proteins (G proteins) in each fraction were purified to near homogeneity and characterized . The most abundant G protein in the membrane fraction had a Mr value of about 22,000 (m22K G) as estimated on sodium dodecyl sulfate-polyacryl-amide gel electrophoresis (SDS-PAGE) . m22K G and the human platelet smg p21, a ras p21 like G protein having the same effector domain as ras p21s, were eluted at the same retention time on C4 reversed-phase high performance liquid chromatography (HPLC) . Moreover, m22K G was specifically recognized by an anti-smg p21 polyclonal antibody . m22K G was phosphorylated by cyclic AMP-dependent protein kinase with a stoichiometry of one phosphate/molecule of protein . The most abundant G protein in the cytosol fraction had a Mr value of about 21,000 (c21K G) as estimated on SDS-PAGE . c21K G was ADP-ribosylated by botulinum ADP-ribosyltransferase and about 0.4 mol of ADP-ribose was maximally incorporated into 1 mol of c21K G . c21K G and the bovine brain rhoA p21, another ras p21 like G protein, were eluted at the same retention time on C4 reversed-phase HPLC and migrated at the same position on two-dimensional gel electrophoresis . These results indicate that the major G proteins in the membrane and cytosol fractions of bovine aortic smooth muscle are smg p21 and rhoA p21, respectively . Possible roles of these G proteins in vascular smooth muscle are discussed.

Naunyn Schmiedebergs Arch Pharmacol, 1991 Oct, 344(4), 387 - 95
Distinct targets for tetanus and botulinum A neurotoxins within the signal transducing pathway in chromaffin cells; Marxen P et al.; Tetanus and botulinum A neurotoxins inhibited exocytosis evoked by various secretagogues in intact and permeabilized chromaffin cells . The block of exocytosis in intact chromaffin cells due to botulinum A neurotoxin could partially be overcome by enhancing nicotine- and veratridine-induced stimulation, whereas the block due to tetanus toxin persisted under the same conditions . The receptor-mediated restoration of 3H-noradrenaline release was specific for nicotinic stimulation, because exocytosis did not occur during muscarinic stimulation . Depolarization of intact chromaffin cells with increasing concentration of K+ failed to restore exocytosis that had been blocked by either toxin . When chromaffin cells, treated with tetanus or botulinum A neurotoxins, were exposed to the Ca2(+)-ionophore A 23187 or permeabilized by staphylococcal alpha-toxin, Ca2(+)-stimulated exocytosis was also inhibited . The inhibition was unaffected by increasing concentrations of free Ca2+ . Activation of proteinkinase C and of G-proteins by phorbolester and GMPPNHP, respectively, increased Ca2(+)-induced exocytosis in control cells as well as in cells treated with tetanus and botulinum A neurotoxins . The block, however, could not be relieved by these manipulations, and it could not be relieved by activating the cGMP or cAMP pathways with analoga of cyclic nucleotides, phosphodiesterases inhibitors, and forskolin either . It is concluded that nicotine and veratridine trigger a mechanism within the sequence of events leading to exocytosis that is located beyond the increase in intracellular Ca2(+)-concentration . This pathway may not be affected by botulinum A neurotoxin . The target of tetanus toxin is probably located even closer to the fusion process, i.e . beyond the step upon which botulinum A neurotoxin acts.

Biochem J, 1991 Oct 1, 279 ( Pt 1), 43 - 8
Redistribution of 23 kDa tubulovesicle-associated GTP-binding proteins during parietal cell stimulation; Basson MD et al.; Small GTP-binding proteins are important regulators of intracellular traffic . The presence of several small GTP-binding proteins was documented in subfractions of rabbit parietal cells . Upon maximal stimulation of the cells with a combination of histamine and forskolin, one 23 kDa GTP-binding band was observed to decrease in a 50,000 g membrane fraction while increasing in 4000 g membranes . The 23 kDa band resolved into one major and two minor species on two-dimensional gels . GTP-binding species of 23 kDa, 24 kDa and 25 kDa were present in purified preparations of tubulovesicles . The three isoelectric species of the 23 kDa proteins observed in parietal cell 50,000 g microsomes were enriched in tubulovesicle preparations . None of the tubulovesicle-associated GTP-binding proteins were substrates for ADP-ribosylation by a preparation of botulinum D toxin . These results indicate that tubulovesicles contain discrete small GTP-binding proteins which redistribute during parietal cell stimulation.

Am J Physiol, 1991 Oct, 261(4 Suppl), 118 - 22
Two types of G proteins involved in regulation of phosphoinositide turnover in pulmonary endothelial cells; Tkachuk VA et al.; The involvement of G proteins in hormonal regulation of phospholipase C in bovine pulmonary arterial endothelial cells and human umbilical vein endothelial cells has been investigated . Histamine and bradykinin stimulated phosphoinositol (PI) turnover in a dose-dependent manner, and phorbol-myristate-acetate inhibited hormone-dependent activation of PI turnover, indicating a feedback control of this process . Activation of PI turnover by histamine and bradykinin is guanine nucleotide-dependent . Stimulation of the endothelial cell G proteins by guanosine 5'-O-(3-thiotriphosphate) leads to the potentiation of hormone-induced activation of PI turnover, whereas guanosine 5'-O-(2-thiodiphosphate), which inactivates G proteins, blocks the hormone-dependent PI turnover . Pertussis toxin blocked the histamine-dependent stimulation but did not affect the bradykinin-dependent stimulation of phospholipase C . By contrast, botulinum toxin (C2 + C3 components) blocked the bradykinin-dependent stimulation of phospholipase C but did not affect the histamine-dependent stimulation of this enzyme . These data suggest that at least two different G proteins are involved in hormone-dependent stimulation of phospholipase C in endothelial cells.

Mol Pharmacol, 1991 Oct, 40(4), 563 - 71
Inhibition of cytoskeletal rearrangement by botulinum C2 toxin amplifies ligand-evoked lipid mediator generation in human neutrophils; Grimminger F et al.; Botulinum C2 toxin, a binary toxin that ADP-ribosylates nonmuscle G-actin, was used as a selective tool to evaluate the role of actin-dependent cytoskeletal rearrangement in ligand-evoked lipid mediator generation . Human neutrophils (PMN) were preincubated with varying concentrations of the toxin for 30 min . Lipoxygenase products of arachidonic acid were measured by chromatographic techniques in the presence of exogenous arachidonic acid to probe PMN 5-lipoxygenase activity . Formation of platelet-activating factor (PAF) was assayed by the bioincorporation of {3H}acetate . Stimulation was performed with the soluble chemotactic ligands formyl-methionyl-leucyl-phenylalanine (FMLP) and PAF, as well as opsonized zymosan . PMN pretreatment with C2 toxin in the range between 200/400 and 800/1600 ng/ml C2I/II caused a dose-dependent suppression of the basal F-actin content and of stimulus-induced actin assembly . Phosphoinositide hydrolysis (measured as liberated inositol phosphates) and PAF generation in response to FMLP and exogenous PAF were markedly increased at these toxin doses . Minor C2 toxin concentrations (range, approximately 25/50 to 200/400 ng/ml C2I/II) were sufficient to amplify stimulus-induced formation of leukotriene B4 and its omega-oxidation products, nonenzymatic hydrolysis products of leukotriene A4, and 5-hydroxyeicosatetraenoic acid (5-HETE) . With increasing toxin doses, leukotriene generation declined and 5-HETE became the predominant metabolite . In contrast to the soluble ligands, the zymosan-effected generation of PAF and leukotrienes was dose-dependently inhibited by C2 toxin concentrations of greater than 200/400 ng/ml, paralleled by a loss of motile and phagocytotic functions in these cells . We conclude that selective inhibition of actin assembly amplifies PAF and 5-lipoxygenase product formation in response to soluble chemoattractants with distinct dose dependences . The augmentation of PAF generation may be linked to amplified second messenger levels at higher doses of C2 toxin, whereas the sensitivity of the 5-lipoxygenase metabolism to low concentrations may indicate toxin effect on a small, functionally specified, actin pool . The present data support an important role of cytoskeletal rearrangement in temporal and/or spatial limitation of chemoattractant-evoked PMN activation.

Tidsskr Nor Laegeforen, 1991 Sep 10, 111(21), 2637 - 9
{Treatment of focal dystonia with botulinum toxin}; Gjerstad L et al.; Focal dystonia and hemifacial spasms are difficult to treat . Medication and surgery may suppress the dystonic movements but the improvement is not satisfactory . The present article reviews use of Botulinum toxin in cases of focal dystonia . Injections of very small doses of Botulinum A toxin into the affected muscles is a new and efficient therapy for patients with focal dystonia . The toxin acts by inhibiting the release of acetylcholine from the nerve terminal, leading to a localized paralysis of the treated muscle . The effect is temporary and gradually diminishes, but the treatment can be repeated . The use of Botulinum toxin must be applied on the basis of a thorough knowledge of its effect and possible side effects.

J Neurol Neurosurg Psychiatry, 1991 Sep, 54(9), 813 - 6
A double blind trial of botulinum toxin "A" in torticollis, with one year follow up; Moore AP et al.; A double blind placebo controlled crossover trial was performed of botulinum toxin "A" in 20 patients with spasmodic torticollis . There was a statistically significant benefit for those treated with toxin; 12 on toxin improved objectively, compared with four on placebo (p less than 0.04) . After a follow up period of one year, 16 still seemed to benefit from repeated toxin injections . The main side effect was dysphagia, which appeared to be dose related in individual patients.

Laryngoscope, 1991 Sep, 101(9), 960 - 4
Quantifying the spread of botulinum toxin through muscle fascia; Shaari CM et al.; Botulinum toxin was recently approved for treating several head and neck dystonias . Paralysis of neighboring muscles is the major complication of its use . Spread of toxin from the injected muscle has been suggested as an etiology . This study examines how botulinum toxin crosses muscle fascia by a novel method of quantifying muscular paralysis . Botulinum toxin (0.2 to 10 U) was placed onto the fascia of rat tibialis anterior (TA) muscles (n = 6) . Toxin was also placed on dose-matched muscles that had their fascia surgically removed (n = 6) . Twenty-four hours later, the nerve to the tibialis anterior was electrically stimulated to deplete the muscle fibers of glycogen . Toxin-paralyzed fibers retained their glycogen and appeared purple on periodic acid-Schiff (PAS) stain . Botulinum toxin easily passed through muscle fascia even at subclinical doses . The presence of fascia reduced the spread of botulinum toxin by 23% . These results suggest that spread of botulinum toxin can be prevented only by delivering small doses to the center of a target muscle.

J Neurochem, 1991 Sep, 57(3), 1024 - 32
Binding of botulinum and tetanus neurotoxins to ganglioside GT1b and derivatives thereof; Schengrund CL et al.; The ability of fragments derived from botulinum neurotoxin (BTx) serotype A to bind to GT1b-coated plastic wells was investigated and compared with the binding characteristics of the parent approximately 150-kDa protein . Although the approximately 50-kDa light chain of BTxA had a marginal binding capacity, the predominant adherence to GT1b-coated wells was exhibited by the approximately 50-kDa carboxy-terminal half of the approximately 100-kDa heavy chain of BTxA; the amino-terminal half of the heavy chain lacked the ability to bind . Binding to GT1b by BTxA and its fragments was compared with that of tetanus neurotoxin (TTx) and the carboxy-terminal half of its heavy chain . Binding of BTxA and the C-terminal half of the heavy chain was optimal in buffers of low ionic strength (mu less than or equal to 0.04 and 0.06, respectively), whereas the heavy chain bound GT1b best at mu greater than or equal to 0.10 . TTx and the approximately 50-kDa C-terminal half of its approximately 100-kDa heavy chain bound GT1b at ionic strengths similar to those of BTxA . Comparison of the binding of BTx serotypes A, B, and E to GT1b (using conditions that were found to be optimal for binding by BTxA) indicated differences in the interaction of the three serotypes with GT1b . Compared with BTxA, adherence to GT1b by serotypes B and E was reduced by approximately 60 and approximately 90%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Klin Oczna, 1991 Sep, 93(9), 264 - 5
{Injection of botulinum toxin into the oculomotor muscles in disorders of ocular motility}; Glasner L et al.; Injections of botulinum toxin into the oculomotor muscles was used in incorrectible diplopia, ocular torticollis, Duane's syndrome and congenital nystagmus . Favourable results were obtained and the sole complications which could be observed were a transitory ptosis and subconjunctival haemorrhages . Frequently 2 to 3 injections were sufficient for a permanent effect.

J Behav Ther Exp Psychiatry, 1991 Sep, 22(3), 221 - 3
Meige's disease misdiagnosed as anxiety disorder; Cairns SL et al.; A woman in her late 40s with a 5 year history of anxiety was treated with relaxation training and cognitive restructuring . Her anxiety was manifested by facial twitching, hand fidgeting, vocal tremor, loss of self-esteem, and depression . Therapy seemed to reduce motor symptoms and improve her self-esteem, confidence, and mood . Six months after the start of therapy the client was found to have Meige's Disease . Following treatment with botulinum toxin, motor symptoms disappeared . This case highlights the need for psychotherapists to be more aware of neurological and medical problems which may mimic psychological ones.

Arch Neurobiol (Madr), 1991 Sep-Oct, 54(5), 210 - 7
{Focal dystonias and facial hemispasm: treatment with botulinum A toxin}; Astarloa R et al.; We report the results of the treatment of 80 patients with various idiopathic focal dystonia and essential hemifacial spasm with Botulinum A toxin . A statistically significant improvement was obtained in our 34 patients with blepharospasm, 19 patients with hemifacial spasm, 59% of 22 patients with cervical dystonia and 60% of 5 patients with hand dystonia . Mean duration of the benefit of each injection was 15.3, 16.3, 7.6 and 8.7 weeks respectively . Adverse effects were local and transient . We concluded that botulinum A toxin is a safe and effective therapy for patients with focal dystonia and hemifacial spasm.

Arch Neurobiol (Madr), 1991 Sep-Oct, 54(5), 206 - 9
{Treatment with botulinum toxin in blepharospasm}; Grandas F; Blepharospasm is a cranial dystonia characterized by forceful spasms of the orbicularis oculi muscle which may lead to functional blindness in approximately two-thirds of patients . Botulinum toxin injection is a simple procedure, very effective and with little morbidity . It is considered as the treatment of choice for patients with disabling blepharospasm.

Arch Neurobiol (Madr), 1991 Sep-Oct, 54(5), 198 - 205
{Advances in the treatment of the dystonias}; Gimenez-Roldan S; Except in Wilson's disease, few secondary dystonias are susceptible to benefit from an aetiological treatment . The somatic distribution of dystonia often determines the therapeutic strategy . Thus, stereotactic surgery may be the treatment of choice for hemidystonia while anticholinergic medication may alleviate generalized dystonia, particularly in childhood . Finally, local infiltrations of botulinum toxin are particularly useful for various forms of local and segmental dystonia . Certain subsyndromes as myoclonic dystonia, levodopa sensitive dystonia and paroxysmal choreoathetosis may benefit from relatively specific treatment strategies.

Ann Ophthalmol, 1991 Sep, 23(9), 326 - 33
A five-year analysis of botulinum toxin type A injections: some unusual features; Balkan RJ et al.; We analyzed patients treated during the past five years with botulinum toxin type A for strabismus and blepharospasm, reviewed our successes, failures, and unusual cases, and drew conclusions based on these treatments . Thirty-seven percent of the strabismus patients were cured, but many patients who were outside the strict definitions, still believed that they were significantly improved . A prominent feature in the treatment of strabismus was variability . Frequently, patients expected to do poorly had encouraging results . One permanent overcorrection occurred, and it converted an esotopic patient into an exotropic one with diplopia . This has persisted for 2.5 years and is the longest reported overcorrection to our knowledge . Our results indicate that larger doses of botulinum toxin produce longer spasm-free intervals in the treatment of blepharospasm . One patient receiving injections for her blepharospasm discovered that its cause was her sedative medication . This is the first reported case of a benzodiazepine inducing blepharospasm to our knowledge.

Neuron, 1991 Sep, 7(3), 421 - 7
TTX-sensitive and TTX-insensitive sodium channel mRNA transcripts are independently regulated in adult skeletal muscle after denervation; Yang JS et al.; The expression of mRNA encoding the TTX-sensitive (SkM1) and TTX-insensitive (SkM2) voltage-dependent sodium channels in adult skeletal muscle is independently regulated . In normal skeletal muscle, only the SkM1 message is expressed and the level varies with muscle fiber type . After surgical denervation, the steady-state SkM1 mRNA level declines transiently, but returns to control levels within 5 days . Expression of SkM2 transcripts is markedly activated, reaching a peak 3 days after axotomy and then declining to a maintained level at approximately 30% of peak . Chemical denervation with botulinum toxin results in higher levels of SkM2 mRNA, which by 7 days posttreatment are 7-fold greater than levels in paired axotomized muscles . SkM2 expression subsequently declines as functional reinnervation appears . Quantal acetylcholine release appears to play a major role in suppression of SkM2 expression in adult innervated or reinnervated muscle, whereas nonquantal factors in toxin-treated, but not axotomized, muscle may sustain high level SkM2 mRNA expression.

J Pharmacol Exp Ther, 1991 Sep, 258(3), 830 - 6
Lectins from Triticum vulgaris and Limax flavus are universal antagonists of botulinum neurotoxin and tetanus toxin; Bakry N et al.; Lectins from Anguilla anguilla, Artocarpus integrifolia, Canavalia ensiformis, Datora stramonium, Glycine max, Limax flavus, Ricinus communis and Triticum vulgaris were tested for their abilities to antagonize the binding of botulinum neurotoxin and tetanus toxin to rat brain membranes and to antagonize the ability of these toxins to block neuromuscular transmission in mouse phrenic nerve-hemidiaphragm preparations . Lectins from Limax flavus and Triticum vulgaris, both of which have affinity for sialic acid, were antagonists of the various serotypes of botulinum neurotoxin and tetanus toxin . When tested against the high affinity binding site for botulinum neurotoxin type B, the lectin from Limax flavus had a Ki of 3.1 x 10(-7) M and the lectin from Triticum vulgaris had a Ki of 3.75 x 10(-7) M . When tested against the high affinity binding site for tetanus toxin, the lectins from Limax flavus and Triticum vulgaris had Ki values of 1.5 x 10(-7) and 1 x 10(-6) M, respectively . In all cases the lectins behaved as competitive antagonists . In reverse experiments, neither botulinum toxin nor tetanus toxin was a very effective antagonist of lectin binding to brain membranes . Studies on isolated neuromuscular preparations showed that the lectin from Triticum vulgaris did not affect transmission at concentrations of 10(-6) to 10(-3) M, but at a concentration of 3 x 10(-5) M the lectin produced highly statistically significant antagonism of the neuromuscular blocking properties of botulinum neurotoxin types A, B, C, D, E and F as well as tetanus toxin . The lectin did not antagonize beta-bungarotoxin.(ABSTRACT TRUNCATED AT 250 WORDS)

J Immunol, 1991 Aug 15, 147(4), 1139 - 46
Microfilament assembly is required for antigen-receptor-mediated activation of human B lymphocytes; Melamed I et al.; The mechanisms responsible for initiating the conversion of globular to filamentous actin (assembly) after stimulation of B lymphocytes and the role of these cytoskeletal changes in cell activation are incompletely understood . We investigated the molecular basis of the signals leading to actin polymerization and concentrated on the involvement of guanosine triphosphate (GTP)-binding regulatory proteins, and protein kinase C (PKC) . In addition, we related these early events to later events in B-cell activation, including cell proliferation . Cross-linking the Ag receptor with Staphylococcus aureus Cowan I (SAC) or anti-IgM antibodies, or stimulation of PKC with phorbol ester induced a time- and concentration-dependent increase in the filamentous actin content of B cells . Inhibition or depletion of PKC resulted in decreased actin assembly induced by anti-IgM, SAC, and PMA, suggesting that the signal for polymerization is generated distally to PKC activation . Pertussis toxin pretreatment inhibited the responses to anti-IgM and SAC but not PMA, and direct stimulation of permeabilized cells with GTP gamma S induced microfilament assembly, indicating the involvement of a GTP-binding protein for receptor-mediated events . Disruption of actin polymerization with botulinum C2 toxin or cytochalasin D inhibited the assembly of actin and {3H}TdR incorporation induced by all stimuli . We conclude that human B cell activation by receptor-mediated stimuli results in actin polymerization by signaling pathways coupled to GTP-binding proteins . These changes in the cytoskeleton may be involved in the transduction of messages leading to responses such as proliferation in B lymphocytes.

Hosp Pract (Off Ed), 1991 Aug 15, 26(8), 35, 38, 41 - 2
Botulinum toxin A therapy in dystonia; Adler CH; Local injections can decrease posturing and pain in 70% to 90% of patients for up to eight months . Responses can be extended with further injections.

Br J Ophthalmol, 1991 Aug, 75(8), 487 - 90
Surgical management of essential blepharospasm; Bates AK et al.; We have reviewed the surgical management of essential blepharospasm over the last 15 years, comparing the results from facial nerve avulsion with those from orbicularis muscle stripping . After facial nerve avulsion 50% of patients remained free of troublesome spasm for 15 months after surgery, but only 25% remained so for more than two years . Following orbicularis oculi myectomy 50% of patients were free of troublesome spasms for 30 months after surgery and 55% of patients had relief from spasm for more than two years . Secondary effects of the two procedures are compared and are found to be fewer after orbicularis myectomy . There were no major complications after either form of surgery . Botulinum toxin is the treatment of first choice for this condition . If this becomes ineffective or inconvenient, surgical treatment is warranted and should not be deferred for fear of severe side effects of treatment, since these are rare . Protractor myectomy gives longer relief from blepharospasm than facial nerve avulsion and has fewer complications . However, it is technically difficult, time consuming, and has greater peroperative morbidity . Facial nerve avulsion may therefore still have a role in selected patients.

Neurology, 1991 Aug, 41(8), 1185 - 8
Botulinum toxin treatment of tremors; Jankovic J et al.; We report the results of an open trial of botulinum toxin (Botox) in the treatment of 51 patients with disabling tremors, classified as dystonic (14), essential (12), combination of dystonic and essential (22), parkinsonian (1), peripherally induced (1), and midbrain (1) . The average age of the patients was 55.8 years, and duration of symptoms was 13.9 years . During a total of 160 treatment visits, an average of 242 +/- 75 units of Botox was injected per visit in cervical muscles of 42 patients with head tremor and 95 +/- 38 in forearm muscles of 10 patients with hand tremor; one patient was injected in both . The average peak effect for all patients was rated as 3.0 (0 to 4 scale) . Thirty-five (67%) patients improved (peak effect greater than or equal to 1) . The average latency from injection to response was 6.8 days, and the average duration of maximum improvement was 10.5 weeks . Local complications, lasting an average of 20.6 days, were noted in 17 (40%) patients injected for head tremor, consisting chiefly of dysphagia in 12 (29%), transient neck weakness in four (10%), and local pain in two (5%) . Six (60%) patients with hand tremor had transient focal weakness . EMG recordings showed decreased amplitude of EMG bursts after Botox treatment . The results of this pilot study indicate that Botox injections can be used to control tremor in patients in whom other forms of therapy have failed.

Laryngoscope, 1991 Aug, 101(8), 911 - 4
A method for the treatment of abductor spasmodic dysphonia with botulinum toxin injections: a preliminary report; Rontal M et al.; A preliminary technical report of the effective treatment of abductor spasmodic dysphonia with botulinum toxin is presented . Our technique attempts to place the toxin close to the posterior cricoarytenoid muscle to allow diffusion of the material to the PCA . Our pilot study demonstrates that botulinum toxin is an effective approach for reducing or eliminating the abductor glottal spasms during phonation and, thereby, providing functional speech communication.

J Pharmacol Exp Ther, 1991 Aug, 258(2), 613 - 9
Tetanus toxin and neuronal membranes: the relationship between binding and toxicity; Bakry N et al.; Tetanus toxin labeled by the Bolton-Hunter technique possesses high specific activity and retains substantial biological activity . This material can be used to characterize tetanus toxin binding to receptors in brain membrane preparations . In experiments aimed at measuring the absorption of labeled toxin, the displacement of labeled toxin by unlabeled toxin and the on-rate and off-rate constants, the data revealed two binding sites . The high affinity site had a Kd of 0.033 to 0.070 nM and a Bmax of 0.26 to 0.4 pmol/mg of protein; the low affinity site had a Kd of 0.89 to 6.9 nM and a Bmax of 1.55 to 3.0 pmol/mg of protein . The binding of tetanus toxin to brain membranes was enhanced greatly by low pH and ionic strength . Similarly to tetanus toxin, botulinum neurotoxin could be labeled by the Bolton-Hunter technique, and its binding to brain membranes was also enhanced by low pH and ionic strength . In studies with a neutralizing monoclonal antibody against tetanus toxin, the antigen-antibody interaction was not significantly altered by media with low ionic strength and pH . On the other hand, the ability of the antibody to block toxin binding to brain membranes was reduced substantially in nonphysiologic media . In a bioassay aimed at determining the effect of pH and tonicity on tissue association by toxin, low pH and ionic strength did not enhance toxicity . The biological activity of tetanus toxin was unaffected and that of botulinum neurotoxin was greatly diminished . The present findings confirm the widely reported observation that low pH and ionic strength promote tissue association by tetanus toxin, but they challenge the premise that this binding is relevant to the normal process of cell poisoning.

Neurology, 1991 Jul, 41(7), 1088 - 91
Cervical dystonia: clinical findings and associated movement disorders; Jankovic J et al.; We studied 300 patients, 61% women, with mean age 49.7 years and mean duration of dystonia 7.8 years, to determine the demographic and clinical characteristics of cervical dystonia (CD) and its relationships to other movement disorders . Torticollis was present in 82%, laterocollis in 42%, retrocollis in 29%, and anterocollis in 25%; however, the majority (66%) had a combination of these abnormal postures . Scoliosis was present in 39%, local pain reported by 68%, and 32% had evidence of secondary cervical radiculopathy . In addition to CD, 16% of patients had oral dystonia, 12% mandibular dystonia, 10% hand/arm dystonia, and 10% had blepharospasm . Tremor was noted in 71% of patients; head-neck tremor was present in 60%, and tremor in other body regions was present in 32% . A family history of a movement disorder was present in 44% of the CD patients . Tardive dystonia was the cause in 6%; 11% had posttraumatic dystonia . Anticholinergic drugs provided moderate improvement in 33% of patients, but local intramuscular botulinum toxin injections relieved CD, local pain, or both in over 90% of all treated patients.

Behring Inst Mitt, 1991 Jul, (89), 153 - 62
Clostridial neurotoxins: from toxins to therapeutic tools?
Niemann H, Binz T, Grebenstein O, Kurazono H, Thierer J, Mochida S, Poulain B, Tauc L.
Tetanus toxin and botulinum toxins are powerful neurotoxins which block neurotransmitter release through an unknown mechanism my means of their light chains . The heavy chains provide the machinery for neuroselective binding, internalization, retrograde intraaxonal transport, and translocation of the L-chains into the cytosole . We have cloned and sequenced the structural genes of tetanus toxin and of five serologically distinct botulinum toxins to identify structurally and functionally conserved subdomains . The minimum essential domains of the L-chains of tetanus and botulinum toxin type A were identified by combined in vitro transcription and microinjection of L-chain specific mRNA into identified presynaptic neurons of Aplysia californica . In addition, a nontoxic mutant of tetanus was generated by replacing histidine(237) by a proline residue . The development of nontoxic neuroselective transporter molecules carrying various marker enzymes is discussed.

Surv Ophthalmol, 1991 Jul-Aug, 36(1), 28 - 46
Botulinum A toxin (Oculinum) in ophthalmology; Osako M et al.; Botulinum A toxin has been used to treat strabismus and a variety of spasmodic neuromuscular diseases . Botulinum toxin treatment of strabismus is not as definitive and stable as the traditional surgical approach, but it has been found most useful in postoperative overcorrection, small deviations, sensory deviations, and acute sixth nerve palsy . This toxin has been effective in the treatment of essential blepharospasm and hemifacial spasm, for which it produces temporary relief of symptoms . In addition, this treatment has been applied to lower lid entropion, myokymia, aberrant regeneration of the seventh nerve, lid retraction, corneal exposure, nystagmus, spasmodic torticollis, and adductor spastic dysphonia.

Muscle Nerve, 1991 Jul, 14(7), 672 - 5
Distant effects of locally injected botulinum toxin: a double-blind study of single fiber EMG changes; Lange DJ et al.; We used single fiber electromyography (SFEMG) to study 42 patients who had enrolled in a double-blind, placebo-controlled trial undertaken to assess the efficacy of botulinum toxin (BTX) injection of neck muscles to treat torticollis . SFEMG in a limb muscle was performed before treatment, 2, and 12 weeks after injection of placebo or BTX . Before treatment, the mean jitter was 26.8 microsec in patients who were to receive BTX, and 25.7 microsec in the placebo group . Two weeks after injection, mean jitter in the group receiving BTX was 43.6 microsec . In the placebo group, it was 26.5 microsec (P = less than .05) . Twelve weeks after injection, mean jitter in the BTX group was 35.5; for the placebo group it was 24.5 . Fiber density did not change in any patient during the study . There were no remote clinical effects of BTX . Injection of BTX into muscles affected with focal dystonia is a promising and safe treatment, but there are subclinical effects on uninjected muscles.

Rehabil Nurs, 1991 Jul-Aug, 16(4), 184 - 8
Botulinum toxin for blepharospasm: challenges for rehabilitation nurses; Kinash RG et al.; Blepharospasm is a chronic, progressive, involuntary spasmodic closure of the eyelids associated with abnormal facial and oromandibular movements . It is a neurologic disorder whose cause is unknown and whose pathophysiology is poorly understood . Without appropriate treatment, it can result in functional blindness and other disabilities . In the last decade, botulinum toxin has been found to be effective therapy for most individuals . The drug, which is given by local injection, has a denervation effect . It relieves symptoms for several months, allowing patients to resume their former lifestyles between treatments . This new therapy modality challenges rehabilitation nurses to bridge the gap between disabled persons in the community and this new technology . Casefinding, referrals, and patient education are among the interventions that can help meet this challenge . The major purpose of this article is to inform rehabilitation nurses about how to recognize the symptoms of neurologic blepharospasm and how to intervene to prevent disabilities that could result.

Clin Neuropharmacol, 1991 Jun, 14(3), 262 - 7
Electromyographic guidance of botulinum toxin treatment in cervical dystonia; Dubinsky RM et al.; We report the results of electromyographic (EMG) guidance in the treatment of cervical dystonia with botulinum toxin . Eight-four patients received a total of 225 injection sessions . Overall there was moderate objective improvement in 78.7% . The mean dose of toxin was 269 +/- 39 mouse lethal units and the mean duration of maximum effect was 107 +/- 49 days . Complications included excessive neck weakness in 16.0% and dysphagia in 11.1% of the injection sessions . We conclude that EMG guidance is a safe and effective method of administering botulinum toxin in the treatment of cervical dystonia.

Am Fam Physician, 1991 Jun, 43(6), 2113 - 20
Facial dystonia, essential blepharospasm and hemifacial spasm; Holds JB et al.; Movement disorders, or dyskinesias, in the facial region may be categorized in several ways . Dystonic movement disorders in the cranial-cervical region, including essential blepharospasm, Meige syndrome and spasmodic torticollis, are characterized by uncontrollable squeezing movements in the face and neck . These disorders typically present in the fifth and sixth decades of life . Essential blepharospasm is particularly debilitating, as the involuntary eyelid closure that accompanies this condition may result in functional blindness with an otherwise normal visual pathway . Hemifacial spasm is an intermittent, unilateral, spasmodic contraction of the muscles innervated by the facial nerve . This disorder usually presents in the third or fourth decade and has a different underlying pathophysiology than the dystonias . Botulinum A toxin therapy has largely supplanted surgical intervention in the treatment of essential blepharospasm and hemifacial spasm.

Laryngoscope, 1991 Jun, 101(6 Pt 1), 630 - 4
Double-blind controlled study of botulinum toxin in adductor spasmodic dysphonia; Troung DD et al.; The treatment of adductor spasmodic dysphonia using botulinum toxin A was conducted in 13 patients as a double-blind, placebo-controlled study . Patients were diagnosed independently by an interdisciplinary team consisting of speech pathologists, an otolaryngologist, and a neurologist . The toxin or saline was injected into each thyroarytenoid muscle under electromyographic and laryngoscopic guidance . Botulinum toxin A markedly reduced perturbation, decreased fundamental frequency range, and improved the spectrographic characteristics of the voice . Fundamental frequency and phonation time remained unchanged . Patients injected with botulinum toxin A noticed significant improvement in their voices in comparison with the placebo-treated group . Excessive breathiness of the voice occurred in two patients, and mild bleeding in one patient in the botulinum toxin A-treated group . Injection with saline resulted in edema of the vocal cord in one patient . Botulinum toxin A proved to be an effective and safe treatment of adductor spasmodic dysphonia.

