Role of Fatty Acid omega-Hydroxylase 1 and Abscisic Acid in Potato Tuber Suberin Formation

Suberin is a complex biopolymer composed of two distinct but covalently-linked domains. The first domain is composed of polymerized phenolic monomers, whereas the second domain is predominately fatty acid derivatives esterified with glycerol. Deposited in specialized cells during development or in response to abiotic stress, suberin functions as a barrier to water loss and pathogen attack. In potato, more than 65% of suberin monomers undergo ω-hydroxylation, representing a major class of fatty acids in the final biopolymer. The ω-hydroxylation reaction is catalyzed by Cytochrome P450 (CYP) proteins, of which few have been characterized to date. In 2009, CYP86A33 from potato was identified and implicated as the main suberin-associated ω-hydroxylase by another research group through RNAi gene silencing; although functional characterization of in vitro protein was unsuccessful. Simultaneously, I identified and characterized gene expression for three CYP86A and CYP94A ω-hydroxylase genes in potato. From this expression analysis, CYP86A33 was also identified as the primary candidate for a suberin-associated ω-hydroxylase, from which ω-hydroxylase activity was confirmed through an in vitro enzyme assay with recombinant protein. Following an in silico analysis of the CYP86A33 promoter region, which identified many ABA-responsive promoter elements, an extensive analysis of the effects of ABA on gene expression and suberin biosynthetic regulation was conducted. Using the biosynthetic inhibitor of ABA production, fluridone, I was able to investigate the effects of ABA on suberin regulation by inhibiting ABA de novo biosynthesis with or without the addition of exogenous ABA. Using wounded potato tubers, three parallel timecourse experiments were conducted with different treatments to quantify ABA tissue concentration, suberin-associated gene expression, and soluble and insoluble aliphatic monomer deposition into the suberin biopolymer. Expression of suberin-associated genes, including CYP86A33, was reduced post-wounding with fluridone treatment. Similarly, insoluble aliphatic monomer accumulation was nearly eliminated from suberin in fluridone-treated tissues, exhibiting both chain length and monomer class specific effects. These fluridone effects to gene expression and suberin deposition were rescued through the addition of exogenous ABA. Overall, ABA was shown to have a regulatory effect post-wounding on the gene expression of key suberin-associated genes, with concomitant downstream impact on aliphatic suberin deposition

New insights into the lymphovascular microanatomy of the colon and the risk of metastases in pT1 colorectal cancer obtained with quantitative methods and three-dimensional digital reconstruction

Aims: UK faecal occult blood test screening has tripled the proportion of pT1 colorectal cancers. The risk of metastasis is predicted by depth of invasion, suggesting that access to deep lymphovascular vessels is important. The aim of this study was to quantify the distribution and size of the submucosal vasculature, and generate a novel three-dimensional (3D) model to validate the findings. Methods and results: Thirty samples of normal large bowel wall were immunostained with CD31, a vascular endothelium marker, to identify blood vessels, which were quantified and digitally analysed for their number, circumference, area and diameter in the deep mucosa and submucosa (Sm1, Sm2, and Sm3). The model required serial sections, a double immunostain (using CD31 and D2-40), and 3D reconstruction. Significant differences were shown between submucosal layers in the number, circumference and area of vessels (P < 0.001). Blood vessels were most numerous in the mucosa (11.79 vessels/0.2 mm2) but smaller [median area of 247 μm2, interquartile range (IQR) 162–373 μm2] than in Sm2, where they were fewer in number (6.92 vessels/0.2 mm2) but considerably larger (2086 μm2, IQR 1007–4784 μm2). The 3D model generated novel observations on lymphovascular structures. Conclusions: The number and size of blood vessels do not increase with depth of submucosa, as hypothesized. The distribution of vessels suggests that we should investigate the area or volume of submucosal invasion rather than the depth

Intramucosal adenocarcinoma of the ileum originated 40 years after ileosigmoidostomy

<p>Abstract</p> <p>Background</p> <p>Small bowel adenocarcinomas (SBAs) are rare carcinomas. They are asymptomatic and usually neither endoscopy nor contrast studies are performed for screening</p> <p>Case presentation</p> <p>A 72-year-old Japanese male had a positive fecal occult blood test at a regular check-up in 2006. He suffered appendicitis and received an ileosigmoidostomy in 1966. A colonoscopy revealed an irregular mucosal lesion with an unclear margin at the ileum side of the anastomosis. A mucosal biopsy specimen showed adenocarcinoma histopathologically. Excision of the anastomosis was performed for this patient. The resected specimen showed a flat mucosal lesion with a slight depression at the ileum adjacent to the anastomosis. Histological examination revealed a well differentiated intramucosal adenocarcinoma (adenocarcinoma in situ). Immunohistological staining demonstrated the overexpression of p53 protein in the adenocarcinoma.</p> <p>Conclusion</p> <p>Adenocarcinoma of the ileum at such an early stage is a very rare event. In this case, there is a possibility that the ileosigmoidostomy resulted in a back flow of colonic stool to the ileum that caused the carcinogenesis of the small intestine.</p

