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1.  Molecular detection of lymph node metastasis in breast cancer patients treated with preoperative systemic chemotherapy: a prospective multicentre trial using the one-step nucleic acid amplification assay 
British Journal of Cancer  2013;109(6):1693-1698.
Background:
For patients with breast cancer treated with preoperative chemotherapy, residual tumour burden in lymph nodes is the strongest prognostic factor. However, conventional pathological examination has limitations that hinder the accurate and reproducible measurement. The one-step nucleic acid amplification (OSNA) assay is a novel molecular method for detecting nodal metastasis. In this prospective multicentre trial, we assessed the performance of the OSNA assay in detecting nodal metastasis after chemotherapy.
Methods:
In total, 302 lymph nodes from 80 breast cancer patients who underwent axillary dissection after chemotherapy were analysed. Each node was cut into two or four slices. One piece or alternate pieces were evaluated by pathology, and the other(s) were examined using the OSNA assay. The results of the two methods were compared. Stromal fibrosis, histiocytic aggregates, and degenerated cancer cells were regarded as chemotherapy-induced histological changes.
Results:
The overall accuracy, sensitivity, and specificity of the OSNA assay compared with the reference pathology were 91.1%, 88.3%, and 91.7%, respectively. Of the 302 lymph nodes, 66 (21.9%) exhibited chemotherapy-induced histology. For these nodes, the accuracy, sensitivity, and specificity were 90.9%, 88.9%, and 93.3%, respectively.
Conclusion:
The OSNA assay can detect the residual tumour burden as accurately as conventional pathology, although chemotherapy-induced histological changes are present.
doi:10.1038/bjc.2013.503
PMCID: PMC3777008  PMID: 24002597
breast cancer; lymph node metastasis; molecular diagnostic technique; preoperative chemotherapy; OSNA
2.  Feasibility trial for adjuvant chemotherapy with docetaxel plus cisplatin followed by single agent long-term administration of S-1 chemotherapy in patients with completely resected non-small cell lung cancer: Thoracic Oncology Research Group Study 0809 
British Journal of Cancer  2013;109(3):545-551.
Background:
We conducted a multicentre feasibility study for single agent long-term S-1 chemotherapy following docetaxel plus cisplatin in patients with curatively resected stage II–IIIA non-small cell lung cancer.
Methods:
Patients received three cycles of docetaxel (60 mg m−2) plus cisplatin (80 mg m−2) and then received S-1 (40 mg m−2 twice daily) for 14 consecutive days with a 1-week rest for >6 months (maximum, 1 year). The primary end point was feasibility, which was defined as the proportion of patients who completed eight or more cycles of S-1 chemotherapy. If the lower 95% confidence interval (CI) of this proportion was 50% or more, then the treatment was considered as feasible. The sample size was set at 125 patients.
Results:
One hundred and thirty-one patients were enrolled, of whom 129 patients were eligible and assessable. In all, 109 patients (84.5%) completed 3 cycles of docetaxel plus cisplatin and 66 patients (51.2%, 95% CI: 42.5–59.8) completed 8 or more cycles of S-1 treatment. Grade 3/4 toxicities during the S-1 chemotherapy included anaemia (7.3%), neutropaenia (3.7%), and anorexia (3.7%).
Conclusion:
The toxicity level was acceptable, although the results did not meet our criterion for feasibility. Modification of the treatment schedule for S-1 chemotherapy might improve the treatment compliance.
doi:10.1038/bjc.2013.378
PMCID: PMC3738148  PMID: 23868010
S-1; long-term; adjuvant chemotherapy; non-small cell lung cancer
3.  Usefulness of 4D-CTA in the detection of cerebral dural sinus occlusion or stenosis with collateral pathways 
The neuroradiology journal  null;26(4):428-438.
