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Renal cell carcinoma (RCC) associated with Xp11.2 translocation (TFE3-RCC) has been recently defined as a distinct subset of RCC classified by characteristic morphology and clinical presentation. The Xp11 translocations involve the TFE3 transcription factor and produce chimeric TFE3 proteins retaining the basic helix–loop–helix leucine zipper structure for dimerization and DNA binding suggesting that chimeric TFE3 proteins function as oncogenic transcription factors. Diagnostic biomarkers and effective forms of therapy for advanced cases of TFE3-RCC are as yet unavailable. To facilitate the development of molecular based diagnostic tools and targeted therapies for this aggressive kidney cancer, we generated a translocation RCC mouse model, in which the PRCC-TFE3 transgene is expressed specifically in kidneys leading to the development of RCC with characteristic histology. Expression of the receptor tyrosine kinase Ret was elevated in the kidneys of the TFE3-RCC mice, and treatment with RET inhibitor, vandetanib, significantly suppressed RCC growth. Moreover, we found that Gpnmb (Glycoprotein nonmetastatic B) expression was notably elevated in the TFE3-RCC mouse kidneys as seen in human TFE3-RCC tumors, and confirmed that GPNMB is the direct transcriptional target of TFE3 fusions. While GPNMB IHC staining was positive in 9/9 cases of TFE3-RCC, Cathepsin K, a conventional marker for TFE3-RCC, was positive in only 67% of cases. These data support RET as a potential target and GPNMB as a diagnostic marker for TFE3-RCC. The TFE3-RCC mouse provides a preclinical in vivo model for the development of new biomarkers and targeted therapeutics for patients affected with this aggressive form of RCC.Implications:Key findings from studies with this preclinical mouse model of TFE3-RCC underscore the potential for RET as a therapeutic target for treatment of patients with TFE3-RCC, and suggest that GPNMB may serve as diagnostic biomarker for TFE3 fusion RCC.

Supplemental Figure S1. Representative images of TFE3 immunohistochemistry of renal papillae and renal pelvis (a, b), ureters (c, d), and bladders (e, f) from a 10-month-old PRCC-TFE3; KSP-Cre- (a, c, e) mouse and a PRCC-TFE3; KSP-Cre+ (b, d, f) mouse. S2, S3, S4, S5. Supplemental Table S1. (a) List of top 50 significantly upregulated (q-value < 0.05, fold change > 2) genes in kidneys from 4 month old PRCC-TFE3; KSP-Cre+ mice. (b) List of significantly downregulated (q-value < 0.05, fold change < 1/2) genes in kidneys from 4 month old PRCC-TFE3; KSP-Cre+ mice. Supplemental Table S2 (a) List of top 50 significantly upregulated (q-value < 0.05, fold change > 2) genes in kidneys from 7 month old PRCC-TFE3; KSP-Cre+ mice. (b) List of top 50 significantly downregulated (q-value < 0.05, fold change < 1/2) genes in kidneys from 7 month old PRCC-TFE3; KSP-Cre+ mice. Supplemental Fig. S2 Gene Set Enrichment Analysis (GSEA) of microarray data from 4 month old PRCC-TFE3; KSP-Cre+ mouse kidneys (n=4) vs 4 month old PRCC-TFE3; KSP-Cre- mouse kidneys (n=4) (a, b, c, g, h, i) and 7 month old PRCC-TFE3;KSP-Cre+ mouse kidneys (n=4) vs 7 month old PRCC-TFE3; KSP-Cre- mouse kidneys (n=3) (d, e, f, j, k, l). Supplemental Table S3 List of commonly upregulated genes in PRCC-TFE3; KSP-Cre+ mouse kidneys (4 month old and 7 month old) and TFEB overexpressed mouse kidneys (P0 and P14, Calcagni et al. Supplemental Fig. S3 Gene Set Enrichment Analysis (GSEA) of microarray data from 4 month old PRCC-TFE3; KSP-Cre+ mouse kidneys (n=4) vs 4 month old PRCC-TFE3; KSP-Cre- mouse kidneys (n=4) (a) and 7 month old PRCC-TFE3;KSP-Cre+ mouse kidneys (n=4) vs 7 month old PRCC-TFE3; KSP-Cre- mouse kidneys (n=3) (b). Marginal (a) and significant (b) enrichment of genes associated with KRAS activation in kidneys from PRCC-TFE3; KSP-Cre+ mice. Supplemental Fig. S4. The immunohistochemical staining of Tfe3 (a), Gpnmb (b), and Ret (c) on PRCC-TFE3;KSP-Cre- kidneys and PRCC-TFE3;KSP-Cre+ kidneys was semiquantified and scored as (-), (1+) and (2+). Supplemental Fig. S5. Volcano plot of gene expression changes for 4 month-old (a) and 7 month-old (b) PRCC-TFE3; KSP-Cre- kidneys and PRCC-TFE3; KSP-Cre+ kidneys.

