Clinical Study of New Tetravalent (Type A, B, E, and F) Botulinum Toxoid Vaccine Derived from M Toxin in Japan.

Botulinum toxin is the most poisonous substance known, and is believed to be a highly lethal as a biological weapon; researchers of the toxin are exposed to this hazard. Botulinum toxoid vaccines have been produced and used in Japan. However, since clinical studies involving these vaccines were conducted before establishment of the Ethical Guidelines for Clinical Research in Japan, their immunogenicity and safety were not systematically assessed.

Enteropathogenic Escherichia coli Uses NleA to Inhibit NLRP3 Inflammasome Activation.

Enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC) are related strains capable of inducing severe gastrointestinal disease. For optimal infection, these pathogens actively modulate cellular functions through the deployment of effector proteins in a type three secretion system (T3SS)-dependent manner. In response to enteric pathogen invasion, the Nod-like receptor pyrin domain containing (NLRP) inflammasome has been increasingly recognized as an important cytoplasmic sensor against microbial infection by activating caspase-1 and releasing IL-1β.

Simulation and estimation of gene number in a biological pathway using almost complete saturation mutagenesis screening of haploid mouse cells.

Background: Genome-wide saturation mutagenesis and subsequent phenotype-driven screening has been central to a comprehensive understanding of complex biological processes in classical model organisms such as flies, nematodes, and plants. The degree of "saturation" (i.e.

Type A1 but not type A2 botulinum toxin decreases the grip strength of the contralateral foreleg through axonal transport from the toxin-treated foreleg of rats.

The adverse effects of botulinum LL toxin and neurotoxin produced by subtype A1 (A1LL and A1NTX) are becoming issues, as the toxins could diffuse from the toxin-treated (ipsilateral) to contralateral muscles. We have attempted to produce neurotoxin from subtype A2 (A2NTX) with an amino acid sequence different from that of neurotoxin subtype A1. We measured the grip strength on the contralateral foreleg as an indicator of toxin spread from the ipsilateral to contralateral muscles.

NleC, a type III secretion protease, compromises NF-κB activation by targeting p65/RelA.

The NF-κB signaling pathway is central to the innate and adaptive immune responses. Upon their detection of pathogen-associated molecular patterns, Toll-like receptors on the cell surface initiate signal transduction and activate the NF-κB pathway, leading to the production of a wide array of inflammatory cytokines, in attempt to eradicate the invaders. As a countermeasure, pathogens have evolved ways to subvert and manipulate this system to their advantage.

Activation of motility by sensing short-chain fatty acids via two steps in a flagellar gene regulatory cascade in enterohemorrhagic Escherichia coli.

The regulated expression of virulence genes is critical for successful infection by an intestinal pathogen. Bacteria rely on sensing environmental signals to find preferable niches and reach the infectious state. Orally ingested enterohemorrhagic Escherichia coli (EHEC) travels through the gastrointestinal tract and encounters a variety of environmental factors, some of which act as triggering signals for the induction of virulence genes.

Comparison of effects of botulinum toxin subtype A1 and A2 using twitch tension assay and rat grip strength test.

Botulinum toxin type A is used as a therapeutic agent for some spastic neurological disorders. Type A organisms have been classified into four subtypes (A1 to A4) based on the amino acid sequence variability of the produced neurotoxin. At present, commercially available preparations of the toxin belong to subtype A1.

PCR with quenching probes enables the rapid detection and identification of ganciclovir-resistance-causing U69 gene mutations in human herpesvirus 6.

A single-nucleotide polymorphism detection assay using PCR with quenching probes (QP-PCR) was developed for the rapid detection of antiviral drug-resistance mutations of human herpesvirus 6 (HHV-6). The mutations examined were in the HHV-6 U69 gene, and were single-base mutations in sequences known to be associated with ganciclovir (GCV) resistance in HCMV. We previously confirmed that they conferred GCV resistance to recombinant baculoviruses (Nakano et al.

Regulation of virulence by butyrate sensing in enterohaemorrhagic Escherichia coli.

Enterohaemorrhagic Escherichia coli (EHEC) colonizes and proliferates at the mucosal surface, inducing severe diarrhoea. Short-chain fatty acids (SCFAs) are abundant in the intestine owing to the metabolic activity of microflora, and are important for colonic health. We found that, although a high concentration of SCFAs inhibited the growth of EHEC, at low concentrations, the SCFAs markedly enhanced the expression of the virulence genes required for cell adherence and the induction of attaching and effacing (A/E) lesions.

Enterohemorrhagic Escherichia coli effector EspL2 induces actin microfilament aggregation through annexin 2 activation.

Enterohemorrhagic Escherichia coli (EHEC) delivers virulence factors into host cells through the type III secretion system (T3SS) to exert the bacterial pathogenicity. EHEC encodes more than 20 type III secretion system-delivered families of effectors that have different functions at different infectious stages and enable a successful infection. One of them, EspL2, is encoded on the SpLE3 phage-like element in EHEC O157:H7 Sakai and is well conserved among various EHEC strains.

Characterization of the human herpesvirus 6 U69 gene product and identification of its nuclear localization signal.

To elucidate the function of the U69 protein kinase of human herpesvirus 6 (HHV-6) in vivo, we first analyzed its subcellular localization in HHV-6-infected Molt 3 cells by using polyclonal antibodies against the U69 protein. Immunofluorescence studies showed that the U69 signal localized to the nucleus in a mesh-like pattern in both HHV-6-infected and HHV6-transfected cells. A computer program predicted two overlapping classic nuclear localization signals (NLSs) in the N-terminal region of the protein; this NLS motif is highly conserved in the N-terminal region of most of the herpesvirus protein kinases examined to date.

