Characterization of Habu thrombin-like enzyme (THLE) with a new n-terminal sequence from the venom of Trimeresurus flavoviridis (Habu)

A thrombin-like enzyme with a new amino-terminal sequence was isolated from the venom of Trimeresurus flavoviridis using Q-Sepharose, CM-Cellulose, and HW55 column chromatographies. Homogeneity was confirmed by the formation of a single band in polyacrylamide gel electrophoresis. The enzyme has a molecular weight of 29,000 Da. This thrombin-like enzyme was inhibited by p-amidinophenylmethanesulfonyl fluoride hydrochloride (p-APMSF), and dithiothreitol (DTT) suggesting that serines and disulfide bonds are involved in the expression of the enzyme's clotting activity. This thrombin-like enzyme hydrolyzes the Aalpha-chain and Bbeta-chain of bovine fibrinogen. The enzyme was stable to heat treatment.     

Monitoring neutrophil engraftment in allogeneic stem cell transplantation by flow cytometric analysis of neutrophil-specific antigens NA1 and NA2

Neutrophil-specific antigen (NA) expression on neutrophils was analysed in 18 Japanese children before and after allogeneic stem cell transplantation (allo-SCT) with myeloablative regimen. Donor-recipient NA-incompatibility was present in one of eight NA1/NA2 heterozygous patients and eight of 10 NA1/NA1 or NA2/NA2 homozygous patients. After allo-SCTs from NA-incompatible donors, a neutrophil recipient-to-donor conversion was confirmed in all cases. Conversion to donor NA type was complete before the absolute neutrophil count reached 0.1 x 10(9)/l. These observations indicate that flow cytometric analysis of NA antigens is a simple and useful method for monitoring neutrophil engraftment in NA-incompatible allo-SCT.     

Anesthetic management of corpus callosotomy with electrophysiological monitoring: a case report

We report anesthetic management of a 5-year-old girl for corpus callosotomy indicated for the treatment of intractable epilepsy. The procedure mandated intraoperative monitoring of evoked potentials and electrocorticogram. During the first half of the surgery until the corpus callosum was exposed, anesthesia was maintained with continuous infusion of propofol. Motor and somatosensory evoked potentials were monitored and diagnosed as intact throughout the procedure, with no epileptic activity observed in 32-lead electrocorticogram. Then propofol infusion was replaced with the inhalation of sevoflurane, 2.0% in air/oxygen mixture, which induced epileptic spike-and-wave activities, synchronized between the hemispheres, in electrocorticogram. After the completion of corpus callosotomy, we observed interhemispheric desynchronization of epileptic activities indicating successful surgical intervention. The patient emerged from anesthesia uneventfully with no neurological deficits, and thereafter with decreased incidence of generalized epileptic episodes. We suggest that such switch of anesthetic agents between propofol and sevoflurane should be helpful in intraoperative electrophysiological monitoring for ascertaining both functional preservation and successful intervention during epileptic surgery.     

Impact of breast cancer diagnosis and treatment on women's sexuality: a survey of Japanese patients

Objective: Research on the impact of breast cancer on Asian women's sexual lives is extremely scarce. This study investigated the sexual changes experienced by breast cancer patients in Japan following surgery, and their sexuality‐related information needs. Methods: An anonymous, cross‐sectional survey of breast cancer out‐patients was conducted in 2005. Data from 85 subjects, who were without recurrence and reported being sexually active pre‐surgery, were analyzed. Results: Subjects were mainly in their 40's and 50's, and the median time since surgery was 43 months. Seventy‐three (85.9%) had resumed sex after surgery with the median time being 3.5 months after surgery. Among 73 who resumed sex, 43 reported that the frequency of sex decreased, and 72 reported at least one sexually related change. The multiple logistic regression analysis revealed that those who had perceived the sexual relationship with their partner important before surgery (OR, 6.705; 95%CI, 1.320–34.051; p = 0.022) were more likely to maintain the same frequency of sex as before surgery. Perceived changes in respondents' sexual relationship did not necessarily result in deterioration of the couple's overall relationship. Regarding sexuality‐related information needs, respondents wished to have information on treatment‐induced sexual changes as well as sexual and general inter‐couple communication strategies. Conclusion: This research revealed that breast cancer patients in Japan experience various sexual problems following breast cancer treatment. Sexuality‐related information should be provided to all patients, regardless of patients age or type of surgery, as a part of routine treatment information giving. Copyright © 2007 John Wiley & Sons, Ltd.     