Otolaryngol Head Neck Surg, 1991 Jun, 104(6), 849 - 55
Successful treatment of selected cases of abductor spasmodic dysphonia using botulinum toxin injection; Ludlow CL et al.; Ten patients with abductor spasmodic dysphonia, who exhibited spasmodic bursts and heightened activity of the cricothyroid muscle during speech, were selected for participation . Between 5 and 20 U of botulinum toxin type A were injected into both right and left cricothyroid muscles . Six patients benefited substantially, whereas four did not . Acoustic analyses of voice patterns showed similar changes to the clinical impressions . Significant group improvements were found in sentence duration while selected patients improved in the proportion of their speech that was voiced and the duration of their voiceless consonants . Those patients with abductor spasmodic dysphonia and other muscle abnormalities in addition to the cricothyroid and with constant breathiness did not benefit.

Am J Physiol, 1991 Jun, 260(6 Pt 1), L539 - 47
ADP ribosylation of type II pulmonary epithelial cell G proteins; Rybin VO et al.; Secretion of pulmonary surfactant by type II pulmonary epithelial cells (T2P) is regulated by receptor-mediated mechanisms . In other systems, coupling of receptor-linked signals to intracellular events involves guanine nucleotide-binding proteins (G proteins), but the specific role of G proteins in T2P signaling pathways is poorly defined . The present studies begin to address the role of G proteins in transmembrane signaling in these pneumocytes . Membrane preparations from purified T2Ps demonstrated ADP ribosylation of specific substrates by pertussis, cholera, and botulinum toxins (PT, CT, and BT, respectively) . Toxin-dependent T2P substrate labeling from 32P-labeled NAD was dependent on time and membrane protein concentration . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography showed ADP ribosylation of membrane substrates of the following molecular masses: PT, 40/41 kDa; CT, 47/51 kDa; BT, 22 kDa . BT-dependent ADP ribosylation of a 22-kDa cytosolic substrate was also observed . Pretreatment of cultured T2P with the individual toxins led to ADP ribosylation of their respective specific substrates in a time-dependent fashion . In cells pretreated with PT or CT, substrates for the complimentary toxins remained available for subsequent ADP ribosylation in vitro . This result supports the specificity of the toxin effects . Basal secretion of the major phospholipid of pulmonary surfactant, disaturated phosphatidylcholine (DSPC) was unaffected in T2P treated with PT, but was stimulated in cells exposed to CT or BT . Neither CT nor BT altered release of lactate dehydrogenase . In cells treated with AMP or with isoproterenol DSPC secretion was stimulated six- to eightfold; preexposure of the cells to CT reduced the response to either agonist by 70%.(ABSTRACT TRUNCATED AT 250 WORDS)

J Biol Chem, 1991 May 25, 266(15), 9580 - 5
Heterologous combinations of heavy and light chains from botulinum neurotoxin A and tetanus toxin inhibit neurotransmitter release in Aplysia; Poulain B et al.; The neuroparalytic activities of botulinum neurotoxin type A (BoNT A), tetanus toxin (TeTx), or homologous and heterologous combinations of their constituent polypeptides were examined at cholinergic and non-cholinergic synapses of Aplysia californica . When applied extracellularly, BoNT A or a mixture of its heavy (HC) and light (LC) chains were far more potent in blocking transmitter release at cholinergic than non-cholinergic synapses . The reverse was true for TeTx or a mixture its constituent chains . Such selectivity was assigned to differences in neuronal targetting and uptake of the neurotoxins since both exhibited similar potencies when injected directly into the cell body of either cell type . When bath-applied, heterologous combinations of the toxins' HC and LC appeared as effective as the parent neurotoxins from whence each HC was derived . Moreover, targetting/internalization was attributable to the analogous N-terminal moieties, H2 and beta 2, of the HC from BoNT A and TeTx . Thus, it may be postulated that the latter regions possess two functional domains, one being distinct and responsible for the divergent neuronal specificity, whereas the other serves a common role in translocating the LC of either toxin . Also, it was shown that the C-terminal portion of the HC of TeTx is unable to play the intracellular role of its counterpart in BoNT A.

Ned Tijdschr Geneeskd, 1991 May 18, 135(20), 889 - 92
{The treatment of hemifacial spasms using botulin}; Struys MA et al.; At the Academic Medical Center of the University of Amsterdam the results of treatment of hemifacial spasms (HFS) with botulinum toxin type A were evaluated in a pilot study . Five men and 21 women with HFS were treated with toxin injections . The mean age was 65 years . Most patients were referred from other centers and had been previously treated without success with various medications or with surgical treatment . Treatment took place at the outpatient department . The toxin was injected into the M . orbicularis oculi at the medial and lateral sides of the upper and lower lids . The total dose varied between 6 and 16 LD50 . Patients were reinjected on demand . All patients except for one reported satisfying improvement of their spasm . The onset of the beneficial effect was 1-2 days after the injection and the effect lasted about 3 months . All patients were re-examined 1 to 2 weeks after each treatment and showed reduction or disappearance of synkinesias . No systemic side effects occurred and local complications were mild and transient . We conclude that local injection of botulinum toxin appears to be a successful alternative in the current treatment of hemifacial spasm.

J Clin Invest, 1991 May, 87(5), 1575 - 84
Adenosine diphosphate-ribosylation of G-actin by botulinum C2 toxin increases endothelial permeability in vitro; Suttorp N et al.; The endothelial cytoskeleton is believed to play an important role in the regulation of endothelial permeability . We used botulinum C2 toxin to perturb cellular actin and determined its effect on the permeability of endothelial cell monolayers derived from porcine pulmonary arteries . The substrate for botulinum C2 toxin is nonmuscle monomeric actin which becomes ADP-ribosylated . This modified actin cannot participate in actin polymerization and, in addition, acts as a capping protein . Exposure of endothelial cell monolayers to botulinum C2 toxin resulted in a dose- (3-100 ng/ml) and time-dependent (30-120 min) increase in the hydraulic conductivity and decrease in the selectivity of the cell monolayers . The effects of C2 toxin were accompanied by a time- and dose-dependent increase in ADP-ribosylatin of G-actin . G-Actin content increased and F-actin content decreased time- and dose-dependently in C2 toxin-treated endothelial cells . Phalloidin which stabilizes filamentous actin prevented the effects of botulinum C2 toxin on endothelial permeability . Botulinum C2 toxin induced interendothelial gaps . The effects occurred in the absence of overt cell damage and were not reversible within 2 h . The data suggest that the endothelial microfilament system is important for the regulation of endothelial permeability.

J Med Assoc Thai, 1991 May, 74(5), 239 - 47
Writer's cramp: the experience with botulinum toxin injections in 25 patients; Poungvarin N; Twenty-five writer's cramp patients have been attending the Movement Disorder Clinic at the Division of Neurology, Department of Medicine, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok during three years period (between January 1988 - January 1991) . There were 17 male subjects and the male to female sex ratio was 2.125:1 . The mean age of the patient was 36.80 (SD 10.21) years with the range of 18-60 years . The mean duration of illness of all patients was 5.88 (SD 7.14) years with the range of 1 to 30 years . Eighteen patients (72.0%) were classified as simple writer's cramp and seven patients (28.0%) were dystonic writer's cramp . The mean age of the patients of both groups was not different while the duration of illness in the dystonic group was statistically significantly longer than the simple group, i.e . 12.0 (SD 12.1) versus 3.9 (SD 3.1) years . Fourteen patients (56%) had associated pain during writing and 6 patients (24%) had hand tremor . All patients were right handed and had a history of various pharmacological treatments without any consistent benefit . They included muscle relaxants, tranquillisers, antiepileptic drugs, and betablockers . Fourteen patients from 17 available history records (82.4%) had been spending at least 4-10 hours writing each day . Twenty-one patients (84%) had botulinum toxin injections, 40-80 international mouse units were given in 2-4 divided doses over the overactive forearm muscles observed during writing without the electromyographic glidance . There was no loss to the follow-up . Fourteen of the 21 subjects (66.7%) showed definite improvement in hand writing, 4 patients (19.0%) improved minimally and 3 patients (14.3%) revealed no improvement . Arm pain in all 12 patients associated during writing was abolished after the injections . There were complications in 7 patients (33.3%) presented as transient finger drop (5 patients, 23.8%) and easily fatigued arm (2 patients, 9.5%) . These preliminary results confirm that botulinum toxin injections is a successful treatment for many patients with writer's cramp without performing complex electromyographic recordings while the patients are writing . The constraints of this treatment are its high cost (i.e . 1 vial of 100 units costs 300 US dollars) and its benefit lasts for only 4-6 months.

J Biol Chem, 1991 Apr 25, 266(12), 7646 - 50
Purification of GTPase-activating protein specific for the rho gene products; Morii N et al.; A GTPase-activating protein specific for the rho gene products (rho-GAP) was purified from the cytosol of bovine adrenal gland . Purification procedures consisted of ammonium sulfate fractionation, chromatographies on columns of phenyl-Sepharose and CM-Sepharose, gel filtration on a TSK-gel G3000SW, and Mono S fast protein liquid chromatography . By these procedures the activity was purified about 36,000-fold with a recovery of 0.6% . The final preparation showed a major protein band at Mr 28,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and stimulated GTP hydrolysis by the purified rho A protein in a time- and dose-dependent manner . No stimulation was found for ras p21 . The ADP-ribosylation on the rho protein by botulinum C3 exoenzyme did not affect its interaction with the purified rho-GAP.

Dtsch Med Wochenschr, 1991 Apr 12, 116(15), 567 - 72
{Treatment of spasmodic torticollis with local injections of botulinum toxin A}; Erbguth F et al.; Sixty patients (30 men, 30 women, mean age 45.5 {18-71} years) with various forms of spasmodic torticollis were treated with local injections of botulinus A toxin . The results of treatment were assessed on an arbitrary scale (0-4) by the patients, by an examiner and by a neurologist who studied video recordings without knowing at what stage they had been recorded . After the first course of injections (a maximum of three injections of 10-40 ng in four weeks) 50 patients (83%) reported a better than 50% improvement; the mean score on the four point scale fell from 3.6 to 1.85 (P less than 0.001) . The severity of the disorder as judged from the video recordings fell from a mean of 3.23 to 1.71 (P less than 0.001) . The effect lasted for an average of 14 weeks . 6 patients developed mild dysphagia, in two further cases severe dysphagia occurred which lasted for up to four weeks . Twenty patients were followed up for more than one year: in nine of them the interval between courses of injections, the dose being unaltered, remained the same (12-14 weeks), but thereafter the abnormal movements returned with the same severity as before treatment . In eight patients the intervals between courses grew longer and the abnormal movements became less severe . Two patients have been symptom free for 20 and 13 months respectively, but one female patient failed to respond owing to the presence of antibodies against botulinus A toxin . The results indicate that local injection of botulinus toxin is a successful treatment for spasmodic torticollis.

Can J Ophthalmol, 1991 Apr, 26(3), 148 - 51
Occult pontine glioma in a patient with hemifacial spasm; Westra I et al.; Hemifacial spasm due to an intracranial mass lesion is rare . We describe a 29-year-old man with hemifacial spasm successfully treated with botulinum A toxin injections for 2 years . The development of acquired diplopia secondary to acquired sixth cranial nerve palsy prompted investigation . Computed tomography done at the time of original diagnosis and on three other occasions (concentrating on the brain stem and cerebellopontine angle) failed to demonstrate an intracranial mass lesion . Magnetic resonance imaging (MRI) showed a large mass lesion in the pons presumed to be a glioma . Patients with hemifacial spasm who have atypical features, especially those with associated neurologic findings, should be screened for tumours . Our case illustrates the superiority of MRI in demonstrating pontine gliomas causing hemifacial spasm.

Can J Ophthalmol, 1991 Apr, 26(3), 133 - 8
Treatment of blepharospasm and hemifacial spasm with botulinum A toxin: a Canadian multicentre study; Taylor JD et al.; Botulinum A exotoxin was recently approved for use in Canada . We describe the efficacy of botulinum toxin in the management of 235 patients with blepharospasm (mean age 64.3 years) and 130 patients with hemifacial spasm (mean age 60.4 years) treated at three Canadian ophthalmologic centres between 1984 and 1989 . A total of 98% of the patients with blepharospasm and 100% of the patients with hemifacial spasm had significant relief of their symptoms; however, 11% of the former and 2% of the latter did not respond to the usual starting concentrations of the drug and needed stronger dosages for relief . The duration of relief varied widely in both groups . Up to 7% of patients had ineffective treatments but responded to subsequent injections . Analysis of variance and linear trend statistics showed that there were no changes in the mean duration of relief over the first several treatments for individual patients in either group . Side effects were transient and included ptosis, exposure keratitis, epiphora and strabismus.

Ophthalmology, 1991 Apr, 98(4), 509 - 12; discussion 512-3
Use of botulinum toxin in strabismus after retinal detachment surgery; Petitto VB et al.; Botulinum toxin was used to treat 20 patients with strabismus after retinal detachment surgery . Preinjection motility deviations ranged from 10 to 60 prism diopters (D) . Postinjection deviations ranged from 0 to 20 prism D, with 75% being 10 D or less . Eighty-five percent achieved fusion that persisted, with 73% requiring only one or two injections . Only muscles in the eye that had undergone retinal reattachment surgery were injected . The average period of follow-up was 12 months . Complications were rare and all resolved spontaneously . Botulinum toxin appears to be useful as a primary treatment modality for persistent strabismus following retinal detachment surgery, possibly obviating the need for complicated strabismus surgery.

Exp Eye Res, 1991 Apr, 52(4), 445 - 9
Succinylcholine-stimulated muscle tensions following botulinum injection in the domestic cat; Dennehy PJ et al.; Succinylcholine (SCh) selectively stimulates, and can therefore selectively assay, the multiple innervated (MI) fiber system of the extraocular muscles . Since botulinum-A toxin has been observed to induce changes in eye position in humans, SCh was used to assess the effect of botulinum-A on the SCh-sensitive MI fibers of extraocular muscles . Intravenous SCh infusion (40 micrograms-1 kg-1 min-1 was performed in the anesthetized domestic cat . Thirty-eight infusions were performed in 19 normal controls, measuring the peak tensions generated in the four horizontal and four vertical rectus muscles . Succinylcholine-stimulated muscle tensions (SSMT) were then repeated in nine animals, 4 weeks and 10 weeks following injection of botulinum-A toxin into both medial rectus muscles . Mean peak SSMTs were unchanged at 4 and 10 weeks following botulinum injection when compared to controls . We propose that botulinum chemo-denervation has no acute or chronic effect on the MI SCh-sensitive muscle fibers of the medial recti of the domestic cat . This lack of effect on the postsynaptic MI fibers indirectly supports light and electron microscopic studies which show changes predominantly in the singly innervated (SI), rather than the MI fibers following botulinum injection . Mean peak SSMTs were also greater for medial and superior rectus muscles compared to lateral and inferior recti respectively, suggesting a greater number or proportion of MI fibers in medial and superior recti.

Ann Otol Rhinol Laryngol, 1991 Apr, 100(4 Pt 1), 274 - 9
Recurrent laryngeal nerve section for spastic dysphonia: 5- to 14-year preliminary results in the first 300 patients; Dedo HH et al.; This presentation compares the preoperative voice recordings and the latest follow-up voice recordings, made 5 to 14 years postoperatively, of the first 300 patients with various degrees of spastic dysphonia whom we treated with recurrent laryngeal nerve (RLN) sections from 1975 to 1982 . Voice therapy was usually given afterward and in some patients, when necessary, "fine tuning" surgery was performed later . The 243 patients who could be located were asked to answer a questionnaire regarding their voice production and communication abilities, and to make a voice recording . The preoperative and long-term postoperative voice recordings were analyzed by means of perceptual voice evaluation and acoustic analysis of the voice spectra . Fifteen percent developed recurrence of mild to moderate spasticity 6 to 24 months after the RLN section . This was curable with laser vocal cord thinning via direct laryngoscopy . Eighty-two percent of patients had little or no voice spasticity 5 to 14 years after their RLN section . The experimental alternative of injecting botulin directly into the vocal cord to temporarily paralyze it is discussed.

Mayo Clin Proc, 1991 Apr, 66(4), 365 - 71
Selective peripheral denervation for torticollis: preliminary results; Davis DH et al.; Herein we report the preliminary results in nine patients who have undergone selective peripheral denervation for spasmodic torticollis and have been followed up for at least 13 months . All patients had improvement immediately after surgical intervention, and the results have been maintained in five patients . In one patient who had recurrent torticollis, a second procedure in conjunction with injection of botulinum toxin has produced substantial improvement; however, follow-up was brief (6 months) . No surgical complications occurred . We believe that selective peripheral denervation is safe and that it can benefit patients with torticollis who have not responded to other types of therapy . These favorable results confirm other published reports on the efficacy of selective peripheral denervation . Long-term follow-up, however, is necessary for determining the role of this procedure in the management of torticollis.

Ann Neurol, 1991 Apr, 29(4), 370 - 6
Change in pattern of muscle activity following botulinum toxin injections for torticollis; Gelb DJ et al.; Twenty patients with torticollis had electromyographic studies of their neck muscles performed before and after a series of local injections of botulinum toxin . The pattern of muscle activity changed after the injections, and this effect persisted even after head position had returned to baseline . Patients who did not experience any clinical benefit from the injections also demonstrated a change in the pattern of muscle activity . These results suggest that the underlying abnormality in torticollis usually involves a general motor program for head position, rather than the activity of individual neck muscles.

Klin Monatsbl Augenheilkd, 1991 Apr, 198(4), 268 - 70
{Involuntary lid closure caused by defective healing of facial paralysis and its treatment with botulinum toxin}; Roggenkamper P et al.; Unvoluntary lidclosure with movement of the mouth is not so rare in aberrant regeneration of nerve fibers after Bell's palsy . There was no therapy until now . 10 patients with this disease were treated with botulinum-toxin injections into the orbicularis muscle . For an average time of 11 weeks after injection there was a complete absence of synkinesis, followed by a time of 9 weeks of less complaints . Thus 2-3 injections might be sufficient treatment within a year . Complications worth mentioning, especially due to the reduced force of lidclosure, were not observed.

J Neurol Neurosurg Psychiatry, 1991 Apr, 54(4), 310 - 3
Neurophysiological observations on the effects of botulinum toxin treatment in patients with dystonic blepharospasm; Valls-Sole J et al.; Botulinum toxin treatment improves dystonic blepharospasm by inducing transient paresis of the orbicularis oculi muscle . It is not known if it also reduces the enhanced brainstem neuronal excitability found in this disorder . We have performed conventional electromyography (EMG) and blink reflex excitability studies on fifteen patients with blepharospasm before and after botulinum toxin treatment . Denervation signs were found with needle EMG in all treated muscles . Amplitude of the facial compound muscle action potential (CMAP) and R1 response was reduced after botulinum toxin injections . In blink reflex excitability studies, the recovery of R2 response was enhanced after treatment even when patients were tested at the time of maximal benefit from botulinum toxin injections . The results suggest that there is little influence of botulinum toxin treatment upon the enhanced excitability of brainstem interneurons in patients with blepharospasm.

Ophthalmology, 1991 Mar, 98(3), 357 - 66
Clinical doxorubicin chemomyectomy . An experimental treatment for benign essential blepharospasm and hemifacial spasm; Wirtschafter JD; Doxorubicin (DXR) was injected as a treatment for benign essential blepharospasm and hemifacial spasm . The other eyelids were treated concurrently with botulinum toxin (BT) . No DXR-treated eyelid has maintained 0 strength (commonly achieved with BT) . Two patients with benign essential blepharospasm and four patients with hemifacial spasm have achieved major improvement, sustained for more than 6 months . Eyelids have been swollen and inflamed for up to 3 months . No spontaneously irreversible complication has occurred . A single injection at the maximum safe dose (1 mg in the upper lid and 1.5 mg in the lower lid) has not proven sufficient to produce cure . Treatment of each lower eyelid of a muscular male with severe blepharospasm may require cumulative doses of up to 4.0 mg, delivered in three injection events separated by at least 2 months, with each injection no greater than 1.5 mg DXR per site . At the present time, there is no assurance that a permanent cure will result.

Br J Ophthalmol, 1991 Mar, 75(3), 181 - 4
Pathological changes in levator palpebrae superioris muscle treated with botulinum toxin in a case of carotico-cavernous fistula; Adams GG et al.; We describe the case of a patient with carotico-cavernous fistula who had botulinum toxin A to induce a protective ptosis 4.5 days before death . The levator palpebrae superioris muscle from both sides and the superior rectus muscle from the injected side were obtained for examination . The preserved samples were stained with haematoxylin and eosin, Martius scarlet blue, Glees, S100, dehydrogenase, ATPase, and toluidine blue as well as being examined by electron microscopy . Inflammation and oedema were found that were probably due to the carotico-cavernous fistula . Axonal and some myelin sheath damage were also seen.

Arch Ophthalmol, 1991 Mar, 109(3), 396 - 404
Botulinum-induced changes in monkey eyelid muscle . Comparison with changes seen in extraocular muscle; Porter JD et al.; Botulin type A was injected into the eyelids of adult monkeys, and structural alterations in the orbicularis oculi muscle were evaluated after survival times of 7 to 84 days . The most profound change seen at both the light- and electron-microscopic levels was nonselective atrophy of virtually all muscle fibers . Moreover, the botulin-induced blockade of neuromuscular transmission was nonspecific in producing alterations in the three orbicularis fiber types . Muscle structural changes appeared to be reversible, with no apparent long-term consequences . While sprouting of preterminal axons was noted in botulin-treated muscle, formation of collateral sprouts did not appear to be widespread . These changes contrast with the fiber type-specific, long-term alterations induced in extraocular muscle by botulin treatment . However, this differential response may be attributed to the very clear differences in fiber type composition and motor control mechanisms between eyelid and extraocular muscle groups . The efficacy of botulin treatments for strabismus and focal dystonia may then be directly related to both the anatomic fiber type composition and the functional properties of motor control systems of the injected muscle.

Arch Ophthalmol, 1991 Mar, 109(3), 393 - 5
Histologic features of human orbicularis oculi treated with botulinum A toxin; Harris CP et al.; To evaluate muscle histologic features in humans following therapeutic botulinum toxin injections, we studied orbicularis oculi from 11 patients with blepharospasm; nine had previously received botulinum toxin injections and two had not . All muscles had comparable variability in muscle fiber diameter, with no necrosis, inflammation, denervation, or consistent alterations in muscle fiber internal architecture . Botulinum toxin produces no persistent histologic changes in human muscle fibers.

Ann Acad Med Singapore, 1991 Mar, 20(2), 223 - 7
Botulinum toxin in the treatment of facial dyskinesias; Chong PN et al.; Patients with hemifacial spasm (N = 25), blepharospasm (n = 8), and benign eyelid fasciculation (n = 2) were treated with botulinum toxin injections (PHLS, Porton Down, England) . All patients reported substantial symptomatic relief . Marked improvement was seen in fifteen patients with hemifacial spasm and six patients with blepharospasm . Benign eyelid fasciculation was completely abolished . Beneficial effects was evident two to three days after injections, became maximum at one week, and remained effective for up to six months . Side effects were transitory and mild . They included periorbital edema, mild diplopia, ptosis and facial weakness . Only in two patients was ptosis unacceptable . Severity of side effects was dose-related . Reinjections had similar efficacy . Botulinum toxin therapy is a safe and effective treatment for these facial dyskinesias and should be considered a viable alternative to surgical procedures.

J Neurosci, 1991 Mar, 11(3), 657 - 66
Axotomy-like changes in cat motoneuron electrical properties elicited by botulinum toxin depend on the complete elimination of neuromuscular transmission; Pinter MJ et al.; The electrical properties of cat medial gastrocnemius (MG) spinal motoneurons were studied 14-21 d following injection of type A botulinum toxin (BTX) into the MG muscle . Treated MG muscles were atrophic, displayed pronounced fibrillation activity, and were markedly but not completely paralyzed . MG motoneuron electrical properties from animals with the highest MG muscle-twitch forces (greater than 20 gm) appeared normal, while motoneuron properties from animals with the lowest MG muscle-twitch forces (less than 10 gm) exhibited axotomy-like changes, though these changes were less pronounced than after axotomy itself . No changes in the axonal conduction velocity were observed, however . Motoneuron connectivity with MG muscle fibers was determined following intracellular stimulation of MG motoneurons by averaging EMG signals from 3 or 4 pairs of recording electrodes inserted into the BTX-treated MG muscles . Normal electrical properties were observed among motoneurons in which detectable EMG activity linked to the intracellular stimulation pulse was observed . The level of this connectivity, however, indicated that a relatively small number of muscle fibers were activated by individual motoneuron action potentials . Axotomy-like changes of electrical properties were observed in MG motoneurons that could not be associated with detectable EMG activity in the BTX-treated MG muscle following repeated trials of intracellular stimulation . These results indicate that the existence of effective neuromuscular transmission at a small number of motor terminals is sufficient to prevent the appearance of axotomy-like changes in motoneuron electrical properties, and that the absence of such transmission at all motor terminals is associated with the appearance of axotomy-like changes . The results suggest that the effects of axotomy itself on motoneuron properties may be based upon the loss or elimination of a potent interaction between muscle and motoneurons normally mediated by neuromuscular transmission.

J Neurochem, 1991 Mar, 56(3), 827 - 35
Microtubule-dissociating drugs and A23187 reveal differences in the inhibition of synaptosomal transmitter release by botulinum neurotoxins types A and B; Ashton AC et al.; The inhibitory effects of botulinum neurotoxins types A and B on Ca2(+)-dependent evoked release of {3H}noradrenaline from rat cerebrocortical synaptosomes were compared and their molecular basis investigated . A23187, a Ca2+ ionophore, proved more efficacious in reversing the blockade produced by type A than that by B, whereas the actions of neither were changed by increasing intraterminal cyclic GMP levels using 8-bromo-cyclic GMP of nitroprusside . Disruption of the actin-based cytoskeleton with cytochalasin D did not alter the inhibition seen subsequently with either toxin . However, prior disassembly of microtubules with colchicine, nocodazole, or griseofulvin reduced the potency of type B toxin, but not that of type A toxin; stabilization of the microtubules with taxol counteracted this effect of colchicine . Because colchicine treatment of synaptosomes did not interfere with the measurable binding of type B toxin or its apparent uptake, it appears to act intracellularly . Collectively, these data suggest that botulinum neurotoxins types A and B inactivate transmitter release by interaction at different sites in the process . Based on the consistent results observed with four different drugs known to affect selectively microtubules, their involvement in the action of the type B neurotoxin is proposed.

Clin Neuropharmacol, 1991 Feb, 14(1), 62 - 77
Cognitive processes in idiopathic dystonia treated with high-dose anticholinergic therapy: implications for treatment strategies; Taylor AE et al.; Studies utilizing single doses of scopolamine have suggested a role for the cholinergic system in memory . Results are consistent in identifying a selective effect on the early encoding stage of information processing . In terms of long-term administration of anticholinergics, patients with Parkinson's disease often display memory deficits . However, underlying pathology within the forebrain cholinergic system complicates the study of treatment effects in this disorder . We therefore assessed multiple memory routines in 20 cognitively intact patients with dystonia where no such pathology has been identified . Patients were tested before and after 2-4 months of 15-74 mg of trihexyphenidyl daily . Twelve tolerated this regime . Compared to control subjects, matched for age and I.Q., only tests with a single presentation of the material to be remembered were affected at follow-up . The speed of information processing was also significantly reduced . Age was strongly related to memory performance in the patient group alone and interacted with dose and duration of treatment . Results suggest that drug-induced slowing of mentation was responsible for impaired encoding, particularly in older patients . These findings affect treatment strategies, especially now that injections of botulinum toxin have proved to be highly effective for certain forms of focal dystonia.

Neurol Clin, 1991 Feb, 9(1), 205 - 24
Botulinum toxin therapy; Savino PJ et al.; Botulinum toxin therapy has emerged as a treatment modality for a variety of spastic- or contracture-related muscle diseases . Its safety has been proven for long-term use in the treatment of benign essential blepharospasm, hemifacial spasm, and certain types of strabismus . Recent approval from the Federal Drug Administration should make botulinum toxin available for use in a greater number of patients.

Arch Neurol, 1991 Feb, 48(2), 221 - 3
Posttraumatic torticollis; Truong DD et al.; We report six cases of torticollis precipitated by neck trauma . The dystonia began 1 to 4 days after the trauma and differed clinically from idiopathic torticollis by marked limitation of range of motion, lack of improvement after sleep ("honeymoon period"), and absence of geste antagonistique . Worsening with action was not present; nor was there improvement with support as seen with idiopathic torticollis . Onset of pain immediately after the trauma and marked spasms of the paracervical muscles were other predominant features . Anticholinergic therapy was without benefit; however, some improvement occurred with botulinum toxin injection . It is concluded that torticollis can be caused by peripheral trauma and that it has unique clinical characteristics.

Ann Otol Rhinol Laryngol, 1991 Feb, 100(2), 85 - 9
Laryngeal dystonia: a series with botulinum toxin therapy; Blitzer A et al.; Laryngeal dystonia is a syndrome characterized by action-induced, involuntary spasms of the laryngeal muscles . Most patients have involvement of the adductor laryngeal muscles producing uncontrolled spasms during phonation, and a "strain-strangle" speech pattern commonly termed "spastic dysphonia." Other patients have involvement of the abductor muscles producing "whispering dysphonia." Rare patients have paradoxical vocal cord motion during respiration with adductor spasms on inspiration . Over the past 5 years we have used botulinum toxin (BOTOX) to treat more than 200 patients with laryngeal dystonia . This group includes patients with adductor involvement (phonatory dystonia, recurrent laryngeal nerve section failure, respiratory dystonia) and those with abductor involvement (whispering dystonia) . Patients received benefit within 24 to 72 hours, with sustained improvement for 2 to 9 months with an average of 4 months . Patients improved to an average of 90% of normal function . Clinically significant adverse effects included extended breathy dysphonia and mild choking on fluids . BOTOX has become our treatment of choice for dystonic conditions of the larynx.

Plast Reconstr Surg, 1991 Feb, 87(2), 285 - 9
Botulinum A toxin for the treatment of adult-onset spasmodic torticollis; Borodic GE et al.; Thirty-five patients with adult-onset idiopathic torticollis were treated by local injections of botulinum A toxin into dystonic cervical muscles . Substantial improvement with respect to reduction and elimination of pain was found in 81 percent, improvement in posture deformity and involuntary spasms in 70 percent, increased range of motion of the neck in 78 percent, reduction in visible sternocleidomastoid hypertrophy in 86 percent, and improvement in tremor in 65 percent . The syndrome was divided into four subtypes based on pattern of dystonic muscle groups involved in the dystonia, head and shoulder posture, and sternocleidomastoid muscle hypertrophy . Injection strategy based on this subdivision is described.

J Biol Chem, 1991 Jan 15, 266(2), 1289 - 98
Low molecular weight GTP-binding proteins in human neutrophil granule membranes; Philips MR et al.; Degranulation of neutrophils involves the differential regulation of the exocytosis of at least two populations of granules . Low molecular weight GTP-binding proteins (LMW-GBPs) have been implicated in the regulation of vesicular traffic in the secretory pathways of several types of cells . In the present study we identify distinct subsets of LMW-GBPs associated with the membranes of neutrophil-specific and azurophilic granules . Ninety-four percent of total {35S}guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) binding activity was equally distributed between the plasma membrane and cytosol with the remaining 6% localized in the granules . In contrast, the cytosol contained only 10% of the total GTPase activity while the specific granules accounted for 13% . {alpha-32P}GTP binding to proteins transferred to nitrocellulose revealed LMW-GBPs in all fractions except the azurophilic granules . The specific granules contained three out of four bands which were found in the plasma membrane; these ranged from 20 to 23 kDa and all were resistant to alkaline extraction . Photoaffinity labeling with {alpha-32P}8-azido-GTP in the presence of micromolar Al3+ identified proteins of 25 and 26 kDa unique to azurophilic granules; these could not be labeled with {alpha-32P}8-azido-ATP and could be extracted by acidic but not alkaline pH . Botulinum C3-mediated {32P}ADP-ribosylation identified proteins of 16, 20, and 24 kDa both in plasma membranes and those of specific granules . An anti-ras monoclonal antibody, 142-24E5, recognized a 20-kDa protein localized to the plasma and specific granule membranes which could not be extracted by alkaline pH, was not a substrate for botulinum C3 ADP-ribosyltransferase, and was translocated from specific granules to plasma membrane after exposure of neutrophils to phorbol myristate acetate . We conclude that neutrophil-specific and azurophilic granules contain distinct subsets of LMW-GBPs which are uniquely situated to regulate the differential exocytosis of these two compartments.