Pancreatic cancer has an extremely poor prognosis because of its resistance to conventional therapies. Cancer stem cell (CSC)‐targeted therapy is considered a promising approach for this disease. Epithelial‐mesenchymal transition‐inducing transcription factors (EMT‐TFs) contribute to CSC properties in some solid tumors; however, this mechanism has not been fully elucidated in pancreatic cancer. Zinc finger protein, SNAIL2 (also known as SLUG), is a member of the SNAIL superfamily of EMT‐TFs and is commonly overexpressed in pancreatic cancer. Patients exhibiting high SNAIL2 expression have a poor prognosis. In this study, we showed that the suppression of SNAIL2 expression using RNA interference decreased tumorigenicity in vitro (sphere formation assay) and in vivo (xenograft assay) in 2 pancreatic cancer cell lines, KLM1 and KMP5. In addition, SNAIL2 suppression resulted in increased sensitivity to gemcitabine and reduced the expression of CD44, a pancreatic CSC marker. Moreover, experiments on tumor spheroids established from surgically resected pancreatic cancer tissues yielded similar results. A microarray analysis revealed that the mechanism was mediated by insulin‐like growth factor (IGF) binding protein 2. These results indicate that IGFBP2 regulated by SNAIL2 may represent an effective therapeutic target for pancreatic cancer., SNAIL2 knockdown reduces tumorigenicity and resistance to gemcitabine in tumor spheroid established from resected tissue of human pancreatic cancer as well as human pancreatic cancer cell lines.

Renal cell carcinoma (RCC) associated with Xp11.2 translocation (TFE3-RCC) has been recently defined as a distinct subset of RCC classified by characteristic morphology and clinical presentation. The Xp11 translocations involve the TFE3 transcription factor and produce chimeric TFE3 proteins retaining the basic helix–loop–helix leucine zipper structure for dimerization and DNA binding suggesting that chimeric TFE3 proteins function as oncogenic transcription factors. Diagnostic biomarkers and effective forms of therapy for advanced cases of TFE3-RCC are as yet unavailable. To facilitate the development of molecular based diagnostic tools and targeted therapies for this aggressive kidney cancer, we generated a translocation RCC mouse model, in which the PRCC-TFE3 transgene is expressed specifically in kidneys leading to the development of RCC with characteristic histology. Expression of the receptor tyrosine kinase Ret was elevated in the kidneys of the TFE3-RCC mice, and treatment with RET inhibitor, vandetanib, significantly suppressed RCC growth. Moreover, we found that Gpnmb (Glycoprotein nonmetastatic B) expression was notably elevated in the TFE3-RCC mouse kidneys as seen in human TFE3-RCC tumors, and confirmed that GPNMB is the direct transcriptional target of TFE3 fusions. While GPNMB IHC staining was positive in 9/9 cases of TFE3-RCC, Cathepsin K, a conventional marker for TFE3-RCC, was positive in only 67% of cases. These data support RET as a potential target and GPNMB as a diagnostic marker for TFE3-RCC. The TFE3-RCC mouse provides a preclinical in vivo model for the development of new biomarkers and targeted therapeutics for patients affected with this aggressive form of RCC. Implications: Key findings from studies with this preclinical mouse model of TFE3-RCC underscore the potential for RET as a therapeutic target for treatment of patients with TFE3-RCC, and suggest that GPNMB may serve as diagnostic biomarker for TFE3 fusion RCC.

