Norihiko Tabuchi

The 44-kb Linear Plasmid Molecule in the Relapsing Fever Agent Borrelia duttonii Strain Ly Serve as a Preservation of vmp Genes

Borrelia duttonii strain Ly, a causative agent of relapsing fever, contains a linear one megabase chromosome and 12 linear plasmid molecules. Here we report that the sequence of the 44‐kb linear plasmid of strain Ly is found to contain variable major protein (vmp) genes for antigenic variation of relapsing fever borreliae. The determined sequence is of 44,010 bp except for both ends of the molecule. Of 39 open reading frames (ORFs) found in the sequence, 21 ORFs (named vmpA to U) showed moderate similarities with vmp genes for Borrelia hermsii. However, most of the vmp homologues are apparently nonfunctional because of their frameshifts within the sequence and/or absence of promoter and ribosome‐binding signals upstream of their genes. RT‐PCR experiments using the specific primer for each vmp gene revealed that vmpE, one of the vmp genes, was expressed at the location of the 44‐kb plasmid molecule. The result suggests that the plasmid molecule may play a role in the preservation of the serotype switching of vmp genes in a mammalian host.

Ile (476), a constituent of di-leucine-based motif of a major lysosomal membrane protein, LGP85/LIMP II, is important for its proper distribution in late endosomes and lysosomes

Lysosomal membrane glycoprotein termed LGP85 or LIMP II extends a COOH-terminal cytoplasmic tail of R459GQGSMDEGTADERAPLIRT478, in which an L475 I476 sequence lies as a di-leucine-based motif for lysosomal targeting. In the present study, we explored the role of the I476 residue in the localization of LGP85 to the endocytic organelles using two substitution mutants called I476A and I476L in which alanine and leucine are replaced at I476, respectively, and I476R477T478-deleted LGP85 called Delta 476-478. Immunofluorescence analyses showed that I476A and I476L are largely colocalized in intracellular organelles with an endogenous late endosomal and lysosomal marker, LAMP-1, but there were some granules in which staining for the LGP85 mutants was prominent, while Delta 476-478 is detected in LAMP-1-positive and LAMP-1-negative intracellular organelles, and on the cell surface. The subcellular fractionation studies revealed that I476A, I476L, and Delta 476-478 are different from wild-type LGP85 in the distribution of early endosomes, late endosomes, and lysosomes. I476A and I476L are present more in late endosomes than in the densest lysosomes, whereas wild-type LGP85 is mainly lysosomal. Substitution of I476 for A and L differentially modified the ratios of late endosomal to lysosomal LGP85. A major portion of Delta 476-478 resided in the light buoyant density fraction containing plasma membrane and early endosomes. Taken together, these results indicate that the existence of the 476th amino acid residue is essential for localization of LGP85 to late endocytic compartments. The fact that isoleucine but not leucine is in the 476th position is especially of importance in the proper distribution of LGP85 in late endosomes and lysosomes.

Study on Patients Who Underwent Suspected Diagnosis of Allergy to Amide-Type Local Anesthetic Agents by the Leukocyte Migration Test

BACKGROUND There are problems in diagnosis of allergy to amide-type local anesthetic agents (ALAs), because definitive diagnosis is not obtained by in vivo tests, which are used for the diagnosis. Consequently, patients may be exposed to risk. There are few diagnoses based on in vitro tests, and there are almost no relevant studies. METHODS Authors examined involvement of allergic reaction using the leukocyte migration test (LMT) through multiple standpoints in 43 patients who underwent suspected diagnosis of allergy to ALAs. RESULTS Rate of LMT-positives was 54%, and especially the positive rate of lidocaine hydrochloride preparations was significantly high. In 15 positives to lidocaine hydrochloride preparations, all cases were indicated as positive in a test with drugs containing antiseptic agent, but only 3 cases were indicated as positive in a test with lidocaine hydrochloride alone. In addition, test with paraben was conducted in 4 cases; 2 cases were confirmed as positive. In relevance of histories of drug or food allergies, development rates of ALAs-allergies were the highest in both allergies, and were 35% and 13%, respectively. CONCLUSIONS There is a high possibility that these adverse reactions were caused by pseudoallergy to drug. Even by allergic reactions, it was assumed that 80% of them might be caused by antiseptic agents such as paraben. In addition, it was suggested that ALAs, especially lidocaine hydrochloride preparations have high antigenicity (sensitizing property). Furthermore, it was considered that patients with past history of drug or food allergies have a high potential for manifestation of the reactions.

