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.

Effect of botulinum neurotoxin type A heavy chain on the growth-related proteins after spinal cord injury in rats / 中国比较医学杂志

Objective To investigate the effect of recombinant botulinum neurotoxin serotype A heavy chain (BoNT/A heavy chain)on local proteins which are related to nerve growth after spinal cord injury in rats,and to get some experimental evidence to explain the mechanism of BoNT/A heavy chain in stimulating neuritogenesis. Methods Recombinant botulinum neurotoxin serotype A heavy chain was applied locally or intrathecally to rats with ipsilateral semi-dissociated lumbar spinal injury. Local spinal tissue was extracted for general protein expression by two dimension electrophoresis plus nitrate silver staining after different time period of injury. Based on the results of 2-D gel electrophoresis,growth-associated protein 43(GAP-43)and of superior cervical ganglion 10(SCG 10)were selected to examine the changes of their expression and distribution features under BoNT/A heavy chain administration using SDS-PAGE,western blot and immunofluorescence. Results (1)The model of spinal cord injury(SCI)in this study was an ipsilateral semi-dissociated lumbar SCI in rat. The rats showed obvious motor and sensory dysfunction in the ipsilateral hind limb.(2)The results from 2-D gel electrophoresis plus nitrate silver staining showed that the administration of BoNT/A heavy chain based on SCI altered the local protein expression pattern. The decrease or increase in the expression of some protein dots /dots group was clearly seen after single SCI. However, these changes were transformed by BoNT/A heavy chain treatment,which appeared as a reversed pattern turning toward that in control group or further increased expression upon SCI,such as the dots located respectively at 35-45 kDa and 18-25 kDa level,pI between 5-7. In addition,the expression of the two dots located at the level as above increased after SCI only, and showed further increase in their expression with BoNT/A heavy chain intervention.(3)The changes of selective GAP-43 and SCG 10 expression and distribution by western blot and immunofluorescence indicated that the administration of BONT/A heavy chain based on SCI amplified the expression of GAP-43 and SCG 10(P < 0.05). Meanwhile,the positive immuonfluorescent staining for both GAP-43 and SCG 10 mainly distributed nearby the proximal area of injury, both cytoplasm and neuronal processes were positively stained. Conclusions Intrathecal delivery of BoNT/A heavy chain increases the expression of growth-associated proteins GAP 43 and SCG 10 after SCI in rats.

Chlamydia trachomatis serotype A infections in the Amazon region of Brazil: prevalence, entry and dissemination

INTRODUCTION: Chlamydia infection is associated with debilitating human diseases including trachoma, pneumonia, coronary heart disease and urogenital diseases. Serotypes of C. trachomatis show a fair correlation with the group of diseases they cause, and their distribution follows a well-described geographic pattern. Serotype A, a trachoma-associated strain, is known for its limited dissemination in the Middle East and Northern Africa. However, knowledge on the spread of bacteria from the genus Chlamydia as well as the distribution of serotypes in Brazil is quite limited. METHODS: Blood samples of 1,710 individuals from ten human population groups in the Amazon region of Brazil were examined for antibodies to Chlamydia using indirect immunofluorescence and microimmunofluorescence assays. RESULTS: The prevalence of antibodies to Chlamydia ranged from 23.9% (Wayana-Apalai) to 90.7% (Awa-Guaja) with a mean prevalence of 50.2%. Seroreactivity was detected to C. pneumoniae and to all serotypes of C. trachomatis tested; furthermore, we report clear evidence of the as-yet-undescribed occurrence of serotype A of C. trachomatis. CONCLUSIONS: Specific seroreactivity not only accounts for the large extent of dissemination of C. trachomatis in the Amazon region of Brazil but also shows an expanded area of occurrence of serotype A outside the epidemiological settings previously described. Furthermore, these data suggest possible routes of Chlamydia introduction into the Amazon region from the massive human migration that occurred during the 1,700s. .

Promoting effect of botulinum neurotoxin serotype A heavy chain on neu-ritogenesis in cultured Neuro-2a cells / 中国病理生理杂志

AIM:To observe the neuritogenic actions of botulinum neurotoxin serotype A heavy chain ( BoNT/A HC) on cultured Neuro-2a cells and to investigate the related signaling mechanisms for the effect of BoNT /A HC. METHODS:Neuro-2a cells were treated with different doses of BoNT/A HC (0.01, 0.1, 1 and 10 nmol/L), and then the cells were harvested at 24 h, 48 h and 72 h of BoNT/A HC exposure for detecting the neurite length and the percen-tage of the cells with neuronal processes by immunofluorescence staining .The most efficient dose of BoNT/A HC was cho-sen for exposure to Neuro-2a cells as the above.Whole cell protein was harvested at different time points for detecting the protein levels of phosphorylated ERK 1/2 ( p-ERK1/2 ) and phosphorylated Akt ( p-Akt ) by Western blot .RESULTS:Low doses of BoNT/A HC stimulated the neurite outgrowth , and increased the percentage of the cells with neurites com-pared with the negative controls (P<0.05), especially in the group with 1 nmol/L of BoNT/A HC treatment.Meanwhile, the phosphorylation of ERK 1/2 and Akt was increased after treated with BoNT/A HC.There was an increasing tendency for the phosphorylation of ERK1/2 after the exposure of the cells to BoNT/A HC.The obvious increase in p-ERK1/2 was seen from 60 min to 5 h with 1 nmol/L of BoNT/A HC treatment ( P<0.05 ) , and the increased protein level of p-Akt was mainly observed at 15 min and 60 min ( P<0.05 ) .CONCLUSION: BoNT/A HC stimulates the neuritogenesis .The neuritogenic mechanism of BoNT/A HC on Neuro-2a cells might be realized by activation of the phosphorylation of ERK 1/2 and Akt.

