Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.24.69 (botulinum neurotoxin)
 1,901 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Neuronal cells grown in culture were exposed to drugs that stimulate protein kinase C (phorbol myristate acetate), inhibit the catalytic site in protein kinase C (H7, staurosporine) or inhibit the regulatory site in protein kinase C (calphostin, sphingosine). In NG-108 and N1E-115 cells, phorbol myristate acetate produced substantial stimulation of protein kinase C activity (0.1 microM produced approximately 75% stimulation). In these same cells, H7 [100% inhibition concentration (IC100) approximately 1 mM] and staurosporine (IC100 approximately 0.2 microM) inhibited the catalytic site in the enzyme, and calphostin (IC80-IC90 approximately 2.0 microM) and sphingosine (IC80-IC90 approximately 1 microM) inhibited the regulatory site in the enzyme. Phorbol myristate acetate, as well as drugs that inhibit the catalytic and regulatory sites in protein kinase C, were tested for their effects on phrenic nerve-hemidiaphragm preparations. At concentrations that stimulated enzyme activity in neuronal cells in culture, phorbol myristate acetate did not augment normal transmission, nor did it restore transmission to preparations bathed in medium with low calcium (0.4-0.6 mM). At concentrations equivalent to the IC80 to IC100 values in neuronal cells in culture, H7, staurosporine, calphostin and sphingosine did not paralyze short-term transmission, nor did they depress transmission in tissues bathed in low calcium. Pretreatment of neuromuscular preparations with phorbol myristate acetate, H7, staurosporine, calphostin or sphingosine did not alter the amount of time necessary for botulinum neurotoxin type A, botulinum neurotoxin type B or tetanus toxin to paralyze transmission. The data indicate that protein kinase C is not required for short-term neuromuscular transmission.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Role of protein kinase C in short-term transmission at the mammalian neuromuscular junction. 133 62

We have previously described genetic constructs and expression systems that enable facile production of recombinant derivatives of botulinum neurotoxins (BoNTs) that retain the structural and trafficking properties of wt BoNTs. In this report we describe the properties of one such derivative, BoNT/A ad, which was rendered atoxic by introducing two amino acid mutations to the light chain (LC) of wt BoNT/A, and which is being developed as a molecular vehicle for delivering drugs to the neuronal cytoplasm. The neuronal binding, internalization, and intracellular trafficking of BoNT/A ad in primary hippocampal cultures was evaluated using three complimentary techniques: flow cytometry, immunohistochemistry, and Western blotting. Neuronal binding of BoNT ad was significantly increased when neurons were incubated in depolarizing medium. Flow cytometry demonstrated that BoNT/A ad internalized into neurons but not glia. After 24 hours, the majority of the neuron-bound BoNT/A ad became internalized, as determined by its resistance to pronase E-induced proteolytic degradation of proteins associated with the plasma membrane of intact cells. Significant amounts of the atoxic LC accumulated in a Triton X-100-extractable fraction of the neurons, and persisted as such for at least 11 days with no evidence of degradation. Immunocytochemical analysis demonstrated that the LC of BoNT/A ad was translocated to the neuronal cytoplasm after uptake and was specifically targeted to SNARE proteins. The atoxic LC consistently co-localized with synaptic markers SNAP-25 and VAMP-2, but was rarely co-localized with markers for early or late endosomes. These data demonstrate that BoNT/A ad mimics the trafficking properties of wt BoNT/A, confirming that our platform for designing and expressing BoNT derivatives provides an accessible system for elucidating the molecular details of BoNT trafficking, and can potentially be used to address multiple medical and biodefense needs.
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PMID:Atoxic derivative of botulinum neurotoxin A as a prototype molecular vehicle for targeted delivery to the neuronal cytoplasm. 2446 85

Neurological diseases constitute a quarter of global disease burden and are expected to rise worldwide with the ageing of human populations. There is an increasing need to develop new molecular systems which can deliver drugs specifically into neurons, non-dividing cells meant to last a human lifetime. Neuronal drug delivery must rely on agents which can recognise neurons with high specificity and affinity. Here we used a recently introduced 'stapling' system to prepare macromolecules carrying duplicated binding domains from the clostridial family of neurotoxins. We engineered individual parts of clostridial neurotoxins separately and combined them using a strong alpha-helical bundle. We show that combining two identical binding domains of tetanus and botulinum type D neurotoxins, in a sterically defined way by protein stapling, allows enhanced intracellular delivery of molecules into neurons. We also engineered a botulinum neurotoxin type C variant with a duplicated binding domain which increased enzymatic delivery compared to the native type C toxin. We conclude that duplication of the binding parts of tetanus or botulinum neurotoxins will allow production of high avidity agents which could deliver imaging reagents and large therapeutic enzymes into neurons with superior efficiency.
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PMID:Duplication of clostridial binding domains for enhanced macromolecular delivery into neurons. 3214 Jun 81

Botulinum toxin is one of the most potent molecule known to mankind. A neurotoxin, with high affinity for cholinergic synapse, is effectively capable of inhibiting the release of acetylcholine. On the other hand, botulinum toxin is therapeutically used for several musculoskeletal disorders. Although most of the therapeutic effect of botulinum toxin is due to temporary skeletal muscle relaxation (mainly due to inhibition of the acetylcholine release), other effects on the nervous system are also investigated. One of the therapeutically investigated areas of the botulinum neurotoxin (BoNT) is the treatment of pain. At present, it is used for several chronic pain diseases, such as myofascial syndrome, headaches, arthritis, and neuropathic pain. Although the effect of botulinum toxin in pain is mainly due to its effect on cholinergic transmission in the somatic and autonomic nervous systems, research suggests that botulinum toxin can also provide benefits related to effects on cholinergic control of cholinergic nociceptive and antinociceptive systems. Furthermore, evidence suggests that botulinum toxin can also affect central nervous system (CNS). In summary, botulinum toxin holds great potential for pain treatments. It may be also useful for the pain treatments where other methods are ineffective with no side effect(s). Further studies will establish the exact analgesic mechanisms, efficacy, and complication of botulinum toxin in chronic pain disorders, and to some extent acute pain disorders.
Neuronal Signal 2018 Sep
PMID:Therapeutic use of botulinum toxin in pain treatment. 3271 87