Summary
In time-resolved CT angiography (4D-CTA), it is of substantial merit to detect the veins and sinuses of the whole brain with the simultaneous demonstration of the natural drainage flow in order to find occlusion or stenosis of the dural sinuses with collateral pathways. As preoperative information for patients with brain tumors, it is important to detect feeding arteries, incidentally found aneurysms or other vascular lesions, and to detect patency of the dural sinuses and the important cortical veins, whether they are compressed by tumors or not. On the other hand, cerebral venous thrombosis (CVT) may occur in patients due to various causes, which has not been unusual in recent years. For patients with acute symptomatic or chronic non-symptomatic CVT, identification of dural sinus occlusion (DSO) or dural sinus stenosis (DSS) and compensatory collateral pathways is necessary for suitable thrombolytic therapy or careful investigation to avoid further CVT. This study reviews our experiences in 116 cases of 4D-CTA for 90 patients with brain tumors and 26 other patients including 11 with arteriovenous malformation, and four with acute CVT and other conditions. 4D-CTA presented DSO/DSS with compensatory venous collateral pathways, which was helpful to detect the severity of the venous abnormality, and see whether it was compressed by brain tumors, or due to other causes in patients with symptomatic or non-symptomatic CVT. 4D-CTA is a useful non-invasive diagnostic tool to detect cerebral venous abnormalities as an alternative to DSA.
PMCID: PMC4202811  PMID: 24007731
4D-CTA; cerebral dural sinus; sinus occlusion; sinus stenosis; venous thrombosis
4.  Disruption of the temporally regulated cloaca endodermal β-catenin signaling causes anorectal malformations 
Cell Death and Differentiation  2014;21(6):990-997.
The cloaca is temporally formed and eventually divided by the urorectal septum (URS) during urogenital and anorectal organ development. Although congenital malformations, such as anorectal malformations (ARMs), are frequently observed during this process, the underlying pathogenic mechanisms remain unclear. β-Catenin is a critical component of canonical Wnt signaling and is essential for the regulation of cell differentiation and morphogenesis during embryogenesis. The expression of β-catenin is observed in endodermal epithelia, including URS epithelia. We modulated the β-catenin gene conditionally in endodermal epithelia by utilizing tamoxifen-inducible Cre driver line (ShhCreERT2). Both β-catenin loss- and gain-of-function (LOF and GOF) mutants displayed abnormal clefts in the perineal region and hypoplastic elongation of the URS. The mutants also displayed reduced cell proliferation in the URS mesenchyme. In addition, the β-catenin GOF mutants displayed reduced apoptosis and subsequently increased apoptosis in the URS epithelium. This instability possibly resulted in reduced expression levels of differentiation markers, such as keratin 1 and filaggrin, in the perineal epithelia. The expression of bone morphogenetic protein (Bmp) genes, such as Bmp4 and Bmp7, was also ectopically induced in the epithelia of the URS in the β-catenin GOF mutants. The expression of the Msx2 gene and phosphorylated-Smad1/5/8, possible readouts of Bmp signaling, was also increased in the mutants. Moreover, we introduced an additional mutation for a Bmp receptor gene: BmprIA. The ShhCreERT2/+; β-cateninflox(ex3)/+; BmprIAflox/− mutants displayed partial restoration of URS elongation compared with the β-catenin GOF mutants. These results indicate that some ARM phenotypes in the β-catenin GOF mutants were caused by abnormal Bmp signaling. The current analysis revealed the close relation of endodermal β-catenin signaling to the ARM phenotypes. These results are considered to shed light on the pathogenic mechanisms of human ARMs.
doi:10.1038/cdd.2014.21
PMCID: PMC4013517  PMID: 24632946
5.  DNA demethylation in normal colon tissue predicts predisposition to multiple cancers 
Oncogene  2012;31(48):5029-5037.
