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Statins are cholesterol-lowering agents that act as inhibitors of 3-hydroxy-3-methyl-glutaryl-coenzymeA (HMG CoA) reductase. Recently, statins have received a lot of attention, especially regarding how statins act on the immune system. Here, the clinical impact of statin intake was examined in patients with resected pancreatic cancer, and the underlying mechanisms were investigated in vitro and in vivo. We found that statin intake was associated with favorable prognostic outcomes in patients with resectable pancreatic cancer. Statins, especially lipophilic statins, exert anti-proliferative effects on pancreatic cancer cells in vitro (simvastatin > fluvastatin > atorvastatin > rosuvastatin > pravastatin). Simvastatin had an anti-proliferative effect on pancreatic cancer cells with decreased the yes-associated protein (YAP)/PDZ-binding motif (TAZ) expression by activating the JNK pathway, and simvastatin treatment with oxaliplatin revealed additive anti-growth effects. Furthermore, lipophilic and hydrophilic statins suppressed programmed cell death ligand 1 (PD-L1) expression by downregulating TAZ. Simvastatin treatment with an anti-PD-1 drug (BP0273) provided immediate anti-growth effects compared to controls, such as anti-PD-1 only and simvastatin only, and suppressed progressive disease during the early period of anti-PD-1 treatment in vivo. In conclusion, Statins display two distinct anti-cancer effects (direct anti-growth effect and elimination of immune suppression by downregulating PD-L1 expression) by targeting YAP/TAZ expression.

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Excess stroma and cancer-associated fibroblasts (CAFs) enhance cancer progression and faciliate immune evasion. Insights into the mechanisms by which the stroma manipulates the immune microenvironment could help improve cancer treatment. Here, we aimed to elucidate potential approaches for stromal reprogramming and improved cancer immunotherapy. Platelet-derived growth factor C (PDGFC) and D expression were significantly associated with a poor prognosis in patients with gastric cancer (GC), and PDGF receptor beta (PDGFRβ) was predominantly expressed in diffuse-type GC stroma. CAFs stimulated with PDGFs exhibited markedly increased expression of CXCL1, CXCL3, CXCL5 and CXCL8, which are involved in polymorphonuclear myeloid-derived suppressor cell (PMN-MDSC) recruitment. Fibrotic GC xenograft tumors exhibited increased PMN-MDSC accumulation and decreased lymphocyte infiltration, as well as resistance to anti-PD-1. Single-cell RNA sequencing and spatial transcriptomics revealed that PDGFRα/β blockade reversed the immunosuppressive microenvironment through stromal modification. Finally, combining PDGFRα/β blockade and anti-PD-1 treatment synergistically suppressed the growth of fibrotic tumors. These findings highlight the impact of stromal reprogramming on immune reactivation and the potential for combined immunotherapy for patients with fibrotic cancer.

DOI： 10.1158/0008-5472.CAN-22-1890

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Fibroblast activation protein (FAP) generally shows low or undetectable expression in most normal tissues but is highly expressed in fibroblasts in almost all carcinomas. FAP is one of the potential molecules to detect activated fibroblasts and has multiple roles in tumour progression. We generated transgenic mice that specifically expressed tdTomato along with FAP promoter activity. Coculturing a mouse gastric cancer cell line and FAP-tdTomato transgenic mouse-derived fibroblasts showed that tdTomato expression was elevated in the cocultured fibroblasts. Moreover, stomach wall transplanted tumours in mice also showed FAP-tdTomato expression in fibroblasts of the stomach and each metastatic legion. These results indicated that FAP-tdTomato expression in fibroblasts was elevated by stimulation through the interaction with cancer cells. Functionally, collagen production was increased in FAP/tdTomato-positive fibroblasts cocultured with mouse cancer cells. These FAP-tdTomato transgenic mice have the potential to be used to investigate real-time FAP dynamics and the importance of FAP expression in tumour development.