Immunodominant Epitope in the C-Terminus of a Variable Major Protein in Borrelia duttonii, an Agent of Tick-Borne Relapsing Fever

Borrelia duttonii strain Ly was isolated from a child with tick‐borne relapsing fever in Tanzania. B. duttonii produces variable major proteins (Vmps), which undergo antigenic variation. We previously reported transcription of the vmpP gene, which is one of the Vmp genes in strain Ly, detected in vitro cultivation. In the current study, we purified the recombinant non‐lipidated VmpP protein by affinity chromatography and produced VmpP polyclonal antibodies. Antigenicity of VmpP was examined by Western immunoblot analysis and peptide‐based enzyme‐linked immunosorbent assays. Antigenic epitopes were shown to comprise five regions interspersed within the VmpP primary amino acid sequence. Synthetic peptides spanning residues of three of five regions, 232–237 (LASIVD), 280–285 (AGGIAL), and 350–355 (KAADQQ), reacted strongly with the VmpP‐specific antibody and these residues were identified as epitopes. In particular, the C‐terminal domain (KAADQQ) of this protein was immunoreactive. Further research based on our results will promote the development of a recombinant vaccine for B. duttonii infection.

Absence of Transovarial Transmission of Borrelia duttonii, a Tick-Borne Relapsing Fever Agent, by the Vector Tick Ornithodoros moubata

We examined the vector competence of the tick, Ornithodoros moubata, using laboratory-reared gerbils as hosts. Transmission of the relapsing fever agent Borrelia duttonii occurred efficiently from infected ticks to uninfected gerbils and from infected gerbils to uninfected ticks. Spirochetes were maintained stably in the ticks for at least 3 months, but they disappeared from the bloodstream of infected gerbils after three episodes of spirochetemia. We also examined transovarial transmission of B. duttonii during the gonotrophic cycle and filial generation. No spirochetes could be detected from the offspring generation of the ticks by culture and polymerase chain reaction (PCR) methods, although spirochetes were still found in the female ticks. The results indicate that, because of the rarity of transovarial infection, the role of transovarial passage of B. duttonii to eggs and larval O. moubata ticks is limited in maintaining B. duttonii. Our findings strongly suggest that B. duttonii is maintained through the O. moubata tick-human transmission cycle in tick-borne relapsing fever endemic areas.

Rhein 8-O-β-D-Glucopyranoside Elicited the Purgative Action of Daiokanzoto (Da-Huang-Gan-Cao-Tang), Despite Dysbiosis by Ampicillin

Sennoside A (SA), the main purgative constituent of Daiokanzoto (da-huang-gan-cao-tang; DKT), is generally regarded as a prodrug that is transformed into an active metabolite by β-glucosidase derived from Bifidobacterium spp. It has been suggested that antibiotics would promote dysbiosis, and thereby inhibit the purgative activity of DKT. In this study, ampicillin was administered to mice for 8 d, and the changes in the SA metabolism of SA alone and of DKT were investigated. The results showed that the SA metabolism of SA singly continued to be inhibited by ampicillin, but that of DKT was activated from day 3 under the same conditions. In order to investigate the mechanism of SA metabolism activated by DKT in the mice administered ampicillin, changes in the SA metabolism were observed in the presence of rhein 8-O-β-D-glucopyranoside (RG) in rhubarb and liquiritin in glycyrrhiza, both of which accelerated the SA metabolism. In fact, RG achieved an activation of SA metabolism similar to that by DKT. The purgative action of DKT, which was continued treatment of the ampicillin, was significantly greater than that by SA alone, and it was shown that RG was involved in this effect. We also analyzed changes in the intestinal microbiota before and after administration of ampicillin. No Bifidobacteria were detected throughout the treatment, but the population of Bacteroides was significantly increased after 3 d under the same conditions. Taken together, these results strongly suggested that the RG in DKT changed the function of Bacteroides and thereby allowed DKT to metabolize SA.