Some colon cancer (CC) patients present synchronous cancers at diagnosis and others develop metachronous neoplasms, but the risk factors are unclear for non-hereditary CC. We showed previously that global DNA demethylation increased with aging and correlated with genomic damage in CC, and we show now that preferentially associates to CCs with wild-type p53. This study aimed to elucidate the extent of DNA hypomethylation in patients with single and multiple CC, its relationship with aging, and its potential as predictive tool. We compared by real-time methylation-specific PCR the relative demethylation level (RDL) of long interspersed nucleotide element-1 (LINE-1) sequences in matched cancer tissues and non-cancerous colonic mucosa (NCM) from patients with single and multiple right-sided CCs. Although no RDL difference was found in NCM from single CC patients and healthy volunteers (P =0.5), there was more demethylation (higher RDL) in NCM from synchronous cancer patients (P =1.1 × 10−5) multiple CCs also were more demethylated than single CCs (P =0.0014). High NCM demethylation was predictive for metachronous neoplasms (P =0.003). In multivariate logistic regression analyses RDL was the only independent predictor for metachronous (P =0.02) and multiple (P =4.9 × 10−5) tumors. The higher LINE-1 demethylation in NCM from patients with multiple (synchronous and metachronous) tumors (P =9.6 × 10−7) was also very significant in patients with tumors without (P =3.8 × 10−6), but not with (P =0.16) microsatellite instability. NCM demethylation increased with aging in patients with single tumors, but decreased in those with multiple tumors. Moreover, the demethylation difference between patients with single vs multiple tumors appeared higher in younger (P =3.6 × 10−4) than in older (P =0.0016) patients. These results predict that LINE-1 hypomethylation in NCM can be used as an epigenetic predictive biomarker for multiple CC risk. The stronger association of demethylation in NCM with multiple CC risk from younger patients also suggests an inherited predisposition for the apparent field cancerization effect of somatic demethylation.
doi:10.1038/onc.2011.652
PMCID: PMC4013778  PMID: 22310288
DNA demethylation; colon cancer; synchronous; metachronous; epigenetics
6.  PHYSIOLOGICAL AND LEUKOCYTE SUBSET RESPONSES TO EXERCISE AND COLD EXPOSURE IN COLD-ACCLIMATIZED SKATERS 
Biology of Sport  2014;31(1):39-48.
We investigated physiological responses and changes in circulating immune cells following exercise in cold and thermoneutral conditions. Participants were short track skaters (n=9) who were acclimatized to cold conditions, and inline skaters (n=10) who were not acclimatized. All skaters were young, and skating at a recreational level three days per week for at least one year. Using a cross-over design, study variables were measured during 60 min of submaximal cycling (65% V.O2max) in cold (ambient temperature: 5±1°C, relative humidity: 41±9%) and thermoneutral conditions (ambient temperature: 21±1°C, relative humidity: 35±5%). Heart rate, blood lactate and tympanic temperature were measured at rest, during exercise and recovery. Plasma cortisol, calprotectin and circulating blood cell numbers were measured before and after 60 min of cold or thermoneutral conditions, and during recovery from exercise. Heart rate was lower in both groups during exercise in cold versus thermoneutral conditions (P<0.05). The increase in total leukocytes during recovery was primarily due to an increase in neutrophils in both groups. The cold-acclimatized group activated neutrophils after exercise in cold exposure, whereas the non-acclimatized group activated lymphocyte and cortisol after exercise in cold exposure. Lymphocyte subsets significantly changed in both groups over time during recovery as compared to rest. Immediately after exercise in both groups, CD16+ and CD69+ cells were elevated compared to rest or before exercise in both conditions. Acclimatization to exercise in the cold does not appear to influence exercise-induced immune changes in cold conditions, with the possible exception of neutrophils, lymphocytes and cortisol concentration.
doi:10.5604/20831862.1086731
PMCID: PMC3994584  PMID: 24917688
exercise; cold; neutrophils; lymphocyte subset; cortisol
7.  Phytohemagglutinin-induced IL2 mRNA in whole blood can predict bortezomib-induced peripheral neuropathy for multiple myeloma patients 
Blood Cancer Journal  2013;3(10):e150-.
The proteasome inhibitor bortezomib has revolutionized the treatment of multiple myeloma. However, bortezomib-induced peripheral neuropathy (BiPN) is a serious complication that compromises clinical outcome. If patients with a risk of developing BiPN could be predicted, physicians might prefer weekly, reduced-dose, or subcutaneous approaches. To seek biomarkers for BiPN, we conducted a multicenter prospective study using a simple and unique system. Multiple myeloma patients received twice-weekly or weekly 1.3 mg/m2 bortezomib intravenously, and a 2-ml sample of whole blood was obtained before treatment and 2–3 days and 1–3 weeks after the first dose. Induction of gene expression was then quantified by real-time PCR. Of a total of 64 enrolled patients, 53 patient samples qualified for mRNA analysis. The BiPN grade was associated with phytohemagglutinin-induced IL2, IFNG and TNFSF2, as well as with lipopolysaccharide-induced IL6 levels. More importantly, of the 19 patients showing a ⩾3-fold increase in phytohemagglutinin-induced IL2, 14 did not suffer from BiPN (73.7% prediction), whereas of the 34 patients with a <3-fold increase, 23 experienced BiPN (67.6% prediction). Therefore, we concluded that pretreatment of phytohemagglutinin-induced IL2 mRNA levels in whole blood serve as a promising biomarker for predicting BiPN, and this finding warrants validation in a larger study.
