Novel aminopeptidase N (APN) isoform cDNAs, BmAPN3 and PxAPN3, from the midguts of Bombyx mori and Plutella xylostella, respectively, were cloned, and a total of eight APN isoforms cloned from B. mori and P. xylostella were classified into four classes. Bacillus thuringiensis Cry1Aa and Cry1Ab toxins were found to bind to specific APN isoforms from the midguts of B. mori and P. xylostella, and binding occurred with fragments that corresponded to the BmAPN1 Cry1Aa toxin-binding region of each APN isoform. The results suggest that APN isoforms have a common toxin-binding region, and that the apparent specificity of Cry1Aa toxin binding to each intact APN isoform seen in SDS^PAGE is determined by factors such as expression level in conjunction with differences in binding affinity. ß

. 519:215-220, 2002), by using monoclonal antibodies (MAbs) that block binding between the binding site and the receptor. First, we produced a series of MAbs against Cry1Aa and obtained two MAbs, MAbs 2C2 and 1B10, that were capable of blocking the binding between Cry1Aa and BmAPN1 (blocking MAbs). The epitope of the Fab fragments of MAb 2C2 overlapped the BmAPN1 binding site, whereas the epitope of the Fab fragments of MAb 1B10 did not overlap but was located close to the binding site. Using three approaches for epitope mapping, we identified two candidate epitopes for the blocking MAbs on Cry1Aa. We constructed two Cry1Aa toxin mutants by substituting a cysteine on the toxin surface at each of the two candidate epitopes, and the small blocking molecule N-(9-acridinyl)maleimide (NAM) was introduced at each cysteine substitution to determine the true epitope. The Cry1Aa mutant with NAM bound to Cys582 did not bind either of the two blocking MAbs, suggesting that the true epitope for each of the blocking MAbs was located at the site containing Val582, which also consisted of 508 STLRVN 513 and 582 VFTLSAHV 589 . These results indicated that the BmAPN1 binding site overlapped part of the region blocked by MAb 2C2 that was close to but excluded the actual epitope of MAb 2C2 on domain III of Cry1Aa toxin. We also discuss another area on Cry1Aa toxin as a new candidate site for BmAPN1 binding.Bacillus thuringiensis, a gram-positive bacterium, produces various insecticidal proteins called Cry toxins which kill only target insects. This bacterium is used as a microbial insecticide and for the genetic development of insect-resistant plants.Cry toxins are expressed in inclusion bodies as protoxins (70 to 140 kDa) during sporulation. When a protoxin is ingested by target insects, it is solubilized in the insect midgut and digested by proteolytic enzymes in the insect (2). After enzymatic activation, the toxic protease-resistant fragment, which is the 60-to 65-kDa activated toxin, binds to specific receptors located in the columnar cells of the midgut apical brush border membrane (28). The binding of the toxin to receptor molecules leads to a conformational change in the toxin. This allows the toxin to insert into the plasma membrane and subsequently generate pores or ion channels which lead to cellular swelling and lysis (27,35,65,68). Finally, the intoxicated insects die. The binding of the activated toxin to a specific gut receptor is considered to be one of the key factors for insect toxicity. The insecticidal specificity of Cry toxins seems to be largely dependent on this receptor recognition.The three-dimensional structures of Cry1Aa trypsin-activated toxin, Cry2A protoxin, Cry3A, and Cry3B have been analyzed by X-ray diffraction crystallography (14,25,43,51). These proteins are comprised of three domains. The Cry1Aa and Cry3A structures have many similar features. The N-terminal domain I is composed of a seven-alpha-helix bundle in which the alpha-5 helix is encircled by the other helices. Domain II consists of thre...