A Simple and Rapid Colloidal Gold-based Immunochromatogarpic Strip Test for Detection of FMDV Serotype A / 中国病毒学·英文版

A sandwich format immunochromatographic assay for detecting foot-and-mouth disease virus (FMDV) serotypes was developed. In this rapid test, affinity purified polyclonal antibodies from Guinea pigs which were immunized with sucking-mouse adapted FMD virus (A/AV88(L) strain) were conjugated to colloidal gold beads and used as the capture antibody, and affinity purified polyclonal antibodies from rabbits which were immunized with cell-culture adapted FMD virus (A/CHA/09 strain) were used as detector antibody. On the nitrocellulose membrane of the immunochromatographic strip, the capture antibody was laid on a sample pad, the detector antibody was printed at the test line(T) and goat anti-guinea pigs IgG antibodies were immobilized to the control line(C). The lower detection limit of the test for a FMDV 146S antigen is 11.7ng/ml as determined in serial tests after the strip device was assembled and the assay condition optimization. No cross reactions were found with FMDV serotype C, Swine vesicular disease (SVD), Vesicular stomatiti svirus (VSV) and vesicular exanthema of swine virus (VES) viral antigens with this rapid test. Clinically, the diagnostic sensitivity of this test for FMDV serotypes A was 88.7% which is as same as an indirect-sandwich ELISA. The specificity of this strip test was 98.2% and is comparable to the 98.7% obtained with indirect-sandwich ELISA. This rapid strip test is simple, easy and fast for clinical testing on field sites;no special instruments and skills are required, and the result can be obtained within 15 min. To our knowledge, this is the first rapid immunochromatogarpic assay for serotype A of FMDV.

Enhanced Effects of BoNT/A DNA Vaccines by Electric Pulses and Bupivacaine / 中国生物工程杂志

Objective:To determine if suitable electric pulses-mediated DNA and DNA and bupivacaine complexes delivery technologies could enhance effects of botulinum neurotoxin serotype A (BoNT/A) DNA vaccines in mouse model. Methods:Vaccination of mice i.m. with plasmid DNA replicon vaccine pSCARSHc and conventional plasmid DNA vaccine pcDNASHc following electric pulses and with DNA and bupivacaine complexes. AHc-specific group antibody ELISA titers and lymphocyte proliferative responses of mice were detected and IgG1 and IgG2a isotype profiles were assayed. Results:Immune effects of DNA vaccines were enhanced following electric pulses and bupivacaine delivery. Effects of DNA vaccines following electric pulses were better than that of DNA vaccines formulated with bupivacaine,and the combined delivery technology of electric pulses and bupivacaine induced the highest level of specific antibodies and lymphocyte proliferative responses. Plasmid DNA replicon vaccine pSCARSHc induced relatively higher AHc-specific antibodies and lymphocyte proliferative responses in immunized Balb/c mice than conventional plasmid DNA vaccine pcDNASHc in these DNA delivery technologies. And vaccine pSCARSHc induced Th2/Th1-type immune responses with a general bias to Th2-type,and vaccine pcDNASHc induced Th2-type immune responses. Conclusion:Suitable electric pulses-mediated DNA and DNA and bupivacaine complexes delivery technologies could enhance effects of BoNT/A DNA vaccines in mouse model. Therefore,the methods described here potentially provide suitable strategies in developing an efficacious vaccine against botulinum neurotoxin serotype A.

Isolation of Cryptococcus neoformans var. grubii (serotype A) from Pigeon Droppings in Korea

Three hundred and sixty five samples of avian droppings, collected from parks and zoo, were investigated for the occurrence of Cryptococcus neoformans in Korea. Thirteen samples were positive for C. neoformans. All isolates were obtained from withered pigeon droppings. Identification and serotyping of isolates were determined by means of serological test and polymerase chain reaction (PCR) fingerprinting. All isolates belonged to C. neoformans var. grubbi (serotype A).

Affinity Selection of Human ScFv Against Botulinum Neurotoxin Serotype A / 微生物学通报

In this work, rare specific binders to botulinum neurotoxin antigen were selected from human phage immunized library through three rounds of panning in vitro. Of them, clone ScFvB17 is of high affinity and specificity to target antigen. B17 is of 750bp encoding 250 amino acid. Analysis results of gene structure showed V-genes of B17 belonging respectively to VH4 or? chainⅡ family are new antibody Fv genes. More important result showed that recombinant ScFvB17 expressed in E.coli could bind specific for neurotoxin competing with antiserum. Successful selection of human phage antibody library and obtaining specific ScFv against botulinum neurotoxin serotype A provide useful materials for further study on detection of neurotoxin and human engineering therapeutic antitoxin.

Recombinant Design of Fc Fusion Protein of Human ScFv Against BoNTa / 微生物学通报

Human ScFv against botulinum neurotoxin serotype A(BoNTa) was modified by fusing human IgG1 Fc to C terminal of ScFv. ScFv-Fc fusion protein was expressed at high level over 30% of total host cell proteins in E.coli. Recombinant protein existed in inclusion body form. Renatured ScFv-Fc was purified to 90%~95% by Protein G Sepharose column. In vitro ScFv-Fc could bind specific to toxiod BoNTa in ELISA. Recombinant ScFv-Fc had similar relative affinity to parent ScFv and had improved stability.