Symptoms of food-borne diseases and gastroenteritis in Kyushu, Japan

In this study we analyzed the symptoms of gastroenteritis or food-borne disease caused by the 10 most prevalent pathogens: Norovirus, Salmonella, Vibrio parahaemolyticus, Campylobacter jejuni, Clostridium perfringens, Shiga toxin-producing Escherichia coli (STEC), enterotoxigenic E. coli (ETEC), Shigella sonnei/flexneri (Shigella), Staphylococcus aureus, and emetic-type Bacillus cereus. The symptoms diarrhea, vomiting, fever, abdominal pain, and headache, and the incubation period in 646 cases in 10 districts of Kyushu between January 2000 and December 2004 were recorded. The pathogen with the shortest mean incubation period was B. cereus (0.8 h), and was followed by S. aureus (3.3 h), C. perfringens (10.7 h) and V. parahaemolyticus (16.4 h). All the patients infected with B. cereus and S. aureus developed symptoms within 6 hours, and those infected with V. parahaemolyticus and C. perfringens developed symptoms within 24 hours. Bloody diarrhea was associated with STEC and Shigella, but rare with other pathogens. Vomiting was associated with almost all cases of S. aureus and B. cereus infection, and occurred in 71.5% of the Norovirus cases and 56.1% of the V. parahaemolyticus cases. Vomiting was less common in the C. perfringens (22.0%) and the ETEC and STEC (both about 5%). Bloody diarrhea, abdominal pain, and vomiting were statistically significantly more common with STEC 0157 infection than with STEC non-0157 infection. Since the cases analyzed in this study included all degrees of illness, mild to severe, and a wide range of ages, the information obtained will serve as a good reference material for administrative and laboratory work when an outbreak takes place.     

Hydroxychavicol: a potent xanthine oxidase inhibitor obtained from the leaves of betel, <Emphasis Type="Italic">Piper betle</Emphasis>

The screening of Piperaceous plants for xanthine oxidase inhibitory activity revealed that the extract of the leaves of Piper betle possesses potent activity. Activity-guided purification led us to obtain hydroxychavicol as an active principle. Hydroxychavicol is a more potent xanthine oxidase inhibitor than allopurinol, which is clinically used for the treatment of hyperuricemia.     

Distribution of Non-Locus of Enterocyte Effacement Pathogenic Island-Related Genes in Escherichia coli carrying eae from Patients with Diarrhea and Healthy Individuals in Japan

The relationship to diarrhea of genes located on the pathogenicity islands (PAI) other than the locus of enterocyte effacement (LEE) was investigated. Enteropathogenic Escherichia coli (EPEC), the retention of espC on the EspC PAI, the OI-122 genes (efa1/lifA, nleB), the phylogenetic marker gene yjaA, and the bundle-forming pilus gene bfpA on the EPEC adherence factor (EAF) plasmid were studied. E. coli strains carrying the intimin gene (eae) without the Shiga toxin gene, isolated from patients with diarrhea (n = 83) and healthy individuals (n = 38) in Japan, were evaluated using PCR. The genotypes of eae and espC were identified by heteroduplex mobility assay (HMA). The proportions of strains isolated from individuals with and without diarrhea that carried these genes were as follows: bfpA, 13.3 and 7.9%, respectively; espC, 25.3 and 36.8%; efa1/lifA, 32.5 and 13.2%; nleB, 63.9 and 60.5%; yjaA, 42.2 and 55.3%. Statistical significance (P < 0.05) was achieved only for efa1/lifA. The proportion of strains lacking espC and carrying efa1/lifA was higher for patient-derived strains (30.1%) than for strains from healthy individuals (13.2%), but the difference was not significant. Strains carrying both espC and efa1/lifA were rare (2 strains from patients). Statistical analyses revealed significant relationships between espC and yjaA and between efa1/lifA and nleB, as well as significant inverse relationships between espC and efa1/lifA and between efa1/lifA and yjaA. espC was found in eae HMA types a1, a2, and c2, whereas efa1/lifA was found in types b1, b2, and c1. In addition, 6 polymorphisms of espC were found. The espC, yjaA, efa1/lifA, and nleB genes were mutually dependent, and their distributions were related to eae type, findings that should be considered in future epidemiological studies.Enteropathogenic Escherichia coli (EPEC) (16), the first pathotype found in E. coli, induces diarrhea and can be life-threatening in infants. Histopathology of EPEC infections, known as “attaching and effacing” (A/E) lesions, shows that the bacteria attach intimately to intestinal epithelial cells, causing striking cytoskeletal changes, effacing the microvilli of the intestines. It is thought that effector and regulator genes for various functions that induce this A/E histopathology are located in a 35-kb pathogenicity island (PAI) (9), called the locus of enterocyte effacement (LEE), on the EPEC chromosome and on a 70- to 100-kb plasmid, EPEC adherence factor (EAF). The 94-kDa outer membrane protein intimin, which contributes to the adhesion of EPEC to the intestinal epithelial cells, is encoded by the eae gene in the LEE. On the other hand, a type IV pilus, the bundle-forming pilus (BFP), which contributes to interbacterial adherence and possibly to adherence to the intestinal epithelial cells, is encoded by the bfpA gene on the EAF plasmid.In developing countries, in most cases, EPEC isolates recovered from humans with diarrhea are typical EPEC (t-EPEC) strains that have an EAF plasmid; however, in developed industrial countries, such as Japan, most strains lack the EAF plasmid and are identified as atypical EPEC (a-EPEC) (34).Although a-EPEC has been isolated in large outbreaks involving both adults and children, it is often isolated from healthy individuals, and these strains are thought to be nonpathogenic. In the past, in case-control studies from both Japan and elsewhere comparing the retention ratios of adherence-related genes, such as eae, in E. coli isolates obtained from patients with diarrhea and from healthy individuals, no statically significant differences were found. It was therefore determined that the probability of diarrhea inducement could not be estimated solely on the basis of adherence-related genes (12, 27, 31). Hence, elucidation of the closeness of the relation of LEE and non-LEE genes as an indicator of the pathogenicity of a-EPEC was sought around the world.In recent years, various PAIs other than the LEE PAI have been found on some EPEC chromosomes. There have been reports of genes putatively related to pathogenicity (13, 26). Some EPEC strains secrete a 110-kDa protein with serine protease activity called EspC, which has been found to result in increases in short-circuit current and a potential difference in rat jejunal tissue mounted in Ussing chambers, causing cytoskeletal damage as well as hemoglobin proteolysis (5, 25, 29). EspC is encoded by the espC gene in the EspC PAI on the chromosome, outside LEE (25), and belongs to the subfamily of serine protease autotransporters of Enterobacteriaceae (SPATEs) (6, 10, 11). However, the target of EspC in living organisms is unclear, and its distribution in a-EPEC and contribution to the symptoms of diarrhea have not been investigated. The finding of secretory immunoglobulin A antibodies responding to EspC in breast milk from Mexican women living under poor sanitation conditions suggests that EspC probably participates as an enterotoxin in EPEC infections (24).efa1 (30) encodes the large, 385-kDa adhesin protein Efa1 and is located on a PAI that is similar to genomic O island 122 (OI-122) (18, 21) of the enterohemorrhagic E. coli (EHEC) O157:H7 strain EDL933. This gene is the same as lifA, which encodes lymphostatin (LifA) (19, 20), in the EPEC strain E2348/69. LifA inhibits the proliferation of peripheral blood lymphocytes and gastrointestinal lymphocytes as well as the production of lymphokines. The gene was designated efa1/lifA.In a case-control study with Norwegian children (2), where a statistical comparison comprehensively investigated the presence of pathogenicity-related gene sequences in a-EPEC strains isolated from children with and without diarrhea, Afset et al., found that efa1/lifA and nleB (the gene encoding non-LEE effector protein B) in OI-122 were the genes most strongly linked to diarrhea and that the phylogenetic marker gene yjaA was inversely related. The Norwegian study used a microarray composed of 242 different probes for the detection of 182 virulence genes or markers. Seven additional putative virulence genes that were not included in the microarray were detected by PCR.To study the possibility of the relatedness of the genes encoded outside LEE to diarrhea, we investigated the retention of all the genes mentioned above in eae-containing E. coli strains isolated from healthy individuals and patients with diarrhea, in an attempt to establish whether they could provide an indicator for EPEC diarrhea symptoms, including those caused by a-EPEC, in Japan.     