To enrich carotenoids, especially β-cryptoxanthin, in juice sac tissues of fruits via molecular breeding in citrus, allele mining was utilized to dissect allelic variation of carotenoid metabolic genes and identify an optimum allele on the target loci characterized by expression quantitative trait (eQTL) analysis. SNPs of target carotenoid metabolic genes in 13 founders of the Japanese citrus breeding population were explored using the SureSelect target enrichment method. An independent allele was determined based on the presence or absence of reliable SNPs, using trio analysis to confirm inheritability between parent and offspring. Among the 13 founders, there were 7 PSY alleles, 7 HYb alleles, 11 ZEP alleles, 5 NCED alleles, and 4 alleles for the eQTL that control the transcription levels of PDS and ZDS among the ancestral species, indicating that some founders acquired those alleles from them. The carotenoid composition data of 263 breeding pedigrees in juice sac tissues revealed that the phenotypic variance of carotenoid composition was similar to that in the 13 founders, whereas the mean of total carotenoid content increased. This increase in total carotenoid content correlated with the increase in either or both β-cryptoxanthin and violaxanthin in juice sac tissues. Bayesian statistical analysis between allelic composition of target genes and carotenoid composition in 263 breeding pedigrees indicated that PSY-a and ZEP-e alleles at PSY and ZEP loci had strong positive effects on increasing the total carotenoid content, including β-cryptoxanthin and violaxanthin, in juice sac tissues. Moreover, the pyramiding of these alleles also increased the β-cryptoxanthin content. Interestingly, the offset interaction between the alleles with increasing and decreasing effects on carotenoid content and the epistatic interaction among carotenoid metabolic genes were observed and these interactions complexed carotenoid profiles in breeding population. These results revealed that allele composition would highly influence the carotenoid composition in citrus fruits. The allelic genotype information for the examined carotenoid metabolic genes in major citrus varieties and the trio-tagged SNPs to discriminate the optimum alleles (PSY-a and ZEP-e) from the rest would promise citrus breeders carotenoid enrichment in fruit via molecular breeding.

Maoto, a traditional kampo medicine, has been clinically prescribed for influenza infection and is reported to relieve symptoms and tissue damage. In this study, we evaluated the effects of maoto as an herbal multi-compound medicine on host responses in a mouse model of influenza infection. On the fifth day of oral administration to mice intranasally infected with influenza virus [A/PR/8/34 (H1N1)], maoto significantly improved survival rate, decreased viral titer, and ameliorated the infection-induced phenotype as compared with control mice. Analysis of the lung and plasma transcriptome and lipid mediator metabolite profile showed that maoto altered the profile of lipid mediators derived from ω-6 and ω-3 fatty acids to restore a normal state, and significantly up-regulated the expression of macrophage- and T-cell-related genes. Collectively, these results suggest that maoto regulates the host’s inflammatory response by altering the lipid mediator profile and thereby ameliorating the symptoms of influenza.