doi:10.1038/bcj.2013.47
PMCID: PMC3816208  PMID: 24096714
bortezomib; peripheral neuropathy; interleukin 2; interferon-γ; TNF-α; interleukin 6
8.  ING1 induces apoptosis through direct effects at the mitochondria 
Cell Death & Disease  2013;4(10):e837-.
The ING family of tumor suppressors acts as readers and writers of the histone epigenetic code, affecting DNA repair, chromatin remodeling, cellular senescence, cell cycle regulation and apoptosis. The best characterized member of the ING family, ING1, interacts with the proliferating cell nuclear antigen (PCNA) in a UV-inducible manner. ING1 also interacts with members of the 14-3-3 family leading to its cytoplasmic relocalization. Overexpression of ING1 enhances expression of the Bax gene and was reported to alter mitochondrial membrane potential in a p53-dependent manner. Here we show that ING1 translocates to the mitochondria of primary fibroblasts and established epithelial cell lines in response to apoptosis inducing stimuli, independent of the cellular p53 status. The ability of ING1 to induce apoptosis in various breast cancer cell lines correlates well with its degree of translocation to the mitochondria after UV treatment. Endogenous ING1 protein specifically interacts with the pro-apoptotic BCL2 family member BAX, and colocalizes with BAX in a UV-inducible manner. Ectopic expression of a mitochondria-targeted ING1 construct is more proficient in inducing apoptosis than the wild type ING1 protein. Bioinformatic analysis of the yeast interactome indicates that yeast ING proteins interact with 64 mitochondrial proteins. Also, sequence analysis of ING1 reveals the presence of a BH3-like domain. These data suggest a model in which stress-induced cytoplasmic relocalization of ING1 by 14-3-3 induces ING1-BAX interaction to promote mitochondrial membrane permeability and represent a paradigm shift in our understanding of ING1 function in the cytoplasm and its contribution to apoptosis.
doi:10.1038/cddis.2013.398
PMCID: PMC3920937
9.  ING1 induces apoptosis through direct effects at the mitochondria 
Cell Death & Disease  2013;4(9):e788-.
The ING family of tumor suppressors acts as readers and writers of the histone epigenetic code, affecting DNA repair, chromatin remodeling, cellular senescence, cell cycle regulation and apoptosis. The best characterized member of the ING family, ING1, interacts with the proliferating cell nuclear antigen (PCNA) in a UV-inducible manner. ING1 also interacts with members of the 14-3-3 family leading to its cytoplasmic relocalization. Overexpression of ING1 enhances expression of the Bax gene and was reported to alter mitochondrial membrane potential in a p53-dependent manner. Here we show that ING1 translocates to the mitochondria of primary fibroblasts and established epithelial cell lines in response to apoptosis inducing stimuli, independent of the cellular p53 status. The ability of ING1 to induce apoptosis in various breast cancer cell lines correlates well with its degree of translocation to the mitochondria after UV treatment. Endogenous ING1 protein specifically interacts with the pro-apoptotic BCL2 family member BAX, and colocalizes with BAX in a UV-inducible manner. Ectopic expression of a mitochondria-targeted ING1 construct is more proficient in inducing apoptosis than the wild type ING1 protein. Bioinformatic analysis of the yeast interactome indicates that yeast ING proteins interact with 64 mitochondrial proteins. Also, sequence analysis of ING1 reveals the presence of a BH3-like domain. These data suggest a model in which stress-induced cytoplasmic relocalization of ING1 by 14-3-3 induces ING1-BAX interaction to promote mitochondrial membrane permeability and represent a paradigm shift in our understanding of ING1 function in the cytoplasm and its contribution to apoptosis.
doi:10.1038/cddis.2013.321
PMCID: PMC3789179  PMID: 24008732
apoptosis; ING1; mitochondria; BAX
11.  Prevalence and specificity of LKB1 genetic alterations in lung cancers 
Oncogene  2007;26(40):5911-5918.