Neurosci Lett, 1991 Jan 14, 122(1), 132 - 4
Cooperative action of the light chain of tetanus toxin and the heavy chain of botulinum toxin type A on the transmitter release of mammalian motor endplates; Weller U et al.; Purified heavy chain of botulinum toxin type A and light chain of tetanus toxin were combined to form a chimeric toxin . It was active on the mouse phrenic nerve-hemidiaphragm with a potency 6 times higher than that of native tetanus toxin . Electrophysiological data from poisoned neuromuscular junctions revealed that the pattern of nerve-evoked and spontaneous transmitter release was equivalent to that seen with tetanus toxin i.e . asynchronous release, and did not resemble that after botulinum toxin type A poisoning . We conclude that the light chain of tetanus toxin alone is responsible for the characteristic effects on spontaneous and nerve-evoked transmitter release of the native toxin and that these properties can be introduced into a new, more potent complex with the heavy chain of botulinum toxin A.

Eye, 1991, 5 ( Pt 1), 45 - 7
Early botulinum toxin treatment of acute sixth nerve palsy; Murray AD; Eight patients with total sixth nerve palsy were treated with botulinum toxin injection to the antagonist non-paretic medical rectus, within eight weeks of the onset of the palsy . Within a few days seven of the eight gained fusion without the necessity of a marked head turn, and none complained of confusing reversal of diplopia . The same seven recovered full function . The mean follow-up period after the last injection was 20 months . Seven palsies were the result of head trauma and one was due to cerebro-vascular disease . This preliminary report suggests that early botulinum toxin injection of patients with recent onset sixth nerve palsy is beneficial . Although all of the patients may possibly have recovered full lateral rectus function without treatment, the aetiologies of their palsies were of the type that frequently do no resolve . A randomised double-blind study is necessary more precisely to determine the effectiveness of this form of therapy.

Neuroscience, 1991, 41(1), 71 - 88
Characteristics of slow-miniature endplate currents show a subunit composition; Vautrin J et al.; The normal neuromuscular junction shows two classes of spontaneous miniature endplate potentials . These classes are based on a discontinuity in the profile of miniature endplate potential amplitude distributions . The amplitude of one class of miniature endplate potentials from a bell-shaped amplitude distribution and the remaining miniature endplate potentials compose a population which forms a left-hand skew distribution with a mode 1/7 to 1/10 that of the bell-miniature endplate potentials {Kriebel M . E . and Gross C . E . (1974) J . gen . Physiol, 64, 85-103} . Some skew-miniature endplate potentials have a slow time-to-peak and show breaks on the rising phase . Most treatments that alter the miniature endplate potential frequency change the ratio of skew-miniature endplate potentials/bell-miniature endplate potentials {Kriebel M . E . et al . (1976) J . Physiol . 262, 553-581} . The time characteristics of miniature endplate currents were readily altered in the isolated frog and mouse neuromuscular junctions with several agents known to increase the percentage of slow-miniature endplate potentials (heat, botulinum toxin, 4-aminoquinoline and increases in bath osmolarity) . The slow-miniature endplate potential amplitudes were a continuum of amplitudes from skew- to giant miniature endplate potentials . The rising phases of miniature endplate potentials were a continuum from smooth to many with breaks and offsets . In a series of sequentially recorded slow-miniature endplate currents, many had congruent rising phases of constant slope regardless of amplitude or of time-to-peak . The rising phases of congruent slow-miniature endplate currents which showed a change in slope deviated at similar amplitudes . The least value of the slope of a slow-miniature endplate current was that of the sub-miniature endplate current; and, miniature endplate currents with overall lower slope values showed a wave pattern and/or irregular breaks which suggests summation of sequentially delayed sub-miniature endplate currents . Plots of the amplitude vs time-to-peak of miniature endplate currents from identified junctions demonstrated that the normal percentage of slow-miniature endplate currents was greatly increased with the treatments used here and that the time-to-peak of giant miniature endplate currents usually was longer than that of normally occurring bell-miniature endplate currents . Giant miniature endplate currents with short time-to-peak values are probably from two miniature endplate currents occurring, by chance, almost simultaneously . During and/or after treatments, miniature endplate currents formed clusters of similar size miniature endplate currents, not randomly distributed in time, which graded from distinct miniature endplate currents to giant miniature endplate currents.(ABSTRACT TRUNCATED AT 400 WORDS)

Mov Disord, 1991, 6(2), 174 - 6
Jaw dystonia triggered by biting into hard food; Lagueny A et al.; We report an unusual case of eating dystonia induced by attempts to bite into hard food and resulting in prolonged jaw opening spasms . Mastication was severely impeded . Simultaneous bilateral electromyogram (EMG) of the masticatory muscles during mastication of hard food showed an abnormal prolonged activity in the anterior digastrics with an abnormal cocontraction of these muscles during contraction of the jaw-closers and a prolonged inhibition of the jaw-closers until the hard bolus was softened . This action dystonia seems to be triggered when more than a certain pressure is exerted on the peridontal mechanoreceptors and could result from a defective central command control of their sensory inputs . Local injections of botulinum toxin had little effect.

Mov Disord, 1991, 6(2), 145 - 50
Treatment of idiopathic spasmodic torticollis with botulinum toxin A: a double-blind study on twenty-three patients; Lorentz IT et al.; In a double-blind, placebo-controlled study, 23 patients suffering from intractable spasmodic torticollis (ST) were given successively either botulinum toxin A (BTA) or normal saline by intramuscular injections in the affected muscles . Evaluation was carried out by three blinded observers, using a clinical and video assessment of the severity of torticollis, employing a scoring system described by Tsui (1) . Patients were also asked to subjectively comment on changes in the amount of pain and on changes in the activities of daily living (ADL) . BTA was proven to be superior on all forms of assessment to placebo, and these results were statistically significant . Side effects mainly consisted of pain at the injection site . Tiredness occurred at equal frequency with BTA and placebo . No serious or systemic side effects were noted . Botulinum toxin is a safe, effective and relatively simple treatment for spasmodic torticollis.

Toxicon, 1991, 29(2), 181 - 9
The translocation of botulinum A neurotoxin by chromaffin cells is promoted in low ionic strength solution and is insensitive to trypsin; Marxen P et al.; Botulinum A neurotoxin (BoNtx) produced a partial inhibition of carbachol induced 3H-noradrenaline (3H-NA) release from bovine adrenal chromaffin cells in monolayer culture . Each of the polysialogangliosides GD1a, GT1b and GD1b enhanced the block of exocytosis when they were applied prior to the toxin exposure . The monosialoganglioside GM1 was not effective . Chromaffin cells treated with neuraminidase lost their sensitivity to BoNtx . Application of gangliosides to these cells, however, restored their susceptibility to the toxin . Treatment of the cells with trypsin did not affect the BoNtx-blockade of 3H-NA-release . The potency of botulinum A toxin was increased in a solution of low ionic strength in which sodium chloride was replaced by sucrose . In agreement with the potency of botulinum A neurotoxin in blocking exocytosis under the various conditions, binding of 125I-botulinum A neurotoxin to chromaffin cells was enhanced in low ionic strength solution and by pretreatment of the cells with gangliosides . The binding was decreased by digestion of gangliosides with neuraminidase . It is concluded that botulinum A neurotoxin binds exclusively to polysialogangliosides which subsequently serve as carriers for the toxin . The low ionic strength may increase some physico-chemical interaction of the toxin with the polysialogangliosides.

Rinsho Shinkeigaku, 1991 Jan, 31(1), 32 - 7
{Treatment of focal dystonia with botulinum toxin}; Nagamine T et al.; We have studied the effect of botulinum toxin in patients with focal dystonia, not responding sufficiently to medical therapy . Injection of 5-100 units to the muscles, selected by EMG and clinical examination, invariably resulted in reduction of muscle tonus, although to a different degree depending on the dosage . The outcome after the first trial was best when dealing with small muscles in patients with blephalospasm or Meige's syndrome . In contrast, several attempts were required for the treatment of a large muscle as in torticollis, or multiple muscles as in writers' cramps . The effect lasted 1-3 months . With precise selection of the affected muscles and careful regulation of necessary dosages, this type of therapy may contribute to the treatment of focal dystonia.

Invest Radiol, 1991 Jan, 26(1), 58 - 64
Proton magnetic resonance spectroscopic studies of hypertrophied muscle . Effect of botulinum toxin treatment; Narayana PA et al.; In vivo image-guided localized proton magnetic resonance spectroscopy (MRS) studies have been performed in a patient with hypertrophied tibialis anterior (TA) muscle and following two injections of botulinum toxin into the affected muscle . Prior to treatment, lipid levels in hypertrophied muscle were found to be low compared to normal . Lipid levels were observed to approach control levels after the second injection . Three months following treatment the trend in exercise-induced changes in lipids was observed to be similar between the hypertrophied and normal TA muscle . This preliminary report demonstrates the potential clinical utility of MRS in evaluating patient response to medical treatment.

Eur Neurol, 1991, 31(1), 44 - 6
Hyperkinesias after hypoglossofacial nerve anastomosis--treatment with botulinum toxin; Dressler D et al.; Hypoglossofacial nerve anastomosis is a successful method for restoration of facial nerve function . Facial hyperkinesis, however, are a common side effect of this therapy and have been a major therapeutical problem ever since . We are reporting a patient whose facial hyperkinesias responded favourably to botulinum toxin carefully injected into the most affected muscles . EMG studies are illustrating the effect of botulinum toxin on the facial hyperkinesias as well as on voluntary muscle activation.

Wien Klin Wochenschr, 1991, 103(1), 15 - 20
{Therapeutic possibilities in spasmodic torticollis}; Wober C et al.; Spasmodic torticollis is classified as a focal dystonia . It is characterized by involuntary contractions of the muscles of the neck, with consequent deviation of the head from the correct posture . Psychological factors are recognized as important trigger and aggravating mechanisms . The various possibilities of therapeutic management (medical and surgical treatment, psychological methods and psychotherapy) are reviewed . Therapy of spasmodic torticollis should be started with methods such as biofeedback, behaviour therapy, and anticholinergic drugs . If these procedures not successful, local application of botulinum toxin offers a new and highly effective technique . Surgical treatment such as neurotomy, rhizotomy, or stereotaxic operations should be restricted to otherwise intractable cases.

Mov Disord, 1991, 6(1), 55 - 9
Writer's cramp: treatment with botulinum toxin injections; Rivest J et al.; Twelve patients with writer's cramp were treated with injections of botulinum toxin . The overactive muscles were identified by clinical observation of the subjects while they were writing . Repeated injections were given at 2-week intervals until the optimal response was obtained . Eleven patients reported some benefit, which was considered by seven to be significant . Eight of the 10 patients who had pain reported moderate to significant relief . Five patients had local complications, consisting of disabling weakness of target or neighbouring muscles . These preliminary results suggest that this treatment can be successfully applied to many patients with writer's cramp without performing complex electromyographic recordings while the patients are writing.

Mov Disord, 1991, 6(1), 29 - 35
Is focal hand dystonia associated with psychopathology?
Grafman J, Cohen LG, Hallett M.
The purpose of this study was to determine if patients with focal hand dystonia have any significant psychopathology . We studied 20 patients with hand cramps who were participating in a therapeutic trial of botulinum toxin injections . Patients were interviewed and administered the Minnesota Multiphasic Personality Inventory (MMPI) . Beck Depression Inventory, Spielberger State-Trait Anxiety Scale, a finger tapping test, and a choice serial reaction time test . Behavioral ratings were also obtained . Group statistics indicated that all personality scale scores and performances on motor tasks were within normal limits . Four out of 20 patients demonstrated mild depression . Trait anxiety scores were higher than state anxiety scores, suggesting that receiving medical treatment had a beneficial effect on mood . The number of depressive symptoms endorsed on the MMPI was correlated with reaction time speed but not finger dexterity . None of the 20 patients reported a remarkable psychiatric history . These results indicate that hand cramps are not associated with serious psychopathology.

Toxicon, 1991, 29(8), 913 - 36
Cellular and molecular actions of binary toxins possessing ADP-ribosyltransferase activity; Considine RV et al.; Clostridial organisms produce a number of binary toxins . Thus far, three complete toxins (botulinum, perfringens and spiroforme) and one incomplete toxin (difficile) have been identified . In the case of complete toxins, there is a heavy chain component (Mr approximately 100,000) that binds to target cells and helps create a docking site for the light chain component (Mr approximately 50,000) . The latter is an enzyme that possesses mono(ADP-ribosyl)transferase activity . The toxins appear to proceed through a three step sequence to exert their effects, including a binding step, an internalization step and an intracellular poisoning step . The substrate for the toxins is G-actin . By virtue of ADP-ribosylating monomeric actin, the toxins prevent polymerization as well as promoting depolymerization . The most characteristic cellular effect of the toxins is alteration of the cytoskeleton, which leads directly to changes in cellular morphology and indirectly to changes in cell function (e.g . release of chemical mediators) . Binary toxins capable of modifying actin are likely to be useful tools in the study of cell biology.

Acta Neuropathol (Berl), 1991, 82(2), 134 - 42
Prevention of diisopropylphosphorofluoridate-induced myopathy by botulinum toxin type A blockage of quantal release of acetylcholine; Sket D et al.; Botulinum toxin type A (BTx), which blocks quantal and partially reduces spontaneous nonquantal acetylcholine (ACh) release at neuromuscular junctions, was tested for its possible attenuating effect on diisopropylphosphorofluoridate (DFP)-induced muscle lesions . The extent of muscle lesion in extensor digitorum longus and soleus muscle of DFP injected rats with and without BTx pretreatment was evaluated using light and electron microscopic procedures . In parallel experiments, acetylcholinesterase (AChE) activity was measured and the functional state of muscles in experimental groups was determined by electrophysiological methods . The results show that pretreatment with BTx almost completely protects the muscles from DFP-induced spontaneous activity and lesions in spite of critically inhibited synaptic AChE . These results are consistent with the conclusion that the effect is not mediated by direct action of organophosphate on muscle, but by the accumulation of ACh resulting in muscle hyperactivity . Therefore, it is concluded that in conditions of acutely inhibited synaptic AChE, the quantal release of ACh is essential for lesion induction, whereas the spontaneous nonquantal ACh release, which is only partially affected in BTx-blocked nerve endings, seems not to be involved.

J Ocul Pharmacol, 1991 Summer, 7(2), 169 - 73
Reduction of cat eye movements using retrobulbar botulinum toxin; Zimm J et al.; We studied the effects of a single retrobulbar injection of Botulinum toxin on the motility of cat eyes . Four cats were sedated and the opposite eye served as a control . Eye movements were plotted by reflecting a laser beam from a mirror fixed to the cornea . We found the mean degrees of deviation per eye per day and summarized these results as mean degrees of deviation per eye per week +/- standard deviation . Statistical analysis was accomplished using Student's t test for independent measures, since measurement of the treated eye pairs was done in a randomized manner on different test days . (table; see text) These results indicate that a single retrobulbar dose of Botulinum toxin can produce a paralysis of the ocular musculature lasting in excess of four weeks in a specific and reproducible manner . In addition, this methodology should prove useful in future experiments in which ocular motility might prove to be a technical concern.

Stereotact Funct Neurosurg, 1991, 57(3), 113 - 22
Selective peripheral denervation in patients with spasmodic torticollis; Braun V et al.; The results of selective peripheral denervation of the involved muscles in 35 patients with spasmodic torticollis are reported . We modified the operation first described by Bertrand . Follow-up was 3 months to 2.5 years in 34/35 patients . 73% noticed a significant improvement or disappearance of dystonia and pain following surgery and physiotherapy for 3 months postoperatively . Selective peripheral denervation is recommended for patients with spasmodic torticollis of at least 1-2 years duration which is resistant to conservative treatment . It may also be used in patients who do not respond to injection of botulinum A toxin or who develop resistance to this kind of therapy.

Arch Neurobiol (Madr), 1991, 54 Suppl 3, 44 - 51
{Focal dystonias and facial hemispasm: treatment with botulinum A toxin}; Astarloa R et al.; We report the results of the treatment of 80 patients with various idiopathic focal dystonia and essential hemifacial spasm with Botulinum A toxin . A statistically significant improvement was obtained in our 34 patients with blepharospasm, 19 patients with hemifacial spasm, 59% of 22 patients with cervical dystonia and 60% of 5 patients with hand dystonia . Mean duration of the benefit of each injection was 15.3, 16.3, 7.6 and 8.7 weeks respectively . Adverse effects were local and transient . We concluded that botulinum A toxin is a safe and effective therapy for patients with focal dystonia and hemifacial spasm.

Arch Neurobiol (Madr), 1991, 54 Suppl 3, 40 - 3
{Treatment with botulinum toxin in blepharospasm}; Grandas F; Blepharospasm is a cranial dystonia characterized by forceful spasms of the orbicularis oculi muscle which may lead to functional blindness in approximately two-thirds of patients . Botulinum toxin injections is a simple procedure, very effective and with little morbidity . It is considered as the treatment of choice for patients with disabling blepharospasm.

Arch Neurobiol (Madr), 1991, 54 Suppl 3, 32 - 9
{Advances in the treatment of the dystonias}; Gimenez-Roldan S; Except in Wilson's disease, few secondary dystonias are susceptible te benefit from an etiological treatment . The somatic distribution of dystonia often determines the therapeutic strategy . Thus, stereotactic surgery may be the treatment of choice for hemidystonia while anticholinergic medication may alleviate generalized dystonia, particularly in childhood . Finally, local infiltrations of botulinum toxin are particularly useful for various forms of local and segmental dystonia . Certain subsyndromes as myoclonic dystonia, levodopa sensitive dystonia and paroxysmal choreoathetosis may benefit from relatively specific treatment strategies.

Acta Med Austriaca, 1991, 18(5), 125 - 9
{Botulinum toxin A in therapy of craniocervical dystonias and hemifacial spasm}; Thill R et al.; 34 patients with focal dystonias (13 with essential blepharospasm, 3 with Meige's syndrome, 2 with hemifacial spasm, 16 with spasmodic torticollis) were treated with botulinum type A toxin . 4 ng of botulinum type A toxin per eye were applied in the M . orbicularis oculi as first injection in the 18 patients without spasmodic torticollis . The 16 patients with idiopathic spasmodic torticollis received 10 ng botulinum toxin A in the contralateral M . sternocleidomastoideus as well as in the ipsilateral M . splenius capitis as first injection . The effect was monitored for a time period of at least 6 weeks by two subjective rating scores, a visual functional score and a global clinical impression score . Patients with blepharospasm showed a distinct improvement already after 4 days which lasted for 6 weeks . 75% of the patients with spasmodic torticollis experienced a moderate to distinct improvement after 4 days which remained stable for 6 weeks . A second injection was performed in 15 (7 blepharospasm, 8 spasmodic torticollis) patients 9-11 weeks later with a similar success . All observed side effects (weakness; stiffness of local muscles; feeling of dryness of eyes, unilateral ptosis) were mild and of transient nature . We suggest therefore botulinum type A toxin as treatment of first choice in focal dystonias.

Ann Otolaryngol Chir Cervicofac, 1991, 108(8), 477 - 82; discussion 482-3
{Treatment of spasmodic dysphonia with botulinum toxin}; Klap P et al.; Spasmodic dysphonia is a focal laryngeal dystonia, a rare form of dystonia . Videostroboscopy, acoustic analysis, computerized voice analysis and over all electrophysiological analysis allow for the study of the different muscles involved in this dysphonia . There are two types of spasmodic dysphonia: adductor spasmodic dysphonia and abductor spasmodic dysphonia . The most efficient therapy nowadays is the injection of botulinum toxin into the thyroarytenoid muscle under fiberoptic visualization . We report 6 patient's cases of spasmodic dysphonia that we have been treating for about 2 years by direct injection of botulinum toxin in the vocal cords.

Fortschr Ophthalmol, 1991, 88(4), 411 - 5
{Changes in the blink reflex in patients with essential blepharospasm and hemifacial spasm}; Roggenkamper P et al.; Electrophysiological investigations have been carried out in 46 patients with essential blepharospasm and 41 with hemifacial spasm . In both diseases reproducible pathologic alterations in the blink reflex were found . In essential blepharospasm a direct R1/R2 transition and a contralateral early component with and without direct transition into the R2C component were recorded . In patients with hemifacial spasm there was a loss of the R1 component in addition . In repeated treatments with botulinum toxin the amplitude (R1,R2) of the blink reflex was reduced in some cases, although in some cases the intensity of spasm was as high as before treatment once the effect had faded.

Neuroreport, 1991 Jan, 2(1), 33 - 6
Tetanus and botulinum A toxins inhibit stimulated F-actin rearrangement in chromaffin cells; Marxen P et al.; Tetanus and botulinum A neurotoxins block exocytosis of noradrenaline in chromaffin cells . During exocytosis vesicles fuse with the plasma membrane . This requires an intact cytoskeleton . The cytoskeleton can be displayed by rhodamine-labelled phalloidin binding to F-actin . When chromaffin cells are stimulated with carbachol, F-actin forms clusters close to the plasma membrane . On withdrawal of the secretagogue the stimulated cells revert to their original appearance . The conversions, like exocytosis, depend on the presence of Ca2+, indicating the association of exocytosis with F-actin arrangement . When exocytosis is blocked in chromaffin cells by tetanus or botulinum A neurotoxins, F-actin fails to cluster during nicotinic stimulation . Thus, the toxins act somewhere between the rise in intracellular free Ca2+ and the rearrangement of F-actin.

Arch Oral Biol, 1991, 36(11), 827 - 36
Ultrastructural changes in the masseter muscle of Macaca fascicularis resulting from intramuscular injections of botulinum toxin type A; Capra NF et al.; Intramuscular injections of botulinum toxin type A (Oculinum) is used to treat strabismus and focal dystonias affecting orofacial muscles . However, the toxin-induced morphological changes that underlie the therapeutic alterations of tone in the muscles of mastication have not been described . In this study, paired intramuscular injections of botulinum toxin (10 units) were made in three adult monkeys (Macaca fascicularis) allowed to survive 14, 28 and 63 days . Another monkey received multiple injection-pairs over 84 days . Animals were killed by deep pentobarbital anaesthesia before transcardiac perfusion-fixation . Tissue sampled from comparable regions of the injected masseter, the uninjected masseter and an uninjected animal was processed for ultrastructural analysis . Few changes were found 14 days post-injection . However, muscle fibres showed myofibrillar dissolution, aberrations in the Z-line, and enlarged mitochondria in the region of the I-band by 28 days . In the 63-day and 84-day animals, the injected muscle was considerably smaller than the uninjected, contralateral muscle . Regions of the injected muscle contained fibres with markedly reduced cross-sectional area . Internalization of myonuclei, loss of myofibrillar organization, and helical complexes were common . Toxin-induced changes, though similar to those that follow denervation by axotomy, were not accompanied by degeneration of neuromuscular junctions . Instead, morphological evidence for axonal sprouting in the region of the neuromuscular junction, possibly contributing to functional recovery, was seen as early as 14 days in toxin-treated muscles.

Eye, 1991, 5 ( Pt 4), 451 - 5
Treatment of strabismus after retinal detachment surgery with botulinum neurotoxin A; Lee J et al.; Thirty-one consecutive patients were treated with injections of Botulinum Neurotoxin A to rectus muscles for strabismus following retinal detachment surgery . In 14 cases the presenting problem was diplopia and in 17 cases the presenting problem was cosmetic appearance . A total of 67 injections was given . Twenty-seven cases had nine months or more follow-up . Of these, four of 11 cases with diplopia had fusion restored, four were shown to have no fusion potential, and three had temporary improvement only . In 16 cases with a primary cosmetic problem there was no useful effect in two, three had surgery as an alternative, three were realigned long-term, and eight had continuing maintenance therapy with toxin . Over half the series had undergone multiple detachment surgery, often for giant tears and other complex pathology.

Eye, 1991, 5 ( Pt 4), 447 - 50
Binocular diplopia in unilateral aphakia: the role of botulinum toxin; Hakin KN et al.; We have treated 12 unilaterally aphakic patients, with a manifest squint and binocular diplopia, with botulinum toxin injection to the appropriate horizontal rectus muscle, in an attempt to reduce the angle of squint and thereby resolve the diplopia . In all cases a short-term reduction in the angle of squint was achieved . In nine patients, whose aphakia was corrected with a contact lens, and eight of whom had had their lenses removed because of trauma, this reduction was only temporary . In three patients, however, who had had a non-traumatic cataract removed, replaced with a posterior chamber implant, control of the deviation was maintained long after the acute effect of the toxin had disappeared, with the development of coarse binocular single vision, a fusion range, and abolition of all diplopia . The possible reasons for these different responses are discussed and it is suggested that in cases of binocular diplopia following lens extraction, botulinum toxin treatment should be considered prior to any extraocular muscle surgery, as temporary reduction of the deviation may be sufficient to allow recovery of binocular single vision.

Ophthal Plast Reconstr Surg, 1991, 7(1), 54 - 60
Innervation zone of orbicularis oculi muscle and implications for botulinum A toxin therapy; Borodic GE et al.; Motor points (areas of maximal sensitivity to electrical stimulation) were found in constant locations over orbicularis oculi when measured in both eyes of six normal subjects . All subjects had a motor point at the lateral terminus of the upper lid crease and the medial extent of the lower lid crease . A study of the innervation zone {distribution of neuromuscular junctions (NMJ)} was conducted on strips of pretarsal and preseptal portions of the upper eyelid orbicularis that had been removed routinely during involutional ptosis surgery . There was no significant difference in NMJ concentration between the medial and lateral sections, as determined by cholinesterase staining . Therefore, we concluded that the innervation zone is diffuse for the orbicularis muscle within this portion of the upper eyelid . Single-point injections of botulinum toxin were then compared to the conventional multiple injection sites on separate eyes in 10 patients with benign essential blepharospasm . Eight of the 10 patients reported greater relief on the side given injections into multiple points; the other two patients experienced no difference between the two methods . Both histologic data and clinical observation of response to botulinum toxin injection suggest the innervation zone for the upper orbicularis is diffuse . Thus, we conclude that multiple injections are superior to the injection of a single motor point.

Lakartidningen, 1990 Dec 19, 87(51-52), 4408 - 10
{Injection of botulinum toxin as a new treatment of torticollis}; Aquilonius SM et al.; Thirty patients with spasmodic torticollis were injected with Botulinum A toxin into sternomastoid and posterior neck muscles . Neurophysiological investigation showed neuromuscular transmission in the sternomastoid muscle to be impaired already within the first day after injection of 50 U toxin . Signs of denervation were discernible after seven days, and of reinnervation after two weeks . As assessed by 'blind' ratings of videotapes, 78 per cent of the patients improved, as compared with the figure of 87 per cent based on clinical evaluation . When injections were repeated every third month, there was a tendency for symptomatic effects to increase with time . Side effects were mild and transient . Thus local injections of botulinum toxin would seem to be the treatment of choice in this form of dystonia.

FEBS Lett, 1990 Dec 17, 277(1-2), 171 - 4
Light chain of botulinum neurotoxin is active in mammalian motor nerve terminals when delivered via liposomes; de Paiva A et al.; Liposomal encapsulation of the individual light and heavy chain of botulinum neurotoxin A was used to investigate their intra-cellular effects on synaptic transmission at the murine neuromuscular junction . Bath-application to phrenic nerve-hemidiaphragms of liposomes containing heavy chain (up to 75 nM) caused no alteration in neurally-evoked muscle tension . In contrast, liposomes with entrapped light chain (9-20 nM final concentration) gave a pre-synaptic blockade of neuromuscular transmission that could be relieved temporarily by 4-aminopyridine, as for the dichain toxin . Any contribution from contaminating intact toxin was excluded both by the purity and minimal toxicity in mice of the light chain preparations used, and by the lack of neuromuscular paralysis seen with liposomes containing the maximum amount of native toxin that could have been present in the light chain liposomes . As bath-application of high concentrations of light chain in the absence of liposomes failed to affect neurotransmiter release, it is concluded that this chain alone can mimic the action of the whole toxin inside mammalian motor nerve endings, its predominant site of action . Thus, light chain could provide a more effective probe for an intra-cellular component concerned with Ca2(+)-dependent secretion.

Am J Ophthalmol, 1990 Dec 15, 110(6), 674 - 82
Ocular movements in essential blepharospasm; Demer JL et al.; In essential blepharospasm histopathologic and electrophysiologic evidence supports the existence of lesions in proximity to brainstem nuclei controlling ocular movements . We studied horizontal ocular movements in eight patients who had been treated previously with surgery or botulinum toxin injection to control essential blepharospasm (mean age, 58 years) and compared these with seven control subjects who did not have blepharospasm (mean age, 68 years) . We examined fixation stability, saccades, the vestibulo-ocular reflex, visual enhancement and suppression of the vestibulo-ocular reflex, optokinetic nystagmus, and pursuit by using digitally sampled, direct current electro-oculography . Patients with blepharospasm exhibited no ocular movement abnormalities . Since quantitative aspects of ocular movements are sensitive to nonspecific brainstem lesions, the absence of abnormal ocular movements suggests that the lesion in blepharospasm is specifically limited to neurons regulating the facial muscles.

Axone, 1990 Dec, 12(2), 40 - 3
Blepharospasm, hemifacial spasm and the nurse's role; Kinash RG et al.; Blepharospasm and hemifacial spasm are involuntary movement disorders that affect the facial muscles . They are classified as cranial dystonias . Their cause is unknown and the underlying pathophysiology is poorly understood . Both dystonias are more common in women than in men . It is the middle-aged group that is most frequently affected . Because of their high visibility, these disorders may cause considerable distress and embarrassment . Affected persons are often mistakenly considered to have psychiatric problems . In addition, both dystonias may result in severe disability . For example, the person with untreated blepharospasm may experience social isolation and functional blindness . Recently, therapy in the form of botulinum toxin became available in larger centers . Repeated injections of the toxin usually relieves symptoms and enable patients to resume a former lifestyle . Neuroscience nurses who are knowledgeable about cranial dystonias and the resources that are currently available can retard progression of disability and help restore the individual's quality of life . Informed neuroscience nurses can also play an important role in case-finding, counselling and referral . Two examples are presented in order to highlight some of the complexities inherent in the diagnosis and treatment of each type of cranial dystonia and to further clarify the nurse's role . These examples are based on the personal and professional experience of the authors.

Biull Eksp Biol Med, 1990 Dec, 110(12), 637 - 8
{State of coagulation hemostasis and fibrinolysis processes in dynamics of rapidly progressing forms of botulinal intoxication}; Chesnokova NP et al.; Phase disturbances of coagulation blood potential were revealed in experiments on white rats in dynamics of rapidly progressing form of botulinic intoxication, intoxication being caused by intraperitoneal injection of type C toxin . In preclinical period of intoxication activation of procoagulant and anticoagulant parts of hemostasis system, as well as fibrinolysis system, was noted . Similar shifts were revealed in the developmental period of generalized pareses of skeletal musculature . Only in the terminal stage of intoxication insufficiency of mechanism in formation of prothrombinase activity developed by simultaneous activation of anticoagulant mechanism and fibrinolysis system.

Ital J Neurol Sci, 1990 Dec, 11(6), 589 - 93
Botulinum A toxin injection in patients with blepharospasm, torticollis and hemifacial spasm; Berardelli A et al.; Botulinum A toxin was injected into the affected muscles in 20 patients with blepharospasm, 8 with torticollis and 12 with hemifacial spasm . In all cases blepharospasm and hemifacial spasm was abolished or markedly reduced . The only side effect was transient ptosis and diplopia . Patients with torticollis had a mild to moderate improvement of the dystonic posture and pain; dysphagia was the most troublesome side effect . Botulinum A toxin is an effective therapy in patients with focal dystonia and spasms.

J Protein Chem, 1990 Dec, 9(6), 705 - 13
Structural analysis of botulinum neurotoxin types A and E in aqueous and nonpolar solvents by Fourier transform infrared, second derivative UV absorption, and circular dichroic spectroscopies; Singh BR et al.; Two pharmacologically similar but antigenetically distinct botulinum neurotoxins, types A and E with a 1000-fold difference in their toxicity, were examined for nonpolar solvent-induced changes in secondary structures and polypeptide foldings to understand their structural differences and their comparative responsiveness/susceptibility to solvent perturbation . Analysis of far UV circular dichroic spectra in aqueous buffer for types A and E neurotoxins yielded the following: the alpha-helix contents were 27 and 20%; the beta-sheets were 36 and 44%, the beta-turns were 6.0 and 0%, and the random coils were 31 and 36%, respectively . Fourier transform infrared spectra, obtained by using attenuated total reflection technique, indicated high content of alpha-helix and beta-pleated sheet structures for both neurotoxins as judged by strong bands at 1651 and 1633 cm-1 in the amide I frequency region and bands at 1314 and 1245 cm-1 in the amide III frequency region . The peak height ratio of 1314 and 1245 cm-1 bands, suggests that the type A neurotoxin has slightly higher alpha-helical content than the type E neurotoxin . These observations are consistent with the secondary structures estimated from far UV circular dichroic spectra . Fourier transform infrared spectra of the neurotoxins, exposed to methanol, showed sharp increases of the 1651 cm-1 band and a significant increase in the height of the 1314 cm-1 band, suggesting increases in the alpha-helical contents of the proteins . The changes were more in the type A than in the type E neurotoxin . The changes were reversible upon reexposure of the proteins to the aqueous buffer . Second derivative absorption spectroscopy demonstrated that methanol also induced changes in the degree of Tyr exposure to solvent . The results are discussed in terms of structural differences between the single and dichain neurotoxins and in terms of their mode of action.

