Interaction of Botulinum Toxin with the Epithelial Barrier

Botulinum neurotoxin (BoNT) is a protein toxin (~150 kDa), which possesses a metalloprotease activity. Food-borne botulism is manifested when BoNT is absorbed from the digestive tract to the blood stream and enters the peripheral nerves, where the toxin cleaves core proteins of the neuroexocytosis apparatus and elicits the inhibition of neurotransmitter release. The initial obstacle to orally ingested BoNT entering the body is the epithelial barrier of the digestive tract. Recent cell biology and molecular biology studies are beginning to elucidate the mechanism by which this large protein toxin crosses the epithelial barrier. In this review, we provide an overview of the structural features of botulinum toxins (BoNT and BoNT complex) and the interaction of these toxins with the epithelial barrier

Detection and Typing of Genital High-Risk HPV DNAs in Cervical Scrapes Using the E6E7-Specific Consensus PCR

Specific types of human papillomaviruses (HPVs) are closely associated with the development of genital carcinomas. We previousy reported a PCR method which amplifies the E6E7 sequence from 6 different high-risk genital HPVs (HPV16, 18, 31, 33, 52 and 58) (J Gen Virol 1991, 72 : 1039-1044.). To amplify broader types of genital high-risk HPVs, we have modified our consensus primers by extending 9 nucleotides at the 3 end of the sense primer and changing 5 nucleotides at the 5 end of the anitisense primer. Genotype diag-nosis was carried out by AvaII plus Rsal digestion. This modified PCR method enabled the detection of trace levels of at least 11 types of genital high-risk HPVs (HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56 and 58), at subpicogram to sub-nanogram amounts of cloned DNA, amplified after the consensus PCR. We applied this method to analyze 155 cervical scrapes from patients who had been diagnosed with premalignant or malignant cervical lesions. HPVs were detected in 63.0% of mild dysplasia (17/27), 100% of moderate dysplasia (all 12 cases), 91.7% of severe dysplasia (11/12), 95.8% of carcinoma in situ (23/24), and 80.0% of invasive cervical cancer (52/65). HPV16 was present predominant-ly (60.9%), followed by HPV58 (153%), HPV52 (13.9%), HPV31 (13.0%), HPV18 (6.1%), HPV35 (2.6%), HPV51 (2.6%), HPV56 (2.6%), HPV33 (1.7%) and HPV39 (1.7%). Five cases contained unclassified types (4.3%). The results indicate that this modified E6E7 consensus PCR method provides a quick and easy way to detect and diagnose genotypes of the high-risk genital HPVs from scraped cells

Structural and Expressional Alterations of Episomal and Integrated Human Papillomavirus Type 16 in Precancerous Lesions and Carcinomas of the Cervix.

HPV infection has long been implicated in the development of cervical car-cinoma. We have analyzed the HPV 16 genome structure and expression of the viral mRNA in cervical intraepithelial neoplasias (CINs) and cervical car-cinomas by using modified polymerase chain reaction (PCR) methods. Genome structure has been determined by PCR using multi-primer sets which are located in each open reading frames and then these results have been compared with the physical state of the viral DNA determined by two-dimensional electrophoresis. Furthermore, we have analyzed the expression of HPV 16 mRNA and genome structure using DNA and RNA simultaneously extracted from CINs and cervical carcinomas using PCR and reverse transcription (RT-) PCR. Our data showed that the DNA regions from the El to Ll region were delet-ed in two of three CINs containing episomal HPV 16 and three out of seven cervical carcinomas containing integrated HPV 16. However, the E6/E7 region was conserved in all the HPV 16-positive samples. RT-PCR analysis has determined the presence of mRNA species which could encode the E6, E6*I, E6*II, E7, E2, E2 ? C, E1^E4, E1^E2 ? C, E4, E2 ? C-E5 and L2 proteins. The overall results of DNA and mRNA analyses in cervical lesions indicated that the expression patterns of the early and late transcripts studied were not specifically related to the grade of malignancy and the physical state or the deletion of the viral genome. Furthermore, alterations in the splicing pat-terns of HPV 16 transcripts may not be involved in tumor progression

Detection of Human Papillomavirus (HPV) DNA Sequences in Normal Oral Scrapes Using the Nested PCR.

We investigated the prevalence rate of HPV DNAs in normal mucosa in the oral region. The nested PCR method was utilized to detect target DNA sequences using HPV E6/E7 consensus primer pairs. Of 56 patients examined, HPV 6 and HPV 16 DNA sequences were detected in a 46-year-old male and a 35-year-old female, respectively. These results suggest that HPVs are uncom-mon in normal oral epithelium, and that we should carry out careful follow-up in HPV DNA-positive cases

Role of C-Terminal Region of HA-33 Component of Botulinum Toxin in Hemagglutination

Clostridium botulinum D型1873株とC型Yoichi株の産生するプロジェニター毒素から得られた1種類のサブコンポーネントである血球凝集素（HA-33）は、C及びD型参照株のそれより少し小さい分子サイズであるが、他のコンポーネントのの大きさに違いがないことが、SDS-PAGEを用いて見いだされた。すなわち、HA-33のN-及びC-末端シーケンス分析に基つき、両株のHA-33淡白において特異的なサイトでC-末端から33アミノ酸残基の欠落があることが見いだされた。両株のプロジェニター毒素は、参照株毒素の血球凝集力価2かそれ以下の弱い血球凝集活性を示すが、赤血球に吸着することはできない。これらの結果はHA-33の短いC-末端部分が、ボツリヌスプロジェンター毒素の血球凝集活性において重要な役割を果たしていることを示す。さらに、シーケンスモチーフの探索により、HA-33のC-末端部分は炭化水素認識サブドメインを持っていることが推測された

Molecular typing of enterohemorrhagic Escherichia coli O157:H7 isolated in Okayama Prefecture using pulsed field gel electrophoresis and random amplification of polymorphic DNA.

