Carotenoid isomerase is key determinant of petal color of Calendula officinalis

Orange petals of calendula (Calendula officinalis) accumulate red carotenoids with the cis-configuration at the C-5 or C-5' position (5-cis-carotenoids). We speculated that the orange-flowered calendula is a carotenoid isomerase (crtiso) loss-of-function mutant that impairs the cis-to-trans conversion of 5-cis-carotenoids. We compared the sequences and enzyme activities of CRTISO from orange- and yellow-flowered calendulas. Four types of CRTISO were expressed in calendula petals. The deduced amino acid sequence of one of these genes (CoCRTISO1) was different between orange- and yellow-flowered calendulas, whereas the sequences of the other three CRTISOs were identical between these plants. Analysis of the enzymatic activities of the CoCRTISO homologs showed that CoCRTISO1-Y, which was expressed in yellow petals, converted carotenoids from the cis-to-trans-configuration, whereas both CoCRTISO1-ORa and 1-ORb, which were expressed in orange petals, showed no activity with any of the cis-carotenoids we tested. Moreover, the CoCRTISO1 genotypes of the F2 progeny obtained by crossing orange and yellow lines linked closely to petal color. These data indicate that CoCRTISO1 is a key regulator of the accumulation of 5-cis-carotenoids in calendula petals. Site-directed mutagenesis showed that the deletion of Cys-His-His at positions 462-464 in CoCRTISO1-ORa and a Gly-to-Glu amino acid substitution at position 450 in CoCRTISO1-ORb abolished enzyme activity completely, indicating that these amino acid residues are important for the enzymatic activity of CRTISO.     

Intraductal polypoid growth variant of pancreatic acinar cell carcinoma metastasizing to the intrahepatic bile duct 6 years after surgery: a case report and literature review

We present the first reported case of intraductal polypoid growth (IPG) variant of pancreatic acinar cell carcinoma (ACC) metastasizing to the intrahepatic bile duct. A 58-year-old Japanese woman had previously presented with obstructive jaundice and a 7.0 cm mass in the pancreatic head. She underwent biliary drainage for 2 months followed by pancreatectomy. Histological examination revealed a carcinoma with acinar pattern, immunohistochemically positive for trypsin, and acinar cell carcinoma was diagnosed. IPGs were prominent in the main pancreatic duct and its tributaries, extending into the intrapancreatic bile duct with tumor casts in the lumen. Imaging examinations 6 years later revealed a growing lesion within the intrahepatic bile duct. Needle biopsy examination suggested metastasis of ACC, and she underwent chemoradiation therapy and partial hepatectomy. Histological examination demonstrated ACC confined to the intrahepatic bile duct. The localization of metastasis and slow growth may indicate indolent biologic behavior of the IPG variant.     

Binding of an Indenoisoquinoline to the Topoisomerase-DNA Complex Induces Reduction of Linker Mobility and Strengthening of Protein-DNA Interaction

Long-duration comparative molecular dynamics simulations of the DNA-topoisomerase binary and DNA-topoisomerase-indenoisoquinoline ternary complexes have been carried out. The analyses demonstrated the role of the drug in conformationally stabilizing the protein-DNA interaction. In detail, the protein lips, clamping the DNA substrate, interact more tightly in the ternary complex than in the binary one. The drug also reduces the conformational space sampled by the protein linker domain through an increased interaction with the helix bundle proximal to the active site. A similar alteration of linker domain dynamics has been observed in a precedent work for topotecan but the molecular mechanisms were different if compared to those described in this work. Finally, the indenoisoquinoline keeps Lys532 far from the DNA, making it unable to participate in the religation reaction, indicating that both short- and long-range interactions contribute to the drug poisoning effect.     

Seeing is believing: engineering anthocyanin and carotenoid biosynthetic pathways

The biosynthetic pathways of flavonoids and carotenoids have been well established, and the biosynthetic genes have been mostly isolated. Metabolic engineering of their biosynthetic pathways has provided not only novel colored or health-beneficial plants but also excellent models to study the efficacy of such engineering. In order to achieve a specific color by accumulating a corresponding compound, it is necessary to upregulate the pathway leading to the compound and downregulate the competing pathway. The regulation of gene expression has to be optimized in a target crop as well.     

The speciation of conger eel galectins by rapid adaptive evolution

Many cases of accelerated evolution driven by positive Darwinian selection are identified in the genes of venomous and reproductive proteins. This evolutional phenomenon might have important consequences in their gene-products' functions, such as multiple specific toxins for quick immobilization of the prey and the establishment of barriers to fertilization that might lead to speciation, and in the molecular evolution of novel genes. Recently, we analyzed the molecular evolution of two galectins isolated from the skin mucus of conger eel (Conger myriaster), named congerins I and II, by cDNA cloning and X-ray structural analysis, and we found that they have evolved in the rapid adaptive manner to emergence of a new structure including strand-swapping and a unique new ligand-binding site. In this review article we summarize and discuss the molecular evolution, especially the rapid adaptive evolution, and the structure-function relationships of conger eel galectins. Published in 2004.     

Self-Enhancement of Hepatitis C Virus Replication by Promotion of Specific Sphingolipid Biosynthesis

Lipids are key components in the viral life cycle that affect host-pathogen interactions. In this study, we investigated the effect of HCV infection on sphingolipid metabolism, especially on endogenous SM levels, and the relationship between HCV replication and endogenous SM molecular species. We demonstrated that HCV induces the expression of the genes (SGMS1 and 2) encoding human SM synthases 1 and 2. We observed associated increases of both total and individual sphingolipid molecular species, as assessed in human hepatocytes and in the detergent-resistant membrane (DRM) fraction in which HCV replicates. SGMS1 expression had a correlation with HCV replication. Inhibition of sphingolipid biosynthesis with a hepatotropic serine palmitoyltransferase (SPT) inhibitor, NA808, suppressed HCV-RNA production while also interfering with sphingolipid metabolism. Further, we identified the SM molecular species that comprise the DRM fraction and demonstrated that these endogenous SM species interacted with HCV nonstructural 5B polymerase to enhance viral replication. Our results reveal that HCV alters sphingolipid metabolism to promote viral replication, providing new insights into the formation of the HCV replication complex and the involvement of host lipids in the HCV life cycle.