Xist Exon 7 Contributes to the Stable Localization of Xist RNA on the Inactive X-Chromosome

To equalize X-linked gene dosage between the sexes in mammalian females, Xist RNA inactivates one of the two X-chromosomes. Here, we report the crucial function of Xist exon 7 in X-inactivation. Xist exon 7 is the second-largest exon with a well-conserved repeat E in eutherian mammals, but its role is often overlooked in X-inactivation. Although female ES cells with a targeted truncation of the Xist exon 7 showed no significant differences in their Xist expression levels and RNA stability from control cells expressing wild-type Xist, compromised localization of Xist RNA and incomplete silencing of X-linked genes on the inactive X-chromosome (Xi) were observed in the exon 7-truncated mutant cells. Furthermore, the interaction between the mutant Xist RNA and hnRNP U required for localization of Xist RNA to the Xi was impaired in the Xist exon 7 truncation mutant cells. Our results suggest that exon 7 of Xist RNA plays an important role for stable Xist RNA localization and silencing of the X-linked genes on the Xi, possibly acting through an interaction with hnRNP U.     

Intraspecific Genetic Diversity of Undaria pinnatifida in Japan, based on the Mitochondrial cox3 Gene and the ITS1 of nrDNA

The intraspecific genetic diversity of the kelp Undaria pinnatifida (Harvey) Suringar (Laminariales, Phaeophyceae) was investigated using DNA sequences of the mitochondrial cytochrome oxidase subunit 3 (cox3) gene and internal transcribed spacer 1 (ITS1) of nuclear ribosomal DNA in plants collected from 21 localities along the Japanese coast between 2001 and 2003. Morphological variation was also examined and compared with the genetic diversity. Cox3 analyses of 106 plants revealed 9 haplotypes (I–IX) that differed from each other by 1–7 bp (all synonymous substitutions). Haplotype I was distributed in Hokkaido and the northern Pacific coast of Honshu, while haplotype III was found along the Sea of Japan coast of Honshu. Other types were found along the central and southern coast of Honshu. ITS1 analyses of 42 plants revealed 0–1.7% nucleotide differences, but plants from the Sea of Japan coast and northern Japan had similar sequences. The lower genetic differentiation along the Sea of Japan and northern coasts might be due to the recent establishment (after the middle of the last glacial period) of the Sea of Japan flora. The cox3 haplotype of cultivated plants was found in natural populations occurring close to cultivation sites (Naruto, Tokushima Pref., and Hokutan, Hyogo Pref.). This suggests that cultivated plants possibly escaped and spread or crossed with plants of natural populations. Morphological analyses of variation in 10 characters were conducted using 66 plants. The results showed no significant local variation owing to the wide variation in each population and did not support any forma previously described. No correlations between the morphological characters and cox3 haplotypes were detected.     

Investigation of Proteomic Profiles of Lamina of Ecklonia kurome (Laminariales): Homology-Based Cross-Species Protein Identification and Analysis of the Post-translational Processing of Vanadium-Dependent Bromoperoxidases Using MALDI-TOF/TOF

Proteomic profiles of the lamina of Ecklonia kurome Okamura, one of the Japanese dominant laminarialean kelps, were investigated by two-dimensional electrophoresis (2-DE) and MALDI-TOF/TOF. Due to the absence of E. kurome DNA or protein databases, homology-based cross-species protein identification was performed using a combination of three database-searching algorithms, Mascot peptide mass fingerprinting, Mascot MS/MS ion search, and mass spectrometry-based BLAST. Proteins were extracted from the lamina by an ethanol/phenol method and subjected to 2-DE (pI 4–7, 10 % polyacrylamide gel). More than 700 spots were detected in the 2-DE gel with CBB, and 93 spots (24 proteins) were successfully identified by MALDI-TOF/TOF and the cross-species database searching. The identified proteins mainly consisted of cytoplasmic carbohydrate metabolic enzymes, chloroplast proteins involved in photosynthesis, and haloperoxidases. Interestingly, vanadium-dependent bromoperoxidases (vBPO), which is thought to be involved in halogen uptake, synthesis of halogenated products, and detoxification of reactive oxygen species, were separated into at least 23 different spots. By comparing mass spectra, amino acid sequences predicted from tandem mass spectra and haloperoxidase activities of the vBPOs, we found that (1) at least two types of vBPOs were expressed in the lamina of E. kurome and (2) two pro-vBPOs might be activated by specific cleavage at N- and C-terminal regions.     