Among cancer cells, there are specific cell populations of whose activities are comparable to those of stem cells in normal tissues, and for whom the levels of cell dedifferentiation are reported to correlate with poor prognosis. Information concerning the mechanisms that modulate the stemness like traits of cancer cells is limited. Therefore, we examined five gastric cancer cell lines and isolated gastric oncospheres from three gastric cancer cell lines. The gastric cancer cells that expanded in the spheres expressed relatively elevated proportion of CD44, which is a marker of gastric cancer stem cells (CSCs), and displayed many properties of CSCs, for example: chemoresistance, tumorigenicity and epithelial–mesenchymal transition (EMT) acquisition. SNAIL, which is a key factor in EMT, was highly expressed in the gastric spheres. Microarray analysis in gastric cancer cell line HGC27 showed that CCN3 and NEFL displayed the greatest differential expression by knocking down of SNAIL; the former was upregulated and the latter downregulated, respectively. Downregulation of CCN3 and upregulation of NEFL gene expression impaired the SNAIL-dependent EMT activity: high tumorigenicity, and chemoresistance in gastric cancer cells. Thus, approach that disrupts SNAIL/CCN3/NEFL axis may be credible in inhibiting gastric cancer development.

Intron retention (IR) is an important regulatory mechanism that affects gene expression and protein functions. Using klotho mice at the pre-symptomatic state, we discovered that retained-introns accumulated in several organs including the liver and that among these retained introns in the liver a subset was recovered to the normal state by a Japanese traditional herbal medicine. This is the first report of IR recovery by a medicine. IR-recovered genes fell into two categories: those involved in liver-specific metabolism and in splicing. Metabolome analysis of the liver showed that the klotho mice were under starvation stress. In addition, our differentially expressed gene analysis showed that liver metabolism was actually recovered by the herbal medicine at the transcriptional level. By analogy with the widespread accumulation of intron-retained pre-mRNAs induced by heat shock stress, we propose a model in which retained-introns in klotho mice were induced by an aging stress and in which this medicine-related IR recovery is indicative of the actual recovery of liver-specific metabolic function to the healthy state. Accumulation of retained-introns was also observed at the pre-symptomatic state of aging in wild-type mice and may be an excellent marker for this state in general.

Phytoene synthase (PSY) is one of the key regulatory enzyme on the biosynthesis and accumulation of carotenoid in citrus fruits. The transcriptional diversity of PSY is mainly attributed to the structural variation in promoter region among PSY alleles. In aim to clarify how this transcriptional diversity is regulated among them, PSY alleles responsible for carotenoid biosynthesis in the fruits are characterized and their promoter sequences were compared. Based on gene structure and expression pattern of PSY homologues on the clementine mandarin genome sequence, PSY alleles responsible for carotenoid biosynthesis are derived from a single locus in the scaffold 6. AG mapping population possessed four PSY alleles derived from parent lines of A255 and G434, and their F1 individuals with PSY-g2 allele tended to have low transcription level. From sequence comparison of their promoter regions, the cis-motif alternation from MYBPZM to RAV1AAT might be a candidate to influence the transcription level. Among the ancestral pedigree varieties of AG mapping population, the transcription level of PSY correlated with genotypes of MYBPZM and RAV1AAT motifs in the promoter region of PSY alleles, so that homozygous genotype of MYBPZM showed higher transcription level while heterozygous genotype of MYBPZM and RAV1AAT showed lower transcription level.

In tumor-only next-generation sequencing (NGS), identified variants have the potential to be secondary findings (SFs), but they require verification through additional germline testing. In the present study, 194 patients with advanced cancer who underwent tumor-only NGS between April 2015 and March 2018 were enrolled, and the incidences of possible and true SFs were evaluated. Among them, 120 patients (61.9%) harbored at least one possible SF. TP53 was the most frequent gene in which 97 variants were found in 91 patients (49.5%). Nine patients provided informed consent to undergo additional germline testing, and a total of 14 variants (BRCA1, n = 1; BRCA2, n = 2; PTEN, n = 2; RB1, n = 1; SMAD4, n = 1; STK11, n = 1; TP53, n = 6) were analyzed. Three variants (BRCA1, n = 1; BRCA2, n = 2) were confirmed to be SFs, whereas TP53 variants were confirmed to be somatic variants. To confirm the low prevalence of SFs in TP53, we analyzed 24 patients with TP53 variants who underwent a paired tumor–normal NGS assay. As expected, all TP53 variants were confirmed to be somatic variants. A total of 30 patients were tested for germline variants in TP53, but none of them resulted in true SFs, suggesting the low prevalence of SFs in this gene. Therefore, the significance of additional germline testing for TP53 variants appears to be relatively low in daily clinical practice using a tumor-only NGS assay, unless patients have any relevant medical or family history.