Germline LKB1 mutations cause Peutz–Jeghers syndrome, a hereditary disorder that predisposes to gastrointestinal hamartomatous polyposis and several types of malignant tumors. Somatic LKB1 alterations are rare in sporadic cancers, however, a few reports showed the presence of somatic alterations in a considerable fraction of lung cancers. To determine the prevalence and the specificity of LKB1 alterations in lung cancers, we examined a large number of lung cancer cell lines and lung adenocarcinoma (AdC) specimens for the alterations. LKB1 genetic alterations were frequently detected in the cell lines (21/70, 30%), especially in non-small cell lung cancers (NSCLCs) (20/51, 39%), and were significantly more frequent in cell lines with KRAS mutations. Point mutations were detected only in AdCs and large cell carcinomas, whereas homozygous deletions were detected in all histological types of lung cancer. Among lung AdC specimens, LKB1 mutations were found in seven (8%) of 91 male smokers but in none of 64 females and/or nonsmokers, and were significantly more frequent in poorly differentiated tumors. The difference in the frequency of LKB1 alterations between cell lines and tumor specimens was likely to be owing to masking of deletions by the contamination of noncancerous cells in the tumor specimens. These results indicate that somatic LKB1 genetic alterations preferentially occur in a subset of poorly differentiated lung AdCs that appear to correlate with smoking males.
doi:10.1038/sj.onc.1210418
PMCID: PMC3457639  PMID: 17384680
LKB1; lung cancer mutation; deletion; smoking; poor differentiation
12.  Clinical evaluation of autologous gamma delta T cell-based immunotherapy for metastatic solid tumours 
British Journal of Cancer  2011;105(6):778-786.
Background:
Adoptive transfer of ex vivo expanded autologous Vγ9Vδ2 T cells may be of therapeutic benefit for cancer because of their potent direct cytotoxicity towards tumour cells, synergistic cytotoxicity when combined with aminobisphosphonates and enhancement of antibody-dependent cell-mediated cytotoxicity.
Methods:
To determine the feasibility and clinical safety of therapy with ex vivo expanded, activated Vγ9Vδ2 T cells in combination with zoledronate, we enrolled 18 subjects with advanced solid tumours into a phase I clinical study. Administered indium111-oxine-labelled Vγ9Vδ2 T cells were tracked in a cohort of patients.
Results:
Administered Vγ9Vδ2 T cells had an activated effector memory phenotype, expressed chemokine receptors predictive of homing to peripheral tissues and were cytotoxic in vitro against tumour targets. Adoptively transferred Vγ9Vδ2 T cells trafficked predominantly to the lungs, liver and spleen and, in some patients, to metastatic tumour sites outside these organs. No dose-limiting toxicity was observed, but most patients progressed on study therapy. However, three patients administered Vγ9Vδ2 T cells while continuing previously ineffective therapy had disease responses, suggesting an additive effect.
Conclusion:
Therapy with aminobisphosphonate-activated Vγ9Vδ2 T cells is feasible and well tolerated, but therapeutic benefits appear only likely when used in combination with other therapies.
doi:10.1038/bjc.2011.293
PMCID: PMC3171009  PMID: 21847128
gamma delta T cells; Vγ9Vδ2 T cells; immunotherapy; clinical trial
16.  An extraterrestrial trigger for the Early Cretaceous massive volcanism? Evidence from the paleo-Tethys Ocean 
Scientific Reports  2012;2:268.