Three outbreaks and many isolated cases of enterohemorrhagic Escherichia coli O157:H7 occurred in 1996 and 1997 in Okayama Prefecture, Japan. In an attempt to investigate the route of these infections, the strains isolated from the 3 outbreaks (total 33 strains) and 15 isolated cases (total 15 strains) were investigated using random amplification of polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE). In addition, 10 strains from an outbreak in Tojo Cho, Hiroshima Prefecture (June 1996), 2 strains from the particular types of meat in Kochi Prefecture, and 42 strains isolated from bovine feces in a farm in Okayama Prefecture were also investigated in the same manner. PFGE was much more useful than RAPD for molecular typing of the clinical isolates, in that it allowed us to classify them into 10 PFGE groups. We noted that the strains differed according to the time and place of the outbreaks (or isolated cases). This indicates that O157:H7 infections in Okayama Prefecture were caused by different strains (although some cases were aggravated by the same strains as were found in other areas). The isolates from bovine feces were classified into 5 groups by PFGE profiles, but none of them were identical to those of the clinical isolates.</p

The Double Polymerase Chain Reaction with Consensus Primers Permits Rapid and Sensitive Detection of Genital Human Papillomavirus Oncogenes

We have developed a sensitive procedure for the detection of relatively low copy numbers of multiple genital human papillomaviruses (HPVs) using the polymerase chain reaction (PCR). HPV DNAs were detected by agarose gel electrophoresis and ethidium bromide staining after 2 rounds of PCR amplification (double PCR) with outer and inner consensus primer pairs for HPV-6, 11, 16, 18, 31, 33, 52, and 58. The detection limit of this method (i. e., 10?? copy of HPV DNA per cell in 1 μg cell DNA) was sufficient for analysis of cervical intraepithelial neoplasia (CIN) specimens. Overall prevalence rate of HPV was 100% in 20 cases of CIN specimens. HPV typing by restriction enzyme analysis revealed that HPV-16 sequence was present in 11 cases, HPV-18 in 1 case, HPV-31 in 4 cases, HPV-33 in 1 case, HPV-52 in 2 cases, HPV-58 in 3 cases, and an unidentified type(s) in 3 cases. There were 4 cases of mixed infections. This procedure obviates the use of hybridization- based for-mat for identification of at least 8 types of HPV sequences present in a small fraction of cells within a heterogeneous population

Multivalency effects of hemagglutinin component of type B botulinum neurotoxin complex on epithelial barrier disruption

金沢大学医薬保健研究域医学系Hemagglutinin (HA) is one of the components of botulinum neurotoxin (BoNT) complexes and it promotes the absorption of BoNT through the intestinal epithelium by at least two specific mechanisms: cell surface attachment by carbohydrate binding, and epithelial barrier disruption by E-cadherin binding. It is known that HA forms a three-arm structure, in which each of three protomers has three carbohydrate-binding sites and one E-cadherin-binding site. A three-arm form of HA is considered to bind to these ligands simultaneously. In the present study, we investigated how the multivalency effect of HA influences its barrier-disrupting activity. We prepared type B full-length HA (three-arm form) and mini-HA, which is a deletion mutant lacking the trimer-forming domain. Size-exclusion chromatography analysis showed that mini-HA exists as dimers (two-arm form) and monomers (one-arm form), which are then separated. We examined the multivalency effect of HA on the barrier-disrupting activity, the E-cadherin-binding activity, and the attachment activity to the basolateral cell surface. Our results showed that HA initially attaches to the basal surface of Caco-2 cells by carbohydrate binding and then moves to the lateral cell surface, where the HA acts to disrupt the epithelial barrier. Our results showed that the multivalency effect of HA enhances the barrier-disrupting activity in Caco-2 cells. We found that basal cell surface attachment and binding ability to immobilized E-cadherin were enhanced by the multivalency effect of HA. These results suggest that at least these two factors induced by the multivalency effect of HA cause the enhancement of the barrier-disrupting activity. © 2017 The Societies and John Wiley & Sons Australia, LtdEmbargo Period 12 month

Collagen adhesion gene is associated with blood stream infections caused by methicillin-resistant Staphylococcus aureus

Objectives: Methicillin-resistant Staphylococcus aureus (MRSA) causes hospital- and community-acquired infections. It is not clear whether genetic characteristics of the bacteria contribute to disease pathogenesis in MRSA infection. We hypothesized that whole genome analysis of MRSA strains could reveal the key gene loci and/or the gene mutations that affect clinical manifestations of MRSA infection. Methods: Whole genome sequences (WGS) of MRSA of 154 strains were analyzed with respect to clinical manifestations and data. Further, we evaluated the association between clinical manifestations in MRSA infection and genomic information. Results: WGS revealed gene mutations that correlated with clinical manifestations of MRSA infection. Moreover, 12 mutations were selected as important mutations by Random Forest analysis. Cluster analysis revealed strains associated with a high frequency of bloodstream infection (BSI). Twenty seven out of 34 strains in this cluster caused BSI. These strains were all positive for collagen adhesion gene (cna) and have mutations in the locus, those were selected by Random Forest analysis. Univariate and multivariate analysis revealed that these gene mutations were the predictor for the incidence of BSI. Interestingly, mutant CNA protein showed lower attachment ability to collagen, suggesting that the mutant protein might contribute to the dissemination of bacteria. Conclusions: These findings suggest that the bacterial genotype affects the clinical characteristics of MRSA infection. (c) 2019 The Author(s). Published by Elsevier Ltd on behalf of International Society for Infectious Diseases