High Mitochondrial Genome Diversity and Intricate Population Structure of Bursaphelenchus xylophilus in Kyushu, Japan.

Mitogenomic diversity and genetic population structure of the pinewood nematode (PWN) Bursaphelenchus xylophilus inhabiting Kyushu, Japan were analyzed. A method for performing long PCR using single nematodes and sequencing nematode mitochondrial genomes individually is presented here. About 8 kb (∼55%) of the complete mitochondrial genome was successfully obtained from 285 individuals collected from 12 populations. The 158 single nucleotide polymorphisms detected corresponded to 30 haplotypes, clearly classified into two clades. Haplotype diversity was 0.83, evidencing a remarkable high diversity within Kyushu. The high genetic differentiation among the 12 populations (0.331) might be due to past invasion and expansion routes of PWN in northeastern and southeastern Kyushu. The distinct genetic composition of populations within the northwestern, central western, and southwestern Kyushu seems to be mostly related to the extinction of pine forests and long-range migration of PWN due to human activity. Overall, direct long PCR and sequencing of single nematode individuals are effective methods for investigating mitochondrial polymorphisms, and these are effective tools for PWN population genetics and other intraspecific studies.

[A case of small intestinal GIST maintained as a long stable disease by imatinib mesylate 400 mg/day, alternate-day administration for 2 weeks followed by a 2 week interval].

UNLABELLED: Gastrointestinal stromal tumors (GIST) are the most common mesenchymal tumors of the gastrointestinal tract. The recent molecular-targeted therapies (imatinib and sunitinib) have improved the treatment of GIST remarkably. However, it would be ideal if the amount of these drugs could be adjusted according to each patient because they have various side effects and are very expensive. We experienced a case of non-curative resectable GIST maintained as a long, stable disease after operation, despite tapering down the dose of imatinib mesylate for personal reasons. CASE: A woman aged 50. She had received surgery for a lower abdominal tumor, and had been diagnosed with GIST of the small intestine and disseminations. When she suspended taking imatinib (400 mg/day) after her operation, these tumors regrew. After restarting imatinib at 400 mg/day in an alternate-day administration lasting 2 weeks followed by a 2 week interval, the disseminated tumors were in significant for 60 months after the operation.

Isolation and characterization of tetranucleotide microsatellite loci in Pinus massoniana (Pinaceae).

PREMISE OF THE STUDY: Tetranucleotide microsatellite markers were developed for the first time in Pinus massoniana Lamb. to facilitate studies of population and conservation biology in this species. METHODS AND RESULTS: Ten tetranucleotide microsatellite primer pairs were developed using dual suppression PCR. Seven, six, and eight of the primer pairs exhibited cross-species transferability to P. thunbergii, P. densiflora, and P. luchuensis, respectively. The number of alleles ranged from 1 to 31 per locus across four pine species. CONCLUSIONS: Considering its advantage over dinucleotide microsatellites in generating fewer artifacts arising from stutter bands, this tetranucleotide microsatellite panel will facilitate future population and conservation biological studies in P. massoniana. Six to eight markers can also be used in studies of three congeneric species.

Improved AFLP protocol using dual-suppression PCR and its application to species with large genomes.

To improve the amplified fragment length polymorphism assay, dual-suppression PCR was introduced into the preamplification step of the assay. The dual-suppression PCR blocked completely the amplification of fragments with the same sequence (Bsp1407I-Bsp1407I or NlaIII-NlaIII) at both ends and amplified selectively fragments with different adaptor sequences (Bsp1407I-NlaIII) at each end. Two protocols, referred to as A and B, were established for species with medium- and large-sized genomes, respectively. Both protocols incorporated the dual-suppression PCR. Protocol A resulted in high-quality electrophoretic profiles for black cottonwood and rice, which have medium-sized genomes. In protocol B, an intensely selective PCR step was added to protocol A. Protocol B yielded profiles for Japanese black pine and Japanese cedar that were improved significantly relative to protocol A: the number of strong peaks increased and that of low peaks decreased. Japanese black pine and Japanese cedar have large genomes. The optimal profiles were generated with a total of eight or nine selective nucleotides.

Ribosomal DNA haplotype distribution of Bursaphelenchus xylophilus in Kyushu and Okinawa islands, Japan.

Ribosomal DNA region sequences (partial 18S, 28S and complete ITS1, 5.8S, and ITS2) of the pinewood nematode (Bursaphelenchus xylophilus) were obtained from DNA extracted directly from wood pieces collected from wilted pine trees throughout the Kyushu and Okinawa islands, Japan. Either a 2569bp or 2573bp sequence was obtained from 88 of 143 samples. Together with the 45 rDNA sequences of pinewood nematode isolates previously reported, there were eight single nucleotide polymorphisms and two indels of two bases. Based on these mutations, nine haplotypes were estimated. The haplotype frequencies differed among regions in Kyushu island (northwest, northeast and center, southeast, and southwest), and the distribution was consistent with the invasion and spreading routes of the pinewood nematode previously estimated from past records of pine wilt and wood importation. There was no significant difference in haplotype frequencies among the collection sites on Okinawa island.