Schweiz Rundsch Med Prax, 1990 Nov 27, 79(48), 1512 - 4
{The role of botulinum toxin in the treatment of essential blepharospasm}; Safran AB; Benign essential blepharospasm is a focal dystonia consisting in involuntary closure of the eyelids . Until early 80's, therapeutic modalities included only psychotropic drugs, biofeedback and surgery, which showed limited efficiency . Recently, it has been suggested to inject botulinum toxin in affected palpebral orbicularis muscles . Used in several thousands of patients, it has been found that this procedure provides significant temporary relief from spasms of the eyelids . Local side effects are minimal, and no systemic side effects have been demonstrated in patients with blepharospasm treated with botulinum toxin.

FEBS Lett, 1990 Nov 12, 274(1-2), 111 - 4
Interaction of recombinant rho A GTP-binding proteins with photoexcited rhodopsin; Wieland T et al.; The small molecular mass GTP-binding proteins rho A, B and C are targets for ADP-ribosyltransferase activity of the botulinum exoenzyme C3 . The possible interaction of recombinant rho A proteins expressed in E . coli with photoexcited rhodopsin was studied by reconstitution with bovine rod outer segment (ROS) membranes depleted of endogenous GTP-binding proteins by treatment with urea . As reported for C3 substrates present in untreated ROS membranes, ADP-ribosylation of recombinant rho A proteins, both normal and Val-14 mutant, by C3 was inhibited when reconstituted with illuminated compared to dark-adapted ROS membranes pretreated with urea . GDP reduced the light-induced inhibition, while GTP{S} and light inhibited ADP-ribosylation of rho A proteins in a synergistic manner.

Consens Statement, 1990 Nov 12-14, 8(8), 1 - 20
Botulinum toxin; Eyelid movements before and after botulinum therapy in patients with lid spasm; Department of Neurobiology, State University of New York, Stony Brook 11794-5230Quantitative analysis of lid motility is presented for 4 individuals with hemifacial spasm and 1 with Meige's syndrome . The data were obtained, by means of a magnetic search coil technique, prior to and 1 week after injection of botulinum toxin into the orbicularis oculi muscle . Before treatment, the peak velocity of blink-related lid lowering and lid raising was slower than normal, yet lid saccades were normal . After botulinum treatment, significant decreases occurred in (1) the amplitude of blinks and lid saccades, and (2) the peak velocity of the blink down-phase . Botulinum treatment significantly alters blink lid-lowering kinematics, while saccadic lid-lowering kinematics are normal, providing further evidence that the orbicularis oculi muscle does not play a primary role in downward lid saccades.

Ophthalmology, 1990 Nov, 97(11), 1434 - 8
Botulinum treatment of childhood strabismus; Scott AB et al.; Four hundred thirteen children ranging in age from 2 months to 12 years were treated for strabismus by botulinum injection of extraocular muscles . An average of 1.7 injections per patient was given . Follow-up at an average of 26 months after the last injection (minimum, 6 months) was available on 362 children (88%) . The frequency of correction of 10 prism diopters (PD) or less in various groups of strabismus cases was: all 362 cases, 61%; all esotropia, 66%; infantile esotropia, 65%; and exotropia, 45% . Smaller deviations (10-20 PD) were more frequently corrected (73%) than were larger deviations (20-110 PD, 54%) . The frequency of correction to 10 PD or less of previously operated cases was not different from that of unoperated cases . There was no globe perforation, amblyopia, or visual loss produced by the injection treatment in this series.

Otolaryngol Head Neck Surg, 1990 Nov, 103(5 ( Pt 1)), 752 - 8
Indirect laryngoscopic approach for injection of botulinum toxin in spasmodic dysphonia; Ford CN et al.; Spasmodic dysphonia is a focal dystonia that causes a loss of the fine control of intrinsic laryngeal muscles and produces a strained staccato voice . Temporary relief from symptoms has been reported in patients treated with botulinum toxin percutaneously injected into the thyroarytenoid muscle . A newly developed method of treatment differs from reported methods by increasing the accuracy of botulinum toxin placement, reducing soft tissue trauma, and applying basic scientific information about the functional histology of intrinsic laryngeal musculature . Sixteen patients with primarily adductor spasmodic dysphonia were treated . Initial assessment included laryngeal examination by indirect laryngoscopy, videoendoscopy, and stroboscopy, neurology examination (including laryngeal EMG), and vocal function studies with acoustic analysis and aerodynamic studies . A device originally designed for collagen injection allowed the precise microdelivery of toxin to the thyroarytenoid muscle . Indirect laryngoscopy was used to direct the needle, in an attempt to cover a broad area of motor end plates . The minimally effective dose was titrated for each patient, to avoid paralysis and preserve laryngeal function . All patients showed improved voices after treatment . There were no major complications . The basic technique can be performed in the otolaryngologist's office and does not require electromyography equipment or expertise.

Aust N Z J Ophthalmol, 1990 Nov, 18(4), 393 - 6
Symptomatic nocturnal lagophthalmos; Lyons CJ et al.; Nocturnal lagophthalmos producing symptoms of corneal exposure is not uncommon . We diagnosed 40 patients with the condition over a six-month period . In 12 (30%), alcohol intoxication preceded the symptoms . Other factors included the use of hynotics, cosmetic blepharoplasty, botulinum toxin injection for blepharospasm, facial palsy, dysthyroid eye disease and cicatrising skin disease of the lids . In the largest group (17 patients, 42%), no cause could be found, but five of these had either first-degree relatives who slept with their eyes open, or had been observed with nocturnal lagophthalmos . In most patients, symptoms were unilateral . Testing of Bell's phenomenon did not predict eye position during sleep as manifested by distribution of punctate corneal staining . All patients were relieved of their symptoms by simple treatment modalities and none required surgery.

Klin Wochenschr, 1990 Oct 3, 68(19), 935 - 41
New approaches in the treatment of the dystonias; Diederich N et al.; At this point the treatment of dystonias remains highly empirical . Secondary dystonias, especially those related to specific drug treatment, have to be ruled out carefully . A few dystonic subgroups respond well to levodopa medication . In the other syndromes, anticholinergics are the usual first choice . In focal conditions botulinum toxin injections seem to be the most effective regimen, although there are only a few long-term studies . Surgical procedures are an ultimate option.

Ear Nose Throat J, 1990 Oct, 69(10), 704 - 5, 709-11, 715-7
The effects of botulinum toxin on hemifacial spasm: an electrophysiologic investigation; Laskawi R et al.; Forty-one patients with unilateral or bilateral facial spasm were studied by electrophysiologic examination . All patients received local treatment with botulinum toxin . In the patients and controls, the measurements of the blink reflex revealed changes that (1) may indicate a central origin of the spasm in some cases, and (2) make it likely that the trigeminal nerve influences the facial nucleus and is thus involved in the regulation of the spasm.

Singapore Med J, 1990 Oct, 31(5), 469 - 71
Botulinum toxin in the treatment of hemifacial spasm; Chong PN; Thirteen patients with disabling idiopathic hemifacial spasm received botulinum toxin A injections to the affected muscles . Previous treatments, including one posterior fossa decompression, had no sustained benefit . There was excellent response in all the patients with improvement in social disability . Local side effects were mild and transient and include mild facial weakness . One patient had mild diplopia . The benefit lasted more than three months . Reinjection resulted in identical efficacy . Botulinum toxin A injection is a useful therapy for Hemifacial spasm.

Ann Neurol, 1990 Oct, 28(4), 512 - 5
Treatment of spasticity with botulinum toxin: a double-blind study; Snow BJ et al.; We studied the effect of botulinum-A toxin on spasticity of the leg adductors in 9 patients who were either chair-bound or bed-bound with chronic stable multiple sclerosis . We injected botulinum toxin (400 mouse units) or placebo into the adductor muscles in a randomized, crossover, double-blind design . Two physicians, who were unaware of the treatment order, used an objective rating scale and independently assessed the patients; interobserver correlation was excellent (r = 0.93-0.81) . We found that botulinum toxin produced a significant reduction in spasticity (p = 0.009) and a significant improvement in the ease of nursing care (p = 0.009) . There were no adverse effects during this short-term trial . This is the first demonstration of the beneficial effect of botulinum toxin on focal spastic muscle contractions.

Aviat Space Environ Med, 1990 Oct, 61(10), 935 - 7
Cervical dystonia following exposure to high-G forces; Clark JB; Injuries to the cervical region have been associated with high-G loads sustained during air combat maneuvering (ACM) in high performance fighter aircraft . The spectrum of injuries ranges from mild neck pain to musculoskeletal strain, injury to the nerve roots or spinal cord, and fracture of the cervical spine . A 36-year-old fighter pilot with 2,800 h in tactical jet aircraft developed progressive cervical dystonia (spasmodic torticollis), following an ACM flight . The patient was successfully treated with local intramuscular injections of botulinum toxin into the affected cervical muscles, resulting in total relief of his spasmodic torticollis . The aeromedical considerations of this rare complication of exposure to G forces in high performance aircraft are discussed.

Arch Ophthalmol, 1990 Oct, 108(10), 1432 - 5
Botulinum vs adjustable suture surgery in the treatment of horizontal misalignment in adult patients lacking fusion; Carruthers JD et al.; Thirty patients were treated with either botulinum toxin or adjustable suture surgery in a prospective, randomized clinical trial . All patients had horizontal deviations greater than 10 prism diopters and absent fusion . Seventeen patients were assigned to toxin treatment, and 13 were assigned to surgical treatment . Follow-up at 6 months after either procedure indicated that surgery was superior, with patient alignment showing a 92.7% average net change, compared with a 50.50% net change in the botulinum-treated group . There was no difference in response between those patients with a starting deviation of 20 PD or less and greater than 20 PD in the surgery group . However, in the botulinum-treated group, those patients with a starting deviation of 20 PD or less seemed to show better responses than those patients with greater than 20 PD . Patients with esotropia showed an 88.89% change with surgery and a 51.55% change with toxin treatment . Patients with exotropia had a 95.83% change with surgery but a 50.3% change with toxin treatment . Since we had 20 patients with exotropia and 10 patients with esotropia, a more formal comparison would require larger numbers.

J Pharmacol Exp Ther, 1990 Oct, 255(1), 227 - 32
Use of monoclonal antibodies as probes for the structure and biological activity of botulinum neurotoxin; Simpson LL et al.; Experiments were done to help clarify the structure-function relationships that govern the interaction between botulinum neurotoxin and the cholinergic neuromuscular junction . Work was done with type E toxin in three different states: 1) unactivated (post-translational product before proteolytic processing), 2) activated (proteolytically modified product) and 3) denatured . Four different monoclonal antibodies were studied (E3, E14, E17 and E32), three of which were capable of diminishing the potency of the toxin . All four antibodies had approximately equivalent affinity for the unactivated and the activated forms of the toxin . Monoclonals E17 and E32 had little ability to interact with denatured toxin, suggesting they recognized conformational epitopes; monoclonals E3 and E14 retained partial ability to bind to denatured toxin, suggesting they recognized both conformational and linear determinants . When phrenic nerve-hemidiaphragm preparations were exposed to toxin under conditions that allowed binding but retarded internalization, the toxin remained accessible to antibodies . However, when tissues were stimulated in an effort to promote endocytosis, the toxin disappeared from accessibility to antibodies . The data indicate that various antigenic domains remain exposed after binding and suggest that certain parts of the toxin molecule undergo little or no conformational change during binding . The data further indicate that the molecular domains recognized by E14, E17 and E32 are internalized simultaneously.

FEBS Lett, 1990 Oct 1, 271(1-2), 19 - 22
Novel GTP-binding proteins in plasma membranes and zymogen granule membranes from rat pancreas and in pancreatic AR 4-2J cell membranes; Lambert M et al.; Photoaffinity labelling with {alpha-32P}GTP allowed to detect a 54 kDa GTP-binding protein in rat pancreatic plasma membranes and in pancreatic AR 4-2J cell membranes . Like the 42 and 48 kDa Gs alpha subunits and the 41 kDa Gi alpha subunit, this protein was absent from zymogen granule membranes . Contrastingly, a new 28 kDa GTP-binding protein (detected by {alpha-32P}GTP binding on immobilized proteins) and a 25 kDa protein (ADP-ribosylated by botulinum toxin D) were found in all three membrane preparations . This is to our knowledge the first report on GTP-binding proteins in zymogen granule membranes.

Jpn J Med Sci Biol, 1990 Oct, 43(5), 163 - 70
Titration of botulinum antitoxin of low levels by the score method; Takahashi M et al.; Botulinum antitoxin is commonly titrated by injecting a mixture of toxin and antitoxin into mice and by utilizing deaths as a marker to measure the amount of unneutralized toxin . We attempted to titrate antitoxin by converting the severity of symptoms (notably palsy) and time-to-death in days into scores . In neutralization tests with toxin levels at 5.9 LD50 and 23.5 LD50, a linear relationship was obtained for antitoxin dose in a range between 0.03 to 0.003 IU/ml . Statistical analysis showed that homogeneity of variance or slope was not denied for the scores obtained on any day from the first to the fourth days after injection, demonstrating that this method can titrate accurately antitoxin of such a low level as 0.003 IU/ml within 4 days after injection.

Biophys Chem, 1990 Oct, 38(1-2), 123 - 30
Conformational changes associated with the nicking and activation of botulinum neurotoxin type E; Singh BR et al.; Secondary and tertiary structural parameters of type E botulinum neurotoxin in the unactivated single-chain and activated two-chain (i.e., after proteolytic cleavage) forms were analyzed using circular dichroism, derivative absorption and fluorescence spectroscopy . The estimated secondary structures (22 and 20% alpha-helix, 44 and 44% beta-pleated sheets, and 34 and 36% random coils for the single- and two-chain neurotoxins, respectively) indicated that virtually no change occurred upon nicking of the single-chain neurotoxin . About 57% of the 70 Tyr residues were exposed in the single-chain form, which increased to 62% in the two-chain form . Fluorescence quenching experiments with neutral, anionic and cationic quenchers indicated that about 40% of the maximum accessible fluorescent Trp residues were exposed on the surface of the single-chain neurotoxin as compared to only 20% in the case of the two-chain neurotoxin . Acrylamide was the most effective quencher with a fraction accessibility of 0.56 and 0.48 of maximum accessible Trp fluorescence residues in the single and two-chain forms of the neurotoxin, respectively . Native polyacrylamide gel electrophoresis of the two forms of the neurotoxin revealed greater mobility for the two chain form . This indicates that the surface charges in the single-chain neurotoxin were altered upon nicking . These observations suggest that nicking of the single-chain type E neurotoxin results in refolding and redistribution of the surface charges of the neurotoxin.

Wien Klin Wochenschr, 1990 Sep 28, 102(18), 523 - 5
{Treatment of essential blepharospasm}; Gugg E et al.; A rather confusing number of conservative, as well as invasive treatment methods for essential blepharospasm are found in the literature . Depending on the severity of symptoms, two types of method became important because of their obvious success and their low rate of complications: Local injection--treatment with botulinum toxin type A in mild and moderate cases and the neurosurgical method of selective neurotomy in severe forms of blepharospasm or after inefficient application of botulinum-A toxin . The different methods of treatment are reviewed.

Biochem Biophys Res Commun, 1990 Sep 28, 171(3), 1304 - 11
The complete nucleotide sequence of the gene coding for botulinum type C1 toxin in the C-ST phage genome; Kimura K et al.; Two DNA fragments, 3 kbp and 7.8kbp, which encode the type C1 botulinum neurotoxin gene, were obtained from toxigenic bacteriophage DNA by treatment with a restriction enzyme . They were cloned into the plasmid vectors for nucleotide sequence determination . The nucleotide sequence contained a single open reading frame coding for 1,291 amino acids corresponding to a polypeptide with a molecular weight of 149,000 . The amino acid sequence of the C1 toxin has a few regions highly homologous with tetanus toxin.

J Immunol, 1990 Sep 15, 145(6), 1845 - 50
Compartmentalization of low molecular mass GTP-binding proteins among neutrophil secretory granules; Dexter D et al.; Neutrophils contain several distinct classes of secretory granules that may sequentially fuse with the phagosome after the ingestion of particulates, or that may be differentially exocytosed after cellular activation with soluble stimuli . The exocytosis of neutrophil secretory granules has been shown to be GTP-dependent at a step distal to activation of the transductional G proteins . Inasmuch as ras-related low molecular mass GTP-binding proteins have been shown to play regulatory roles in vesicle sorting in the secretory pathway in yeast, the differential mobilization of neutrophil granules might be regulated by distinct GTP-binding proteins . We therefore explored the distribution and identity of low molecular mass GTP-binding proteins in neutrophil secretory granules and other subcellular fractions . After lysis by nitrogen cavitation, four highly resolved fractions were harvested from discontinuous Percoll gradients: a microsomal fraction enriched for plasma membranes, specific granules, primary granules, and cytosol . At least seven bands of distinct Mr were detected by probing protein blots with {32P}GTP . Microsomes contained a prominent GTP-binding band at 26 kDa and weaker ones at 24 and 22.5 kDa; specific granules contained bands at 26, 24, 22, and 20 kDa; primary granules showed bands at 24 and 23 kDa; cytosol showed strong bands at 23.5 and 19 kDa and a weak band at 26 kDa . Antiserum against ADP-ribosylation factor reacted strongly with the 19-kDa band in cytosol but with none of the membrane fractions . None of these proteins was recognized by antibodies against ras or against Sec4p . Botulinum exoenzyme C3 labeled bands of molecular mass 20 and 21 kDa in cytosol and microsomes that have distinct mobilities from all the blotted {32P}GTP-binding proteins . The highly compartmentalized subcellular distribution of the blotted {32P}GTP-binding proteins in neutrophils is consistent with a regulatory role in the differential mobilization of granule compartments during cellular activation.

Head Neck, 1990 Sep-Oct, 12(5), 392 - 9
Botulinum A toxin for the treatment of spasmodic torticollis: dysphagia and regional toxin spread; Borodic GE et al.; Chemodenervation of cervical muscles with botulinum A toxin, although useful in treating spasmodic torticollis, has been associated with dysphagia . Retrospective analysis of dose and injection site (sternomastoid vs . posterior cervical muscle groups) in 26 patients (49 injections) suggested that dysphagia was related to the quantity of toxin injected into the sternomastoid muscle: dysphagia, median 150 IU (7 injections); and no dysphagia, median 100 IU (42 injections; p = 0.026 Wilcoxon test) . In a prospective study (31 injections to 24 patients), limiting the dose administered to the sternomastoid to 100 IU, substantially reduced the incidence of dysphagia (0 of 31, p = 0.27, Fisher's exact test) . Denervation of human orbicularis muscle fibers, 5 weeks to 4 months after injection of botulinum A toxin for the treatment of blepharospasm, was successfully demonstrated by intense, diffuse acetylcholinesterase staining . A weight-adjusted dose similar to that given for torticollis was injected into longissimus dorsi muscle in 6 albino rabbits . Using the acetylcholinesterase stain as a marker, a diffusion gradient was noted over a distance of 30 to 45 mm from the point of injection and in contralateral muscle 15 to 25 mm from this point . Thus, denervation was demonstrated to occur within a definable area which crossed anatomic barriers, such as fascia and bone . These clinical and laboratory data suggest that dysphagia following botulinum toxin therapy results from toxin spread to pharyngeal musculature from the sternocleidomastoid injection site . Limiting of the injection dose to 100 IU or less to the sternomastoid substantially decreases the incidence of this complication.

J Neurochem, 1990 Aug, 55(2), 468 - 72
Phorbol esters induce neurotransmitter release in cholinergic synaptosomes from Torpedo electric organ; Guitart X et al.; The effect of phorbol esters and so the involvement of Ca2+/phospholipid-dependent protein kinase (protein kinase C;PKC) in the release of acetylcholine (ACh) was studied using Torpedo electric organ synaptosomes . 12-O-Tetradecanoylphorbol 13-acetate (TPA), a known activator of PKC, induced neurotransmitter release in a concentration-dependent manner and increased the potassium-evoked release of ACh . The effect of TPA was shown to be independent of the extrasynaptosomal calcium concentration . TPA-induced ACh release was reversed by H-7, an inhibitor of PKC activity . This drug showed no effect on potassium-evoked ACh release . Botulinum toxin, a strong blocker of potassium-induced ACh release in that synaptosomal preparation, showed no inhibitory effect on the TPA-induced ACh release . Our results suggest that activation of PKC potentiates the release of an ACh pool that is not releasable by potassium depolarization, independently of the extracellular calcium concentration.

HNO, 1990 Aug, 38(8), 295 - 7
{Botulinum toxin treatment of synkinesia following facial paralysis}; Roggenkamper P et al.; We report ten patients with unilateral synkinesias after facial palsy treated with botulinum toxin injections . All patients suffered from extensive mass movements around the eye . After periocular injections all patients showed much improvement: a period of 11 weeks free of symptoms was followed by a 9-week period of minimal symptoms . There were no important complications . The use of botulinum toxin injections is an effective therapy for mass movements after defect healing of facial palsy.

J Neurol Neurosurg Psychiatry, 1990 Aug, 53(8), 640 - 3
Botulinum toxin treatment in spasmodic torticollis; Blackie JD et al.; Botulinum toxin A was administered to 19 patients in a double-blind placebo controlled trial . Toxin was more effective than placebo for improving both head position and pain which was measured by an objective rating scale and videofilm assessments . Following the controlled trial, treatment with botulinum toxin was continued in an open fashion . A total of 60 patients with torticollis received toxin in a total of 117 treatment periods . The mean follow up period was 8.4 months . In 39 patients with pain there was benefit in 77% of treatment periods . Some improvement in neck posture occurred in 83% of the treatment periods with a mean duration of 12 weeks . Side effects were frequent with dysphagia being the most common (28% of treatment periods) . Botulinum toxin is an effective treatment for toticollis but treatment should be initiated with doses at the lower end of the range used in this study (400-600 mouse units).

J Neurol Neurosurg Psychiatry, 1990 Aug, 53(8), 633 - 9
Botulinum toxin treatment of cranial-cervical dystonia, spasmodic dysphonia, other focal dystonias and hemifacial spasm; Jankovic J et al.; In the past five years, 477 patients with various focal dystonias and hemifacial spasm received 3,806 injections of botulinum A toxin for relief of involuntary spasms . A definite improvement with a global rating greater than or equal to 2 on a 0-4 scale, was obtained in all 13 patients with spasmodic dysphonia, 94% of 70 patients with blepharospasm, 92% of 13 patients with hemifacial spasm, 90% of 195 patients with cervical dystonia, 77% of 22 patients with hand dystonia, 73% of 45 patients with oromandibular dystonia, and in 90% of 21 patients with other focal dystonia who had adequate follow up . While the average duration of maximum improvement lasted about 11 weeks after an injection (range seven weeks in patients with hand dystonia to 15 weeks in patients with hemifacial spasm), some patients benefited for over a year . Only 16% of the 941 treatment visits with follow up were not successful . Except for transient focal weakness, there were very few complications or systemic effects attributed to the injections . This study supports the conclusion that botulinum toxin injections are a safe and effective therapy for patients with focal dystonia and hemifacial spasm.

Clin Neuropharmacol, 1990 Aug, 13(4), 355 - 8
Failure of fixed-dose, fixed muscle injection of botulinum toxin in torticollis; Koller W et al.; We studied the effect of botulinum toxin injection in 30 patients with torticollis in a double-blind, placebo-controlled, crossover study . A fixed dose of toxin was injected into the contralateral sternocleidomastoid and both trapezius muscles . Clinical improvement was assessed by a rating scale and by patient self-evaluation . Subjective rating noted improvement in some patients, but there was no change in objective measures . The use of larger doses and injection of additional muscles may be necessary to achieve increased efficacy of botulinum toxin in the treatment of torticollis.

Neurology, 1990 Aug, 40(8), 1213 - 8
Double-blind, placebo-controlled trial of botulinum toxin injections for the treatment of spasmodic torticollis; Greene P et al.; We enrolled 55 patients in a double-blind, placebo-controlled, parallel design study of the effectiveness of botulinum toxin (Botox) injections for the treatment of spasmodic torticollis . Patients received a standard series of injections, either placebo or Botox . We determined the sites of injection and dose per muscle by the nature of head deviation . Compared with placebo, Botox produced statistically significant improvement in the severity of torticollis, disability, pain, and degree of head turning . There were no serious side effects . During the double-blind phase, 61% of patients injected with Botox improved; 74% of patients subsequently improved during a later open phase at a higher dose of Botox . Direction of head turning, severity of torticollis, and presence or absence of jerky movements did not significantly influence the response rate . We conclude that Botox is a valuable treatment for spasmodic torticollis.

Biochem Biophys Res Commun, 1990 Jul 31, 170(2), 673 - 83
Identification of a major GTP-binding protein in bovine aortic smooth muscle cytosol as the rhoA gene product; Kawahara Y et al.; In bovine aortic smooth muscle, about 50% of total GTP-binding activity was present in the cytosol fraction . A major GTP-binding protein (G protein) with a Mr value of about 21,000 (21K G) in this fraction was purified to near homogeneity and characterized . 21K G bound maximally about 0.8 mol of {35S}guanosine 5'-(3-O-thio)triphosphate/mol of protein with a Kd value of about 20 nM . 21K G showed GTPase activity with a turnover number of about 0.007 min-1 . 21K G was ADP-ribosylated by botulinum ADP-ribosyltransferase and about 0.4 mol of ADP-ribose was maximally incorporated into 1 mol of 21K G . 21K G and the bovine brain rhoA gene product (rhoA p21) were eluted at the same retention time on C4 reversed-phase high performance liquid chromatography and migrated at the same positions on two-dimensional gel electrophoresis . These results indicate that the major G protein in bovine aortic smooth muscle cytosol is rhoA p21.

Wien Klin Wochenschr, 1990 Jul 13, 102(14), 403 - 7
{The treatment of blepharospasm with botulinum toxin A}; Koltringer P et al.; 12 patients suffering from essential blepharospasm were treated by injection of 4 ng botulinum toxin A into the Musculus orbicularis oculi of each eye . The effect was followed up over a period of 6 weeks by means of a subjective rating scale, a visual function score and a score for the overall clinical impression . After 4 days an improvement was observed in all patients and it was still present after 6 weeks . After a time interval of 12 weeks reinjection was performed in 8 cases with half the initial dose, since the beneficial effect had decreased to under 50% . Follow-up showed an impressive response to reinjection of botulinum toxin A, which remained undiminished still after 6 weeks . No serious side effects were observed . Hence, this form of therapy appears to be an effective alternative to all other therapeutic measures in this disease.

Br J Pharmacol, 1990 Jul, 100(3), 487 - 90
Tetrahydroaminoacridine (tacrine) stimulates neurosecretion at mammalian motor endplates; Thesleff S et al.; 1 . Tacrine (20 microM) induced, like 4-aminoquinoline (4-AQ, 200 microM), the appearance of a population of miniature endplate potentials (m.e.p.ps) with more than twice the normal amplitude or time-to-peak . The times-to-peak of nerve impulse-evoked endplate potentials were not similarly affected . 2 . Cholinesterase inhibition by edrophonium (25 microM) did not prevent tacrine or 4-AQ from inducing this population of m.e.p.ps . 3 . Nerve-muscle preparations in which the normal calcium-sensitive quantal release of acetylcholine had been blocked by botulinum neurotoxin type A also responded to tacrine by an increase in the frequency of giant or slow m.e.p.ps . 4 . Reduction of the temperature from 30 degrees to 14 degrees C reduced the frequency of giant or slow m.e.p.ps induced either by tacrine or by 4-AQ . A similar effect was obtained by colchicine (5 mM) . This supports the idea that proximo-distal axonal transport is required for the secretory activity . 5 . The neurosecretion evoked by tacrine could explain the therapeutic effects of the drug claimed in the treatment of Alzheimer's type of dementia.

J Ophthalmic Nurs Technol, 1990 Jul-Aug, 9(4), 138 - 40
The role of the office assistant or operating room nurse in botulinum injection as an alternative to strabismus surgery; Renier S et al.; Botulinum injection of eye muscles as an alternative to strabismus surgery can be performed in young children with low dose ketamine sedation, or reassurance without sedation for older children . The OR nurse or office assistant should provide a calm, restful environment rapport with the patient prior to surgery, a connection with her voice through the procedure and rapid reunion of the child and parent in a restful environment . If a child over 6 years is unable to cooperate with the office procedure, it quickly abandoned and it is done in the operating room under sedation.

Rinsho Shinkeigaku, 1990 Jul, 30(7), 718 - 22
{Local alcoholisation treatment of spasmodic torticollis}; Hasegawa O et al.; Idiopathic spasmodic torticollis is a type of focal dystonia . Major muscles which rotate the neck are M . sternocleidomastoideus (SCM) and M . splenius capitis (Spl) on both sides . In torticollis patients, its clinical characteristics could be understood as a vectorial summing up of tonus in bilateral SCM and Spl at rest . There is not any curative treatment for dystonia yet . A variety of medications and many types of surgical interventions have been tried without consistent or satisfactory results . In recent days local injection of botulinum-A toxin has shown to be effective in weakening focal dystonias . We used pure ethanol for local injection . Fourteen patients aged between 20-77 years (mean 48.9) were treated by alcoholisation . Disease duration ranged from 5 months to 12 years (mean 4.9 years) . All had torticollis alone or had segmental dystonia containing spasmodic torticollis . Patients were recorded electromyographically using surface electrodes to make sure which neck muscles were hypertonic, and were rated before and after treatment according to the stages (0; normal-5; most severe) . On the bases of these recordings the two most active muscles were selected for injection . Into the motor point of these muscles 1 ml of 1% lidocaine, and then the same dose of 99% ethanol were injected . This procedure was repeated on the mean ten times (6-14 times) every other week . The number of times of injection was decided in each case . Using the paired Student t test, there was a significant (p less than 0.01) improvement of the stage for the patients after injection, with a mean of 3.7 before treatment and 2.3 after treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Membr Biochem, 1990 Jul-Sep, 9(3), 203 - 14
Effect of botulinum D toxin on human neutrophilic leukocytes and localization of its substrates; Andre P et al.; Botulinum D toxin has been shown to ADP-ribosylate 22-kD proteins in neutrophilic leukocytes, but the function of these GTP-binding proteins remains unknown . In analogy to small GTP-binding proteins like SEC4 to YPT1, it has been suggested that botulinum D toxin substrates might be involved in secretory process of myeloid cells . Three main findings lead to the opposite conclusion . First of all, in human neutrophils, botulinum D toxin does not modify the release of azurophilic and specific granules induced by a chemoattractant (a formylpeptide) or a phorbol ester . Second, botulinum D toxin ADP-ribosylates 24 to 26-kD proteins that are only present in plasma membranes of human neutrophils . The membrane location of these substrates differs largely from that of the GTP-binding proteins involved in exocytosis and located in granules . Finally, since the same quantity of the toxin substrates is present in neutrophils as in their precursors, HL60 cells (which are devoid of specific granules and characterized by immature azurophilic granules and NADPH oxidase), it is unlikely that endogenous botulinum D toxin substrates are directly involved in the secretory responses of neutrophils.

J Pharmacol Exp Ther, 1990 Jul, 254(1), 98 - 103
Isolation and characterization of a novel human monoclonal antibody that neutralizes tetanus toxin; Simpson LL et al.; A human monoclonal antibody, designated 53-2-4, has been isolated and characterized in terms of its ability to interact with clostridial neurotoxins . In enzyme-linked immunosorbent assay assays the antibody reacted with native tetanus toxin, tetanus toxoid and the C fragment obtained from the carboxyterminus of the toxin (AA 864-1314) . The antibody did not react with the B fragment of tetanus toxin (AA 1-863) or with six serotypes of botulinum neurotoxin (A to F) . Both culture supernatant from the clonal line producing the antibody as well as homogeneous protein obtained by affinity purification of the antibody neutralized tetanus toxin . When tested in vivo, the antibody provided complete production against a supralethal injection of toxin; when tested in vitro, the antibody produced at least 99% inactivation of a 1 x 10(-9) M solution of toxin . The exceptional neutralizing activity of the antibody was attributed to its high affinity for the toxin (4.2 x 10(-10) mol/liter) . Animal experiments revealed a novel phenomenon that has been labeled delayed intoxication . At the appropriate ratio of antibody to antigen, the toxin was retained in the host in a latent form . After several days the biological activity of the toxin became apparent and there was onset of nervous system poisoning . Isolated tissue experiments showed that each antibody molecule is capable of associating with two antigen molecules . The antibody has greater neutralizing activity when mixed with free toxin than when mixed with toxin already bound to plasma membrane receptors.