DOI： 10.1111/febs.16712

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BACKGROUND: Remodeling the tumor microenvironment (TME) to benefit cancer cells is crucial for tumor progression. Although diffuse-type gastric cancer (DGC) preferentially interacts with the TME, the precise mechanism of the complicated network remains unknown. This study aimed to investigate the mutual activation mechanism underlying DGC progression. METHODS: Mass cytometry analysis of co-cultured macrophages, noncancerous fibroblasts (NFs), and DGC cells was performed. RNA sequencing was applied to examine gene expression in fibroblasts. DGC cells were treated with cytokines to examine their effect on characteristic changes. The TCGA and Kumamoto University cohorts were used to evaluate the clinical relevance of the in vitro findings. RESULTS: Cohort analysis revealed that DGC patients had a poor prognosis. The fibroblasts and macrophages interacted with DGC cells to form a cell cluster in the invasive front of DGC tissue. The original 3D triple co-culture system determined the promotional effects of nonmalignant cells on DGC invasive growth. We notably identified a mixed-polarized macrophage cell type with M1/M2 cell surface markers in a triple co-culture system. IL-1β from mixed-polarized macrophages induced the conversion of NFs to cancer-associated fibroblast-like (CAF-like) cells, promoting the malignant phenotype of DGC cells by inducing the secretion of IL-6, IL-24, and leukemia inhibitory factor (LIF). Moreover, IL-6 and colony stimulating factor 2 (GM-CSF) cooperated to maintain the stable state of mixed-polarized macrophages. Finally, we found that mixed-polarized macrophages were frequently detected in DGC tissues. CONCLUSION: These findings demonstrated that mixed-polarized macrophages exist as a novel subtype through the reciprocal interaction between DGC cells and nonmalignant cells.

DOI： 10.1007/s10120-022-01352-3

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Accumulating evidence suggests that high levels of Fusobacterium nucleatum in colorectal tumor tissues can be associated with poor prognosis in patients with colorectal cancer (CRC); however, data regarding distinct prognostic subgroups in F. nucleatum-positive CRC remain limited. Herein, we demonstrate that high-iron status was associated with a worse prognosis in patients with CRC with F. nucleatum. Patients with CRC presenting elevated serum transferrin saturation exhibited preferential iron deposition in macrophages in the tumor microenvironment. In addition, F. nucleatum induced CCL8 expression in macrophages via the TLR4/NF-κB signaling pathway, which was inhibited by iron deficiency. Mechanistically, iron attenuated the inhibitory phosphorylation of NF-κB p65 by activating serine/threonine phosphatases, augmenting tumor-promoting chemokine production in macrophages. Our observations indicate a key role for iron in modulating the NF-κB signaling pathway and suggest its prognostic potential as a determining factor for interpatient heterogeneity in F. nucleatum-positive CRC.

DOI： 10.1172/jci.insight.156802

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BACKGROUND: Some reports showed the immune tolerance of soluble human leukocyte antigen E (HLA-E), but the role that soluble HLA-E plays in gastric cancer (GC) is unknown. We aimed to clarify the molecular mechanism and clinical significance of soluble HLA-E in GC. METHODS: We examined the expression of HLA-E on GC cells and soluble HLA-E under co-culture with natural killer (NK) cells in a time-dependent manner. Changes in NK cell activity were investigated using anti-NK group 2 member A (NKG2A) antibodies in the presence of soluble HLA-E. Expression of soluble HLA-E in the serum of GC patients was determined. RESULTS: Whereas HLA-E expression on GC cells peaked with interferon (IFN)-γ secretion by NK cells in a time-dependent manner, soluble HLA-E was upregulated in conditioned medium. Pre-incubation with anti-NKG2A antibodies increased the activation of NKG2A+ NK cells in the presence of soluble HLA-E. Expression of soluble HLA-E in the serum of GC patients correlated with disease progression. CONCLUSIONS: HLA-E expression dynamically changes on GC cells and in conditioned medium. Furthermore, soluble HLA-E can contribute to immune escape in GC cell lines, which may have significance in clinical practice. Moreover, soluble HLA-E may be a potential prognostic biomarker.