To explore the transcription factors associated with carotenoid metabolism in citrus fruit, one transcription factor (CubHLH1) was selected through microarray screening in Satsuma mandarin (Citrus unshiu Marc.) fruit, which was treated with exogenous ethylene or gibberellin (GA), accelerating or retarding carotenoid accumulation in peel, respectively. The amino acid sequence of CubHLH1 has homology to Arabidopsis activation-tagged bri1 suppressor 1 (ATBS1) interacting factor (AIF), which is functionally characterized as a negative regulator of the brassinolide (BR) signalling pathway. Yeast two-hybrid analysis revealed that protein for CubHLH1 could interact with Arabidopsis and tomato ATBS1. Overexpression of CubHLH1 caused a dwarf phenotype in transgenic tomato (Solanum lycopersicum L.), suggesting that CubHLH1 has a similar function to Arabidopsis AIF. In the transgenic tomato fruit at ripening stage, the lycopene content was reduced along with the changes in carotenoid biosynthetic gene expression. The abscisic acid (ABA) content of all the transgenic tomato fruit was higher than that of the wild type. These results implied that CubHLH1 is considered to have a similar function to Arabidopsis AIFs and might be directly involved in carotenoid metabolism in mature citrus fruit.

Background Somatic embryogenesis in nucellar tissues is widely recognized to induce polyembryony in major citrus varieties such as sweet oranges, satsuma mandarins and lemons. This capability for apomixis is attractive in agricultural production systems using hybrid seeds, and many studies have been performed to elucidate the molecular mechanisms of various types of apomixis. To identify the gene responsible for somatic embryogenesis in citrus, a custom oligo-DNA microarray including predicted genes in the citrus polyembryonic locus was used to compare the expression profiles in reproductive tissues between monoembryonic and polyembryonic varieties. The full length of CitRKD1, which was identified as a candidate gene responsible for citrus somatic embryogenesis, was isolated from satsuma mandarin and its molecular function was investigated using transgenic ‘Hamlin’ sweet orange by antisense-overexpression. Results The candidate gene CitRKD1, predominantly transcribed in reproductive tissues of polyembryonic varieties, is a member of the plant RWP-RK domain-containing protein. CitRKD1 of satsuma mandarin comprised two alleles (CitRKD1-mg1 and CitRKD1-mg2) at the polyembryonic locus controlling embryonic type (mono/polyembryony) that were structurally divided into two types with or without a miniature inverted-repeat transposable element (MITE)-like insertion in the upstream region. CitRKD1-mg2 with the MITE insertion was the predominant transcript in flowers and young fruits where somatic embryogenesis of nucellar cells occurred. Loss of CitRKD1 function by antisense-overexpression abolished somatic embryogenesis in transgenic sweet orange and the transgenic T1 plants were confirmed to derive from zygotic embryos produced by self-pollination by DNA diagnosis. Genotyping PCR analysis of 95 citrus traditional and breeding varieties revealed that the CitRKD1 allele with the MITE insertion (polyembryonic allele) was dominant and major citrus varieties with the polyembryonic allele produced polyembryonic seeds. Conclusion CitRKD1 at the polyembryonic locus plays a principal role in regulating citrus somatic embryogenesis. CitRKD1 comprised multiple alleles that were divided into two types, polyembryonic alleles with a MITE insertion in the upstream region and monoembryonic alleles without it. CitRKD1 was transcribed in reproductive tissues of polyembryonic varieties with the polyembryonic allele. The MITE insertion in the upstream region of CitRKD1 might be involved in regulating the transcription of CitRKD1. Electronic supplementary material The online version of this article (10.1186/s12870-018-1369-3) contains supplementary material, which is available to authorized users.

