Botulinum neurotoxin serotype A heavy chain intervenes in the H3 acetylation: A preliminary study / 中国比较医学杂志

Objective To investigate the effect and molecular mechanism of botulinum neurotoxin serotype A (BoNT/A) heavy chain on neuron regeneration. Methods Cell culture, rats, immunofluorescence, SDS-PAGE and western blot, etc. were adopted in this study to explore the alterations of histone-3 acetylation (acetyl-H3 ) by local treatment of BoNT/A heavy chain to spinal cord injury (SCI) in rats (in vivo) or by adding it into cell culture (in vitro). Meanwhile, the relevance of acetyl-H3 to neurite out-growth based on SCI and cell culture with BoNT/A heavy chain application was approached as well. Results The application of BoNT/A heavy chain to cultured Neuro-2a cells increased the level of H3 acetylation. The increase of H3 acetylation was paralleled with the growth of neuritogenesis. Also, the neuronal treatment of BoNT/A heavy chain to SCI promoted the re-growth of neuronal processes surrounding the lesions. The growth of neuronal processes was positively correlated to the level of H3 acetylation. During the periods of BoNT/A heavy chain treatment in vivo or in vitro, the increase of H3 acetylation showed two peaks. Conclusions BoNT/A heavy chain increased the H3 acetylation, which might be one of its neuritogenic mechanisms.

Antinociceptive Effects of Transcytosed Botulinum Neurotoxin Type A on Trigeminal Nociception in Rats

We examined the effects of peripherally or centrally administered botulinum neurotoxin type A (BoNT-A) on orofacial inflammatory pain to evaluate the antinociceptive effect of BoNT-A and its underlying mechanisms. The experiments were carried out on male Sprague-Dawley rats. Subcutaneous (3 U/kg) or intracisternal (0.3 or 1 U/kg) administration of BoNT-A significantly inhibited the formalin-induced nociceptive response in the second phase. Both subcutaneous (1 or 3 U/kg) and intracisternal (0.3 or 1 U/kg) injection of BoNT-A increased the latency of head withdrawal response in the complete Freund's adjuvant (CFA)-treated rats. Intracisternal administration of N-methyl-D-aspartate (NMDA) evoked nociceptive behavior via the activation of trigeminal neurons, which was attenuated by the subcutaneous or intracisternal injection of BoNT-A. Intracisternal injection of NMDA up-regulated c-Fos expression in the trigeminal neurons of the medullary dorsal horn. Subcutaneous (3 U/kg) or intracisternal (1 U/kg) administration of BoNT-A significantly reduced the number of c-Fos immunoreactive neurons in the NMDA-treated rats. These results suggest that the central antinociceptive effects the peripherally or centrally administered BoNT-A are mediated by transcytosed BoNT-A or direct inhibition of trigeminal neurons. Our data suggest that central targets of BoNT-A might provide a new therapeutic tool for the treatment of orofacial chronic pain conditions.

Rehabilitation for Upper Limb Hemiparesis after Stroke: / The Japanese Journal of Rehabilitation Medicine

A multi-institutional study using our protocol of low-frequency repetitive transcranial magnetic stimulation (rTMS) and intensive occupational therapy (OT) showed significant improvement of motor function of the affected upper limb in poststroke patients. The response to the treatment was not influenced by age or time after stroke onset. Our protocol is a safe, feasible, and potentially useful neurorehabilitative intervention for upper limb hemiparesis after stroke. The extent of the improvement seems to be influenced by the baseline severity of upper limb hemiparesis. The results suggest that patients with Brunnstrom stage 4 or 5 upper limb hemiparesis are best suited for this protocol. Botulinum toxin type A (BoNT-A) has been reported to be an effective treatment for limb spasticity after stroke. However, the spasticity reduction after BoNT-A injection alone does not ensure an improvement in the active motor function of the affected limb. Our proposed protocol of a BoNT-A injection, followed by home-based functional training seems to have the potential to improve the active motor function of the affected upper limb after stroke.

Development of Capture ELISA Using a Biotinylated Monoclonal Antibody for Detection of Botulinum Neurotoxin Type A

A capture enzyme-linked immunosorbent assay (capture ELISA) was developed to detect Clostridium botulinum neurotoxin type A (BoNT/A) in assay buffer and human serum. The assay is based upon affinity-purified rabbit polyclonal and biotinylated monoclonal antibodies directed against the BoNT/A complex purified from C. botulinum ATCC19397. For the capture ELISA, the optimized amount (2 microgram/ml) of rabbit polyclonal antibody was immobilized on ELISA plates to detect BoNT/A (ranging from 0 to 500 ng/ml), which was recognized by 2 microgram/ml of the monoclonal antibody. From three independent repeated experiments, standard curves were linear over the range of 0~31.25 ng/ml BoNT/A and the coefficients (r(2)) ranged from 0.9951~0.9999 for all assays. The inter-variations were typically 0.50~6.93% and the specificity was confirmed by showing no cross-reactivity against BoNT/B and /E. The detection limit of capture ELISA was 0.488 ng/ml, which was close to mouse LD(50). In addition, application with BoNT/A-spiking human sera showed a possibility to detect BoNT/A with capture ELISA from the contaminated human sera. Taken together, the newly developed capture ELISA could serve as a rapid and sensitive screening tool for detecting BoNT/A simultaneously from massive specimens.

Soluble expression of reconstructed heavy chain fragment of botulinum neurotoxin serotype A in Escherichia coli / 解放军医学杂志

Objective To clone the reconstructed Hc gene of botulinum neurotoxin type A(BoNT/AHc)and to explore the soluble expression of the reconstructed gene in E.coli.Methods The gene of Hc fragment was synthesized by replacing rare codons with frequent ones in E.coli,while the components of amino acids didn't change,and the contents of AT decreased from 76.4 % to 57.3%.The reconstructed gene was then cloned into the prokaryotic expression vector pQE-60.The recombinant plasmid pQE-60Hc was introduced into E.coli Origami(DE3)that was induced to express the protein.The soluble expression products were then detected by Western Blot.Finally the expressed product of recombination plasmid pQE-60Hc was analyzed with SDS-PAGE after purification through Ni-NTA column.Results The reconstructed Hc gene of BoNT/AHc was amplified by PCR.The expression vector pQE-60Hc was constructed successfully with BamH Ⅰ and Nco Ⅰto ingest both BoNT/AHc and vector pQE-60.Reconstructed gene could be expressed effectively in E.coli in soluble form.The molecular weight of expressed product of recombination plasmid pQE-60Hc analyzed by SDS-PAGE was 52 000,which was the same as anticipated.And the soluble expression product accounted for to 11.5 % of the total bacterial protein.Western blot assay showed that the expression product could bind to specific antibody agent BoNT/A.Conclusion The expression vector has been constructed and the reconstructed gene was expressed successfully and effectively in E.coli,which may provide a foundation for further study on antitoxin and vaccine.