The Early Cretaceous Greater Ontong Java Event in the Pacific Ocean may have covered ca. 1% of the Earth's surface with volcanism. It has puzzled scientists trying to explain its origin by several mechanisms possible on Earth, leading others to propose an extraterrestrial trigger to explain this event. A large oceanic extraterrestrial impact causing such voluminous volcanism may have traces of its distal ejecta in sedimentary rocks around the basin, including the paleo-Tethys Ocean which was then contiguous with the Pacific Ocean. The contemporaneous marine sequence at central Italy, containing the sedimentary expression of a global oceanic anoxic event (OAE1a), may have recorded such ocurrence as indicated by two stratigraphic intervals with 187Os/188Os indicative of meteoritic influence. Here we show, for the first time, that platinum group element abundances and inter-element ratios in this paleo-Tethyan marine sequence provide no evidence for an extraterrestrial trigger for the Early Cretaceous massive volcanism.
doi:10.1038/srep00268
PMCID: PMC3280599  PMID: 22355780
17.  Downregulated plasma miR-92a levels have clinical impact on multiple myeloma and related disorders 
Blood Cancer Journal  2012;2(1):e53-.
Recent studies have demonstrated that one-third of known microRNAs (miRNAs) are stably detectable in plasma. Therefore, we assessed plasma miRNAs to investigate the dynamics of oncomir 17-92a, which is highly expressed in multiple myeloma (MM) patients. The plasma miR-92a level in symptomatic MM patients was significantly downregulated compared with normal subjects (P<0.0001), regardless of immunoglobulin subtypes or disease stage at diagnosis. In contrast, miR-92a levels in peripheral blood CD8+ or CD4+ cells from MM patients were lower than those of normal subjects, and the miR-92a levels of the cells tended to correlate with plasma miR-92a levels. The plasma miR-92a level in the complete remission group became normalized, whereas the partial response (PR) and very good PR groups did not reach the normal range. In smoldering MM, the plasma miR-92a level did not show a significant difference compared with normal subjects. Our findings suggest that measurement of the plasma miR-92a level in MM patients could be useful for initiation of chemotherapy and monitoring disease status, and the level may represent, in part, the T-cell immunity status of these patients.
doi:10.1038/bcj.2011.51
PMCID: PMC3270255  PMID: 22829237
multiple myeloma; plasma miR-92a; T lymphocytes
21.  MEK–ERK-dependent multiple caspase activation by mitochondrial proapoptotic Bcl-2 family proteins is essential for heavy ion irradiation-induced glioma cell death 
Cell Death & Disease  2010;1(7):e60-.
Recently developed heavy ion irradiation therapy using a carbon beam (CB) against systemic malignancy has numerous advantages. However, the clinical results of CB therapy against glioblastoma still have room for improvement. Therefore, we tried to clarify the molecular mechanism of CB-induced glioma cell death. T98G and U251 human glioblastoma cell lines were irradiated by CB, and caspase-dependent apoptosis was induced in both cell lines in a dose-dependent manner. Knockdown of Bax (BCL-2-associated X protein) and Bak (BCL-2-associated killer) and overexpression of Bcl-2 or Bcl-xl (B-cell lymphoma-extra large) showed the involvement of Bcl-2 family proteins upstream of caspase activation, including caspase-8, in CB-induced glioma cell death. We also detected the activation of extracellular signal-regulated kinase (ERK) and the knockdown of ERK regulator mitogen-activated protein kinase kinase (MEK)1/2 or overexpression of a dominant-negative (DN) ERK inhibited CB-induced glioma cell death upstream of the mitochondria. In addition, application of MEK-specific inhibitors for defined periods showed that the recovery of activation of ERK between 2 and 36 h after irradiation is essential for CB-induced glioma cell death. Furthermore, MEK inhibitors or overexpression of a DN ERK failed to significantly inhibit X-ray-induced T98G and U251 cell death. These results suggested that the MEK–ERK cascade has a crucial role in CB-induced glioma cell death, which is known to have a limited contribution to X-ray-induced glioma cell death.
doi:10.1038/cddis.2010.37
PMCID: PMC3039836  PMID: 21364665
apoptosis; heavy ion radiation; carbon beam; glioma; MAPK
22.  Actions anchored by concepts: defective action comprehension in semantic dementia 
Objective
To study the ability of patients with semantic dementia to understand actions, in order to examine the contribution of semantic memory to action comprehension.