J Biol Chem, 1990 Jun 5, 265(16), 9153 - 8
The complete sequence of botulinum neurotoxin type A and comparison with other clostridial neurotoxins; Binz T et al.; The seven serologically different botulinum neurotoxins are highly potent protein toxins that inhibit neurotransmitter release from peripheral cholinergic synapses . The activated toxins consist of the toxifying A-subunits (Mr approximately 50,000) linked by a disulfide bond to the receptor-binding BC-subunits (Mr approximately 100,000) . We have established the complete sequence of botulinum neurotoxin type A (BoNT/A; 1,296 amino acid residues, Mr = 149,425) and a partial sequence of botulinum neurotoxin type E (273 amino acid residues) as deduced from the corresponding nucleotide sequences of the chromosomally located structural genes . The promoter of the BoNT/A gene is inactive in Escherichia coli . Primer extension experiments indicated that initiation of transcription of the BoNT/A gene occurred 118 nucleotides upstream from the ATG codon . A comparison of the protein sequence revealed an overall identity of 33.8% to that of tetanus toxin . No significant similarity to other known proteins including ADP-ribosylating toxins could be detected . Three of the six histidine residues of the A-subunit of BoNT/A were found in the peptide sequence H223ELIHXXH230 within a domain of predicted alpha-helical secondary structure . This motif is also found in similar positions of the A-subunits of tetanus toxin and BoNT/E.

Ital J Neurol Sci, 1990 Jun, 11(3), 275 - 80
Botulinum A toxin treatment for eyelid spasm, spasmodic torticollis and apraxia of eyelid opening; Defazio G et al.; Botulinum-A toxin (botAtox) was used in the treatment of blepharospasm (BS), idiopathic hemifacial spasm (HFS), idiopathic spasmodic torticollis (ST) and apraxia of eyelid opening (AEO) . The injection of 7.5-30 U botAtox per eye spread over 3 or 4 sites in the palpebral part of orbicularis palpebrae (OP) reduced palpebral spasm in 12/13 cases of BS and in 7/8 cases of HFS . The effect lasted for 14.5 weeks on average (range 4-30 weeks) . Palpebral ptosis (lasting 1-3 weeks) was the most frequent side effect (16/107 eyes treated) but was not related to dose of botAtox or number of inoculation sites . Injection of 60-160 U botAtox into the sternocleidomastoid, trapezius and splenius capitis muscles reduced ST objectively in 1/4 patients for about 4 weeks . In the other patients the reduction or abolition of the hypertrophy of the previous hyperactive muscles was accompanied by persistence or rearrangement of the dystonia pattern, suggesting a change in the pattern of activity of the neck muscles after botAtox . 5 U botAtox per eye spread over 4 sites in the OP significantly reduced the frequency of the episodes of involuntary eyelid closure in 2 patients with AEO but not BS . The therapeutic effect lasted for 7 months after the first treatment and for 8 months after the second in a 46 year old woman with a 6 month history while the second patient (72 year old parkinsonian) has now completed her 3rd month of treatment.

Neurosci Lett, 1990 May 31, 113(2), 211 - 6
Response of the chick ciliary ganglion-iris neuromuscular preparation to botulinum neurotoxin; Lomneth R et al.; Response of the chick ciliary ganglion-iris muscle neuromuscular junction (NMJ) preparation to the botulinum neurotoxin (NT) was investigated . The 150 kDa serotypes A and E NTs inhibited muscle contraction in a dose dependent fashion . Neurotoxicity of type E NT increased 20-40 fold after mild digestion with trypsin . The 50 kDA light and 100 kDa heavy chains of type A NT, following separation, applied individually, did not paralyze the tissues . Preincubation of the NMJ preparations with the isolated type A heavy chain delayed (antagonized) the paralytic action of the 150 kDa dichain type A NT . Sequential administration of type A heavy chain, followed by type A light chain mimicked the action of the parent NT . The chick ciliary preparation therefore is a useful NMJ preparation to study neurotoxicity of botulinum neurotoxins.

Med J Aust, 1990 May 21, 152(10), 528 - 30
Treatment of idiopathic spasmodic torticollis with botulinum-A toxin: a pilot study of 19 patients; Lorentz IT et al.; Nineteen patients with spasmodic torticollis, unresponsive to standard therapy, were administered local injections of botulinum-A toxin into the affected muscles . During an average follow-up period of 11.5 months, a more than 25% improvement was noted in 14 of 19 patients . All those with purely focal dystonia and 9 of 10 patients with a disease history of less than three years benefited from treatment . Side effects were insignificant and transient . Botulinum toxin is a very effective and safe method of treatment for spasmodic torticollis.

Biochim Biophys Acta, 1990 May 16, 1034(2), 176 - 9
Hemagglutinating and binding properties of botulinum C2 toxin; Sugii S et al.; To characterize the binding substance(s) for botulinum C2 toxin, the hemagglutinating activity of component II of botulinum C2 toxin (C2II) was studied by hemagglutination and hemagglutination inhibition . Human and animal erythrocytes were agglutinated by trypsinized C2II much more strongly than by untreated C2II . Trypsinized C2II agglutinated neuraminidase-treated erythrocytes more strongly than intact, trypsin- and pronase-treated ones . On the other hand, trypsin- and pronase-treated erythrocytes were more weakly hemolyzed by trypsinized C2II than intact and neuraminidase-treated ones, and trypsinized C2II showed both hemagglutinating and hemolytic activities to these erythrocytes . Hemagglutination of trypsin-treated human type B erythrocytes was inhibited by galactose, N-acetylgalactosamine, N-acetylglucosamine, L-fucose and mannose . Thyroglobulin and bovine salivary mucin were much stronger inhibitors . From these findings, the binding substance(s) for botulinum C2 toxin on erythrocytes is(are) suggested to be glycoprotein(s).

Ophthalmic Surg, 1990 May, 21(5), 335 - 8
Side effects of the use of botulinum toxin for treatment of benign essential blepharospasm and hemifacial spasm; Kalra HK et al.; From April 1983 to April 1988, 381 botulinum toxin injections for lid spasms were administered to 106 patients . Sixty-nine had bilateral blepharospasm and 37 had hemifacial spasm . Of the 381 injections, 308 had been given to patients who returned for follow-up examinations . No systemic effects were noted at any of these visits; all side effects were temporary; there were no serious complications . Ptosis, the most frequently encountered problem, occurred after 26 (8.4%) of the injections . Other complications included: corneal exposure (after eight injections, 2.59%); face droop (after 11 injections, 3.57%); diplopia (after five injections, 1.62%); and subtle visual blurring (after eight injections, 2.59%) . One patient noted jaw tenseness, another mentioned tearing, one reported brow droop, and another complained of crossed eyes . Ten injections had minimal effect; in these cases a repeat injection usually was effective . Only four patients chose surgery after beginning injections . We conclude that botulinum toxin injections are a safe, effective means of treating lid spasms.

Br J Ophthalmol, 1990 May, 74(5), 309 - 10
Acute angle-closure glaucoma following botulinum toxin injection for blepharospasm; Corridan P et al.; Botulinum toxin inhibits acetylcholine release and therefore could cause mydriasis . We report a case of acute angle-closure glaucoma which occurred shortly after a series of injections of botulinum toxin round the eyelids for blepharospasm.

Invest Ophthalmol Vis Sci, 1990 May, 31(5), 964 - 7
Motor nerve sprouting in human orbicularis muscle after botulinum A injection; Holds JB et al.; The paralytic properties of botulinum A toxin have led to its use in humans in the treatment of strabismus and facial dystonias such as essential blepharospasm . Examination of orbicularis muscle from 10 patients with essential blepharospasm who received 2-18 injections of botulinum toxin 6 weeks to 3 years prior to surgery revealed characteristic nodal, terminal and ultraterminal "sprouting" of the motor axons . Orbicularis muscle from five individuals never exposed to botulinum failed to demonstrate these changes . The significance of persistent motor nerve sprouting in response to botulinum exposure remains to be elucidated.

Int J Food Microbiol, 1990 May, 10(3-4), 269 - 89
Growth and toxigenesis of C . botulinum type E in fishes packaged under modified atmospheres; Baker DA et al.; Modified atmosphere packaging of fresh fish is used to market high quality products in some European countries . The potential risk of C . botulinum growth in these extended shelf-life foods is still a concern; especially since toxigenesis may precede organoleptic spoilage . This paper will present toxigenic data from rockfish, salmon and sole muscle tissues which were inoculated with a pool of non-proteolytic C . botulinum type E at seven levels (10(-2)-10(4) spores/sample), and stored under vacuum and 100% CO2, at incubation temperatures between 30 and 4 degrees C, for up to 60 days . Factorial experimental design allowed predictive formulae to be developed able to describe the lag time prior to C . botulinum toxigenesis and the probability of one spore to initiate toxigenesis based upon the storage conditions . Accurate characterization of the microbial ecology of C . botulinum in modified atmosphere-packaged fish, will support safe exploitation of these packaging systems in the market place, and identify critical control points for potential product or process abuses.

Biokhimiia, 1990 May, 55(5), 878 - 87
{Identification and purification of GTP-binding regulatory proteins from plasma membranes of swine heart}; Goffenberg SI et al.; A 23 kDa GTP-binding protein was purified from pig heart sarcolemma . This protein was not ADP-ribosylated by cholera, pertussis and botulinum C3 toxins . In pig heart sarcolemma pertussis toxin ADP-ribosylated 40 kDa subunit of Gi-protein, cholera toxin--45 kDa subunit of Gs-protein, botulinum C3 toxin ADP-ribosylated a group of proteins with Mr 22, 26 and 29 kDa . Antiserum generated against the peptide common for all alpha-subunits of G-proteins did not react with purified 23 kDa protein . Trypsin cleaved the 23 kDa protein in the presence of guanyl nucleotides into a 22 kDa fragment . Proteolysis of the 39 kDa alpha 0-subunit from bovine brain plasma membranes and ADP-ribosylated 40 kDa alpha i-subunit from pig heart sarcolemma in the presence of GTP gamma S yielded the 37 and 38 kDa fragments, respectively . In the presence of GTP and GDP the proteolysis of alpha 0 yielded the 24 and 15 kDa fragments, while the proteolysis of ADP-ribosylated alpha i-subunit yielded a labelled 16 kDa peptide . Irrespective of nucleotides trypsin cleaved the ADP-ribosylated 26 kDa substrate of botulinum C3 toxin into two labelled peptides with Mr 24 and 17 kDa . The data obtained indicate the existence in pig heart sarcolemma of a new 23 kDa GTP-binding protein with partial homology to the alpha-subunits of "classical" G-proteins.

FEBS Lett, 1990 Apr 24, 263(2), 195 - 8
Interaction of small G proteins with photoexcited rhodopsin; Wieland T et al.; Bovine rod outer segment (ROS) membranes contain in addition to the heterotrimeric G protein transducin, several small GTP-binding proteins (23-27 kDa) . Furthermore, these membranes contain two substrate proteins (about 22 and 24 kDa) for botulinum C3 ADP-ribosyltransferase known to ADP-ribosylate small G proteins in any mammalian cell type studied so far . Most interestingly, {32P}ADP-ribosylation of ROS membrane small G proteins by C3 is regulated by light and guanine nucleotides in a manner similar to pertussis toxin-catalyzed {32P}ADP-ribosylation of the alpha-subunit of transducin . These findings suggest that not only the heterotrimeric G protein transducin but also the C3 substrate small G proteins present in ROS membranes interact with photoexcited rhodopsin and thus contribute to its signalling action.

Arch Ophthalmol, 1990 Apr, 108(4), 509 - 10
Botulinum treatment of strabismus following retinal detachment surgery; Scott AB; Twenty patients with strabismus and diplopia following surgery for retinal detachment were treated by botulinum toxin injection of the eye muscles . Twelve patients had regained fusion with elimination of diplopia in the primary position at the time of examination, 5 to 96 months after treatment (mean, 24 months) . Three patients had partial diplopia elimination, and five patients continued to have diplopia.

Ther Umsch, 1990 Apr, 47(4), 320 - 8
{Use of botulinum toxin in ophthalmology}; Huber A; Botulinum toxin A injection in essential blepharospasmus and hemifacial spasmus is an important alternative to surgical therapy . The toxin is injected into the lateral parts of the lower and upper lid in single doses of one to two nanograms under electromyographic control . The effect is visible after a few days and lasts for several months . The procedure can be repeated several times . A second important application of botulinum toxin A is strabismus . In paralytic strabismus the contracture of the antagonist of the paralyzed muscle can be weakened by local injection of botulinum toxin by means of a needle electrode under electromyographic control . Thus the contracture of the homolateral antagonist can be overcome and not seldom singular binocular vision obtained again . In cases of moderate pareses which recover spontaneously the muscle weakening effect of the toxin on the antagonist helps to restore binocular single vision . In cases of chronic paralytic strabismus the toxin injection into the antagonist facilitates the surgical intervention on the paralyzed muscle . In concomitant strabismus botulinum toxin A is above all valuable in small angle cases, in sensory strabismus, in cases of over- or undercorrection after surgery, and these especially in adults . In congenital esotropia, in commitant squints with large angles and in chronic or intermittent exotropia surgery is the preferred modality of treatment . The best results are obtained with repeated small doses . Generally a 65% reduction of the strabismus angle after two to three injections can be expected . With injections into the lids and the extraocular muscles no general systemic side effects has been observed.

J Immunol, 1990 Apr 1, 144(7), 2690 - 5
Functional maturation of human T lymphocytes is accompanied by changes in the G-protein pattern; Pessa-Morikawa T et al.; The putative guanine nucleotide binding (G)-protein involved in transduction of signals from the TCR/CD3 complex has not been identified . We have used a UV-photoaffinity labeling technique to covalently attach {alpha-32P}GTP to human lymphocyte and thymocyte membrane proteins . Ten bands specifically labeled with {32P}GTP were detected by SDS-PAGE and autoradiography in T lymphocyte membranes . Among these, a 40-kDa protein was identified by immunoblotting as the alpha-subunit of the adenylate cyclase-inhibiting G-protein, Gi, and two proteins of 44 and 46 kDa were identified as the alpha-subunits of adenylate cyclase stimulating G-protein (Gs) . These proteins also served as substrates for ADP-ribosylation by pertussis toxin and cholera toxin, respectively . Comparison of GTP-labeled membrane proteins from immature and more mature thymocytes and blood T lymphocytes, revealed that bands of 26, 30, 34, 40, 44 and 46 kDa were absent or weakly labeled in immature thymocytes, intermediate in mature thymocytes, and strongest in blood T cells . Similar increases were seen in ADP ribosylation of the substrates for pertussis, cholera, and botulinum C3 toxin . However, corresponding quantitative changes in Gi and Gs were not detected by immunoblotting, which suggests that the increased labeling is caused by enhanced affinity of the proteins for GTP rather than by increased amount of protein during thymic maturation . A concomitant maturation of GTP-induced cAMP production was seen in the cell populations, but no such change occurred in direct activation of adenylate cyclase by forskolin . The changes in some (but not all) GTP-binding proteins during acquisition of immunocompetence indicates their importance in T lymphocyte physiology.

Biochimie, 1990 Apr, 72(4), 213 - 7
Botulinum neurotoxin type E fragmented with endoproteinase Lys-C reveals the site trypsin nicks and homology with tetanus neurotoxin; Gimenez JA et al.; Botulinum neurotoxin type E, a 150 kDa single chain protein, cleaved with endoproteinase Lys-C yielded 113, 73, and 50 kDa fragments . The N-terminal sequence of the 113 kDa fragment, Gly-Ile-Arg-Lys-Ser-Ile-Cys-Ile, overlaps the N-terminal sequence, Lys-Ser-Ile-Cys-Ile, of the 103 kDa heavy chain produced by nicking the neurotoxin with trypsin . The -Arg-Lys- bond is therefore the site on the single chain type E NT where trypsin nicks generating the 50 kDa light and 103 kDa heavy chains of the dichain NT . The sequence of the first 50 N-terminal residues of the 73 kDa fragment were determined . This fragment is a segment of the heavy chain; 50% of the 50 residues are present in identical positions in a similar segment of the heavy chain of tetanus neurotoxin.

Biochem Biophys Res Commun, 1990 Feb 28, 167(1), 265 - 72
ADP-ribosylation of the rho/rac proteins induces growth inhibition, neurite outgrowth and acetylcholine esterase in cultured PC-12 cells; Nishiki T et al.; Botulinum ADP-ribosyltransferase C3 (C3 exoenzyme) was purified to homogeneity and added to cultured rat pheochromocytoma PC-12 cells . Incubation with this exoenzyme caused inhibition of cell growth and induced neurites as well as acetylcholine esterase in these cells . These changes were dependent on the amount of the enzyme added to the culture, which correlated with the in situ ADP-ribosylation of the rho/rac proteins in the cells . Preincubation with a specific anti-C3 exoenzyme monoclonal antibody inhibited both the ADP-ribosyltransferase activity and the neurite-inducing activity of the enzyme preparation . These results suggest that C3 exoenzyme affected the cellular function of the rho/rac proteins by ADP-ribosylation to induce these changes in the cells.

Neurology, 1990 Feb, 40(2), 277 - 80
Botulinum toxin injections for cervical dystonia; Jankovic J et al.; We followed 205 of 232 patients with medically intractable cervical dystonia for at least 3 months and up to 4 years, during which time they received 1,074 injections in 505 visits . One hundred forty-five of the 205 patients (71%) improved substantially (global rating greater than or equal to 2; from 0 = no response to 4 = marked improvement in severity and function) after 1 or more visits . Of the 89 patients who reported pain, 68 (76%) had almost complete relief of their pain . While most patients noted improvement within the 1st week after injection, some had a latency of up to 8.5 weeks . Duration of maximum benefit lasted up to 12.5 months in some, but the average was 11.2 weeks . Only 58 of 205 (28%) patients, seen in 76 of 505 visits (15% of all visits), had complications, primarily mild dysphagia (35 patients) or neck weakness (17 patients) . We conclude that botulinum toxin is a safe and effective therapy for most patients with cervical dystonia.

Otolaryngol Head Neck Surg, 1990 Feb, 102(2), 122 - 31
Spasmodic dysphonia: botulinum toxin injection after recurrent nerve surgery; Ludlow CL et al.; The purpose of this study was to determine if botulinum toxin injections into the thyroarytenoid muscle would reduce symptoms in adductor spasmodic dysphonic patients who had experienced symptom recurrence after recurrent laryngeal nerve surgery . Five patients were seen between 3 to 10 years after surgery with a return of speech symptoms and persistent unilateral vocal fold paralysis . Before injection, comparisons with controls on spectrographic measures of pitch and voice breaks, aperiodicity, and sentence length demonstrated significant symptoms of spasmodic dysphonia (p less than or equal to 0.02) . Electromyographic measures demonstrated equal levels of thyroarytenoid muscle activation on the operated and non-operated sides with bipolar needle electrodes, and heightened activity in both muscles relative to normal . Therefore, symptom return was associated with thyroarytenoid innervation after recurrent nerve surgery . In all patients, the thyroarytenoid muscle on the side operated on was injected with type A botulinum toxin . In two patients, toxin was also injected on the side not operated on . Significant (p less than or equal to 0.002) reductions in all speech symptoms occurred after injection . Electromyographic measures demonstrated significant reductions in the percent activation levels of both the injected muscle and noninjected muscles (p less than or equal to 0.01) . Botulinum toxin injections were an effective treatment of post-surgical symptom recurrence in adductor spasmodic dysphonia.

Biomed Sci, 1990 Feb, 1(2), 160 - 4
Regulation by bradykinin of phosphoinositide metabolism in the endothelial cells of the pulmonary artery; Voino-Yasenetskaya TA et al.; The effect of the vasodilatory peptide bradykinin on the regulation of phosphoinositide metabolism in endothelial cells was investigated . Activation of phosphoinositide metabolism by bradykinin in the endothelium of the bovine pulmonary artery was not blocked by pertussis toxin, which ADP-ribosylates a membrane protein of molecular mass 40 kDa, but botulinum toxin, which ADP-ribosylates a membrane protein of molecular mass 24 kDa, fully blocked bradykinin-stimulated phosphoinositide metabolism . The effect of bradykinin was potentiated by guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S), an activator of GTP-binding proteins, and inhibited by guanosine 5'-O-(2-thiodiphosphate) (GDP-beta-S), an inhibitor of GTP-binding proteins . Activation of phosphoinositide metabolism by bradykinin was fully blocked by a B2-receptor antagonist, whereas a B1-receptor antagonist did not affect bradykinin action . It is concluded that the B2-receptor in endothelial cells is coupled to phospholipase C via a GTP-binding protein, which is a substrate for botulinum toxin.

Rev Neurol (Paris), 1990, 146(6-7), 440 - 3
{Treatment of spasmodic torticollis by local injections of botulinum toxin}; Jedynak CP et al.; Injections of botulinum toxin into the main cervical muscles responsible for abnormal posture and movements in spasmodic torticollis reduced pain and attenuated dystonia for a period of 2 months on average . After several sessions 9 out of 36 patients (25 p . 100) felt they had improved by at least 50 percent, 16 (44 p.100) by 50 to 75 percent, and 6 (17 p . 100) by more than 75 percent . There were six failures.

Neuroscience, 1990, 35(1), 85 - 91
An antibody to neural cell adhesion molecule impairs motor nerve terminal sprouting in a mouse muscle locally paralysed with botulinum toxin; Booth CM et al.; We have investigated the possible role of neural cell adhesion molecule, in promoting motor nerve terminal sprouting in the paralysed mouse gluteus maximus muscle, using a rabbit polyclonal antiserum to mouse brain neural cell adhesion molecule (donated by Dr C . Goridis) . This antiserum recognizes neural cell adhesion molecule in the basal lamina and extracellular matrix of denervated mouse muscles (1988, Booth C . M . and Brown M . C., Neuroscience 27, 699-709) . As a single dose of antibody injected in vivo over the surface of denervated gluteal muscles bound for 12-48 h to over 80% of fibres expressing neural cell-adhesion molecule, once daily injections were used to see if there was any action on terminal sprouting . Injections of antibody were made for four days, starting three days after muscles were paralysed with botulinum toxin . To avoid non-specific complement-mediated attack on neural cell adhesion molecule bearing motor nerve terminals, mice of the DBA/2 strain were used as these lack complement component C5 . They were further complement depleted by daily intraperitoneal injections of colloidal inulin . The percentage of end plates bearing terminal sprouts (visualized in whole mounts of the gluteus maximus with zinc iodide-osmium staining) was reduced from a mean of 60.5% +/- 3.2 (n = 11) for mice treated with botulinum toxin alone and 61.5% +/- 1.2 (n = 8) for those treated with non-immune serum, to 37.3% +/- 3.6 (n = 8) in the anti-neural cell adhesion molecule injected series.(ABSTRACT TRUNCATED AT 250 WORDS)

J Pediatr Ophthalmol Strabismus, 1990 Jan-Feb, 27(1), 3 - 9
An investigation of the clinical use of botulinum toxin A as a postoperative adjustment procedure in the therapy of strabismus; McNeer KW; Attempts to circumvent multiple surgical procedures are an integral part of the history of investigations in strabismus . In 1973, Botulinum toxin A (Oculinum) injections into human EOM was proposed as an alternate method to surgery for strabismus and has since proved to be effective . This article reports the results from a retrospective investigation of EOM Oculinum injection used in the postoperative period as an alternate to additional surgery . It is difficult to establish whether Oculinum altered the time span of the clinical course in these patients, but when it was effective, it provided unprecedented convenience.

HNO, 1990 Jan, 38(1), 29 - 32
{Essential blepharospasm and botulinum toxin . An electrophysiologic study}; Laskawi R et al.; Thirty-five patients with essential blepharospasm were investigated electrophysiologically after local treatment with subcutaneous periocular injections of botulinum toxin . In all patients the blink reflex was measured on both sides . The results showed in some cases reproducible changes in the reflexes, indicating a central supranuclear origin for the spasm . Furthermore, an influence of afferent information on the "spasm threshold" via the trigeminal nerve is suggested.

Arch Phys Med Rehabil, 1990 Jan, 71(1), 24 - 6
Treatment of detrusor-sphincter dyssynergia with botulinum A toxin: a double-blind study; Dykstra DD et al.; The ability of botulinum A toxin to denervate and relax a spastic external urethral sphincter was evaluated in a double-blind study involving five men with high spinal cord injuries and detrusor-sphincter dyssynergia . The sphincter was injected with either a low dose of botulinum A toxin or normal saline once per week for three weeks . Electromyography of the external urethral sphincter indicated denervation in the three patients who received toxin injections . The urethral pressure profile decreased an average of 25cm of water, postvoiding residual volume of urine decreased an average of 125cc, and bladder pressure during voiding decreased to an average of 30cm of water . Bulbosphincteric reflexes were more difficult to obtain, and they showed a decreased amplitude with normal latency . In the two patients who received normal saline injections, parameters were unchanged from baseline values until subsequent injection with botulinum A toxin once per week for three weeks when their responses were similar to those of the other three patients . Mild generalized weakness lasting two to three weeks was noted by three patients after initial toxin injections . The duration of the toxin's effect averaged two months . The results suggest that botulinum A toxin, an inhibitor of acetylcholine release at the neuromuscular junction, may be useful in the treatment of detrusor-sphincter dyssynergia.

J Physiol (Paris), 1990, 84(2), 180 - 7
Membrane interactions of tetanus and botulinum neurotoxins: a photolabelling study with photoactivatable phospholipids; Schiavo G et al.; Tetanus and botulinum neurotoxins (TeNT and BoNT) bind strongly and specifically to the nervous tissue, as it can be inferred from their potency and from their effects restricted to the nervous system . The molecular basis of these properties are presently unknown . As a first approach, we have investigated the interaction of TeNT and BoNT with model membranes by photolabelling with phospholipid analogues carrying the photoreceptor group at different positions of the lipid molecule in order to probe different membrane regions . We found that at neutral pH TeNT and BoNTs (type A, B and E) adsorb onto the surface of negatively charged liposomes . Polysialogangliosides increase this interaction only slightly thus suggesting that they provide a minor contribution to toxin lipid binding . On this basis we propose that clostridial neurotoxins bind to lipids via both a predominant unspecific interaction with negatively charged lipids (including gangliosides) and a specific, but weaker, interaction with polysialogangliosides . At acidic pH values both chains of these neurotoxins are labelled strongly by photogroups located in the hydrophobic milieu of the membrane with a pH dependence that overlaps the range of pH values reached in the endosomal lumen . This result is consistent with their insertion into the lipid bilayer in agreement with the idea that clostridial neurotoxins may penetrate into cells via intracellular low pH compartments.

J Physiol (Paris), 1990, 84(2), 167 - 73
Trophic interrelations at the neuromuscular junction as revealed by the use of botulinal neurotoxins; Thesleff S et al.; 1 . From denervation studies the trophic influence of the motor nerve on the muscle cell is well documented while little is known about the influence of the muscle on the nerve . Sectioning the axon invariably destroys the nerve terminals and produces nerve degeneration products which themselves may affect nerve and muscle properties . With regard to those difficulties we believe that the botulinal neurotoxins (BoTx) are valuable complements to denervation since they selectively interrupt impulse transmission across the synapse without damaging its morphology . 2 . Paralysis of mouse or rat skeletal muscle in vivo with BoTx type A causes marked growth of motor nerve terminals . The sprouting terminals are rich in large dense-core synaptic vesicles containing various neuropeptides and they spontaneously release large quanta of ACh . Thus, it appears that paralysis by BoTx is a strong stimulus for motor nerve growth and the delivery of "trophic" substances to the nerve terminals . 3 . Postsynaptically, in extrajunctional areas, paralysis by BoTx induces all the changes observed following denervation, i.e . atrophy, appearance of extra-junctional ACh receptors, TTX-resistant action potentials, a fall of resting membrane potential, fibrillation potentials and the disappearance of extrajunctional acetylcholinesterase activity . Endplate properties are, however, largely maintained . 4 . BoTx blockade delays and prevents the retraction of polyneuronal innervation and motoneurone death during development . This supports the suggestion that the paralysed muscle secretes factors essential for growth and for the survival of motoneurones . 5 . Like denervated muscle, BoTx paralysed ones, express a high endocytotic activity restricted to a segment in the endplate region.(ABSTRACT TRUNCATED AT 250 WORDS)

J Physiol (Paris), 1990, 84(2), 152 - 66
Presynaptic actions of botulinal neurotoxins at vertebrate neuromuscular junctions; Molgo J et al.; 1 . In the present paper we review some presynaptic aspects of the mode of action of botulinal toxins (BoTxs) at vertebrate neuromuscular junctions with emphasis on studies carried out in our laboratories using electrophysiological and morphological techniques . 2 . Spontaneous quantal transmitter release recorded as miniature end-plate potentials is drastically affected by BoTxs . The low probability of release at poisoned terminals can be enhanced by carbonyl cyanide m-chlorophenylhydrazone (CCCP), Cd2+ and La3+ . However, CCCP and La3+ which drastically deplete clear synaptic vesicles from unpoisoned terminals failed to markedly affect the density of synaptic vesicles at poisoned terminals . It is concluded that poisoned terminals have a reduced sensitivity to the release-promoting action of Ca2+, Cd2+ and La3+ . 3 . When comparing the effect of the various BoTxs on nerve-impulse evoked transmitter release it appears that increasing phasic Ca2+ entry into the terminals enhances evoked synchronized quantal release only from terminals poisoned with serotypes A and E . In contrast, enhanced Ca2+ entry into terminals poisoned with serotypes B, D and F induced a period of high frequency asynchronous release suggesting that these BoTxs may affect a presynaptic step beyond the influx of Ca2+, that may be involved in the synchronization of transmitter quanta . These data suggest that the actions of BoTxs involve several steps of the acetylcholine release process . 4 . The analysis of presynaptic currents which depend on both Ca2+ entry and intraterminal background Ca2+ levels strongly suggests that neither Ca2+ entry nor intraterminal Ca2+ levels are altered by BoTxs . Furthermore, poisoned terminals are no more efficient than unpoisoned ones in dealing with Ca2+ overloads . 5 . Finally, the morphological examination of junctions paralysed by BoTx-A indicates that the toxin triggers a particularly important overgrowth of the nerve terminals and suggests that the in vivo functional recovery may occur from an extension of the original nerve terminal arborization and the concomitant remodelling of postsynaptic structures.

J Physiol (Paris), 1990, 84(2), 143 - 51
The study of clostridial and related toxins . The search for unique mechanisms and common denominators; Simpson LL; Experiments have been conducted that deal with the structure and biological activity of clostridial toxins . Studies have dealt mainly with botulinum neurotoxin, but work has also been done with tetanus toxin and with the binary toxin . Structural studies indicate that proteolytic processing of botulinum neurotoxin induces two major outcomes: activation and aging . The first is associated with a marked increase in toxicity and with conversion from a single chain to a dichain structure . The second is associated with nominal changes in toxicity and with molecular rearrangements in the dichain structure . Immunological studies have resulted in isolation and characterization of a monoclonal antibody that neutralizes tetanus toxin . Monoclonal antibodies have also been raised against botulinum neurotoxin, and these antibodies have been used to demonstrate that: i) activation is not due to marked conformational changes in the relevant epitopes, ii) binding of the toxin to cholinergic nerve endings does not produce detectable conformational changes, and iii) all functional domains of the toxin appear to be internalized simultaneously . Immunological studies done in vivo and in vitro suggest that certain antibodies may enter cholinergic nerves and neutralize subsequently internalized toxin . Additional work on clostridial toxins has produced the following results: i) the ligand binding assay typically used with tetanus toxin (i.e., low pH and ionic strength) is of questionable biological significance, ii) the binary toxin, like the clostridial neurotoxins, enters cells by receptor-mediated endocytosis, and iii) tetanus toxin can alter the disposition of protein kinase C in one neuroblastoma cell line.

J Urol (Paris), 1990, 96(7), 375 - 80
{Effects of Botulinum A Toxin on the periurethral striated sphincter of the neurogenic bladder . Preliminary study}; Schurch B et al.; The goal of this study was to try to determine the effects of the Botulinum A Toxin on the spasticity of the rhabdosphincter in 9 men with spinal cord injury and detrusor-sphincter dyssynergia . The cystometrography, before and after the endoscopic injection of 100 units of Botulinum A Toxin, consisted of recording the bladder, urethral and rectal pressures with microtip transducers the anatomical position of which was radiographically controlled . The subjective and objective results of that study allow us to conclude that the Botulinum A Toxin has a place in the treatment of spinal injuries with detrusor-sphincter dyssynergia . Due to his blocking effect on the release of acetylcholine in the motor nerve endings, the Botulinum A Toxin suppresses or decreases the spasticity of the rhabdosphincter and improves voiding . Although its relatively short living action (2-3 months) may require renewed injections, it has the advantage to hold off a surgical treatment such as a sphincterotomy and to give the patient another chance to reach a balanced bladder function secondary to the postinjection changes of reflexes which may have taken place between the bladder and the rhabdosphincter and vice versa.