Maturation of functional type III secretion machinery by activation of anaerobic respiration in enterohaemorrhagic Escherichia coli.

Enterohaemorrhagic Escherichia coli (EHEC) is a gastrointestinal pathogen that causes diarrhoea and more severe diseases in humans. A key feature of EHEC is the type III secretion system (TTSS), which translocates virulence factors (effectors) directly into host cells. In this study, the expression and secretion of effectors in EHEC grown under anaerobic conditions were examined.

Real-time PCR determination of human herpesvirus 6 antiviral drug susceptibility.

A quantitative real-time PCR assay was developed to determine the antiviral drug susceptibility of human herpesvirus 6 (HHV-6). After short-term culture of the virus, HHV-6 isolates' susceptibility to the antiviral ganciclovir (GCV) was determined by measuring the HHV-6 variant B (HHV-6B) DNA levels in culture supernatants and infected cells using real-time PCR. A total of 12 well-characterized GCV-sensitive or -resistant strains and clinical isolates were used.

ppGpp with DksA controls gene expression in the locus of enterocyte effacement (LEE) pathogenicity island of enterohaemorrhagic Escherichia coli through activation of two virulence regulatory genes.

For a new pathogen to emerge, it must acquire both virulence genes and a system for responding to changes in environmental conditions. Starvation of nutrients or growth arrest induces the stringent response in Escherichia coli, via increased ppGpp. We found the adherence capacity of enterohaemorrhagic E.

Dendritic cells that endocytosed antigen-containing IgG-liposomes elicit effective antitumor immunity.

Liposomes represent a promising vehicle to deliver exogenous antigens to dendritic cells (DCs) for tumor immunotherapy. Targeting exogenous antigens to Fcgamma receptors on DCs has been shown to result in efficient presentation of antigen-derived peptides on major histocompatibility complex (MHC) class I and class II molecules. In this study, it was investigated whether DCs that endocytosed physicochemically optimized antigen-containing liposomes conjugated with IgG efficiently present antigens on MHC class I and class II molecules, and consequently induce strong antitumor immune responses.

Dual regulatory pathways integrating the RcsC-RcsD-RcsB signalling system control enterohaemorrhagic Escherichia coli pathogenicity.

Bacterial pathogenesis is strictly regulated in response to changes in environmental conditions. A His-Asp phosphorelay system consisting of a sensor kinase and response regulator is used by Gram-positive and Gram-negative bacteria to control gene expression in response to environmental stimuli. We screened His-Asp phosphorelay systems for their effect on virulence expression in enterohaemorrhagic Escherichia coli (EHEC), and found rcsD or rcsB overexpression enhanced locus for enterocyte effacement (LEE) gene transcription and adherence to Caco-2 cells through transcriptional activation of the ler regulatory gene.

Comparison of the therapeutic indexes of different molecular forms of botulinum toxin type A.

Botulinum toxin is produced by Clostridium botulinum in three different molecular-weight forms: LL toxin, 900 kDa; L toxin, 500 kDa; and M toxin, 300 kDa. We isolated the M toxin, then compared its muscle-weakening efficacy with those of L+LL toxin and BOTOX both in vitro and in vivo. The twitch tension of the mouse isolated phrenic nerve-hemidiaphragm was used for the in vitro study.

Mammalian PIG-X and yeast Pbn1p are the essential components of glycosylphosphatidylinositol-mannosyltransferase I.

Within the endoplasmic reticulum (ER), mannoses and glucoses, donated from dolichol-phosphate-mannose and -glucose, are transferred to N-glycan and GPI-anchor precursors, and serine/threonine residues in many proteins. Glycosyltransferases that mediate these reactions are ER-resident multitransmembrane proteins with common characteristics, forming a superfamily of >10 enzymes. Here, we report an essential component of glycosylphosphatidylinositol-mannosyltransferase I (GPI-MT-I), which transfers the first of the four mannoses in the GPI-anchor precursors.

Cytotoxicity of Clostridium septicum alpha-toxin: its oligomerization in detergent resistant membranes of mammalian cells.

Alpha-toxin is an important agent of the virulence of Clostridium septicum. We examined cytotoxicity for alpha-toxin to various mammalian cells with recombinant toxin fused with a histidine-tag at the amino-terminal. The recombinant toxin retained the activity indistinguishable from the native form.

Sensitivity of polarized epithelial cells to the pore-forming toxin aerolysin.

Aerolysin is one of the major virulence factors produced by Aeromonas hydrophila, a human pathogen that produces deep wound infection and gastroenteritis. The toxin interacts with target mammalian cells by binding to the glycan core of glycosylphosphatidyl inositol (GPI)-anchored proteins and subsequently forms a pore in the plasma membrane. Since epithelial cells of the intestine are the primary targets of aerolysin, we investigated its effect on three types of polarized epithelial cells: Caco-2 cells, derived from human intestine; MDCK cells, a well-characterized cell line in terms of protein targeting; and FRT cells, an unusual cell line in that it targets its GPI-anchored proteins to the basolateral plasma membrane in contrast to other epithelial cells, which target them almost exclusively to the apical surface.

Requirement of N-glycan on GPI-anchored proteins for efficient binding of aerolysin but not Clostridium septicum alpha-toxin.

Aerolysin of the Gram-negative bacterium Aeromonas hydrophila consists of small (SL) and large (LL) lobes. The alpha-toxin of Gram-positive Clostridium septicum has a single lobe homologous to LL. These toxins bind to glycosylphosphatidylinositol (GPI)-anchored proteins and generate pores in the cell's plasma membrane.