DOI： 10.1245/s10434-022-12505-0

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The arachidonic acid cascade is a major inflammatory pathway that produces prostaglandin E2 (PGE2). Although inhibition of 15-hydroxyprostaglandin dehydrogenase (15-PGDH) is reported to lead to PGE2 accumulation, the role of 15-PGDH expression in the tumor microenvironment remains unclear. We utilized Panc02 murine pancreatic cancer cells for orthotopic transplantation into wild-type and 15-pgdh+/- mice and found that 15-pgdh depletion in the tumor microenvironment leads to enhanced tumorigenesis accompanied by an increase in cancer-associated fibroblasts (CAFs) and the promotion of fibrosis. The fibrotic tumor microenvironment is widely considered to be hypovascular; however, we found that the angiogenesis level is maintained in 15-pgdh+/- mice, and these alterations were also observed in a genetically engineered PDAC mouse model. Further confirmation revealed that fibroblast growth factor 1 (FGF1) is secreted by pancreatic cancer cells after PGE2 stimulation, consequently promoting CAF proliferation and vascular endothelial growth factor A (VEGFA) expression in the tumor microenvironment. Finally, in 15-pgdh+/-Acta2-TK mice, depletion of fibroblasts inhibited angiogenesis and cancer cell viability in orthotopically transplanted tumors. These findings highlighted the role of 15-pgdh downregulation in enhancing PGE2 accumulation in the pancreatic tumor microenvironment and in subsequently maintaining the angiogenesis level in fibrotic tumors along with CAF expansion.

DOI： 10.1111/cas.15495

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Immune checkpoint inhibitors have shown efficacy in various cancers. Although programmed death ligand 1/2 (PD-L1/L2) expressions have been demonstrated as predictive biomarkers of response to immune checkpoint inhibitors and prognostic markers, whether PD-L1/L2 expression is altered in esophageal squamous cell carcinoma during the therapeutic course is unclear. Whether PD-L1/L2 expression in metastatic or recurrent lesions is consistent with that in primary tumors is also unknown. This study included 561 surgically resected esophageal squamous cell carcinomas and PD-L1/L2 expression was evaluated by immunohistochemistry. We investigated the influence of chemotherapeutic drugs (cisplatin and fluorouracil) on PD-L1/L2 expression and PD-L1/L2-related pathways in vitro. We also examined PD-L1/L2 expression in 18 surgically resected lymph node metastases and 10 recurrent lesions compared with primary lesions. The positive rate of PD-L1 was significantly higher in patients with preoperative chemotherapy than in those without preoperative therapy. The positive rate of PD-L2 expression showed no significant difference between patient groups. Cisplatin increased PD-L1 expression in cancer cell lines in vitro, but decreased PD-L2 in some cell lines. The effects of cisplatin on phosphorylated signal transducer and activator of transcription 1/3 (pSTAT1/3) also differed depending on cell lines. Fluorouracil increased PD-L1 and PD-L2 expression. PD-L1/L2 expression in lymph node metastases and recurrent lesions did not always match expression in primary lesions. PD-L1/L2 expression may be altered by preoperative chemotherapy, and PD-L1 /L2 expression in primary lesions does not always match that of metastatic/recurrent lesions. Thus, one-time evaluation is not sufficient to evaluate PD-L1/L2 expression as a biomarker in esophageal cancer.

DOI： 10.1111/cas.15198

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BACKGROUND: Signet ring cell carcinoma (SRCC) is a particular histologic variant of gastric cancer (GC). However, the critical factor related to the aggressive characteristics of SRCC has not been determined. METHODS: We collected surgically resected tissues from 360 GC patients in the Kumamoto University cohort and generated survival curves via the Kaplan-Meier method. In vitro, we identified the specific transcript variant of MUC20 in SRCC cells by direct sequencing and investigated the role of MUC20 in GC progression using GC cells with MUC20 silencing and forced expression. In vivo, we examined chemoresistance using MUC20 variant 2 (MUC20v2)-overexpressing non-SRCC cells to construct a xenograft mouse model. RESULTS: We analyzed a comprehensive GC cell line database to identify the specifically expressed genes in gastric SRCC. We focused on MUC20 and investigated its role in GC progression. Survival analysis revealed that GC patients with high MUC20 expression exhibited a poor prognosis and that MUC20 expression was significantly correlated with SRCC histological type. Moreover, we found that gastric SRCC cells specifically expressed MUC20v2, which was dominantly expressed in the cytoplasm. Silencing MUC20v2 caused cell death with characteristic morphological changes in gastric SRCC cells. To further determine the types of cell death, we examined apoptosis, pyroptosis and ferroptosis by detecting cleaved PARP, gasdermin E-N-terminal (GSDME-N), and lipid reactive oxygen species (ROS) levels, respectively. We found that apoptosis and pyroptosis occurred in MUC20-silenced gastric SRCC cells. In addition, MUC20v2-overexpressing GC cells exhibited chemoresistance to cisplatin (CDDP) and paclitaxel (PTX). RNA sequencing revealed that the pathways involved in intracellular calcium regulation were significantly upregulated in MUC20v2-overexpressing GC cells. Notably, forced expression of MUC20v2 in the cytoplasm of GC cells led to the maintenance of mitochondrial calcium homeostasis and mitochondrial membrane potential (MMP), which promoted cell survival and chemoresistance by suppressing apoptosis and pyroptosis. Finally, we investigated the significance of MUC20v2 in a xenograft model treated with CDDP and showed that MUC20v2 overexpression caused chemoresistance by inhibiting cell death. CONCLUSION: These findings highlight the novel functions of MUC20v2, which may confer cell survival and drug resistance in GC cells. SIGNIFICANCE: MUC20v2 protects GC cells from apoptosis and pyroptosis by maintaining mitochondrial calcium levels and mitochondrial membrane potential and subsequently induces drug resistance.