Eur Arch Otorhinolaryngol, 1990, 247(6), 391 - 2
Botulinum toxin to suppress hyperkinesias after hypoglossal-facial nerve anastomosis; Dressler D et al.; Facial hyperkinesias are a common side effect of hypoglossal-facial nerve anastomoses . We report a patient whose facial hyperkinesias were suppressed by botulinum toxin injections a treatment recently introduced in the therapy of craniocervical dystonias . EMG studies are used to document the effect of botulinum toxin on the facial hyperkinesias as well as on voluntary muscle activation.

Ophthal Plast Reconstr Surg, 1990, 6(4), 252 - 9
Botulinum A toxin injection . Failures in clinical practice and a biomechanical system for the study of toxin-induced paralysis; Holds JB et al.; Botulinum A toxin injection has great utility in the treatment of essential blepharospasm and other facial spasm disorders . Several investigators have noted the failure of botulinum toxin injections to relieve lid spasm in occasional patients and a decrease in effectiveness or duration of effect following multiple injections in other patients . We reviewed the charts of 30 consecutive patients presenting for the evaluation or treatment of facial dystonia . Of 20 patients who had received multiple injections of botulinum toxin, 10 patients were felt to be treatment failures . A new biomechanical system was developed to investigate the duration and degree of paralysis induced in the gastrocnemius muscle of the rat . Animals were treated with four sequential injections at 6-week intervals to the same muscle, resulting in muscle atrophy and an increase in the duration and degree of muscle paralysis, contrary to clinical findings in humans . The review of patient data confirms that, for many patients, repeated injection of botulinum toxin results in a decrease in duration and degree of effect despite an increased toxin dose . An opposite effect was noted in our experimental model because of progressive muscle atrophy.

Neurologija, 1990, 39(1), 29 - 33
Local treatment of spasmodic torticollis with botulinum toxin; Kostic V et al.; Fifteen patients with spasmodic torticollis were treated with local injections of botulinum--A toxin . All the patients were followed for a period of 4 to 7 months . Thirteen out of 15 patients (86%) improved in the amount of sustained movements of torticollis . In six out of 9 patients presenting with pain complete relief was noted . Beneficial effects of botulinum toxin injections lasted from 2 to 3 months, with reproducible efficacy after repeated injections . The most frequent side effect was dysphagia, presented in 10 patients.

J Fr Ophtalmol, 1990, 13(5), 259 - 64
{Treatment of facial spasm with botulin A toxin}; Adenis JP et al.; The authors use injections of botulinum A toxin in treatment of facial spasms . 105 patients (70 patients with blepharospasm, 35 patients with hemispasm) were treated during 4 years . The toxin Wisconsin relieved spasms for an average of 10 weeks . Local complications such as ptosis and diplopia appeared in less than 1/5 of the cases . No general complications were observed . Repeated treatments are necessary . The authors noted a decrease of lacrimal secretion on the treated side in hemifacial spasm and concluded that botulinum A toxin injections could be used as a treatment of epiphora.

Ann Otolaryngol Chir Cervicofac, 1990, 107(6), 363 - 5
{Objective tinnitus and palatal myoclonus . A new therapeutic approach}; Le Pajolec C et al.; The syndrome combining velar myoclonia with objective tinnitus is rare and, in adults, it corresponds most of the times to a neurological lesion of the dento-olivary tractus . Its functional manifestations are often invalidating and propounded medical treatments uneffective . The authors report a case in which local injection of botulinus toxin allowed to obtain satisfactory functional improvement . Such therapy may prove useful when conventional management is failing.

Graefes Arch Clin Exp Ophthalmol, 1990, 228(5), 401 - 6
Vertical rectus muscle transposition with intraoperative botulinum injection for treatment of chronic sixth nerve palsy; McManaway JW 3rd et al.; Six adult patients with acquired, chronic, complete sixth nerve palsy had vertical rectus muscle transposition to the lateral rectus muscle insertion with intraoperative injection of botulinum toxin into the ipsilateral medial rectus muscle . Five of six patients were orthophoric in primary gaze, and the remaining patient achieved single binocular vision with a small head turn . This method compares favorably with previously described transposition procedures for sixth nerve palsy in terms of amount of correction and size of the window of single binocular vision with a lower risk of anterior segment ischemia.

Eye, 1990, 4 ( Pt 4), 538 - 42
Botulinum toxin therapy in dysthyroid strabismus; Lyons CJ et al.; We report our experience with the use of Botulinum toxin injection in 38 patients (64 injections) with severe dysthyroid strabismus . Three quarters of the injections led to a decrease in the angle of the squint by a mean 75% of the initial deviation . The average duration of effect was two months . Twenty six patients went on to surgery after stabilisation of their squint and endocrine status . Six patients achieved a stable long-term result with Botulinum toxin only . We suggest these results of treatment of early dysthyroid myopathy are more consistent with the characteristics of inflammatory spasm than contracture . The value of Botulinum toxin as a temporary means of maintaining binocularity in these young patients is discussed.

Clin Neurol Neurosurg, 1990, 92(2), 165 - 8
Responsiveness of idiopathic spasmodic torticollis to botulinum A toxin injection . A critical evaluation of five cases; Maurri S et al.; Five patients with Idiopathic Spasmodic Torticollis (IST), one associated with Meige's disease, have been cured by local injection of purified botulinum "A" toxin (BT) . The therapeutic effect on the different subjects treated proved to be variable, and often the effect varied as well on the same subject from session to session . The response was only partly related to the dose of BT injected, and appeared inversely proportional to the number of hyperactive muscles . Transient dysphagia was the major untoward effect of the treatment . The inconstant response to BT makes us consider BT worth to be used selectively in the so-called "agonistic" cases of IST.

Brain Res Mol Brain Res, 1990 Jan, 7(1), 9 - 16
Purification and characterization from bovine brain membranes of a GTP-binding protein with a Mr of 21,000, ADP-ribosylated by an ADP-ribosyltransferase contaminated in botulinum toxin type C1--identification as the rhoA gene product; Hoshijima M et al.; We have previously shown that there are multiple GTP-binding proteins (G proteins) with Mr values of about 20,000 in bovine brain membranes and identified one G protein with a Mr of 20,000 as the rho gene product . We have also shown that this rho gene product is ADP-ribosylated by an ADP-ribosyltransferase contaminated in botulinum toxin type C1 . In the present studies, we have purified another G protein with a Mr of about 21,000 to near homogeneity from bovine brain membranes by several column chromatographies and identified it as the rhoA gene product . Further analysis of the amino acid sequence of the G protein, which we have purified and identified as the rho gene product previously, has revealed that this G protein is the rhoB gene product . The rhoA gene product binds maximally about 0.9 mol of {35S}guanosine 5'-(3-O-thio) triphosphate (GTP gamma S)/mol of protein with a K d value of about 20 nM . {35S}GTP gamma S-binding to the rhoA gene product is inhibited by pretreatment with N-ethylmaleimide . The rhoA gene product hydrolyzes GTP to liberate Pi with a turnover number of about 0.01 min-1 . Moreover, the rhoA gene product is ADP-ribosylated by an ADP-ribosyltransferase contaminated in botulinum toxin type Cl . About 0.3 mol of ADP-ribose is maximally incorporated into 1 mol of the rhoA gene product . The ADP ribosylation of the rhoA gene product does not affect its GTP gamma S-binding or GTPase activity . These properties of the rhoA gene product are similar those of the rhoB gene product described previously . These results together with the earlier observations indicate that there are at least two rho gene products (rhoA, B) among three members of the rho gene family (rhoA, B, C) in bovine brain membranes and that both of them are ADP-ribosylated by an ADP-ribosyltransferase contaminated in botulinum toxin type C1.

Toxicon, 1990, 28(8), 992 - 6
Identification of specific domains in botulinum and tetanus neurotoxins; Singh BR; Specific domains of botulinum and tetanus neurotoxins have been identified by computing the bias in the use of Lys over Arg in their respective polypeptide chains (E . London and C.L . Luongo, Biochem . biophys . Res . Commun . 160, 333-339, 1989) . A strong bias was noted in the C-terminal domain of the light chains of both the neurotoxins (bRK', -8.0 to -12.0) suggesting that this domain could represent a 'catalytic domain' similar to that present in other dichain toxins such as diphtheria . Interestingly, this domain has a segment which has significant homology with the partial sequence of botulinum exoenzyme C3, and ADP-ribosyl transferase, implying a possible relationship with an enzymatic activity.

J Neurosci, 1990 Jan, 10(1), 317 - 22
Multiple GTP-binding proteins from cholinergic synaptic vesicles; Ngsee JK et al.; Cholinergic synaptic vesicles purified from the electric organ of the marine ray, Discopyge ommata, contain 2 different size classes of GTP-binding proteins: one or more with an apparent molecular weight (MW) between 37 and 41 kDa, and 3 major and at least 2 minor proteins with MW between 20 and 29 kDa . These GTP-binding proteins were detectable using the alpha 32P-GTP overlay technique and covalent modification with bacterial toxins . The higher MW GTP-binding proteins are ADP-ribosylated by pertussis toxin and 2 of the lower MW GTP-binding proteins are sensitive to botulinum toxin.

Medicina (B Aires), 1990, 50(2), 129 - 34
{Treatment of blepharospasm with botulinum toxin}; Pikielny RT et al.; Blepharospasm is a relatively frequent cranial dystonia which may be seen either alone or related to orofacial-mandibular dystonia (Meige's syndrome) . In its maximum degree it can cause functional blindness.Twelve patients with blepharospasm (4 essential and 8 Meige's syndrome) who had been previously treated unsuccessfully with drugs (trihexyphenidyl, biperiden, carbamazepine, lithium, baclofen, lisuride, imipramine, clonazepam and butyrophenones) were treated for 12 months with periocular injections of botulinum toxin (BOTOX) . A "low" dose of 12,5 U per eye was employed . With this dose, eleven out of twelve patients experienced significant improvement which lasted from five to fifteen weeks . The only nonresponder obtained complete relief upon duplicating the dose . The only side effect was uni or bilateral ptosis in six patients which improved completely in seven to twenty one days . One patient developed a peripheral facial palsy with complete remission in nineteen days . No systemic side effects were noted . There was only one desertion from this study due to depression enhanced by prolonged (21 days) ptosis . All patients (including the deserter) agreed that treatment with BOTOX provided more relief than any other previous therapeutic method . Our results confirm those obtained by others but a more prolonged study is needed to better evaluate long term effects.

Neuroscience, 1990, 39(3), 711 - 5
Release of vasopressin from isolated permeabilized neurosecretory nerve terminals is blocked by the light chain of botulinum A toxin; Dayanithi G et al.; The intracellular action on exocytosis of botulinum A toxin and constituent chains was studied using permeabilized isolated nerve endings from the rat neural lobe . The release of the neuropeptide vasopressin was measured by radioimmunoassay . In the presence of the reducing agent dithiothreitol, the two-chain form of botulinum A toxin inhibited vasopressin release induced by 10 microM free calcium . Half maximal inhibition was obtained with 15 nM botulinum A toxin . In the absence of the heavy chain the light chain of the toxin strongly inhibited exocytosis with a half maximal effect of 2.5 nM . The inhibitory effects on secretion could be prevented by incubating the light chain with an immune serum against botulinum A toxin . The heavy chain of botulinum A toxin did not affect vasopressin release . However, it prevented the inhibitory effects of the light chain on stimulated exocytosis . It is concluded that botulinum A toxin inhibits the calcium-dependent step leading to exocytosis by interfering with a target present in the isolated and permeabilized nerve terminals . The functional domain of this neurotoxin, which is responsible for the inhibition of vasopressin release, is present in its light chain.

J Physiol (Paris), 1990, 84(4), 285 - 9
Botulinum toxin A in clinical medicine; Elston JS; The results of the treatment with botulinum toxin A injection of 234 patients with blepharospasm and 73 with hemifacial spasm were reviewed . Visual function improved in the majority of patients with blepharospasm, and the improvement was sustained for up to 40 sets of injections . Mid and lower facial movements were also reduced in a minority of patients . However, a sub-group with pre-tarsal blepharospasm or persistent levator inhibition after treatment had a poor response . An average 75% reduction in abnormal movements was seen in cases of hemifacial spasm . Side effects of the treatment were usually mild and short lived.

J Physiol (Paris), 1990, 84(3), 229 - 36
Chains and fragments of tetanus toxin, and their contribution to toxicity; Ahnert-Hilger G et al.; 1 . Single-chain toxin is enzymatically converted into two-chain isotoxins which differ from the precursor by their higher pharmacological activity, acidity and hydrophilicity . The interchain disulfide bridge and the disulfide loop within fragment C have been located at the amino acid level . 2 . Independent of the enzymes used, the nicking sites are positioned within a region spanning no more than 17 amino acids . The N- and C-termini of the primary gene product are preserved in the two-chain toxin . The chains have been separated by isoelectric focussing and can be reconstituted to functionally intact toxin . 3 . Light chain inhibits neurotransmitter release on different systems . First, permeabilized bovine adrenal chromaffin cells and rat pheochromocytoma (PC 12) cells release catecholamines when exposed to micromolar {Ca2+} . Inhibition is achieved with light chain or reduced two-chain toxin, but not with single-chain toxin or heavy chain . Washing away the light chain does not restitute the Ca2(+)-evoked release . The light chains of tetanus and botulinum A toxin act in a apparently similar, however not identical manner . Second, light but not heavy chain inhibits the release of acetylcholine when injected into Aplysia neurones . 4 . The pharmacology of heavy chain is quite different . Ganglioside binding is mediated by its fragment C moiety, and modulated by the adjoining beta 2 piece and by light chain . Heavy chain and to a lesser degree its N-terminal beta 2-fragment promote the loss of calcein from liposomes indicating pore formation . Its C-terminal fragment C is inactive in this respect.(ABSTRACT TRUNCATED AT 250 WORDS)

J Physiol (Paris), 1990, 84(3), 220 - 8
Structure and biological activity of botulinum neurotoxin; DasGupta BR; Botulinum neurotoxin appears to undergo structural alterations after synthesis and also before it inhibits neurotransmitter release . Discussions and conjectures are presented in this context: 1 . At what sites on the 150 kDa neurotoxin does posttranslational proteolytic processing occur? 2 . Does neurotransmitter inhibition depend on separation of a segment of the neurotoxin from the rest of the molecule? 3 . At what step in the intoxication pathway does activation of neurotoxin (enhanced lethality following limited proteolysis) manifest? 4 . Can the receptor binding parameters (based on bovine brain synaptosome and lipid membrane), channel forming property (lipid bilayer membrane) and intracellular inhibitory activity (based on permeabilized chromaffin and PC 12 cells) provide clues to differences in the lethal potency between the neurotoxin serotypes? In addition, the following issues are considered: 5 . The spontaneous fragmentation of isolated 50 kDa light chain, after its separation from 100 kDa heavy chain, 6 . Effect of specific chemical modification of Arg, His, Lys, Trp, Tyr and Asp/Glu residues of types A, B and E neurotoxins on lethality and antigenicity, and 7 . Development of second generation toxoids.

J Physiol (Paris), 1990, 84(4), 247 - 61
Inhibition of neurotransmitter release by botulinum neurotoxins and tetanus toxin at Aplysia synapses: role of the constituent chains; Poulain B et al.; 1 . The effects on the release of transmitter by botulinum neurotoxins (BoNT; types A, B, E), tetanus toxin (TeTx), constituent chains or fragments were studied on identified cholinergic and non-cholinergic synapses in Aplysia . 2 . Cholinergic synapses in the buccal ganglion were found to be greater than 100 fold more sensitive to extracellular application of BoNT than to TeTx whereas in non-cholinergic synapses of the cerebral ganglion the potencies of the toxins were reversed . When intracellularly applied TeTx and BoNT were found nearly equipotent . This disparity in the susceptibilities of BoNT and TeTx to inhibit transmission was attributed to differences in the toxin's acceptors or uptake systems in the two neurone types . 3 . Micro-injection into cholinergic neurones of the isolated renatured toxins' chains showed that both light and heavy chains of BoNT are intracellularly required whereas the light chain of TeTx alone is sufficient . 4 . The heavy chain of BoNT as well as that of TeTx were found to mediate internalization of active moieties via its amino-terminal half . Furthermore the heavy chain of one toxin could internalize the light chain of the other.

J Pharmacol Exp Ther, 1990 Jan, 252(1), 135 - 9
Pharmacologic characterization of the changes in cholinergic sensitivity of rat jejunal circular muscle after myenteric plexus ablation; Herman JR et al.; Neurons located in the myenteric plexus are generally believed responsible for motor control of intestinal circular muscle . The in vitro isometric responses of naive and myenterically denervated (MD) rat jejunal circular muscle to bethanechol and carbachol, alone and in the presence and absence of neuronal antagonists (hexamethonium bromide, tetrodotoxin and Botulinum toxin A) 15 and 30 days after myenteric plexus ablation, were determined . The responses to bethanechol indicated no differences in muscarinic sensitivity between naive and MD tissue . The relative potency of carbachol, which acts at both muscarinic and nicotinic receptors, in MD tissue 15 days after denervation was significantly higher than that in naive tissue . However, 30 days after denervation, the relative potencies of carbachol in naive and MD circular muscle were comparable . The presence of neuronal antagonists had no effect on the relative potency of carbachol 15 days after myenteric denervation, but altered significantly the responses 30 days after denervation . The effects produced by the neuronal antagonists 30 days after myenteric denervation were qualitatively and quantitatively different than those produced in naive tissue, suggesting that the nature of the innervation in these tissues was different . These results demonstrate that circular muscle was denervated initially after myenteric plexus ablation but reinnervation occurred within 30 days . The reinnervation observed is likely due to neurons located in the submucosal plexus.

J Physiol (Paris), 1990, 84(3), 237 - 46
Clues to the multi-phasic inhibitory action of botulinum neurotoxins on release of transmitters; Dolly JO et al.; 1 . With the aim of gaining insight into the mechanism of Ca2(+)-dependent secretion, inhibition of transmitter release by botulinum neurotoxins or their fragments was studied at mammalian motor nerve terminals, cerebrocortical synaptosomes and PC-12 cells . 2 . Relative to BoNT type A, the feeble neuromuscular paralytic activity of its two chains and the lack of activity observed with a proteolytic fragment, H2L (lacking H1, the C-terminal half of the heavy chain) highlight a requirement of the intact, disulphide-linked dichain protein for efficient targetting (binding/uptake) to peripheral cholinergic nerve endings . 3 . In PC-12 cells, the renatured light chain alone proved equally potent as the whole toxin in reducing Ca2(+)-evoked noradrenaline release, when digitonin-permeabilization was used to overcome the uptake barrier . Treatment of BoNT A with 10 mM dithiothreitol, under non-denaturing conditions, was not very effective in reducing its inter-chain disulphide bond(s) and had little influence on the level of inhibition seen . 4 . Altering the intra-synaptosomal concentrations of cyclic nucleotides (c-AMP, c-GMP) or protein kinase C activity failed to affect the reduction of Ca2(+)-dependent K(+)-stimulated noradrenaline release caused by BoNT A or B . On the other hand, raising the cytosolic Ca2+ concentration with the ionophore A23187 reversed the inhibitory effect of BoNT A to a greater extent than that of type B, revealing differences in their actions . 5 . Whereas BoNT-induced decrease of Ca2(+)-dependent K(+)-evoked release of noradrenaline was unaffected by destruction of the actin-based cytoskeleton in synaptosomes with cytochalasin D, disassembly of microtubules with colchicine, nocodazole or griseofulvin antagonised the intracellular action of type B but not A . It is speculated that BoNT B blocks transmitter release by interfering with the proposed detachment of synaptic vesicles from microtubules . Establishing the precise involvement of tubulin in the toxin's action may provide a valuable clue to the mechanism of neurotransmitter release or its control.

J Physiol (Paris), 1990, 84(3), 211 - 9
Type A botulinum toxin disorganizes quantal acetylcholine release and inhibits energy metabolism; Dunant Y et al.; The physiological, morphological and biochemical effects of type A Botulinum toxin (BoTX) were analysed in the electric organ of Torpedo, a modified neuromuscular system . The quantal content of the postsynaptic potential, or electroplaque potential (EPP), was reduced by BoTX but the quantum size remained unchanged till complete failure of the neurally evoked transmission . BoTX also suppressed the occurrence of spontaneous electroplaque potentials (MEPPs) of a quantal size but potentials of a smaller amplitude still kept on occurring in the intoxicated synapses . BoTX inhibited the evoked release of acetylcholine (ACh; biochemically measured) but the rate of spontaneous ACh release transiently increased during the period when evoked release went down . On the other hand, there were no significant change of ACh content, of ACh turnover, of ACh repartition in the vesicular and free compartments, or in the number of synaptic vesicles . Surprisingly, the amount of ATP was reduced to 50% in BoTX treated tissue at the time of transmission failure; also the level of creatine phosphate (CrP) was lowered to less than 20% and the rate of activity of creatine kinase was reduced . It was concluded that, electrophysiologically, BoTX affects synaptic transmission in a very similar way in the electric organ and in the neuromuscular junctions . On the other hand, the shortage of ATP supply found in the present study may play a role in the pathophysiology of intoxication and should be taken into account in investigations designed to see whether BoTX affects various phosphorylations in cholinergic nerve terminals.

J Physiol (Paris), 1990, 84(2), 174 - 9
The action of botulinum toxin on cholinergic nerve terminals isolated from the electric organ of Torpedo marmorata . Detection of a putative toxin receptor; Solsona C et al.; Botulinum neurotoxin type A (BoNTx) inhibits the release of acetylcholine (ACh) from Torpedo electric organ synaptosomes . We have studied several biochemical and morphological aspects in order to characterize the molecular interactions of BoNTx intoxication in our preparation . 1 . We are describing for the first time an electrophoretic band from cholinergic presynaptic plasma membrane (PSPM) that is recognized by 125I-BoNTx as a putative BoNTx receptor . 2 . Furthermore we describe direct interaction of botulinum toxin-gold complexes with synaptic vesicles through the three-step model of the BoNTx intoxication.

J Physiol (Paris), 1990, 84(2), 188 - 90
Channels formed in phospholipid bilayer membranes by diphtheria, tetanus, botulinum and anthrax toxin; Finkelstein A; Diphtheria, tetanus, botulinum, and anthrax toxin are multipartate toxins, one of the domains of which is (or is presumed to be) an enzyme . Cell intoxication requires that the enzymatic portion gain access to the cytosol via endocytosis into an acidic vesicle compartment of the cell . Translocation of the enzyme across the vesicular membrane is dependent on the low pH of the vesicle and involves another domain of the toxin; for each of these toxins, that domain is capable of forming channels in phospholipid bilayer membranes . These channels are large (greater than 12 A diameter) and voltage-gated, and the pH conditions required for their formation in lipid bilayers are similar to those existing in acidic vesicles and required for cell intoxication.

J Biol Chem, 1989 Dec 25, 264(36), 21928 - 33
Multiple domains of botulinum neurotoxin contribute to its inhibition of transmitter release in Aplysia neurons; Poulain B et al.; The binding, internalization, and inhibition of transmitter release by botulinum neurotoxin (BoNT) was investigated using the intact toxin, its heavy (HC) or light (LC) chains, and a proteolytic fragment thereof . In Aplysia neurons, blockade of acetylcholine release upon external application of BoNT types A or E was prevented by reducing the temperature to 10 degrees C, due to arresting intoxication at the membrane binding step . At this low temperature, type A HC, H2 (comprised of the N-terminal of HC), or H2L (H2 disulfide-linked to LC) antagonized the neuroparalytic action of BoNT A or E, indicating that the latter bind saturably to common ecto-acceptor via the H2 region . In contrast, H2L was unable to counteract BoNT-induced paralysis at the murine neuromuscular junction . In accordance with this species difference, unlike native BoNT, saturable binding of 125I-labeled H2L could not be detected in mammalian peripheral or central nerve terminals . Possibly, more stringent structural requirements form the basis of the toxin's greater effectiveness in inhibiting neurotransmission at mouse nerve muscle synapses than Aplysia nerve terminals . In further identification of functional domains in the toxin, an unprocessed single-chain form of BoNT type E was found to be ineffective when applied extra- or intracellularly to Aplysia neurons . Notably, bath application of the latter to a neuron preinjected with HC, but not H2L or LC, resulted in a blockade of release . This shows that the single-chain species can become internalized and requires, not only LC, but also processed HC for its inhibitory action; consistently, the proteolyzed form of BoNT E was active.

FEBS Lett, 1989 Dec 18, 259(1), 67 - 70
Histamine and bradykinin stimulate the phosphoinositide turnover in human umbilical vein endothelial cells via different G-proteins; Voyno-Yasenetskaya TA et al.; The G-proteins which regulate hormonal turnover of phosphoinositide (PI) in human umbilical vein endothelial cells have been investigated . A 40-41 kDa doublet present in the membranes of these cells was selectively ADP ribosylated by pertussis toxin (PTx), and this doublet was Gi alpha 2 and Gi alpha 3 according to immunoblotting with specific antisera . By contrast, a doublet of 24-26 kDa proteins in the same membrane preparations was ADP ribosylated by the C3 component of botulinum toxin (BoTx) . PTx-dependent ADP ribosylation blocked stimulation of PI turnover by histamine, but did not affect stimulation by bradykinin, whereas BoTx (C2 + C3 components) had the opposite effect . Thus two different groups of G-proteins may be involved in hormone-dependent stimulation of PI turnover in human umbilical vein endothelial cells.

Clin Chim Acta, 1989 Dec 15, 185(3), 347 - 55
Small molecular weight GTP-binding proteins and signal transduction; Yamamoto K et al.; We have separated multiple GTP-binding proteins (G proteins) having Mr values of about 20,000 (small Mr G proteins) from bovine brain membranes, purified to near homogeneity and characterized two novel G proteins designated as smg p25A and smg p21, the c-Ki-ras protein (c-Ki-ras p21) and the two rho proteins (rho p20 and rho p21) . smg p25A is present abundantly in brain and adrenal medulla . This G protein is also found in rat pheochromocytoma PC-12 cells, and its mRNA level increased after differentiation of the cells into neuron-like cells in response to nerve growth factor or dibutyryl cyclic AMP . These results suggest that smg p25A plays an important role in the regulation of neuronal functions . In contrast, smg p21 is found in most tissues . This G protein has the same putative effector domain as ras p21s, suggesting that smg p21 exerts the actions similar and/or antagonistic to those of ras p21s . In fact, smg p21 has been found to be identical with the protein encoded by the Krev-1 gene recently isolated as a gene suppressing the transforming action of Ki-ras p21 in NIH/3T3 cells . On the other hand, rho p20 and rho p21 are ADP-ribosylated by an ADP-ribosyltransferase contained or contaminated in botulinum toxin type C1, presumably C3 . Botulinum ADP-ribosyltransferase C3 has recently been shown to induce morphological changes similar to those induced by ras p21 in fibroblasts . Thus, small Mr G proteins are part of a huge network of intracellular regulatory systems and play important roles in the regulation of various cell functions including cell transformation, proliferation and differentiation.

Biull Eksp Biol Med, 1989 Dec, 108(12), 707 - 10
{The effect of bacterial antigens on lymphocyte immune receptors in mice}; Burlakov GV; It has been shown that the treatment of a suspension of lymphocytes obtained from spleens in mice by LPS S . sonnei as well as by exotoxins Cl . tetani and Cl . botulinum has led to a notable reduction in the number of luminescent cells by comparison with control samples . The action of tested remedies was found to increase with their concentration . These was complete correlation between these results and the data obtained by the immunity rosette formation method . A reliable reduction was also noted in the number of lymphocytes forming rosettes with the sheep erythrocytes . It has been inferred that the bacterial products tested possessed a marked capacity for blocking the immune receptors of lymphocytes.

Mo Med, 1989 Dec, 86(12), 815 - 7
Neurological application of botulinum toxin; Kumar P et al.; Botulinum toxin, a potent biological toxin with paralytic properties, has found use as a therapeutic agent in various diseases . The authors review the toxin's therapeutic value in three neurological diseases.

Acta Physiol Scand, 1989 Dec, 137(4), 497 - 501
Characterization of the actions of botulinum neurotoxin type E at the rat neuromuscular junction; Molgo J et al.; Botulinum neurotoxin (BoTx) serotype E blocks spontaneous and evoked quantal release of acetylcholine at the rat neuromuscular junction . Increasing extracellular Ca2+ to 8 mmol l-1 or substituting Ca2+ with La3+ (0.1 and 1.0 mmol l-1) or depolarizing the nerve terminals by 20 mmol l-1 K+ markedly increases miniature end-plate potential frequency in normal muscle, but in BoTx-E poisoned preparations none of these ions, with the exception of 1 mmol l-1 La3+, was able to restore spontaneous quantal transmitter release to levels recorded at unpoisoned junctions . In absolute values the enhancement with La3+ was much less than that reported at normal junctions . Nerve stimulation in the presence of 3,4-diaminopyridine (10-20 mumol l-1) and high calcium (8 mmol l-1) evoked multiquantal end-plate potentials and muscle twitches . We conclude that the neuromuscular block produced by BoTx serotype E is similar to that previously described for BoTx serotype A but differs from that produced by BoTx serotypes B, D and F in not causing desynchronization of nerve impulse-evoked transmitter release . 3,4-Diaminopyridine might be useful in the treatment of poisoning by BoTx serotype E since it markedly enhanced synchronous transmitter release from poisoned motor nerve terminals.

Ital J Neurol Sci, 1989 Dec, 10(6), 553 - 60
Facial dystonia: clinical features, prognosis and pharmacology in 31 patients; Defazio G et al.; The natural history and response to different treatments were assessed in 31 consecutive patients with blepharospasm (BS) and/or oromandibular dystonia (OMD) . The mean age at onset was 52.4 years and there was a female preponderance of 2.5 to 1 . Ocular symptoms preceded the onset of blepharospasm in more than 50% of the affected patients, whereas psychiatric and dental problems prior to the onset of focal dystonia were found in 10% and 13% of the cases respectively . Dystonia elsewhere, mainly in the craniocervical area, was found in 23% of patients and appeared to follow a somatotopic progression . The first 2-3 years of history were crucial for the spread of dystonia to other face and body parts . When OMD was the first symptom, a lower tendency of dystonia to progress elsewhere was observed . A putative cause was found in 14% of patients who showed clinical and radiographic evidence of basal ganglia or rostral brainstem-diencephalon lesions . The response to different drugs was inconsistent although transient improvement was induced by haloperidol in 6 patients, by L-Dopa plus deprenyl in 3 patients, by trihexyphenidyl in 2 patients and by clonazepam in 2 patients . One, apparently spontaneous, remission was observed . Botulinum A toxin was injected in the orbicularis oculi of 8 patients affected by BS: moderate to marked improvement lasting 5 to 30 weeks (mean 14.5 weeks) was achieved in all cases; transient ptosis, lasting 1 to 3 weeks, occurred in 3 cases.

Biophys Chem, 1989 Nov, 34(3), 259 - 67
Changes in the molecular topography of the light and heavy chains of type A botulinum neurotoxin following their separation; Singh BR et al.; Botulinum neurotoxin serotype A, an approx . 150 kDa protein, is composed of two subunits, the light and heavy chains (approximately 50 and approximately 100 kDa, respectively) . The neurotoxin's mode of action is believed to depend on coordinated but independent actions of the two subunit chains . The molecular environments of the aromatic amino acid residues of the dichain neurotoxin and the two isolated subunit chains were analyzed using near-ultraviolet circular dichroism (CD) (between 250 and 320 nm) and second-derivative ultraviolet absorption spectroscopy (between 240 and 320 nm) to investigate the conformational variations of the subunit chains in separated and conjugated forms . The mean residue weight ellipticities showed virtually no change (i.e., 1.7%) in the vicinities of Phe (268 nm), and only a small change (11%) around Tyr (279 nm) residues following dissociation of the subunit chains . However, significant changes (23-26%) at 286 nm as well as at 292 nm were noted, suggesting considerable alteration in the conformation of the subunits . Second-derivative ultraviolet absorption spectra indicated the degree of Tyr exposure in the dichain neurotoxin, isolated heavy and light chains at 70.7, 81.5 and 46.4%, respectively . A weighted mean of the degree of exposed Tyr residues in the separated heavy and light chains was 69.6%, virtually same as the 70.7% exposed Tyr residues observed in the intact dichain neurotoxin, indicating no difference in their Tyr exposure upon separation of the two chains . This was corroborated by the CD data which revealed only small changes in the CD signals of Tyr residues, and no alteration in those of the Phe residues following separation of the subunit chains . However, a change in the CD signal at 292 nm suggested that the conformations of Trp-containing segments of the two chains were significantly influenced upon their separation . The heavy and light chains of the neurotoxin therefore appear to exist as two semi-independent domains, in spite of being linked by disulfide and noncovalent bonds, and at least part of their conformations depends on interactions between them.

Br J Clin Pract, 1989 Nov, 43(11), 401 - 3
Botulinum toxin in treating spasticity; Das TK et al.; The value of locally injected botulinum toxin is emphasised . The toxin was injected directly into the skeletal muscles of eight patients with severe spasticity due to stroke-related hemiplegia . It produced both subjective and objective improvement . The toxin injections were well tolerated and no significant side effects were noted.