Aminopeptidase N (APN) and cadherin-like protein (BtR175) from Bombyx mori larvae were examined for their roles in Cry1Aa-and Cry1Ac-induced lysis of B. mori midgut epithelial cells (MECs). APNs and BtR175 were present in all areas of the midgut, were particularly abundant in the posterior region, and were found only on columnar cell microvilli and not on the lateral membrane that makes cell^cell contacts. This distribution was in accordance with the distribution of Cry1A-susceptible MECs in the midgut. The lytic activity of Cry1Aa and Cry1Ac on collagenase-dissociated MECs was linearly dependent on toxin concentration. Although pre-treatment of MECs with anti-BtR175 antibody was observed to partially inhibit the lytic activity exerted by 0.1^1 nM Cry1Aa toxin or 5 nM Cry1Ac toxin, no signi¢cant inhibition was observed when MECs were pre-treated with anti-APN antibody. These results suggest that BtR175 functions as a major receptor for Cry1A toxins in the midgut of B. mori larvae. ß

Congenital

MDVP can be used for snoring sound analysis as a noninvasive examination of sleep-related breathing disorders for differential diagnosis. However, a suitable option that is rapid and has an easy-to-use interface would be more advantageous for analyzing snoring sounds.

Regional differences in human papillomavirus (HPV) genotypes and the presence of mixed HPV infections may affect adversely the efficacy of the HPV vaccine. Therefore, a simple and high-throughput HPV genotyping system is required. Recently, a novel HPV genotyping kit (the Mebgen™ HPV kit) was developed. This kit uses multiplex PCR and Luminex xMAP™ technology to detect 13 types of high-risk HPVs and an internal control in a 96-well format. In the present study, the analytical performance of the kit was examined using HPV plasmid DNA. All 13 types of HPVs were detected with a minimum detection sensitivity of 250 copies/test, and highly specific signals were observed. HPV 16 plasmid was detected in samples containing mixtures with other HPV-type plasmids in ratios ranging from 1:1 to 1:1000. No cross reactivity was observed with DNA from 27 types of other infectious microbes. A clinical evaluation was carried out using cervical samples from 356 patients with persistent abnormal smears diagnosed at mass public health screenings for cervical cancer. The samples were preserved in Tacas™ medium until analysis. HPV was detected in 162 (45.5%) samples including 110 (67.9%) with single infections and 52 (32.1%) with multiple infections. The type distribution of the 13 high-risk HPVs was as follows: 28.4% HPV 16, 11.7% HPV 18, 6.8% HPV 31, 3.1% HPV 33, 3.7% HPV 35, 9.3% HPV 39, 1.9% HPV 45, 8.6% HPV 51, 37.0% HPV 52, 9.3% HPV 56, 16.7% HPV 58, 3.7% HPV 59, and 1.9% HPV 68. To evaluate sample stability over time, changes in the detection of HPV DNA derived from HeLa and SiHa cells were measured in 3 types of liquid-based cytology media. HPV DNA was detected in Tacas and Thinprep™ samples after storage at 4°C or 30°C for 4 weeks and within 1 week of collection in Surepath™ samples. These results suggest that this newly developed HPV genotyping kit is suitable for use in both clinical applications and large-scale epidemiological studies.

No abstract

Background and purpose Botulinum toxin (BT) injection into the laryngeal muscles has been a standard treatment for spasmodic dysphonia (SD). However, few high‐quality clinical studies have appeared, and BT is used off‐label in most countries. Methods We performed a multicenter, placebo‐controlled, randomized, double‐blinded, parallel‐group comparison/open‐label clinical trial to obtain approval for BT (Botox) therapy in Japan. Twenty‐four patients (22 with adductor SD and two with abductor SD) were enrolled. The primary end point was the change in the number of aberrant morae (phonemes) at 4 weeks after drug injection. The secondary end points included the change in the number of aberrant morae, GRBAS scale, Voice Handicap Index (VHI), and visual analog scale (VAS) over the entire study period. Results In the adductor SD group, the number of aberrant morae at 4 weeks after injection was reduced by 7.0 ± 2.30 (mean ± SE) in the BT group and 0.2 ± 0.46 in the placebo group (p = 0.0148). The improvement persisted for 12 weeks following BT injections. The strain element in GRBAS scale significantly reduced at 2 weeks after BT treatment. The VHI and VAS scores as subjective parameters also improved. In the abductor SD group, one patient responded to treatment. Adverse events included breathy hoarseness (77.3%) and aspiration when drinking (40.9%) but were mild and resolved in 4 weeks. Conclusions Botulinum toxin injection was safe and efficacious for the treatment of SD. Based on these results, BT injection therapy was approved as an SD treatment in Japan.