We developed 708 gene-based markers for citrus genome analysis. Sequence-tagged site (STS) primers were designed that were located in conserved exon regions and whose PCR products spanned genomic introns. Of these, 79.7 % comprised cleaved amplified polymorphic sequence markers. The gene-based markers and their annotation and position on Clementine scaffolds ver. 1.0 permitted comparison of the genetic map and the Clementine genome sequence. The 708 gene-based markers were used to construct a genetic map using the 87 progenies (AG population) from the cross between ‘Okitsu 46 gou’ (‘Sweet Spring’ (‘Ueda unshiu’ (Citrus unshiu) × Hassaku (Citrus hassaku Hort. ex Tanaka)) × ‘Trovita’ orange) × ‘Kankitsu Chukanbohon Nou 5 gou’ (‘Lee’ (Citrus clementina × tangelo) × Citrus kinokuni). The markers were integrated using common STSs on different phase maps in cross-pollination mode. The integrated map (AGI map) comprised 706 loci, including two morphological traits, and spanned 990.9 centimorgans (cM) with an average marker distance of 1.40 cM. These markers formed nine linkage groups (LGs) (corresponding to citrus physical chromosomes): LG-01 to LG-09 corresponded to Scaffold_01, Scaffold_07, Scaffold_09, Scaffold_06, Scaffold_03, Scaffold_02, Scaffold_04, Scaffold_08, and Scaffold_05, respectively. LG-08 and LG-09 contained morphological traits controlling embryo color and seedlessness. Eighty-eight loci comprised three or more alleles on the AGI map; 36.4 % of them were related to transcription factors and DNA-binding proteins. The 708 gene-based markers and the AGI map are valuable for integrating various citrus genetic maps, alignment of genomic sequences, chromosome assignment, and understanding the diversity of citrus germplasms.

Citrus fruits contain significant amounts of various carotenoids and some of them are known to benefit human health. Approximately 115 different carotenoids have been reported in citrus fruits, and the color of the fruit and peel are caused by carotenoid accumulation (Stewart and Wheaton, 1973). The carotenoid content and composition in

Susceptibly to the induction of rat tongue cancer (TC) by oral 4-nitroquinoline 1-oxide (4NQO) exposure is a polygenic trait. Among several quantitative trait loci identified by crosses between TC-susceptible Dark Agouti (DA) rats and TC-resistant Wistar-Furth (WF) rats, we focused on tongue cancer susceptibility locus (Tcas3) of chromosome 4. We examined tongue carcinogenesis in the reciprocal congenic strains DA.WF-Tcas3 and WF.DA-Tcas3 and in their parental strains. The Tcas3DA allele, and not the Tcas3WF allele, significantly favored tumor latency, incidence and TC number/size. In genomic DNA of TCs induced in (DA x WF) F1 rats, the resistant Tcas3WF allele was frequently and selectively lost, particularly in larger tumors. Thus, we searched the possible candidate genes in the Tcas3 region using microarray analysis of TCs in F1 rats and revealed significant upregulation of 2 cancer-related genes, parathyroid hormone-like hormone (Pthlh) and Kras2. The relevance of the WF allele of Pthlh as a cancer modifier was indicated by 3 single nucleotide polymorphisms specific to this strain. In contrast, no consistent strain-specific variations were found in Kras2. Moreover, the plasma Ca2+ level was consistently higher in DA rats when compared to the level in WF rats bearing TCs; moreover, the Pthlh-mRNA expression level was >30-fold higher in TCs when compared to this level in the normal tongue mucosa. Immunostaining experiments showed strong PTHrP protein expression in TCs of DA rats, and the signal was intensified in larger TCs. Kras2 was also upregulated in TCs, but to a lesser degree than PTHrP. Thus, Pthlh is a promising candidate modifier gene in the development and progression of rat TCs.