DOI： 10.1007/s10120-022-01283-z

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Cancer cells craftily adapt their energy metabolism to their microenvironment. Nutrient deprivation due to hypovascularity and fibrosis is a major characteristic of pancreatic ductal adenocarcinoma (PDAC); thus, PDAC cells must produce energy intrinsically. However, the enhancement of energy production via activating Kras mutations is insufficient to explain the metabolic rewiring of PDAC cells. Here, we investigated the molecular mechanism underlying the metabolic shift in PDAC cells under serine starvation. Amino acid analysis revealed that the concentrations of all essential amino acids and most nonessential amino acids were decreased in the blood of PDAC patients. In addition, the plasma serine concentration was significantly higher in PDAC patients with PHGDH-high tumors than in those with PHGDH-low tumors. Although the growth and tumorigenesis of PK-59 cells with PHGDH promoter hypermethylation were significantly decreased by serine starvation, these activities were maintained in PDAC cell lines with PHGDH promoter hypomethylation by serine biosynthesis through PHGDH induction. In fact, DNA methylation analysis by pyrosequencing revealed that the methylation status of the PHGDH promoter was inversely correlated with the PHGDH expression level in human PDAC tissues. In addition to PHGDH induction by serine starvation, PDAC cells showed enhanced serine biosynthesis under serine starvation through 3-PG accumulation via PGAM1 knockdown, resulting in enhanced PDAC cell growth and tumor growth. However, PHGDH knockdown efficiently suppressed PDAC cell growth and tumor growth under serine starvation. These findings provide evidence that targeting the serine biosynthesis pathway by inhibiting PHGDH is a potent therapeutic approach to eliminate PDAC cells in nutrient-deprived microenvironments.

DOI： 10.1016/j.canlet.2021.09.007

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BACKGROUND: The predictive significance of programmed death ligand 1 (PD-L1) for programmed death 1 (PD-1) inhibitors remains unclear in gastric cancer (GC) due to the dynamic alteration by treatments. We aimed to elucidate the effects of trastuzumab (Tmab) on PD-L1 expression in GC. METHODS: PD-L1 expression was evaluated by multicolour flow cytometry analysis after co-culturing GG cell lines and immune cells with Tmab. IFN-γ in the co-culture experiments was quantified. Immunohistochemistry (IHC) for PD-L1 expression using clinical samples was also performed to confirm PD-L1 alteration by Tmab. RESULTS: PD-L1 expression was significantly upregulated by Tmab in HER2-amplified GC cell lines co-cultured with peripheral blood mononuclear cells (PBMCs). PD-L1 upregulation by Tmab was also observed in the GC cells co-cultured with NK cells in time-dependent manner, but not with monocytes. IFN-γ concentration in conditioned media from co-cultured PBMCs and NK cells with Tmab was significantly higher and anti-IFN-γ significantly suppress the Tmab-induced PD-L1 upregulation. IHC also suggested PD-L1 upregulation after Tmab treatment. CONCLUSIONS: Tmab can upregulate PD-L1 expression on GC cells through interaction with NK cells. These results suggest clinical implications in the assessment of the predictive significance of PD-L1 expression for PD-1 inhibitors.