[A case of severe hand-foot syndrome caused by capecitabine].

A 55-year-old woman underwent total mastectomy and axillary lymphnode dissection in 2001. Widespread lymphnode metastasis was found histologically (26/33). Neither PgR nor ER was positive. She underwent an AC regimen and paclitaxel chemotherapy. As CEA began to rise in 2002, she was given paclitaxel and docetaxel chemotherapy sequentially. As CEA rose again in 2004, capecitabine was begun. Painful erythema of the palms and soles of the feet appeared at the end of the second cycle. After admission, severe bone marrow suppression and jaundice were found. The bilateral hands, palms and soles of the feet became bullous and erosive with desquamation. The erosive lesions began to heal with epithelization in the third week. After general conditions had improved, capecitabine was restarted at a reduced dose. This patient had continued taking capecitabine even though she noticed the occurrence of the adverse effect. Patients and doctors must share confidential information when performing chemotherapy at the outpatient clinic.

[Method of levofolinate.5-FU administration by hepatic arterial infusion therapy for hepatic metastasis from colorectal cancer].

Hepatic arterial infusion chemotherapy with levofolinate (l-leucovorin) and fluorouracil regimen was performed using an implanted port system on unresectable hepatic metastasis patients with colorectal cancer. A comparative study was performed on two groups in which the levofolinate was administered arterially or intravenously. Levofolinate 200-250 mg/m(2) was infused for two hours intra-arterially or intravenously, and 5-FU 400-600 mg/m(2) was administered as a bolus in midinfusion. The regimen was repeated weekly for six weeks, followed by no medication for two weeks. Six patients were administered intra-arterially and 7 patients intravenously. The response rate was higher in the group in which levofolinate was given intravenously. The adverse effect was lower in the former than in the latter group. When 5-FU and levofolinate was performed using an implanted port system, it seemed better to administer levofolinate intravenously.

Prognostic factors and scoring system for survival in colonic perforation.

BACKGROUND/AIMS: No ideal and generally accepted prognostic factors and scoring systems exist to determine the prognosis of peritonitis associated with colonic perforation. This study was designed to investigate prognostic factors and evaluate the various scoring systems to allow identification of high-risk patients. METHODOLOGY: Between 1996 and 2003, excluding iatrogenic and trauma cases, 26 consecutive patients underwent emergency operations for colorectal perforation and were selected for this retrospective study. Several clinical factors were analyzed as possible predictive factors, and APACHE II, SOFA, MPI, and MOF scores were calculated. RESULTS: The overall mortality was 26.9%. Compared with the survivors, non-survivors were found more frequently in Hinchey's stage III-IV, a low preoperative marker of pH, base excess (BE), and a low postoperative marker of white blood cell count, PaO2/FiO2 ratio, and renal output (24h). According to the logistic regression model, BE was a significant independent variable. Concerning the prognostic scoring systems, an APACHE II score of 19, a SOFA score of 8, an MPI score of 30, and an MOF score of 7 or more were significantly related to poor prognosis. CONCLUSIONS: Preoperative BE and postoperative white blood cell count were reliable prognostic factors and early classification using prognostic scoring systems at specific points in the disease process are useful to improve our understanding of the problems involved.

Sequence characterized amplified region markers tightly linked to the mating factors of Lentinula edodes.

Detecting the mating types in shiitake, Lentinula edodes (Berk.) Pegler, is important for making progress in the breeding of this mushroom and determining the compatibility of the pair to cross. Shiitake is a tetrapolar fungus with two unlinking mating factors, A factor and B factor. We screened molecular markers linked to the mating factors using the randomly amplified polymorphic DNA (RAPD) method to develop the mating type identification procedure. Using 147 oligonucleotide primers, a total of 6 linkage markers for the shiitake mating factors, 4 markers for the A factor and 2 markers for the B factor, were discovered with a logarithm of the odds threshold of 3.0 for linkage. Two RAPDs that perfectly segregated with each mating factor among 72 basidiospore strains were detected. Both of these RAPDs were cloned and sequenced to convert them to the sequence characterized amplified region (SCAR) markers. Four primers, two sets of primers, were designed according to the internal sequences of two RAPDs tightly linking to the A factor or B factor. Consequently, we determined the polymerase chain reaction condition for multiplex analyses of these SCAR markers.