Advances in next-generation sequencing (NGS) technologies have enabled physicians to test for genomic alterations in multiple cancer-related genes at once in daily clinical practice. In April 2015, we introduced clinical sequencing using an NGS-based multiplex gene assay (OncoPrime) certified by the Clinical Laboratory Improvement Amendment. This assay covers the entire coding regions of 215 genes and the rearrangement of 17 frequently rearranged genes with clinical relevance in human cancers. The principal indications for the assay were cancers of unknown primary site, rare tumors, and any solid tumors that were refractory to standard chemotherapy. A total of 85 patients underwent testing with multiplex gene assay between April 2015 and July 2016. The most common solid tumor types tested were pancreatic (n = 19; 22.4%), followed by biliary tract (n = 14; 16.5%), and tumors of unknown primary site (n = 13; 15.3%). Samples from 80 patients (94.1%) were successfully sequenced. The median turnaround time was 40 days (range, 18–70 days). Potentially actionable mutations were identified in 69 of 80 patients (86.3%) and were most commonly found in TP53 (46.3%), KRAS (23.8%), APC (18.8%), STK11 (7.5%), and ATR (7.5%). Nine patients (13.0%) received a subsequent therapy based on the NGS assay results. Implementation of clinical sequencing using an NGS-based multiplex gene assay was feasible in the clinical setting and identified potentially actionable mutations in more than 80% of patients. Current challenges are to incorporate this genomic information into better therapeutic decision making.

CuSTS1 cDNA clone was isolated from Satsuma mandarin ( Citrus unshiu Marc.) and functionally characterized. Genomic clone corresponding to CuSTS1 consists of 7 exons and 6 introns which is typical structure of angiosperm sesquiterpene synthase genes. Their predicted proteins possess a general feature of plant sesquiterpene synthases and RR motif and DDXXD motifs were conserved. Phylogenetic tree analysis showed that the molecular evolutions of citrus germacrene synthase might occur according to enzymatic function before the divergence of Citrus species, in contrast to citrus monoterpene synthase genes. Functionally analysis indicated that CuSTS1 protein produces mainly germacrene A from farnesyl diphosphate (FPP) in accompany with side product. In Satsuma mandarin, transcription of CuSTS1 were comparatively abundant in flowers and young fruit at 60 days after flowering (DAF), and peel at 120 DAF. The transcription was decreasing toward fruit maturing. In young fruit at 120 DAF, CuSTS1 is induced by methyl jasmonic acid, salicylic acid and ethylene treatments, suggesting it would be involved in plant defense response.

Gibberellin 3 (GA 3 )-responsive genes were investigated with a citrus 22K oligoarray 1 to further the understanding of transcriptional regulation by GA 3 treatment in Satsuma mandarin fruit ( Citrus unshiu Marc.). 213 GA 3 -responsive genes were identified that showed a 3-fold or greater expression change after 72 h GA 3 treatment, compared to expression after 72 h air treatment. GA 3 treatment induced expression of pathogenesis-related (PR) proteins and genes that function in photosynthesis, chloroplast biogenesis, resistance, defense and stress. Also, GA 3 treatment reduced the transcription of several ethylene-inducible genes, such as carotenoid metabolic genes, which are associated with fruit ripening. Contrasting effects between GA 3 and ethylene were observed on photosynthesis and chloroplast biogenesis, chlorophyll metabolism, and carotenoid metabolism, indicating that the endogenous GA 3 level might be important for the endogenous regulation of maturation and senescence in mature citrus fruit. It was also found that the GA response pathway was likely to take part in cross-talk with the pathogen-related pathway in mature citrus fruit.