DOI： 10.1038/s41416-020-01138-3

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In the tumor microenvironment, senescent non-malignant cells, including cancer-associated fibroblasts (CAFs), exhibit a secretory profile under stress conditions; this senescence-associated secretory phenotype (SASP) leads to cancer progression and chemoresistance. However, the role of senescent CAFs in metastatic lesions and the molecular mechanism of inflammation-related SASP induction are not well understood. We show that pro-inflammatory cytokine-driven EZH2 downregulation maintains the SASP by demethylating H3K27me3 marks in CAFs and enhances peritoneal tumor formation of gastric cancer (GC) through JAK/STAT3 signaling in a mouse model. A JAK/STAT3 inhibitor blocks the increase in GC cell viability induced by senescent CAFs and peritoneal tumor formation. Single-cell mass cytometry revealed that fibroblasts exist in the ascites of GC patients with peritoneal dissemination, and the fibroblast population shows p16 expression and SASP factors at high levels. These findings provide insights into the inflammation-related SASP maintenance by histone modification and the role of senescent CAFs in GC peritoneal dissemination.

DOI： 10.1016/j.celrep.2021.108779

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BACKGROUND: Pancreatic cancer has an extremely poor prognosis, even after curative resection. Treatment options for pancreatic cancer remain limited, therefore new therapeutic targets are urgently needed. We searched for genes predictive of poor prognosis in pancreatic cancer using a public database and validated the survival impact of the selected gene in a patient cohort. METHODS: We used a public database to search for genes associated with early pancreatic cancer recurrence. As a validation cohort, 201 patients who underwent radical resection in our institution were enrolled. Expression of the target gene was evaluated using immunohistochemistry (IHC). We evaluated growth and invasiveness using small interfering RNAs, then performed pathway analysis using gene set enrichment analysis. RESULTS: We extracted ARHGEF2 from GSE21501 as a gene with a high hazard ratio (HR) for early recurrence within 1 year. The high ARHGEF2 expression group had significantly poorer recurrence-free survival (RFS) and poorer overall survival (OS) than the low ARHGEF2 expression group. Multivariate analysis demonstrated that high ARHGEF2 expression was an independent poor prognostic factor for RFS (HR 1.92) and OS (HR 1.63). In vitro, ARHGEF2 suppression resulted in reduced cell growth and invasiveness. Bioinformatic analysis revealed that ARHGEF2 expression was associated with MYC, G2M, E2F, and CDC25A expression, suggesting that c-Myc and cell cycle genes are associated with high ARHGEF2 expression. IHC revealed a positive correlation between ARHGEF2 and c-Myc expression. CONCLUSIONS: High ARHGEF2 expression is associated with cell cycle progression, and predicts early recurrence and poor survival in patients with pancreatic cancer.

DOI： 10.1245/s10434-020-09383-9

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For the production of tumor-specific vaccines, including dendritic cell (DC) vaccines, the tumor cells themselves are an ideal source. Floating tumor cells in the ascites fluid from patients with malignant ascites are a good candidate source, but it is not easy to obtain pure tumor cells from ascites because of various types of cell contamination as well as protein aggregates. We here report an effective method to recover pure tumor cells from malignant ascites. We used lavage fluid from 13 patients with malignant ascites who were treated with modified cell-free and concentrated ascites reinfusion therapy (KM-CART). Cellular components were separated from the lavage fluid by centrifugation, enzymatic digestion and hemolysis. Tumor cells were purified by depleting CD45(+) leukocytes with antibody-conjugated magnetic beads. The tumor cell lysate was extracted by freeze-and-thaw cycles. The mean obtained total cell number was 7.50 x 10(7) cells (range 4.40 x 10(6)-2.48 x 10(8) cells). From this fraction, 6.39 x 10(6) (range 3.23 x 10(5)-2.53 x 10(7)) CD45(-) cells were collected, and the tumor cell purity was over 80 % defined as CD45(-)CD326(+). A sufficient amount of tumor lysate, average = 2416 mu g (range 25-8743 mu g), was extracted from CD45(-)CD326(+) tumor cells. We here established an effective method to produce highly purified tumor cells from KM-CART lavage fluid. The clinical feasibility of this simple preparation method for generating tumor lysate should be examined in clinical studies of DC vaccines.