Expression profiles of MUC1, MUC2, and MUC4 mucins in human neoplasms and their relationship with biological behavior

Mucins are high molecular weight glycoproteins that play important roles in carcinogenesis or tumor invasion. To clarify the relationship of the expression patterns of mucins in human neoplasms with their biological behavior, we examined the expression profiles of MUC1, MUC2, and MUC4 mucins in various human neoplasms using immunohistochemistry and in situ hybridization, and compared them with clinicopathologic factors including outcome of the patients. MUC1 or MUC4 expression is related with the aggressive behavior of human neoplasms and a poor outcome of the patients. In contrast, MUC2 expression tends to be related with the indolent behavior of human neoplasms and a favorable outcome of the patients, although indolent pancreatobiliary neoplasms sometimes show invasive growth with MUC1 expression in the invasive areas. The expression of MUC2 mucin in indolent pancreatobiliary neoplasms coincided with expression of MUC2 mRNA. Our recent studies to clarify the MUC2 gene regulation mechanism disclosed that DNA methylation and histone modification in the 5' flanking region of the MUC2 promoter may play an important role. Further studies of the epigenetics also in MUC1 and MUC4 gene expression may be needed to understand the relationship between the expression of mucins in human neoplasms with their biological behavior.     

Temperature stress-induced changes in the proteomic profiles of Ecklonia cava (Laminariales, Phaeophyceae)

Proteomic profiling on Ecklonia cava Kjellman grown under various seawater temperatures was conducted to search for biomarkers that were useful to evaluate the health of the colonies and formulate actions for the maintenance of marine forests. In the cultivated strains, protein expression was not significantly changed when the cultivation temperature was lowered from 15°C (control) to 10°C. On the contrary, it was markedly changed, i.e., photosynthesis-related proteins were up-regulated and metabolic enzymes were down-regulated, when the temperature was heightened to 20°C. With the cultivation at 30°C, 25 spots within 27 spots expressed at this temperature peculiarly could be identified and classified into ten proteins. Of the distinctive 27 spots at 30°C, 20 spots were detected in the wild strains cultured at the same temperature for a brief time. It is presumed that the proteins including vanadium-dependent bromoperoxidase are heat stress-induced proteins.     

Expression of MUC17 Is Regulated by HIF1α-Mediated Hypoxic Responses and Requires a Methylation-Free Hypoxia Responsible Element in Pancreatic Cancer

MUC17 is a type 1 membrane-bound glycoprotein that is mainly expressed in the digestive tract. Recent studies have demonstrated that the aberrant overexpression of MUC17 is correlated with the malignant potential of pancreatic ductal adenocarcinomas (PDACs); however, the exact regulatory mechanism of MUC17 expression has yet to be identified. Here, we provide the first report of the MUC17 regulatory mechanism under hypoxia, an essential feature of the tumor microenvironment and a driving force of cancer progression. Our data revealed that MUC17 was significantly induced by hypoxic stimulation through a hypoxia-inducible factor 1α (HIF1α)-dependent pathway in some pancreatic cancer cells (e.g., AsPC1), whereas other pancreatic cancer cells (e.g., BxPC3) exhibited little response to hypoxia. Interestingly, these low-responsive cells have highly methylated CpG motifs within the hypoxia responsive element (HRE, 5'-RCGTG-3'), a binding site for HIF1α. Thus, we investigated the demethylation effects of CpG at HRE on the hypoxic induction of MUC17. Treatment of low-responsive cells with 5-aza-2'-deoxycytidine followed by additional hypoxic incubation resulted in the restoration of hypoxic MUC17 induction. Furthermore, DNA methylation of HRE in pancreatic tissues from patients with PDACs showed higher hypomethylation status as compared to those from non-cancerous tissues, and hypomethylation was also correlated with MUC17 mRNA expression. Taken together, these findings suggested that the HIF1α-mediated hypoxic signal pathway contributes to MUC17 expression, and DNA methylation of HRE could be a determinant of the hypoxic inducibility of MUC17 in pancreatic cancer cells.     

Effective DNA extraction method for fragment analysis using capillary sequencer of the kelp, Saccharina

Deoxyribonucleic acid (DNA) fragment analysis can become an effective tool to study genetic differences between species and individuals on saccharinan kelp from which the little genetic diversity has been reported. Here, extraction methods of DNA suitable for use in analysis with a capillary sequencer is examined on Saccharina japonica var. diabolica which contains abundant polysaccharide. When amplified fragment length polymorphism was performed using genomic DNA extracted by seven different methods: (1) commercial kit, (2) original cetyl trimethylammonium bromide (CTAB) method, (3)–(5) three types of modified CTAB method, (6) modified sodium dodecyl sulfate (SDS) method, (7) combination of CTAB method and SDS method, a high reproducible peak that was suitable for analysis was noticeable in the electropherogram in the experiment with the last combination method (7). It is considered that the pretreatment washing of polysaccharide and the subsequent purification for protein and ribonucleic acid in SDS method and for polysaccharide in CTAB method are effective to obtain the high-purity DNA.