Methods
The ability to comprehend symbolic and instrumental actions was assessed in 6 patients with semantic dementia and 10 healthy controls. The patients were also given the imitation test of meaningful and meaningless actions.
Results
In all patients with semantic dementia, comprehension of both symbolic and instrumental actions was defective. The comprehension of symbolic actions was more impaired than that of instrumental actions. Their ability to imitate other's actions was well preserved.
Conclusion
This study showed that comprehension of action was impaired in semantic dementia, suggesting that semantic memory has an important role in comprehension of human action.
doi:10.1136/jnnp.2006.096297
PMCID: PMC2077425  PMID: 16891383
24.  Fine mapping and DNA fiber FISH analysis locates the tobamovirus resistance gene L3 of Capsicum chinense in a 400-kb region of R-like genes cluster embedded in highly repetitive sequences 
The tobamovirus resistance gene L3 of Capsicum chinense was mapped using an intra-specific F2 population (2,016 individuals) of Capsicum annuum cultivars, into one of which had been introduced the C. chinenseL3 gene, and an inter-specific F2 population (3,391 individuals) between C. chinense and Capsicum frutescence. Analysis of a BAC library with an AFLP marker closely linked to L3-resistance revealed the presence of homologs of the tomato disease resistance gene I2. Partial or full-length coding sequences were cloned by degenerate PCR from 35 different pepper I2 homologs and 17 genetic markers were generated in the inter-specific combination. The L3 gene was mapped between I2 homolog marker IH1-04 and BAC-end marker 189D23M, and located within a region encompassing two different BAC contigs consisting of four and one clones, respectively. DNA fiber FISH analysis revealed that these two contigs are separated from each other by about 30 kb. DNA fiber FISH results and Southern blotting of the BAC clones suggested that the L3 locus-containing region is rich in highly repetitive sequences. Southern blot analysis indicated that the two BAC contigs contain more than ten copies of the I2 homologs. In contrast to the inter-specific F2 population, no recombinant progeny were identified to have a crossover point within two BAC contigs consisting of seven and two clones in the intra-specific F2 population. Moreover, distribution of the crossover points differed between the two populations, suggesting linkage disequilibrium in the region containing the L locus.
Electronic supplementary material
The online version of this article (doi:10.1007/s00122-008-0848-6) contains supplementary material, which is available to authorized users.
doi:10.1007/s00122-008-0848-6
PMCID: PMC2755798  PMID: 18663424
25.  Adolescent smoking behaviour and cigarette brand preference in Japan 
Tobacco Control  2006;15(3):172-180.
Objectives
As part of efforts to develop a smoking control strategy for Japanese adolescents, the results of two nationwide surveys on adolescent smoking behaviour were compared.
Design
Descriptive study on smoking behaviour among high school students was conducted. Self‐reporting anonymous questionnaires were administered to 115 814 students in 1996 and 106 297 in 2000 through randomly sampled junior and senior high schools throughout Japan.
Main outcome measures
Smoking prevalence, proportion of smokers by usual sources of cigarettes, national estimated cigarettes consumed by minors, share of cigarette brands smoked by high school students.
Results
The experiment rate among junior high school boys decreased in 2000 compared with that in 1996, whereas current and daily smoking rates did not. Although prevalence among Japanese girls was much lower than that among boys, prevalence among girls increased in 2000. The main source of cigarettes among high school smokers was vending machines. The proportion of smokers who usually purchased cigarettes from vending machines increased in 2000, in spite of the 1998 introduction of restrictions on night‐time operations. Japanese adolescents were more likely than adults to smoke American cigarette brands, and the adolescent market share of American brands has increased rapidly, especially for menthol brands.
Conclusions
This survey revealed the seriousness of the problem of smoking behaviour among Japanese high school students, and suggested that this behaviour may be influenced by social environmental factors, including the marketing strategies of the tobacco industry. Action should be taken to reduce the prevalence and impact of pro‐tobacco marketing messages and to abolish cigarette vending machines.
doi:10.1136/tc.2005.013060
PMCID: PMC2564654  PMID: 16728747
Japan; adolescent behaviour; brand preference; cigarette smoking; smoking behaviour

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