DOI： 10.1186/s40064-015-1508-3

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Anthocyanins and flavones are biosynthesized from flavanones as a common intermediate, which is the common metabolic branch point for anthocyanin and flavone biosynthesis. The conversion of dihydroflavonols from flavanones for anthocyanin biosynthesis and the conversion of flavones from flavanone for flavone biosynthesis are catalyzed by flavanone 3-hydroxylase (F3H) and flavone synthase (FNSI, II), respectively. Therefore, to elucidate the molecular genetic mechanism of copigmentation between anthocyanins and flavones, the F3H and FNS genes and their transcriptional factors should be characterized, and in this study, 3 cDNA clones encoding F3H named IhF3H1, IhF3H2, and IhF3H3 were isolated and characterized from the flower buds of Dutch iris, Iris x hollandica. Nucleotide sequence analysis showed that IhF3H1 (Genbank accession no. AB183826) is 1,313 bp long and contains an open reading frame (ORF) encoding 370 amino acids with a calculated molecular mass of 40,995 Da and an isoelectric point (pI) of 5.47. IhF3H2 (Genbank accession no. AB265225) is 1,295 bp long and contains an ORF encoding 372 amino acids with a calculated molecular mass of 41,139 Da and a pI of 6.42. IhF3H3 (Genbank accession no. AB265226) is 1,335 bp long and contains an ORF encoding 372 amino acids with a calculated molecular mass of 41,117 Da and a pI of 5.69. The soluble crude protein extracts of Escherichia colt cells expressing IhF3H1, IhF3H2, and IhF3H3 were subjected to flavanone 3-hydroxylation assays in the presence of naringenin as a substrate and 2-oxoglutarate, ascorbate, and FeSO4 as cofactors. Heterologous expression demonstrated that each IhF3H cDNA encodes functional flavanone 3-hydroxylase, which catalyzes 3-hydroxylation from naringenin to dihydroflavonol.

DOI： 10.1508/cytologia.77.359

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Iris setosa var. hondoensis and L setosa var. nasuensis are endemic to Japan, and their origins still remain to be clarified. In this study, we report the characterization of interspecific hybrids (allotriploids) from the cross between autotetraploid I. setosa var. setosa (4x) and diploid I. laevigata (2n=32) and their relationship to I. setosa var. hondoensis or L setosa var. nasuensis. In reciprocal crosses between I. setosa var. setosa (2x, 4x) and I. laevigata, progeny plants were obtained only in the cross between I. setosa var. setosa (4x) and I. laevigata (2x). These progenies were identified as allotriploids (2n=54) and their aneuploids (2n=53) of I. setosa var. setosa (4x)xI laevigata (2x) by flow cytometric (FCM), cytological and random amplified polymorphic DNA (RAPD) analyses, and the five hybrids (SLI-5) except SL6 were characterized. Furthermore, these characteristics of I. setosa var. hondoensis and L setosa var. nasuensis were compared with those of SLI-5. These varieties were also examined by FCM, cytological, PAPD and cleaved amplified polymorphic sequence (CAPS) analyses, which comparative analyses showed that varieties have the hybrid nature between T setosa var. setosa and I. laevigata, and maternal plants are organelle DNAs of the former species.

DOI： 10.1508/cytologia.73.401

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In plants, O-methyltransferases (OMTs) play an important role in methylation of secondary metabolites, especially flavonoids and other phenylpropanoids, and two cDNA clones, IhOMT1 and IhOMT2 (Iris hollandica OMT), encoding OMTs were successfully isolated from a cDNA library of flower buds of I. hollandica. IhOMT1 encodes an open reading frame (ORF) of 365 amino acids with calculated molecular mass of 40,193Da and isoelectric point (pI) of 5.54, while IhOMT2, which shares 31.5% amino acid sequence identity with IhOMT1, encodes 369 amino acids with calculated molecular mass of 40,385Da and pI of 5.50. In addition, the molecular masses of both recombinant IhOMT1 and IhOMT2 proteins were estimated to be about 40kDa by protein gel blot analysis. Characterization of the enzymatic properties using the recombinant IhOMT1 protein confirmed that IhOMT1 cDNA encodes a S-adenosyl-L-methionine (SAM)-dependent caffeic acid 3-OMT, which catalyzes the transfer of the methyl moiety from SAM to caffeic acid to form ferulic acid. Its optimum activity was observed at pH 7.5-8.0 and at 35 degrees C. This is the first report of the isolation and characterization of a COMT cDNA clone involved in the phenylpropanoid biosynthesis of Iridaceae plants. In contrast, IhOMT2 showed no activity in SAM-dependent assays for various phenylpropanoids.