Eur J Biochem, 1989 Oct 20, 185(1), 197 - 203
Inhibition of transmitter release by botulinum neurotoxin A . Contribution of various fragments to the intoxication process; Poulain B et al.; 1 . The contribution of a proteolytic fragment (H2L) of botulinum neurotoxin type A (comprised of the aminoterminal region of the heavy-chain disulphide-linked to the light chain) to inhibition of neurotransmitter release was investigated, using central cholinergic synapses of Aplysia, rodent nerve-diaphragm preparations and cerebrocortical synaptosomes . 2 . No reduction in neurotransmitter release was observed following external application to these preparations of highly purified H2L or after intracellular injection into Aplysia neurons . 3 . The lack of activity was not the result of alteration in the light chain of H2L during preparation of the latter because (a) renaturation of this light chain with intact heavy chain produced a toxic di-chain form and (b) simultaneous application of heavy chain and light chain from H2L inhibited transmitter release in Aplysia . 4 . Bath application of H2L and heavy chain together inhibited release of transmitter; however, at the neuromuscular junction the potency of this mixture was much lower than that of native toxin . A similar blockade resulted when heavy chain was applied intracellularly and H2L added to the bath, demonstrating that H2L is taken up into cholinergic neurons of Aplysia . This uptake is shown to be mediated by the amino-terminal moiety of heavy chain (H2), because bath application of light chain plus H2 led to a decrease in acetylcholine release from a neuron that had been injected with heavy chain . 5 . A role within the neuron is implicated for a carboxy-terminal portion of heavy chain (H1) since intracellular injection of light chain and H2 did not affect transmitter release . Although the situation is unclear in mammalian nerves, these collective findings indicate that blockade of transmitter release in Aplysia neurons requires the intracellular presence of light chain and H1 (by inference), whilst H2 contributes to the internalization step.

Biochem Biophys Res Commun, 1989 Oct 16, 164(1), 562 - 6
Novel GTP-binding proteins in plasma membranes of the fungus Metarhizium anisopliae; St Leger RJ et al.; We report the existence of several families of GTP-binding proteins in plasma membranes of Metarhizium anisopliae . Two proteins (18.4 and 24 kDa) resemble mammalian Gn-proteins in their being toxin insensitive, binding {alpha-32P}GTP on nitrocellulose blots of sodium dodecyl sulfate (SDS)-polyacrylamide gels, and also in their immunological properties . Four other proteins (31-38.2 kDa) were similar except that they did not bind {alpha-32P}GTP after treatment with sodium dodecyl sulfate . An 18.2 kDa cholera toxin substrate and three toxin insensitive bands (18.6, 18.8, and 24 kDa) are novel proteins antigenically related both to mammalian G-proteins and ras gene products . An additional 23 kDa pertussis toxin substrate (the major G-protein in a crude mycelial extract) reacted strongly with antisera to G-proteins but not with anti-ras serum . Other substrates ADP ribosylated by cholera toxin or botulinum D toxin were immunologically unreactive . Analysis of the structural and functional characteristics of these multiple GTP-binding proteins will promote a better understanding of signal transduction in fungi.

Biochem Biophys Res Commun, 1989 Oct 16, 164(1), 333 - 8
Demonstration of GTP-binding proteins and ADP-ribosylated proteins in rat liver Golgi fraction; Toki C et al.; A Golgi-rich fraction isolated from rat liver was found to contain GTP-binding proteins with 20-25 kDa, which were tightly bound to the Golgi membrane . The Golgi fraction also contained two species of proteins which were ADP-ribosylated by bacterial toxins . Protein(s) which was ADP-ribosylated by botulinum toxin had a similar molecular mass as those with GTP-binding activity but was easily released from the membrane . Another protein with 46 kDa which was ADP-ribosylated by pertussis toxin was tightly bound to the membrane but had no significant GTP-binding activity under conditions tested here . These proteins were much less or negligible in the plasma membrane and the endoplasmic reticulum.

J Biol Chem, 1989 Oct 5, 264(28), 16378 - 82
rac, a novel ras-related family of proteins that are botulinum toxin substrates; Didsbury J et al.; A new family of ras-related proteins, designated rac (ras-related C3 botulinum toxin substrate) has been identified . rac1 and rac2 cDNA clones were isolated from a differentiated HL-60 library and encode proteins that are 92% homologous and share 58% and 26-30% amino acid homology with human rhos and ras, respectively . Nucleotide sequence analysis predicts both rac1 and rac2 proteins to contain 192 amino acids with molecular masses of 21,450 and 21,429 daltons, respectively . rac1 and rac2 possess four of the five conserved functional domains in ras associated with binding and hydrolysis of guanine nucleotides . They also contain the COOH-terminal consensus sequence Cys-X-X-X-COOH which localizes ras to the inner plasma membrane and the residues Gly12 and Ala59, at which sites mutations elicit transforming potential to ras . The rac transcripts, particularly rac2, display relative myeloid tissue selectivity . Both rac1 transcripts (2.4 and 1.1 kilobases (kb} increase when HL-60 cells differentiate to neutrophil-like morphology . In contrast, differentiation of U937 cells to monocyte-like morphology causes no change in the 2.4-kb mRNA and a decrease in the 1.1-kb mRNA species . rac2 mRNA (1.45 kb) increases 7-9-fold and 3-fold upon differentiation of HL-60 and U937 cells, respectively . Neither rac mRNAs are present in a Jurkat T cell line, and unlike rac1, rac2 mRNA is absent in human brain and liver tissue . Transfection experiments permitted the demonstration that rac1 and rac2 are substrates for ADP-ribosylation by the C3 component of botulinum toxin . The data suggest that racs are plasma membrane-associated GTP-binding proteins which could regulate secretory processes, particularly in myeloid cells.

J Biol Chem, 1989 Oct 5, 264(28), 16383 - 9
Identification of the ral and rac1 gene products, low molecular mass GTP-binding proteins from human platelets; Polakis PG et al.; Identification of the GTP-binding proteins from human platelet particulate fractions was attained by their purification via successive column chromatography steps followed by amino acid sequencing . To enhance the likelihood of identifying the GTP-binding proteins, two assays were employed to monitor GTP-binding activities: (i) guanosine 5'-(3-O-{35S}thio)triphosphate (GTP gamma S)-binding followed by rapid filtration and ii) {alpha-32P}GTP-binding following sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotting onto nitrocellulose membranes . The latter assay permitted the isolation of a 28-kDa GTP-binding protein that bound {alpha-32P}GTP prominently but was only poorly detected with the GTP gamma S-binding assay . The amino acid sequences of three peptide fragments derived from the 28-kDa protein were identical to regions of the amino acid sequence deduced from a simian ral cDNA with the exception of one conservative substitution (Asp147----Glu) . A full length human ral cDNA was isolated from a placental cDNA library, and its deduced amino acid sequence, compared with simian ral, also contained the Asp----Glu substitution along with two other substitutions and an additional three NH2-terminal amino acids . In addition to the 28-kDa protein, two distinct 25-kDa GTP-binding proteins were purified from platelets . One of these proteins has been previously characterized as G25K, an abundant low molecular mass GTP-binding protein . Partial amino acid sequence obtained from the second unidentified 25-kDa protein indicates that it is the product of the rac1 gene; a member of a newly identified gene family which encode for low molecular mass GTP-binding proteins (Didsbury, J., Weber, R.F., Bokoch, G . M., Evans, T., and Snyderman, R . (1989) J . Biol . Chem . 264, 16378-16382) . These results identify two new GTP-binding proteins in human platelets, ral, the major protein that binds {alpha-32P}GTP on nitrocellulose transfers, and rac1, a substrate for botulinum C3 ADP-ribosyltransferase.

Arch Biochem Biophys, 1989 Oct, 274(1), 235 - 40
Botulinum neurotoxin type A radiolabeled at either the light or the heavy chain; Dekleva ML et al.; Botulinum neurotoxin (NT) has two distinct structural regions called L and H chains (approximately 50 and approximately 100 kDa, respectively) . Although the H chain is responsible for binding of the NT to neuronal cells, it is not known which of the subunits is internalized and therefore responsible for causing the blockage of acetylcholine release in susceptible neuronal cells . In this report we describe for the first time the preparation of type A NT which is selectively radiolabeled at either the L or the H chain subunit . Such NT preparations will be useful as tools for determining the distribution of L and H chains in poisoned neuronal cells and the role that each subunit plays in inducing toxicity . The L and H chains of the NT (approximately 150 kDa) were separated, purified, and then individually radiolabeled by reductive methylation of the lysine residues using {3H}- or {14C}formaldehyde . The labeled L and H chains were reconjugated with the complementary unlabeled L and H chains . Formation of -S-S- and noncovalent bonds between the L and H chains regenerated the approximately 150 kDa NT . Autoradiographs of sodium dodecyl sulfate polyacrylamide gels confirmed that each reconstituted NT preparation was labeled at only one subunit chain . NT selectively labeled at either the L or the H chain had specific radioactivities of ca . 25-30 and 45-55 microCi/mumol, respectively, and toxicity (mouse LD50/mg protein) values of 2.2 +/- 1.1 X 10(7) and 3.0 +/- 1.0 X 10(7), respectively . A linear increase in the specific radioactivity of L and H chain subunits was observed with increasing concentrations of 3H- or 14C-labeled formaldehyde in the reaction mixture and with increasing concentrations of L or H chain in the reaction mixture.

Infect Immun, 1989 Oct, 57(10), 3053 - 7
Inactivation of botulinum and tetanus toxins by chelators; Bhattacharyya SD et al.; Purified type A botulinum toxin of about 10(6) mouse 50% lethal doses per ml was greater than 99.9% inactivated when incubated at pH 7.4 for 30 min at 37 degrees C in 20 mM 1,10-phenanthroline (PTL) or 2,2'-dipyridyl (DPD) and was 96% inactivated when incubated in 70 mM 8-hydroxyquinoline-5-sulfonic acid (HQL), but was not affected when incubated in 200 mM EDTA . When used as a representative of the chelating agents, PTL inactivated greater than or equal to 99.9% of toxicity in the culture filtrate of C . botulinum type A, B, and E strains . Highly purified tetanus toxin at 2.5 x 10(5) 50% lethal doses per ml lost all toxicity in 40 mM PTL or 150 mM DPD but was not detectably affected by 100 mM HQL (the highest concentration possible) . Toxin inactivation by 20 mM PTL was completely blocked when the PTL was prereacted with an equimolar amount of Zn2+ and significantly reduced when it was preincubated with one-third its molar amount of Fe2+ . DPD at 20 mM had little toxin-inactivating potency when preincubated with an equimolar amount of Zn2+ and only some of this potency when preincubated with an equimolar amount of Fe2+ . Toxicity was not recovered by adding Zn2+ or Fe2+ to PTL-treated toxin . Neutron activation analysis of type A toxin showed that for each toxin molecule present, there was 1 atom of Fe, 0.4 atom of Zn, and 22 to 55 atoms each of Ca and Mg . The biological activity of botulinum toxin seems to depend on a metal component, which is likely to be Fe.

FEBS Lett, 1989 Sep 25, 255(2), 391 - 4
The light chain but not the heavy chain of botulinum A toxin inhibits exocytosis from permeabilized adrenal chromaffin cells; Stecher B et al.; The heavy and light chains of botulinum A toxin were separated by anion exchange chromatography . Their intracellular actions were studied using bovine adrenal chromaffin cells permeabilized with streptolysin O . Purified light chain inhibited the Ca2+-stimulated {3H}noradrenaline release with a half-maximal effect at about 1.8 nM . The inhibition was incomplete . Heavy chain up to 28 nM was neither effective by itself nor did it enhance the inhibitory effect of light chain . It is concluded that the light chain of botulinum A toxin contains the functional domain responsible for the inhibition of exocytosis.

Biochem Biophys Res Commun, 1989 Sep 15, 163(2), 1175 - 81
cDNA cloning of Gb, the substrate for botulinum ADP-ribosyltransferase from bovine adrenal gland and its identification as a rho gene product; Ogorochi T et al.; A 1.5 kilobase cDNA coding for the complete amino acid sequence of Gb, the substrate for ADP-ribosyltransferase in C1 and D botulinum toxins from bovine adrenal gland, has been isolated from a cDNA library of bovine adrenal gland . This cDNA encodes a polypeptide of 21,770 Da consisting of 193 amino acid residues, and the deduced amino acid sequence contains all the partial amino acid sequences reported previously (Narumiya, S., Sekine, A., and Fujiwara, M . (1988) J . Biol . Chem., 263, 17255-17257) . Sequence comparison revealed that Gb is identical with the product of human rho clone 12 (rho A) . The present results also confirmed our suggestion that the ADP-ribosylation occurs at Asn41 in the putative effector domain of the rho gene product.

Naunyn Schmiedebergs Arch Pharmacol, 1989 Sep, 340(3), 345 - 51
Inhibition of the contraction of the isolated longitudinal muscle of the guinea-pig ileum by botulinum C2 toxin: evidence for a role of G/F-actin transition in smooth muscle contraction; Mauss S et al.; The effect of botulinum C2 toxin was studied on the contractions of the guinea pig ileum myenteric plexus longitudinal muscle preparation . Botulinum C2 toxin inhibited the muscle contraction induced by electrical stimulation (60 V; 0.5 ms; 0.33 Hz) in a time and concentration dependent manner . The inhibitory effect occurred with a time lag of about 1 h, and depended on the presence of both toxin components . After 4 h of incubation with 1.7 micrograms/ml of component I and 6.7 micrograms/ml of component II of botulinum C2 toxin, the smooth muscle contraction was inhibited by about 60% . At these toxin concentrations, about 55% of the modifiable smooth muscle actin was ADP-ribosylated . Smooth muscle contraction induced by bradykinin and bethanechol were similarly inhibited . Moreover, the C2 toxin inhibited muscle contraction induced by Ba2+, and by direct muscle membrane depolarization (60 V; 10 ms; 0.33 Hz) after suppression of acetylcholine release by normorphine . Also cytochalasin D inhibited the electrically evoked contraction of the ileum longitudinal muscle . In contrast to botulinum C2 toxin, inhibition of contractility by cytochalasin D occurred without a lag phase, and was reversed by washing off the toxin . In contrast of guinea pig ileum longitudinal muscle, botulinum C2 toxin did not reduce the contraction of the rabbit aortic smooth muscle stimulated by K+-depolarization or noradrenaline.

Mayo Clin Proc, 1989 Sep, 64(9), 1085 - 90
Treatment of blepharospasm with botulinum toxin; Kennedy RH et al.; Many therapeutic modalities, including medications, excision of the muscles used in closure of the eyelids (myectomy), and selective extirpation of branches of the facial nerve (neurectomy), have been used for the management of blepharospasm . Because of limited effectiveness and undesirable side effects, none of these treatments has been completely satisfactory . Recent reports about injection of botulinum toxin indicate that it is safe and effective for most patients . Relief from blepharospasm, however, is usually transient, and repeated injections are usually necessary . The current availability of effective therapy for blepharospasm emphasizes the importance of prompt diagnosis and referral of affected patients to physicians knowledgeable in the use of botulinum toxin and other therapeutic approaches.

J Neurol Sci, 1989 Sep, 92(2-3), 181 - 92
Early terminal and nodal sprouting of motor axons after botulinum toxin; Pamphlett R; Axonal sprouting in distal motor axons was studied in an attempt to answer two questions: (a) is the cell body required for early axonal sprouting?, and (b) do nodal, as well as terminal, axonal sprouts arise after muscle inactivity not caused by nerve injury? Botulinum toxin (BT) was used to induce axonal sprouting without nerve trauma . Mice were injected in the right calf with a sublethal dose of BT and the soleus muscle examined ultrastructurally at times varying from 3 h to 5 days post-injection . Terminal axonal sprouts were seen 2 days after injection, and based on the time taken for BT to act and the growth rate of sprouts, axons were calculated to sprout within 24 h of muscle inactivity . This short time suggests that early axonal regrowth is initiated and controlled at the distal axon . Sprouts were seen arising from the intramuscular nodes of Ranvier from 2 days after BT injection . Unlike the terminal sprouts which elongated over time, the nodal sprouts remained short and confined by the basal lamina overlying the node, probably because without structural denervation there were no empty perineural sheaths to act as pathways to the motor endplates . The finding of terminal and nodal sprouts after botulinum toxin supports the hypothesis that muscle inactivity gives rise to a single growth factor for both terminal and nodal sprouting.

Ophthalmology, 1989 Sep, Pt 2, 37 - 41
Botulinum toxin therapy of eye muscle disorders . Safety and effectiveness . American Academy of Ophthalmology; Influence of botulinum C2 toxin on F-actin and N-formyl peptide receptor dynamics in human neutrophils; Department of Immunology, Scripps Clinic and Research Foundation, La Jolla, California 92037Stimulation of human neutrophils with the chemotactic N-formyl peptide causes production of oxygen radicals and conversion of monomeric actin (G-actin) to polymeric actin (F-actin) . The effects of the binary botulinum C2 toxin on the amount of F-actin and on neutrophil cell responses were studied . Two different methods for analyzing the actin response were used in formyl peptide-stimulated cells: staining of F-actin with rhodamine-phalloidin and a transient right angle light scatter . Preincubation of neutrophils with 400 ng/ml component I and 1,600 ng/ml component II of botulinum C2 toxin for 30 min almost completely inhibited the formyl peptide-stimulated polymerization of G-actin and at the same time decreased the amount of F-actin in unstimulated neutrophils by an average of approximately 30% . Botulinum C2 toxin preincubation for 60 min destroyed approximately 75% of the F-actin in unstimulated neutrophils . Right angle light scatter analysis showed that control neutrophils exhibited the transient response characteristic of actin polymerization; however, after botulinum C2 toxin treatment, degranulation was detected . Single components of the binary botulinum C2 toxin were without effect on the actin polymerization response . Fluorescence flow cytometry and fluorospectrometric binding studies showed little alteration in N-formyl peptide binding or dissociation dynamics in the toxin-treated cells . However, endocytosis of the fluorescent N-formyl peptide ligand-receptor complex was slower but still possible in degranulating neutrophils treated with botulinum C2 toxin for 60 min . The half-time of endocytosis, estimated from initial rates, was 4 and 8 min in control and botulinum C2 toxin-treated neutrophils, respectively.

Surv Ophthalmol, 1989 Sep-Oct, 34(2), 123 - 32
Essential blepharospasm and related dystonias; Jordan DR et al.; Essential blepharospasm is an idiopathic disorder of progressive involuntary spasms of the orbicularis oculi and upper facial (corrugator, procerus) muscles . Blepharospasm literally means spasm of the eyelids; however, most patients with blepharospasm also have or will develop squeezing in the lower face and neck muscles (Meige's syndrome, orofacial dystonia, or oromandibular dystonia) . Some patients develop dystonic, uncontrolled movements in areas outside the facial nerve distribution (segmental cranial dystonia or craniocervical dystonia) . Chronic, forceful squeezing by the periocular muscles becomes debilitating for the patient and leads to functional and cosmetic eyelid deformities . Treatment has included a variety of modalities and oral medications that are of limited efficacy . Botulinum-A toxin injections have delivered the best temporary relief from this disorder, while the periorbital myectomy operation has been shown to give the best long-term results.

Muscle Nerve, 1989 Sep, 12(9), 716 - 22
Botulinum toxin therapy in hemifacial spasm: clinical and electrophysiologic studies; Geller BD et al.; Three patients with idiopathic hemifacial spasm were studied clinically and electrophysiologically before and after injections of botulinum toxin into the involved periocular and facial muscles . The spasms were improved for approximately 3 months, and the effect was repeatable on reinjection . The spasms diminished only as long as the muscles were clinically weak, and spasms were observed electromyographically even though therapy eliminated the clinical spasms . Uninjected muscles continued to have spasms . Transmission of excitation from the zygomatic branch to the marginal mandibular branch of the facial nerve and vice versa in all patients was unaltered after therapy, but the amplitude of the response was decreased . The efficacy of botulinum toxin in hemifacial spasm appears to be related to the production of muscle weakness; there is no demonstrable effect on phenomena believed to be ectopic excitation or ephaptic transmission in the facial nerve.

Biochem Biophys Res Commun, 1989 Aug 15, 162(3), 1388 - 95
Characterization of botulinum type A neurotoxin gene: delineation of the N-terminal encoding region; Betley MJ et al.; A 456 basepair HindIII fragment that encoded a portion of the type A botulinum neurotoxin gene was cloned into Escherichia coli using a plasmid vector . DNA sequence analysis revealed that this botulinum DNA insert encoded an open reading frame of 35 amino acid residues of which 34 corresponded to the N-terminal residues of botulinum neurotoxin type A.

Aust N Z J Ophthalmol, 1989 Aug, 17(3), 239 - 45
Early and late botulinum toxin treatment of acute sixth nerve palsy; Murray AD; Ten patients with sixth nerve palsy were treated with botulinum toxin injection to the antagonist nonparetic medial rectus . All patients were followed for a minimum of 14 months after the last injection . Six patients were treated within eight weeks of the onset of the palsy . Within a few days five of the six gained fusion, without the necessity of a marked head turn and none complained of confusing reversal of diplopia . The same five recovered full function . Four patients with no medial rectus contracture on forced duction testing were treated six months or longer after the onset of the palsy and none recovered full function . One of the four patients was permanently over-corrected despite persistent weakness of the lateral rectus . This preliminary report suggests that early botulinum toxin injection of patients with recent onset (acute) sixth nerve palsy is beneficial . Since some patients may recover spontaneously a randomised double-blind study is necessary to more precisely determine the effectiveness of this form of therapy . Botulinum toxin does not appear to be effective in facilitating recovery in those cases with poor lateral rectus function six months or longer after the onset of the palsy, even when there is no contracture of the medial rectus.

J Clin Microbiol, 1989 Aug, 27(8), 1906 - 8
Evaluation of neutralizing antibodies to type A, B, E, and F botulinum toxins in sera from human recipients of botulinum pentavalent (ABCDE) toxoid; Siegel LS; Twenty-five serum specimens from personnel immunized with botulinum pentavalent toxoid (ABCDE) had titers of neutralizing antibodies to type A (5.7 to 51.6 IU/ml), type B (0.75 to 18 IU/ml), and type E (0.61 to 10 IU/ml) botulinum toxins . Titers for one type could not be used to predict titers for another type in individuals receiving the toxoid . Cross-neutralizing antibodies to type F botulinum toxin were not detected (less than 0.0125 IU/ml).

Plast Reconstr Surg, 1989 Aug, 84(2), 353 - 5
Botulinum toxin: a treatment for facial asymmetry caused by facial nerve paralysis; Clark RP et al.; Injury to the frontal or other facial nerve branches can result in an asymmetry that can be very distressful to both patient and surgeon . This is especially true following cosmetic procedures such as rhytidectomy . We propose a means to create temporary symmetry while awaiting the possible return of nerve function . Botulinum neurotoxin causes a muscle paralysis lasting for approximately 3 months, and it is well established as the preferred treatment for blepharospasm . A case is presented in which botulinum toxin type A was injected into the opposite functioning frontalis muscle of a patient with unilateral frontal nerve paralysis . The patient experienced satisfactory relief of the asymmetry caused by onesided forehead wrinkling and brow elevation . Botulinum toxin therapy should be considered for both temporary and permanent facial asymmetries due to facial nerve paralysis as well as spasm.

J Neurochem, 1989 Aug, 53(2), 428 - 35
Calcium is released by exocytosis together with catecholamines from bovine adrenal medullary cells; von Grafenstein HR et al.; We have tested the hypothesis that exocytosis is a possible export route for calcium from bovine adrenal medullary cells . After prelabelling cells in primary tissue culture with 45Ca, evoked 45Ca export and catecholamine secretion show the same time course, a similar fraction of the total pool of 45Ca and catecholamine is released, and the same concentrations of carbamylcholine or KCl are required for half-maximal triggered release . Increasing the osmolarity of the extracellular medium or treating the cells with botulinum toxin type D inhibits both evoked catecholamine secretion and 45Ca export to the same extent without inhibiting 45Ca influx . Incorporation of 45Ca into chromaffin granules is very slow, however, and incorporated 45Ca is not immediately releasable . 45Ca entering the cell during short-term stimulation is not found in the releasable pool during a second period of triggered secretion . Our data suggest that chromaffin granules are the largest pool of intracellular calcium in bovine adrenal medullary cells and that most of the calcium in chromaffin granules does not rapidly exchange with cytoplasmic Ca, but can be released directly by exocytosis . Exocytosis does not appear to play a major role in exporting Ca that enters the cell during short-term stimulation.

Can J Physiol Pharmacol, 1989 Aug, 67(8), 879 - 82
Comparison of the effects of botulinum toxin in adult and neonatal rats: neuromuscular blockade and toxicity; Bambrick LL et al.; A single dose of botulinum toxin (BoTX) was injected subcutaneously to induce neuromuscular blockade in the triceps surae muscles of the hindlimbs of neonatal and adult rats . The efficacy of the toxin in producing complete neuromuscular blockade of the lower limb muscles, assessed by blockade of (a) postural and flexor reflexes and (b) muscle contraction in response to nerve stimulation, was dose dependent at all ages over a BoTX dose range of 10-60 ng/kg . However, BoTX was dramatically more toxic in adult animals resulting in a decline in body weight and lethal consequences in 25% of adult animals 1 week after administration of BoTX doses as low as 40 ng/kg . In contrast, neonatal animals, given the same dosage, continued to grow and no mortalities were observed . The differences in toxicity of BoTX in adult and neonatal rats are readily accounted for by the short duration of effect in the younger animals, which, in turn, is probably the result of more rapid generation of new and functional nerve terminals.

Muscle Nerve, 1989 Aug, 12(8), 613 - 26
AAEE Minimonograph #33: electrodiagnostic approach to defects of neuromuscular transmission; Keesey JC; Clinical testing for neuromuscular dysfunction is supported by an extensive amount of excellent basic information about normal and abnormal subcellular physiology and ultrastructure . This information provides an essential frame of reference for describing the rationale of single-fiber electromyography (SFEMG) . SFEMG in turn helps to explain the more conventional clinical testing of neuromuscular function by repetitive nerve stimulation (RNS) . Electrical findings in myasthenia gravis, Lambert-Eaton myasthenic syndrome, and botulinum intoxication are discussed from the subcellular level via the cellular level (SFEMG) to the integrated responses of whole muscle (RNS) as a rational means of understanding the technique of clinical repetitive nerve stimulation.

J Neurosci, 1989 Aug, 9(8), 2902 - 6
Neurotransmission regulates stability of acetylcholine receptors at the neuromuscular junction; Avila OL et al.; The majority of acetylcholine receptors (AChRs) at normally innervated neuromuscular junctions are stable, with a half-life averaging about 12 d in most rodent muscles . Following denervation, the AChRs turn over much more rapidly after a lag period . The mechanism by which motor nerves normally maintain stabilization of junctional AChRs is not yet known . In order to determine whether synaptic transmission plays a role in this process, we have compared the effects of pre-and postsynaptic chloinergic blockade with those of surgical denervation . 125l-alpha-bungarotoxin was used to label junctional AChRs and follow their loss over time . Presynaptic blockade of quantal ACh transmission was produced in the soleus (SOL) and flexor digitorum brevis muscles of mice by repeated injections of type A botulinum toxin . Postsynaptic blockade of quantal and nonquantal ACh transmission was produced by continuous infusion of alpha-bungarotoxin in the SOL . Our findings show that treatment with botulinum toxin resulted in an accelerated loss of junctional AChRs that was similar to the effects of surgical denervation, though briefly delayed in its onset . Treatment with alpha-bungarotoxin produced an effect that was quantitatively equivalent to the accelerated loss of junctional AChRs following surgical denervation, with an identical time course . These results support the concept that cholinergic transmission is a mediator of the neural control of stability of junctional AChRs . The possibility that receptor stabilization may represent a mechanism of long-term postsynaptic "memory" dependent on neural transmission is discussed.

Exp Neurol, 1989 Aug, 105(2), 171 - 6
Neural regulation of mRNA for the alpha-subunit of acetylcholine receptors: role of neuromuscular transmission; Lipsky NG et al.; Levels of mRNA for acetylcholine receptor (AChR) subunits are relatively low in innervated skeletal muscles . Following denervation they rise rapidly, leading to increased AChR synthesis . The mechanism by which motor nerves normally regulate these mRNA levels is not yet known . In order to determine the possible role of synaptic transmission in this process, we have compared the effect of blockade of cholinergic ACh transmission with that of surgical denervation . Blockade of quantal ACh transmission was produced by injection of type A botulinum toxin into the soleus muscles of rats . We measured mRNA for the alpha-subunit of the AChR (alpha-AChR mRNA) in RNA extracts of botulinum-treated, denervated, and normal control muscles by hybridization with a highly specific cDNA probe . Our findings show that treatment with botulinum toxin resulted in an increase in alpha-AChR mRNA which was similar to the effect of surgical denervation, although slower in its time course . Since botulinum toxin specifically inhibits quantal ACh release, these results support the concept that cholinergic synaptic transmission plays a key role in mediating the neural control of the alpha-AChR message . The difference between the effects of denervation and botulinum-treatment may be explained by the fact that botulinum toxin does not block the spontaneous non-quantal component of ACh transmission, which has previously been shown to have a partial influence in regulating certain properties of muscles . The present results suggest that synaptic transmission has an important influence in regulating gene expression in the target cell.

J Leukoc Biol, 1989 Aug, 46(2), 161 - 8
Effect of granulocyte-macrophage colony-stimulating factor on superoxide production in cytoplasts and intact human neutrophils: role of protein kinase and G-proteins; Mege JL et al.; Granulocyte-macrophage colony-stimulating factor, GM-CSF, potentiates superoxide generation produced by human neutrophils stimulated with fMet-Leu-Phe and platelet-activating factor, PAF, but not by phorbol 12-myristate 13-acetate (PMA) or opsonized zymosan . The potentiation is greatest in fMet-Leu-Phe-stimulated cells . This indicates that the actions of only certain receptors are potentiated by GM-CSF . Incubation of the cells with the protein kinase inhibitor H-7 or with the protein synthesis inhibitor cyclohexamide before the addition of GM-CSF does not affect the observed potentiation . The rationales behind these studies are to examine the roles of protein kinase C and protein synthesis in the action of GM-CSF . The data suggest that neither protein kinase C nor protein synthesis is necessary for GM-CSF action . On the other hand, no potentiation can be seen in the presence of cytochalasin B . Unlike intact cells, GM-CSF does not enhance superoxide production by cytoplasts stimulated with fMet-Leu-Phe . The rationale behind the use of cytoplasts is to examine the role of granules and/or nucleus in GM-CSF action, and the data indicate that one or more of these two components is necessary for the priming effect of GM-CSF . The amount of actin associated with the cytoskeleton under control of fMet-Leu-Phe-stimulated condition is the same in normal and GM-CSF-treated human neutrophils . Botulinum D toxin ADP-ribosylates a protein with a molecular weight of 22 kDa . This ribosylation is reduced in homogenates obtained from cells pretreated with botulinum D toxin or GM-CSF . Botulinum D toxin does not affect the basal or the fMet-Leu-Phe-induced rise in the intracellular concentration of free calcium in human neutrophils . GM-CSF also increases the rise in intracellular concentration of free calcium in human neutrophils stimulated with PAF or fMet-Leu-Phe . The increases are inhibited by pertussis toxin . Several important conclusion can be drawn from these data . 1) GM-CSF potentiates the rise in Ca2+i produced by PAF and fMet-Leu-Phe, and these potentiations are inhibited in pertussis-toxin-treated cells . 2) GM-CSF does not prime cytoplasts to stimulation by fMet-Leu-Phe . This suggests that the granules and/or nucleus are necessary for the priming action . 3) The priming by GM-CSF is not mediated by the H-7-sensitive protein kinase C, botulinum D-sensitive G-protein, or protein synthesis.

Harefuah, 1989 Jul, 117(1-2), 9 - 10
{Use of botulinum toxin in ophthalmology}; Nemet P et al.; We have used botulinum toxin (Oculinum) for the past 2 years to treat strabismus and other ophthalmic conditions . It was effective for blepharospasm and hemifacial spasm, and in producing pharmacological ptosis . There were a few local complications, such as ptosis and diplopia, which disappeared within a few weeks . This is a new treatment modality with specific indications in ophthalmology.

Ophthalmology, 1989 Jul, 96(7), 935 - 43
Management of strabismus with botulinum A toxin; Biglan AW et al.; Three hundred eight patients with strabismus were treated with botulinum A toxin (Oculinum) chemodenervation; 153 were followed by the authors for at least 6 months . In this study group, 97 received botulinum A toxin injections as the primary method of treatment of their ocular deviation . Fifty-six received injections after traditional extraocular muscle surgery . Botulinum A toxin was useful for management of patients with recent surgical overcorrections and for management of some patients with sixth cranial nerve palsy . Chemodenervation of an extraocular muscle was not as successful as traditional strabismus surgery for treatment of infantile esotropia and other comitant deviations . Botulinum A toxin injection was ineffective in patients who had restrictive strabismus . This drug has limited application in the management of patients with strabismus.