A comprehensive transcriptome analysis using a citrus 22K oligoarray was performed to identify ethylene-responsive genes and gain an understanding of the transcriptional regulation by ethylene in mandarin fruit (Citrus unshiu Marc.). In the 72 h after ethylene treatment, 1493 genes were identified as ethylene-responsive with more than 3-fold expression change, and an interesting aspect of gene regulation by ethylene was observed, namely, that more than half of the ethylene-responsive genes were repressed. This aspect might suggest that ethylene demotes numerous biological processes and plays an important role in fruit ripening and senescence. Ethylene repressed the transcription of most genes involved in photosynthesis, chloroplast biogenesis, and sugar metabolism, while it induced the transcription of several genes related to resistance, defense, stress, amino acid synthesis, protein degradation, and secondary metabolism. In carotenoid metabolism, the sensitivity and responsive patterns to exogenous ethylene were significantly different among carotenoid biosynthesis genes. Ethylene might cause a change of their transcriptional balance and influence carotenoid composition of fruits. Besides, most of ethylene biosynthesis genes and its signal transduction components did not show any significant expression change (

A series of quinoline-3-carbothioamides and their analogues was prepared via four synthetic routes and evaluated for their antinephritic and immunomodulating activities. The optimal compound 9g strongly inhibited the T-cell independent antibody production in mice immunized with TNP-LPS and was highly effective in two nephritis models, namely chronic graft-versus-host disease and autoimmune MRL/l mice.

We have developed a CiFT co-expression system for the rapid evaluation of transgenic citrus flowers and fruit. In order to co-express a target gene and the CiFT gene, a CiFT co-expression vector was constructed by inserting the fragment containing a chimeric target gene into the binary vector harboring P35S::CiFT. This system was applied to metabolically engineer aroma components in trifoliate orange (Poncirus trifoliata L. Raf). In order to reduce the limonene level, a gene encoding limonene synthase (CitMTSE1) from satsuma mandarin (Citrus unshiu Marc.) was introduced into trifolliate orange in an anti-sense orientation with the CiFT co-expression vector. Transgenic plants flowered extremely early, and began to produce normal fruit within 2 years of Agrobacterium infection. Transcripts of transgenes were accumulated in reproductive tissues of transgenic plants, and endogenous transcripts for limonene synthase were reduced. The ratio of limonene to other monoterpenes was significantly decreased in flowers and fruit of transgenic plants. These results clearly indicated that the CiFT co-expression system is a useful tool for the functional analysis of genes in citrus flowers and fruit. Such analyses are important for several applications, including the metabolic engineering of aroma components in citrus.

A series of quinoline-3-carbothioamides and their analogues was prepared via four synthetic routes and evaluated for their antinephritic and immunomodulating activities. The optimal compound 9g strongly inhibited the T-cell independent antibody production in mice immunized with TNP-LPS and was highly effective in two nephritis models, namely chronic graft-versus-host disease and autoimmune MRL/l mice.

A series of linomide-related quinoline-3-carboxamides and their analogues was prepared and evaluated for antinephritic activities. The 6-MeS derivative 7a was highly effective in two nephritis models, namely chronic graft-versus-host disease and autoimmune MRL/l mice.

To obtain genetic information on carotenoid content in Citrus fruit for a breeding program, we investigated quantitative trait loci (QTL) in a mapping population derived from a cross between ‘Okitsu-46’ and ‘Nou-5’. Among individual progeny, total and each carotenoid contents were segregated in a transgressive manner. QTLs for total and each carotenoid contents were detected by Kruskal-Wallis analysis (P 2.0) from 51 individuals. Most of the QTLs for each carotenoid component were mapped to different locations on the linkage map. In β-cryptoxanthin, the strongest QTL (LOD 3.4) was detected on linkage group 6 of ‘Nou-5’, and accounted for 26.9% of explainable variance. LOD scores of QTLs for total carotenoid content were lower than those for each carotenoid content. There were one QTL for total carotenoid content at the criteria of LOD > 2.0, and seven weaker QTLs, which overlapped with QTLs for each carotenoid component. The effects of QTLs detected for each carotenoid content worked cumulatively to increase total carotenoid content. This study provides preliminary data because we obtained data in only one season and from a limited number of individuals (n = 51). However, the results suggested that QTL information could be used to generate DNA markers to select progeny with higher carotenoid content.