Impact of hiatal hernia on histological pattern of non-erosive reflux disease

BACKGROUND: Hiatus hernia (HH) has major pathophysiological effects favoring gastroesophageal reflux and hence contributing to esophageal mucosa injury, especially in patients with severe gastroesophageal disease. However, prospective studies investigating the impact of HH on the esophageal mucosa in non-erosive reflux disease (NERD) are lacking. This study evaluated the association between the presence of (HH) and the histological findings in symptomatic patients with NERD. METHODS: Fifty consecutive patients with gastroesophageal reflux disease (GERD) were enrolled. After conventional endoscopy, Lugol solution was applied and biopsy specimens were obtained. Histological parameters including basal zone hyperplasia, papillary length and cellular infiltration were evaluated. The chi-square test with Yates' correlation was used for comparing discrete parameters between groups. However, Fisher's exact probability test was used where the expected frequencies were lower than 5. Wilcoxon's test for unpaired samples was preferred in cases of semi-quantitative parameters. RESULTS: The presence of HH along with more severe findings (0.01 <P < 0.05) was confirmed in 18 patients. NERD was observed in 29 (58%) patients. Basal zone hyperplasia and loss of glycogen accompanied HH in all cases, and the correlation was significant in NERD (P < 0.001). The remaining histological patterns were similar between erosive reflux disease and NERD in the presence of HH. CONCLUSION: The presence of HH is correlated with more severe endoscopy findings, and predisposes for severe histological abnormality in cases of NERD

DNA index determination with Automated Cellular Imaging System (ACIS) in Barrett's esophagus: Comparison with CAS 200

BACKGROUND: For solid tumors, image cytometry has been shown to be more sensitive for diagnosing DNA content abnormalities (aneuploidy) than flow cytometry. Image cytometry has often been performed using the semi-automated CAS 200 system. Recently, an Automated Cellular Imaging System (ACIS) was introduced to determine DNA content (DNA index), but it has not been validated. METHODS: Using the CAS 200 system and ACIS, we compared the DNA index (DI) obtained from the same archived formalin-fixed and paraffin embedded tissue samples from Barrett's esophagus related lesions, including samples with specialized intestinal metaplasia without dysplasia, low-grade dysplasia, high-grade dysplasia and adenocarcinoma. RESULTS: Although there was a very good correlation between the DI values determined by ACIS and CAS 200, the former was 25% more sensitive in detecting aneuploidy. ACIS yielded a mean DI value 18% higher than that obtained by CAS 200 (p < 0.001; paired t test). In addition, the average time required to perform a DNA ploidy analysis was shorter with the ACIS (30–40 min) than with the CAS 200 (40–70 min). Results obtained by ACIS gave excellent inter-and intra-observer variability (coefficient of correlation >0.9 for both, p < 0.0001). CONCLUSION: Compared with the CAS 200, the ACIS is a more sensitive and less time consuming technique for determining DNA ploidy. Results obtained by ACIS are also highly reproducible

Efficacy of capillary pattern type IIIA/IIIB by magnifying narrow band imaging for estimating depth of invasion of early colorectal neoplasms

<p>Abstract</p> <p>Background</p> <p>Capillary patterns (CP) observed by magnifying Narrow Band Imaging (NBI) are useful for differentiating non-adenomatous from adenomatous colorectal polyps. However, there are few studies concerning the effectiveness of magnifying NBI for determining the depth of invasion in early colorectal neoplasms. We aimed to determine whether CP type IIIA/IIIB identified by magnifying NBI is effective for estimating the depth of invasion in early colorectal neoplasms.</p> <p>Methods</p> <p>A series of 127 consecutive patients with 130 colorectal lesions were evaluated from October 2005 to October 2007 at the National Cancer Center Hospital East, Chiba, Japan. Lesions were classified as CP type IIIA or type IIIB according to the NBI CP classification. Lesions were histopathologically evaluated. Inter and intraobserver variabilities were assessed by three colonoscopists experienced in NBI.</p> <p>Results</p> <p>There were 15 adenomas, 66 intramucosal cancers (pM) and 49 submucosal cancers (pSM): 16 pSM superficial (pSM1) and 33 pSM deep cancers (pSM2-3). Among lesions diagnosed as CP IIIA 86 out of 91 (94.5%) were adenomas, pM-ca, or pSM1; among lesions diagnosed as CP IIIB 28 out of 39 (72%) were pSM2-3. Sensitivity, specificity and diagnostic accuracy of the CP type III for differentiating pM-ca or pSM1 (<1000 μm) from pSM2-3 (≥1000 μm) were 84.8%, 88.7 % and 87.7%, respectively. Interobserver variability: κ = 0.68, 0.67, 0.72. Intraobserver agreement: κ = 0.79, 0.76, 0.75</p> <p>Conclusion</p> <p>Identification of CP type IIIA/IIIB by magnifying NBI is useful for estimating the depth of invasion of early colorectal neoplasms.</p