Molecular mechanisms of high level tetracycline-resistance in group A streptococcal isolates, T serotypes 4 and 11

The molecular mechanism of high level tetracycline resistance in T serotypes 4 and 11 group A streptococcal (GAS) isolates was examined in 61 tetracycline-resistant isolates in Japan. PCR and sequencing analyses revealed that the T serotype/emm genotype, T4/4 isolates carried tet(O) genes, which were genetically homogenous. The T11/11 and T11/89 isolates carried different subtypes of tet(M) genes, which were present on transposons Tn916 and Tn1545, respectively. In addition, these T11 isolates may have obtained the tet(M) gene after the 1990s, because resistance to tetracycline in T11 isolates was rarely found before then. These results strongly suggested that the T4 and T11 GAS isolates acquired tetracycline-resistance via different molecular mechanisms.     

Human neutrophil antigen-2a expression on neutrophils from healthy adults in western Japan

BACKGROUND: Human neutrophil antigen-2a (HNA-2a)-specific MoAb, which is necessary to prepare panel neutrophils and to determine the phenotype of patients' neutrophils, has been produced in many laboratories in Japan. The frequency of the HNA-2a-positive population in western Japan is unknown. STUDY DESIGN AND METHODS: A cell line secreting an HNA-2a-specific MoAb, TAG4, was established. TAG4 was characterized by flow cytometry, and the reactivities of TAG4 and another HNA-2a-specific MoAb, 7D8, were compared in an inhibition assay and two-color flow cytometry. TAG4 was tested against granulocytes from 408 healthy adult volunteers in western Japan. RESULTS: TAG4 reacted with neutrophils, but not with the other blood cells. The reactivity of TAG4 with granulocytes treated with glycosyl-phosphatidylinositol-specific phospholipase C was markedly reduced. The reactivity of TAG4 and 7D8 was similar, but 7D8 did not prevent TAG4 from binding. A total of 406 of 408 donors were TAG4 positive. The mean percentage of HNA-2a expression of females was 70.2 percent, and that of males was 66.8 percent. CONCLUSION: TAG4 and 7D8 seem to bind to different epitopes of HNA-2a. The frequency of the HNA-2a-positive population is 99.5 percent of healthy adults in western Japan. The mean percentage of HNA-2a expression of females was greater than in males.