DOI： 10.1016/j.jplph.2006.12.002

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Among anthocyanin modifications, acylation is very important for increasing water solubility and stabilizing anthocyanins, and two cDNA clones, Ih3AT1 and Ih3AT2, encoding anthocyanin acyltransferase (AAT) were successfully isolated from a cDNA library constructed from flower buds of Iris hollandica. Ih3AT1 encodes an open reading frame (ORF) of 429 amino acids with a calculated molecular mass of 47,706 Da and pI of 6.61, while Ih3AT2 encodes an ORF of 429 amino acids with a molecular mass of 47,670 Da and pI of 6.61. The identity of their deduced amino acid sequences was 95.8%. Reverse transcription-PCR (RT-PCR) analyses of Ih3AT1 and Ih3AT2 transcripts showed that these genes were not expressed in leaves, but were expressed in the flower buds and fully opened flowers. The molecular masses of both reccmbinant Ih3AT1 and Ih3AT2 proteins were estimated to be about 50 kDa by Western blotting. Characterization of the enzymatic properties using the recombinant Ih3AT1 and Ih3AT2 proteins confirmed that Ih3AT1 and Ih3AT2 cDNAs encode anthocyanin 3-p-coumaroyltransferase, which catalyzes the transfer of the p-coumaroyl moiety from p-coumaroyl-CoA to anthocyanidin 3-rhamnosylglucoside-5-glucoside (antbocyanidin 3RG5G) to form anthocyanidin 3-(p-coumaroyl)rhammnosylglucoside-5-glucoside (anthocyanidin 3pCRG5G). This is the first report of AAT gene cloning from a monocot plant. (c) 2006 Elsevier Ireland Ltd. All rights reserved.

DOI： 10.1016/j.plantsci.2006.06.005

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In anthocyanin biosynthesis, UDP-glucose: anthocyanidin 3-O-glucosyltransferase (3GT) catalyzes the transfer of the glucosyl moiety from UDP-glucose to the 3-hydroxyl group of anthocyanidins, producing the first stable anthocyanins. A cDNA clone (Ih3G7) encoding 3GT was isolated from a cDNA library of flower buds from Iris hollandica by screening with a 3GT cDNA clone of Antirrhinum majus. The cDNA encodes an open reading frame of 460 amino acids with a calculated molecular mass of 49,558 Da and an isoelectric point of 5.54. The molecular mass of recombinant Ih3GT protein from Escherichia coli was also estimated to be ca. 50 kDa by Western blotting. A molecular phylogenetic analysis of the deduced amino acid sequences of glycosyltransferases from various plants showed that Ih3GT is a member of 3GT group, which is further classified into monocot and dicot subgroups. Characterization of the enzymatic properties using the recombinant Ih3GT protein showed that Ih3GT cDNA encodes anthocyanidin 3-O-glucosyltransferase. This is the first report of 3GT gene cloning from an ornamental monocot plant. (c) 2005 Elsevier Ireland Ltd. All rights reserved.

DOI： 10.1016/j.plantsci.2005.04.007

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UDP-glucose: anthocyanin 5-O-glucosyltransferase (5GT) catalyzes the transfer of the glucosyl moiety from UDP-glucose to the 5-hydroxyl group of anthocyanin. A putative full-length cDNA encoding 5GT was isolated from a cDNA library from flower buds of Iris hollandica by screening with a cDNA clone of UDP-glucose: anthocyanidin 3-O-glucosyltranseferase from Antirrhinum majus as a probe. The cDNA encodes an open reading frame of 463 amino acids with a calculated molecular mass of 50, 100 Da. Heterologous expression of the cDNA in Escherichia coli demonstrated that it encoded 5GT. This is the first report of 5GT gene cloning from monocotyledonous plants. A molecular phylogenetic analysis of the amino acid sequences of glycosyltransferases from various plants showed that Ih5GT is a member of the 5GT group, although distant from dicot subgroup. (C) 2004 Elsevier Ireland Ltd. All rights reserved.

DOI： 10.1016/j.plantsci.2004.06.020

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Language：English