Comparison of DNA histograms by standard flow cytometry and image cytometry on sections in Barrett's adenocarcinoma

<p>Abstract</p> <p>Background</p> <p>The purpose of this study was to compare DNA histograms obtained by standard flow cytometry (FC) and high fidelity image cytometry on sections (ICS) in normal gastrointestinal mucosa and Barrett's adenocarcinoma (BAC).</p> <p>Methods</p> <p>Archival formalin-fixed paraffin-embedded tissue blocks of 10 normal controls from 10 subjects and 42 BAC tissues from 17 patients were examined. DNA FC was performed using standard techniques and ICS was carried out by Automated Cellular Imaging System (ACIS). DNA ploidy histograms were classified into diploid with peak DNA index (DI) at 0.9–1.1, and aneuploid with peak DI > 1.1. DI values of aneuploid peaks were determined. Additionally, for DNA ICS, heterogeneity index (HI) representing DNA content heterogeneity, and histograms containing cells with DI > G2 were also identified.</p> <p>Results</p> <p>All control samples were diploid by both FC and ICS analyses. In BAC, FC showed diploid peaks in 29%, diploid peaks with additional aneuploid or tetraploid peaks in 57%, and 14% of the samples, respectively. In contrast, ICS showed aneuploid peaks in all the cases with peak DI > 1.25; 37 cases had peak DI between 1.25 and 2.25; and 5 cases had peak DI > 2.25. HI values (mean ± SD) were 11.3 ± 1.1 in controls and 32.4 ± 8.5 in BAC (p < 0.05). Controls had no G2 exceeding cells. However, 19/37 (51%) of the cases with primary peak DI < 2.25 had cells exceeding 9N.</p> <p>Conclusion</p> <p>ICS detects DNA aneuploidy in all BAC samples while FC missed the diagnosis of aneuploidy in 29%. In addition, ICS provides more information on HI and G2 exceeding rates.</p

Frequent Occurrence of Mitochondrial DNA Mutations in Barrett’s Metaplasia without the Presence of Dysplasia

BACKGROUND: Barrett's esophagus (BE) is one of the most common premalignant lesions and can progress to esophageal adenocarcinoma (EA). The numerous molecular events may play a role in the neoplastic transformation of Barrett's mucosa such as the change of DNA ploidy, p53 mutation and alteration of adhesion molecules. However, the molecular mechanism of the progression of BE to EA remains unclear and most studies of mitochondrial DNA (mtDNA) mutations in BE have performed on BE with the presence of dysplasia. METHODS/FINDINGS: Thus, the current study is to investigate new molecular events (Barrett's esophageal tissue-specific-mtDNA alterations/instabilities) in mitochondrial genome and causative factors for their alterations using the corresponding adjacent normal mucosal tissue (NT) and tissue (BT) from 34 patients having Barrett's metaplasia without the presence of dysplasia. Eighteen patients (53%) exhibited mtDNA mutations which were not found in adjacent NT. mtDNA copy number was about 3 times higher in BT than in adjacent NT. The activity of the mitochondrial respiratory chain enzyme complexes in tissues from Barrett's metaplasia without the presence of dysplasia was impaired. Reactive oxygen species (ROS) level in BT was significantly higher than those in corresponding samples. CONCLUSION/SIGNIFICANCE: High ROS level in BT may contribute to the development of mtDNA mutations, which may play a crucial role in disease progression and tumorigenesis in BE

Depressed-Type of Early Colon Cancer with Extensive Lymph Node Metastasis

Early colorectal cancer (ECC) is defined as invasive tumor limited to the colonic and rectal mucosa or submucosa, regardless of the presence or absence of lymph node metastasis. The incidence of lymph node metastasis in ECC ranges from 0 to 15.4%, and risk factors include depth of submucosal invasion, growth patterns (polypoid or non-polypoid), histologic subclassification, and lymphatic invasion. Of non-polypoid growth patterns, the depressed types of colorectal cancer have higher malignant potential than polypoid types, even for small sizes. Unfortunately, this type is also difficult to detect on colonoscopic examination. In this report, we describe a case of depressed type ECC with extensive lymph node metastasis without regional lymph node involvement