Ophthalmology, 1989 Jul, 96(7), 931 - 4
Chemodenervation of strabismic children . A 2- to 5-year follow-up study compared with shorter follow-up; Magoon EH; To determine longer-term efficacy of botulinum treatment, the author examined 85 children younger than 14 years of age who had been treated from November 1982 to February 1984, comparing shorter follow-up (range, 6-24 months) with longer follow-up (range, 2-5.5 years) as of last examination before March 1988 . Fifty esotropes meeting the 2-year criteria for follow-up had an average of 35 prism diopters (PD) before and 5 PD after treatment . Twelve exotropes averaged 30 and 5 PD . No long-term complications were discovered . The results are similar to the shorter follow-up and suggest that botulinum is effective in creating a 2- to 5-year stable improvement for strabismic children.

Nervenarzt, 1989 Jul, 60(7), 401 - 6
{Clinical and therapeutic aspects of essential blepharospasm}; Russegger L et al.; The rare disease of blepharospasm which is nowadays believed to be an extrapyramidal dystonic movement disorder is discussed in clinical, differential diagnostic and therapeutic viewpoints . The abundant number of treatment methods proposed in the literature are critically reviewed . 13 cases are described, which were treated by our own surgical procedure - a modified neurotomy of branches of the parotid plexus . The importance of botulinum-toxin treatment in mild and moderate cases of blepharospasm, as well as the success and low rate of complications in neurotomy-treated patients with severe eyelid spasms, is stressed.

J Pediatr Ophthalmol Strabismus, 1989 Jul-Aug, 26(4), 162 - 4
Botulinum toxin injection into the superior rectus muscle of the non-dominant eye for dissociated vertical deviation; McNeer KW; A variety of surgical procedures on the superior rectus muscles have been proposed as a remedy for Dissociated Vertical Deviation (DVD); Botulinum toxin (Oculinum) has been one such suggestion as a surgical alternative . We selected five older patients with DVD whose age placed them beyond a risk of amblyopia to evaluate Oculinum injection into the superior rectus muscle as therapy for DVD . The patients achieved a reduction from the mean value of 20 prism diopters to the mean value of less than 5 pd hypertropia at 172 weeks following injection . Our study suggests that Oculinum is effective in treating DVD, but caution should be exercised because transient ptosis invariably accompanies injection.

J Laryngol Otol, 1989 Jul, 103(7), 698 - 9
Pharyngeal paralysis due to botulinum toxin injection; Koay CE et al.; Botulinum toxin injection is now recognized as an effective treatment for spasmodic torticollis . Complications of this increasingly popular method of treatment include mild and transient dysphagia, with or without dysphonia, lasting up to four weeks . Two cases of paralysis of vocal cord contralateral to the injected sternomastoid have also been reported . A case of severe dysphagia lasting six weeks associated with ipsilateral vocal cord palsy following botulinum toxin injection is presented . The probable mechanism for these complications is discussed.

Nervenarzt, 1989 Jul, 60(7), 386 - 93
{Botulinum toxin in therapy of craniocervical dystonia}; Dressler D et al.; Craniocervical dystonias are a major therapeutic problem, since in the majority of cases various drug regimens as well as surgical interventions either fail or are accompanied by serious side effects . Botulinum toxin, well known as a biological toxin which blocks the cholinergic neuromuscular synapse, proved to be a new and successful concept in the treatment of these disorders . In the present study, mechanisms of action, clinical indications and practical use of botulinum toxin are described.

Neurologia, 1989 Jul-Aug, 4(6), 194 - 9
{Pharmacologic, surgical and infiltration of botulin toxin treatment in blepharospasm}; Grandas F et al.; The response to different therapeutic methods was evaluated in a series of 264 patients with blepharospasm . The most effective drug therapy were anticholinergic agents, which resulted in the initial improvement of symptoms in 20% of treated patients . There was improvement in isolated cases treated with levodopa, dopaminergic agonists, neuroleptics, benzodiazepines and tricyclic antidepressants . Bilateral avulsion of the facial nerve was carried out in 29 patients; 27 of these (93%) improved . Spasms relapsed in 22 cases, after a mean period of 12 months . Myectomy of the orbicular muscle was performed in 8 patients . In only 2 cases some improvement was obtained . One hundred and fifty-one patients were treated with infiltrations of botulinum toxin A in the orbicular muscle . 78% of cases improved . The mean duration of the benefit of each injection was 9.2 weeks . The most common secondary effects were local (ptosis, diplopia) and transient.

Arzneimittelforschung, 1989 Jul, 39(7), 762 - 5
Effects of new 4-aminopyridine derivatives on neuromuscular transmission and on smooth muscle contractility; Berger SG et al.; The effects of new 4-aminopyridine (4-AP) derivatives were investigated in the isolated mouse phrenic nerve-hemidiaphragm preparation under single impulse stimulation and tetanic conditions . The basic 4-AP structure was modified in position 3 on the pyridine nucleus by introducing different substituents . Results were compared to those obtained with 4-AP and 3,4-diaminopyridine . The compounds were tested for their antagonistic effect against calcium antagonists and botulinum toxin A . The effect on smooth muscle was investigated on the isolated guinea-pig ileum . Physico-chemical parameters of the test compounds were determined by the partition coefficient and ionization constant . Finally structure-activity relationship analysis revealed that the activity was highly related to lipophilicity and the steric volume . So far 4-AP itself provided the most advantageous molecular structure.

J Biol Chem, 1989 Jun 25, 264(18), 10354 - 60
Isolated light chains of botulinum neurotoxins inhibit exocytosis . Studies in digitonin-permeabilized chromaffin cells; Bittner MA et al.; The effects of botulinum neurotoxins or their light and heavy chain subunits were investigated in digitonin-permeabilized adrenal chromaffin cells . Because these cells are permeable to proteins, the toxin had direct access to the cell interior . Botulinum type A neurotoxin and its light chain subunit inhibited Ca2+-dependent catecholamine secretion in a dose-dependent manner . The heavy chain subunit had no effect . Inhibition required introduction of the neurotoxin or light chain into the cell and was not seen when intact cells were incubated with these proteins . The inhibition of secretion by type A neurotoxin and light chain was incomplete, the maximal response being 65% . The inhibition was not overcome by increasing Ca2+ concentrations . The action of the light chain was irreversible and rapid . Botulinum type E neurotoxin also inhibited secretion in a dose-dependent manner . Its potency was increased 30-fold following mild trypsinization, which nicked the single chain protein to the dichain form . In contrast to the results seen with types A and E, botulinum type B neurotoxin did not inhibit secretion, while its light chain totally abolished secretion . Trypsinization of the neurotoxin produced the dichain form, which did not inhibit secretion . Reduction of the trypsinized neurotoxin with dithiothreitol produced inhibition equivalent to that seen with the purified light chain subunit . Isolated type A heavy chain had no effect on the inhibitory action of type A or B light chains . The data demonstrate that the ability of botulinum neurotoxins to inhibit secretion is confined to the light chain region of these proteins . Furthermore, while the botulinum neurotoxin types A, B, and E have similar macrostructures, they are not identical with respect to their biological activities.

Arch Ophthalmol, 1989 Jun, 107(6), 820 - 3
Vertical rectus muscle transposition and botulinum toxin (Oculinum) to medial rectus for abducens palsy; Rosenbaum AL et al.; Ten adult patients developed sixth-nerve palsy after trauma or a cerebral tumor . No clinical evidence of recovery of function was noted by at least 8 months after onset . All patients underwent total transposition of the superior and inferior rectus muscle insertions to the area of the lateral rectus insertion, accompanied by botulinum toxin (Oculinum) injection of the ipsilateral medial rectus . These patients developed a mean diplopia-free field of 51 degrees, with a diplopia-free field in the abducted field of 20 degrees . This procedure involved surgery on only two rectus muscles, but the results compared favorably with surgical strategies involving three rectus muscles . Thus, the risk of developing anterior segment ischemia was greatly reduced.

Doc Ophthalmol, 1989 Jun, 72(2), 189 - 98
Effect of botulinum toxin on extraocular muscle proprioception; Manni E et al.; Injections of botulinum toxin type A (BoTox) in one extraocular muscle (EOM) induce long lasting paretic lengthening of the muscle permitting realignment to occur in strabismus, while eye movements appear to be unaffected after the transitory period of induced paresis . It has been hypothesized a BoTox-induced change in the spindle discharge of EOMs to explain the effect in EOM length . In decerebrate lambs and goats, first order neurons of eye muscle spindles were identified in a cellular pool located in the medial dorsolateral portion of the semilunar ganglion . The belly of the muscle to which the recorded unit belonged was infiltrated with BoTox . A decrease in afferent discharge of the spindle and in its stretch sensitivity was observed . This effect began 10-15 minutes after the injection . There was no corresponding decrease in muscle tension during the first 45 minutes . This finding suggests that the block of release of acetylcholine at motor endings is earlier and more efficacious in gamma- than in alpha-motoneurons . As a result of the proprioceptive input reduction, an unbalance between the agonist and antagonist muscles should occur favouring the ocular realignment.

J Biol Chem, 1989 May 25, 264(15), 8602 - 5
Asparagine residue in the rho gene product is the modification site for botulinum ADP-ribosyltransferase; Sekine A et al.; We reported previously that the ADP-ribosyltransferase in C1 and D botulinum toxins specifically catalyzes ADP-ribosylation of an Mr 22,000 guanine nucleotide-binding protein and that this substrate named Gb (b = botulinum) has an amino acid sequence homologous to that deduced from the rho gene (Narumiya, S., Sekine, A., and Fujiwara, M . (1988) J . Biol . Chem . 263, 17255-17257) . In this study we have determined the amino acid sequence at its ADP-ribosylation site . Purified substrate was {32P}ADP-ribosylated by C1 botulinum toxin and digested with trypsin . The radioactive peptides were isolated by reversed-phase high performance liquid chromatography and digested further either with protease V8, with proteases V8 and thermolysin, or with proline endopeptidase and thermolysin . By this procedure three radioactive peptides were obtained, and their amino acid sequences were X-Tyr-Val-Ala-Asp-Ile-Glu, X-Tyr, and Val-Phe-Glu-X-Tyr in which no amino acid peak was found in X . During the sequencing the radioactivity quantitatively adhered to the sequencing filter and was not eluted with either of the identified amino acid residues . Analysis of the protein without the ADP-ribosylation yielded the corresponding sequence as Thr-Val-Phe-Glu-Asn-Tyr which corresponds to Thr37-Tyr42 in the amino acid sequence deduced from the Aplysia rho gene . These results strongly suggest that the asparagine residue is the ADP-ribosylation site in the rho gene product . This ADP-ribose protein bond was stable in 0.5 M hydroxylamine at pH 7.5 at 37 degrees C for at least 5 h . The ADP-ribosylation of this protein affected neither its GTPase- nor its {35S}guanosine 5'-O-thiotriphosphate-binding activity.

FEBS Lett, 1989 May 8, 248(1-2), 23 - 7
Reductive chain separation of botulinum A toxin--a prerequisite to its inhibitory action on exocytosis in chromaffin cells; Stecher B et al.; Cleavage of the disulfide bond linking the heavy and the light chains of tetanus toxin is necessary for its inhibitory action on exocytotic release of catecholamines from permeabilized chromaffin cells {(1989) FEBS Lett . 242, 245-248; (1989) J . Neurochem., in press} . The related botulinum A toxin also consists of a heavy and a light chain linked by a disulfide bond . The actions of both neurotoxins on exocytosis were presently compared using streptolysin O-permeabilized bovine adrenal chromaffin cells . Botulinum A toxin inhibited Ca2+-stimulated catecholamine release from these cells . Addition of dithiothreitol lowered the effective doses to values below 5 nM . Under the same conditions, the effective doses of tetanus toxin were decreased by a factor of five . This indicates that the interchain S-S bond of botulinum A toxin must also be split before the neurotoxin can exert its effect on exocytosis.

Eur J Pharmacol, 1989 May 2, 164(1), 45 - 53
Effects of botulinum A toxin on presynaptic modulation of evoked transmitter release; Nakov R et al.; A possible influence of botulinum A toxin on the modulation of evoked neurotransmitter release was investigated in hippocampus tissue . Rabbit hippocampal slices prelabelled with {3H}noradrenaline ({3H}NA), {3H}5-hydroxytryptamine ({3H}5-HT) or {3H}choline were superfused with physiological medium and were stimulated electrically during superfusion . The evoked release of {3H}NA, {3H}5-HT and {3H}acetylcholine {( 3H}ACh) was inhibited by botulinum A toxin in a concentration- and time-dependent manner . Neither the inhibition of release of {3H}NA and {3H}5-HT by the alpha 2-adrenoceptor agonist clonidine nor facilitation of release in the presence of alpha 2-antagonists were influenced by pretreatment of the tissue with botulinum toxin . The toxin caused no {32P}ADP ribosylation of synaptosomal proteins of hippocampus . The facilitation of the stimulation-induced {3H}NA and {3H}5-HT release by the specific protein kinase C (PKC) activator 4 beta-phorbol-12,13-dibutyrate (PDB) was significantly diminished by botulinum A toxin . These results show that the evoked transmitter release is inhibited by botulinum A toxin by a mechanism which does not involve ADP ribosylation or an interaction with the alpha 2-adrenoceptor mechanism.

Am J Otol, 1989 May, 10(3), 220 - 9
Bell's palsy: management of sequelae using EMG rehabilitation, botulinum toxin, and surgery; May M et al.; Fifteen percent of patients who have had an acute episode of Bell's palsy will be left with debilitating facial dysfunction . This chapter describes our approach to managing a variety of hypo- and hyperkinetic disorders caused by injury and faulty regeneration of the facial nerve, using electromyographic rehabilitation (EMGR) (13 patients), Oculinum toxin injection (14 patients), or surgical reanimation (72 patients) . Improvement was noted after EMGR in 12 of 13 patients (92%), all 14 patients treated with Oculinum experienced temporary improvement, and improvement was noted in 66 of 72 patients who underwent surgery (92%) . The indications, techniques, and results of these three rehabilitative methods are discussed.

J Neurol Neurosurg Psychiatry, 1989 May, 52(5), 652 - 5
Jaw closing spasm--a form of focal dystonia? An electrophysiological study; Lagueny A et al.; The case of a 36 year old man suffering from unilateral right jaw closing spasms over two years is reported . Permanent spasm with trismus severely impeding mouth-opening was combined with paroxysms triggered by various sensory stimuli . The diagnosis of temporo-mandibular joint syndrome was considered but treatment failed to improve the symptoms . Neurological investigation two years after onset of the spasms showed by electrophysiological studies excessive co-contraction of the antagonistic jaw-closers, mainly the right masseter during attempts at jaw opening and absence of the silent period in the right masseter and anterior temporalis following jaw tap and trigeminal exteroceptive stimulation . Jaw dystonia was therefore considered and Botulinum A toxin was injected into the right masseter and temporalis which dramatically improved the patient's condition.

J Pediatr Ophthalmol Strabismus, 1989 May-Jun, 26(3), 106 - 8
Long-term results: botulinum for sixth nerve palsy; Wagner RS et al.; Eight patients with intracranial malignancies or vascular lesions and sixth nerve palsies were treated with botulinum toxin chemodenervation of the antagonist medial rectus muscle . Primary deviation ranged from 20 to 75 prism diopters (pd) of esotropia . Six were treated acutely (within 3 months of onset) and two, which demonstrated partial recovery of lateral rectus function but with residual esotropia and diplopia, were treated after 6 months . After a mean follow-up of 20.6 months, seven were diplopia-free with excellent rotations . Five had complete resolution of the esotropia and diplopia, with near complete recovery of abduction . One had 6 pd residual esotropia, while another, whose sixth nerve had been resected, required a modified Jensen procedure, resulting in full rotations . The single case of bilateral sixth nerve palsy had a functional improvement but was lost to follow-up . One patient had a vertical strabismus induced with the injection and had a gradual return of the esotropia.

Ann Otol Rhinol Laryngol, 1989 May, 98(5 Pt 1), 339 - 45
Transmucosal electrical stimulation of laryngeal muscles; Sanders I et al.; A new technique is described that enables discrete activation of individual laryngeal muscles by electrical stimulation across overlying mucosa . In 15 dogs, we defined six distinct motor points by transmucosal stimulation at 3 mA while observing the resulting characteristic position of the arytenoid and true vocal cord . Five dogs were then paralyzed with succinylcholine in order to simulate bilateral vocal cord paralysis . Application of a 3-mA stimulus at each motor point yielded no motion of the cords, but when the current was increased to 20 mA, characteristic responses were elicited . In five other dogs, botulinum toxin was injected directly into laryngeal muscles . Stimulation was used in an attempt to quantify the degree of neuromuscular blockade . In the last group of five dogs, we simulated cricoarytenoid arthritis by scarifying the joint . The extent and nature of the joint's impairment could be demonstrated by stimulation . Transmucosal stimulation appears promising as a clinical technique for correlating particular vocal cord movements and thresholds of activation with specific laryngeal disorders . Additionally, such a technique may be useful in clarifying how each laryngeal muscle acts upon the cricoarytenoid joint.

Mol Cell Biol, 1989 May, 9(5), 2239 - 43
Effects of botulinum toxin type D on secretion of tumor necrosis factor from human monocytes; Imamura K et al.; Botulinum toxins are potent neurotoxins which block the release of neurotransmitters . The effects of these toxins on hematopoietic cells, however, are unknown . Monocytes secrete a variety of polypeptide growth factors, including tumor necrosis factor (TNF) . In the study reported here, the effects of botulinum toxin type D on the secretion of TNF from human monocytes were examined . The results demonstrate that botulinum toxin type D inhibits the release of TNF from monocytes activated by lipopolysaccharide (LPS) but not by 12-O-tetradecanoylphorbol-13-acetate . Botulinum toxin type D had no detectable effect on intracellular TNF levels in LPS-treated monocytes, indicating that the effects of this toxin involve the secretory process . This inhibitory effect of botulinum toxin type D on TNF secretion from LPS-treated monocytes was partially reversed by treatment with 12-O-tetradecanoylphorbol-13-acetate or introduction of guanosine 5'-{gamma-thio}triphosphate into these cells . The results demonstrate that TNF secretion is regulated by at least two distinct guanine nucleotide-binding proteins, one responsible for the activation of phospholipase C and another which acts as a substrate for botulinum toxin type D . ADP-ribosylation of monocyte membranes by botulinum toxin type D demonstrated the presence of three substrates with Mrs of 45,000, 21,000, and 17,000 . While the role of these substrates in exocytosis is unknown, the results suggest that the Mr 21,000 substrate is involved in a process other than TNF secretion.

FEBS Lett, 1989 Apr 24, 247(2), 221 - 6
Identification of rho as a substrate for botulinum toxin C3-catalyzed ADP-ribosylation; Quilliam LA et al.; Recombinant Aplysia rho and a GTP-binding protein purified from human neutrophil membranes (G22K) were ADP-ribosylated by botulinum toxin C3 with stoichiometries of 0.8 and 0.6, respectively . Rho and G22K appeared to be different proteins since (i) rho migrated faster on polyacrylamide gels, (ii) unlike G22K, rho did not require the presence of cytosol to be ADP-ribosylated, (iii) G22K was not recognized by an anti-rho antiserum, and (iv) antibody 142-24E05 recognized G22K effectively but only poorly cross reacted with rho . ADP-ribosylation had no effect on the ability of rho to bind or hydrolyse GTP . Therefore, it appears that there are multiple botulinum toxin C3 substrates and that the toxin exerts its effects on cell function by a mechanism other than modulating the GTPase activity of rho.

Ophthalmic Surg, 1989 Apr, 20(4), 280 - 3
Intractable orbicularis myokymia: treatment alternatives; Jordan DR et al.; Orbicularis myokymia frequently occurs in young, otherwise health individuals . The intermittent muscle fasciculations are transient and generally disappear with time . If the myokymia is persistent or progressive, neurologic assessment and investigation may be necessary . Muscle relaxants, botulinum-A toxin, and surgical myectomy are methods of treatment that only occasionally need to be considered . We present limited orbicularis myectomy and botulinum-A toxin injections as efficacious treatments in five selected intractable cases of orbicularis myokymia.

Biochem J, 1989 Apr 1, 259(1), 47 - 53
Effect of pH on the interaction of botulinum neurotoxins A, B and E with liposomes; Montecucco C et al.; The interaction of botulinum neurotoxin serotypes A, B and E with membranes of different lipid compositions was examined by photolabelling with two photoreactive phosphatidylcholine analogues that monitor the polar region and the hydrophobic core of the lipid bilayer . At neutral pH the neurotoxins interacted both with the polar head groups and with fatty acid chains of phospholipids . At acidic pHs the neurotoxins underwent structural changes characterized by a more extensive interaction with lipids . Both the heavy and light chain subunits of the neurotoxins were involved in the process . The change in the nature and extent of toxin-lipid interaction occurred in the pH range 4-6 and was not influenced by the presence of polysialogangliosides . The present data are in agreement with the idea that botulinum neurotoxins enter into nerve cells from a low pH intracellular compartment.

J Physiol, 1989 Apr, 411, 195 - 205
A study of synchronization of quantal transmitter release from mammalian motor endings by the use of botulinal toxins type A and D; Molgo J et al.; 1 . The effects of botulinum toxin (BoTx) types A and D on spontaneous and evoked phasic transmitter release were studied in the isolated extensor digitorum longus muscle of the rat or the levator auris longus muscle of mice . 2 . The toxins were injected subcutaneously into the hindleg of adult rats or the dorsal aspect of the neck of mice . At various times after the injection the muscles were removed from the anaesthetized animal and neuromuscular transmission examined in vitro by conventional intracellular techniques . 3 . Both toxins reduced spontaneous transmitter release recorded as the frequency of miniature end-plate potentials but BoTx type D was less effective in that respect than the type A toxin . 4 . With both toxins the block of evoked phasic transmitter release, recorded as end-plate potentials, was almost complete . As previously reviewed by Simpson (1986) the block produced by BoTx type A was partially reversed by procedures which elevate the intraterminal level of calcium ions . However, in BoTx type D-paralysed muscles such procedures failed to restore phasic transmitter release but caused a period of high-frequency asynchronous transmitter release following each nerve impulse . 5 . To investigate if the lack of synchronization of evoked transmitter release observed in BoTx type D-paralysed muscles was due to alterations in presynaptic currents we examined, by perineural recordings, the Na+, fast K+, slow K+, K+-Ca2+-dependent and the Ca2+ currents in BoTx type D-paralysed muscles . These presynaptic currents were not altered as compared to unpoisoned controls . 6 . We suggest that there exists a presynaptic process, which in addition to Ca2+ influx participates in transmitter synchronization and which is a main target for BoTx type D action.

Proc Natl Acad Sci U S A, 1989 Apr, 86(7), 2209 - 13
Anthrax toxin: channel-forming activity of protective antigen in planar phospholipid bilayers; Blaustein RO et al.; The three separate proteins that make up anthrax toxin--protective antigen (PA), edema factor (EF), and lethal factor (LF)--act in binary combinations to produce two distinct reactions in experimental animals: edema (PA + EF) and death (PA + LF) . PA is believed to interact with a membrane receptor, and after proteolytic processing, to mediate endocytosis and subsequent translocation of EF or LF into the cytosol . PA can be separated, after mild trypsinolysis, into two fragments, PA65 (65 kDa) and PA20 (20 kDa) . We demonstrate that trypsin-cleaved PA is capable of forming cation-selective channels in planar phospholipid bilayer membranes and that this activity is confined to the PA65 fragment; PA20, LF, and EF are devoid of channel-forming activity . These PA65 channels exhibit pH-dependent and voltage-dependent activity--a property reminiscent of the channels formed by the two-chain proteins diphtheria, tetanus, and botulinum toxins.

FEBS Lett, 1989 Mar 27, 246(1-2), 181 - 4
Nonmuscle actin ADP-ribosylated by botulinum C2 toxin caps actin filaments; Weigt C et al.; The effect of nonmuscle actin ADP-ribosylated by botulinum C2 toxin on the polymerization of nonmuscle actin was investigated in order to clarify whether nonmuscle actin is converted into a capping protein by ADP-ribosylation . ADP-ribosylated actin was found to decrease the rate of polymerization of actin filaments which are free at both ends . ADP-ribosylated actin turned out to have no effect on the rate or extent of polymerization at the pointed ends of actin filaments the barbed ends of which were capped by gelsolin . The monomer concentration reached at the final stage of polymerization was similar to the critical concentration of the pointed ends of actin filaments . The results suggest that nonmuscle actin ADP-ribosylated by botulinum C2 toxin acts as a capping protein which binds to the barbed ends to inhibit polymerization.

Mol Cell Biochem, 1989 Mar 16, 86(1), 87 - 95
Molecular topography and secondary structure comparisons of botulinum neurotoxin types A, B and E; Singh BR et al.; Botulinum neurotoxin (NT) serotypes A, B and E differ in microstructure and biological activities . The three NTs were examined for secondary structure parameters (alpha-helix, beta-sheet, beta-turn and random coil content) on the basis of circular dichroism; degree of exposed Tyr residues (second derivative spectroscopy) and state of the Trp residues (fluorescence and fluorescence quantum yield) . The proteins are high in beta-pleated sheet content (41-44%) and low in alpha-helical content (21-28%) . About 30-36% of the amino acids are in random coils . The beta-sheet contents in the NTs are similar irrespective of their structural forms (i.e . single or dichain forms) or level of toxicity . About 84%, 58% and 61% of Tyr residues of types A, B, and E NT, respectively, were exposed to the solvent (pH 7.2 phosphate buffer) . Although the fluorescence emission maximum of Trp residues of type B NT was most blue shifted (331 nm compared to 334 for types A and E NT, and 346 nm for free tryptophan) the fluorescence quantum yields of types A and B were similar and higher than type E . In general the NTs have similar secondary (low alpha-helix and high beta-sheets) and tertiary (exposed tyrosine residues and tryptophan fluorescence quantum yield) structures . Within this generalized picture there are significant differences which might be related to the differences in their biological activities.

J Neurol Neurosurg Psychiatry, 1989 Mar, 52(3), 355 - 63
Treatment of focal dystonias of the hand with botulinum toxin injections; Cohen LG et al.; The effects of botulinum toxin injections have been studied on 19 patients with hand dystonia . The dystonic muscles were identified by clinical examination and EMG findings of localised bursts of muscle activation with fine wire electrodes during the tasks that precipitated the dystonia . Injections into the most active muscles were given to each patient every 2 weeks in increasing doses (up to 20 U the first week, up to 40 U the second week, and up to 80 U the third week) until performance improvement was achieved . Subjective improvement of cramping, pain and/or tension was associated with temporary weakness in injected muscles . Benefit was seen in 16 patients, lasted between 1 and 6 months, and was reproducible.

Ann Otol Rhinol Laryngol, 1989 Mar, 98(3), 213 - 6
Botulinum toxin for relief of bilateral abductor paralysis of the larynx: histologic study in an animal model; Cohen SR et al.; We previously reported the effectiveness of botulinum toxin injections in the cricothyroid muscle under electromyographic guidance for lateralization of the true vocal cord in the mongrel dog . These additional experiments were performed to substantiate the effectiveness of botulinum toxin injections in laryngeal muscle to overcome airway obstruction produced by bilateral abductor vocal cord paralysis . A predetermined aliquot of the toxin was injected into the cricothyroid muscle of ten dogs, the duration of its effectiveness was noted, and repeated injections were given the animals . Biopsies of the injected muscles were obtained in three of the animals for routine histologic and electron microscopic studies . The effects of the toxin were recorded by cinelaryngoscopy and videotape documentation . In all the dogs, the true vocal cord was lateralized effectively and there was no morbidity, dysphagia, aspiration, or deaths . This study also confirms that the effects of the toxin in the canine larynx are spontaneously reversed and that multiple injections do not cause irreparable damage to the laryngeal muscles.

Pediatr Neurol, 1989 Mar-Apr, 5(2), 121 - 3
Use of botulinum toxin to treat blepharospasm in a 16-year-old with a dystonic syndrome; Seiff SR et al.; A 16-year-old boy with generalized dystonia had continuous, severe blepharospasm and facial grimacing . Local intradermal injections of botulinum A toxin greatly reduced the spasms and improved function . No side effects were observed . Local botulinum A toxin injections may be useful in the treatment of eyelid and facial spasms in patients with generalized dystonias.

Gan To Kagaku Ryoho, 1989 Mar, 16(3 Pt 2), 499 - 508
{ras p21-like small MW GTP-binding proteins in transmembrane signaling}; Takai Y et al.; We have separated multiple GTP-binding proteins (G proteins) having Mr values of about 20,000 (small MW G proteins) from bovine brain membranes, purified to near homogeneity and characterized two novel G proteins designated as smg p25A and smg p21, c-Ki-ras p21 and rho p20 . smg p25A is specifically found in neural tissue and adrenal medulla . This G protein is also found in rat pheochromocytoma PC-12 cells, and its mRNA level increases after differentiation of the cells into neuron-like cells in response to nerve growth factor or dibutyryl cyclic AMP . These results suggest that smg p25A plays an important role in the regulation of neural functions . In contrast, smg p21 is found in most tissues . This G protein has the same putative effector domain as ras p21s, suggesting that smg p21 exerts actions similar and/or antagonistic to those of ras p21s . In fact, smg p21 has been found to be identical with the ras-suppressor gene, designated as Krev-1, recently isolated by Noda's group . On the other hand, rho p20 is ADP-ribosylated by botulinum toxin . This toxin, known to be a neurotoxin, has recently been shown to induce the morphological changes similar to those induced by ras p21 in fibroblasts . Thus, ras p21-like small Mr G proteins are part of a huge network of intracellular regulatory systems and play important roles in the regulation of various cell functions including cell transformation, proliferation and differentiation.

Mol Cell Biochem, 1989 Feb 21, 85(2), 159 - 69
Effect of tetranitromethane on the biological activities of botulinum neurotoxin types A, B and E; Woody M et al.; Botulinum neurotoxin serotypes A, B and E were modified at pH 7.9 with tetranitromethane, a reagent highly specific for tyrosine residues . The type B and E neurotoxins were completely detoxified without significant damage to their serological activities . Under similar modification conditions, the type A neurotoxin was incompletely detoxified with some alteration in its serological reactivity . Modification of only tyrosine residues to nitrotyrosine was evident from amino acid analysis of the acid hydrolysates of the modified proteins . The completely detoxified type B and E neurotoxins, used as toxoid, elicited antibodies in rabbits . The antisera precipitated and neutralized the homologous neurotoxin . The two toxoids, type B and E, were prepared with greater than 99% pure neurotoxins as tested by sodium dodecyl sulfate-polyacrylamide gel electrophoresis whereas the traditional toxoids produced with formaldehyde are very crude preparations of the neurotoxin (approximately 90% impure) . Chemical modification using tetranitromethane is more specific than products that form during approximately 7 days of reaction between a protein and formaldehyde . The toxoids produced with tetranitromethane may be considered second-generation toxoids, compared with the first-generation toxoids (crude preparation of neurotoxins detoxified with formaldehyde).

Brain Res, 1989 Feb 6, 479(1), 167 - 71
Is the internal calcium regulation altered in type A botulinum toxin-poisoned motor endings?
Mallart A, Molgo J, Angaut-Petit D, Thesleff S.
The hypothesis according to which botulinum A toxin blocks acetylcholine release from motor endings by stimulating intracellular Ca2+ disposal systems was tested by recording presynaptic membrane currents from poisoned muscles . Calcium and calcium-activated potassium currents displayed amplitudes, time courses and stimulation frequency-dependent inactivation similar to those observed in unpoisoned preparations . This indicates that poisoned endings are no more efficient than normal ones in dealing with Ca2+ overloads.

Ann Otol Rhinol Laryngol, 1989 Feb, 98(2), 93 - 7
Botulinum toxin injection for the treatment of oromandibular dystonia; Blitzer A et al.; Dystonia is a neurologic disorder characterized by abnormal, involuntary movements causing twisting and turning postures; it is postulated to be a disorder of central motor processing . The dystonias, when classified by region of the body involved, have been characterized as focal, segmental, and generalized . Focal dystonia can affect jaw mechanics, leading to forceful contraction of the jaw muscles and resulting in inappropriate deviation of the jaw . Localized injections of botulinum toxin have been used successfully in the management of other focal or segmental dystonias . We have treated 20 oromandibular dystonia patients with botulinum toxin . Six patients had only jaw and tongue involvement; 11 had blepharospasm and jaw involvement; and three had jaw involvement as part of a more generalized dystonia . Five patients had been diagnosed originally and treated as having temporomandibular joint syndrome . All but one of the patients had improvement of their symptoms with the toxin injections . The patients averaged 47% improvement with the injections.

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