Otolith matrix proteins OMP-1 and Otolin-1 are necessary for normal otolith growth and their correct anchoring onto the sensory maculae

Fish otoliths are highly calcified concretions deposited in the inner ear and serve as a part of the hearing and balance systems. They consist mainly of calcium carbonate and a small amount of organic matrix. The latter component is considered to play important roles in otolith formation. Previously, we identified two major otolith matrix proteins, OMP-1 (otolith matrix protein-1) and Otolin-1, from salmonid species. To assess the function of these proteins in otolith formation, we performed antisense morpholino oligonucleotide (MO)-mediated knockdown of omp-1 and otolin-1 in zebrafish embryos. We first identified zebrafish cDNA homologs of omp-1 (zomp-1) and otolin-1 (zotolin-1). Whole-mount in situ hybridization then revealed that the expression of both zomp-1 and zotolin-1 mRNAs is restricted to the otic vesicles. zomp-1 mRNA was expressed from the 14-somite stage in the otic placode, but the zOMP-1 protein was detected only from 26-somite stage onwards. In contrast, zotolin-1 mRNA expression became clear around 72 hpf. MOs designed to inhibit zomp-1 and zotolin-1 mRNA translation, respectively, were injected into 1-2 cell stage embryos. zomp-1 MO caused a reduction in otolith size and an absence of zOtolin-1 deposition, while zotolin-1 MO caused a fusion of the two otoliths, and an increased instability of otoliths after fixation. We conclude that zOMP-1 is required for normal otolith growth and deposition of zOtolin-1 in the otolith, while zOtolin-1, a collagenous protein, is involved in the correct arrangement of the otoliths onto the sensory epithelium of the inner ear and probably in stabilization of the otolith matrix.     

Interaction of auxin and ERECTA in elaborating Arabidopsis inflorescence architecture revealed by the activation tagging of a new member of the YUCCA family putative flavin monooxygenases

The aboveground body of higher plants has a modular structure of repeating units, or phytomers. As such, the position, size, and shape of the individual phytomer dictate the plant architecture. The Arabidopsis (Arabidopsis thaliana) ERECTA (ER) gene regulates the inflorescence architecture by affecting elongation of the internode and pedicels, as well as the shape of lateral organs. A large-scale activation-tagging genetic screen was conducted in Arabidopsis to identify novel genes and pathways that interact with the ER locus. A dominant mutant, super1-D, was isolated as a nearly complete suppressor of a partial loss-of-function allele er-103. We found that SUPER1 encodes YUCCA5, a novel member of the YUCCA family of flavin monooxygenases. The activation tagging of YUCCA5 conferred increased levels of free indole acetic acid, increased auxin response, and mild phenotypic characteristics of auxin overproducers, such as elongated hypocotyls, epinastic cotyledons, and narrow leaves. Both genetic and cellular analyses indicate that auxin and the ER pathway regulate cell division and cell expansion in a largely independent but overlapping manner during elaboration of inflorescence architecture.     

Inhibitory effects of glycolipids fraction from spinach on mammalian DNA polymerase activity and human cancer cell proliferation

We succeeded in purifying the fraction containing the major glycolipids in monogalactosyl diacylglycerol, digalactosyl diacylglycerol and sulfoquinovosyl diacylglycerol (SQDG) from dried vegetables. This glycolipids fraction was an inhibitor of DNA polymerase alpha (pol alpha) in vitro and also the proliferation of human cancer cells. In this study, eight common vegetables were investigated in terms of the glycolipids fraction, the amounts of major glycolipids, mammalian DNA polymerase inhibitory activity and antiproliferative activity toward human cancer cells. Green tea possessed the largest amount of glycolipids overall. Spinach contained the largest amount of SQDG, followed by parsley, green onion, chive, sweet pepper, green tea, carrot and garlic. Spinach had the strongest inhibitory effect on pol alpha activity and human cancer cell proliferation. A significant correlation was found between SQDG content and inhibition of DNA polymerase. Therefore, the inhibition of pol alpha activity by SQDG may lead to cell growth suppression. Of the six subspecies of spinach (Spinacia oleracea) tested, "Anna" had the largest amount of SQDG, strongest inhibitory activity toward DNA polymerase and greatest effect on human cancer cell proliferation. Based on these results, the glycolipids fraction from spinach is potentially a source of food material for a novel anticancer activity.     

Effects of DNA polymerase inhibitory and antitumor activities of lipase-hydrolyzed glycolipid fractions from spinach

We succeeded in purifying the major glycolipid fraction in the class of sulfoquinovosyl diacylglycerol, monogalactosyl diacylglycerol and digalactosyl diacylglycerol (DGDG) from a green vegetable, spinach (Spinacia oleracea L.). This glycolipid fraction was an inhibitor of DNA polymerases and a growth inhibitor of NUGC-3 human gastric cancer cells, and, interestingly, the activities were much stronger when the fraction was hydrolyzed by lipase. Glycolipids in the hydrolyzed fraction consisted of sulfoquinovosyl monoacylglycerol (SQMG), monogalactosyl monoacylglycerol (MGMG) and DGDG. In the in vivo antitumor assay using Greene's melanoma, the fraction containing SQMG, MGMG and DGDG showed to be a promising suppressor of solid tumors. Spinach glycolipid fraction might be a potent antitumor compound if directly injected into a tumor-carrying body, and this fraction may be a healthy food material that has antitumor activity.     

Exacerbation of granuloma formation in IL-1 receptor antagonist-deficient mice with impaired dendritic cell maturation associated with Th2 cytokine production

Dendritic cell (DC) maturation at the site of inflammation and migration into draining lymph nodes is fundamental to initiate Ag-specific immune responses. Although several proinflammatory cytokines, including IL-1, are known to promote DC maturation in vitro, their contributions to DC activation and migration within peripheral inflamed tissue compartments are not yet fully understood. We show here that endogenous IL-1 receptor antagonist (IL-1ra) controls the activation state of liver-recruited DCs and their migration in a Propionibacterium acnes-induced murine granulomatous liver disease model. After P. acnes treatment, formation of portal tract-associated lymphoid tissue was conversely impaired in IL-1ra-deficient mice. IL-1ra-deficient mice developed hepatic granulomas within 3 days after P. acnes administration and showed a more pronounced granuloma formation than wild-type mice. Although sinusoidal granulomas contained numerous CD11c+ DCs at day 7, expressions of CCR7, IL-12p40 by these DCs were dramatically decreased in IL-1ra-deficient mice, suggesting aberrant DC maturation and sinusoid portal migration in the absence of endogenous IL-1ra. This was accompanied with enhanced intrahepatic Th2 cytokine production and severe hepatocellular damage. Thus, hepatocyte-derived IL-1ra may control optimal activation and migration of inflammatory DCs within the liver and thereby determine the local immune responses in granulomatous liver disease.     

Aspergillus parasiticus cyclase catalyzes two dehydration steps in aflatoxin biosynthesis

In the aflatoxin biosynthetic pathway, 5'-oxoaverantin (OAVN) cyclase, the cytosolic enzyme, catalyzes the reaction from OAVN to (2'S,5'S)-averufin (AVR) (E. Sakuno, K. Yabe, and H. Nakajima, Appl. Environ. Microbiol. 69:6418-6426, 2003). Interestingly, the N-terminal 25-amino-acid sequence of OAVN cyclase completely matched an internal sequence of the versiconal (VHOH) cyclase that was deduced from its gene (vbs). The purified OAVN cyclase also catalyzed the reaction from VHOH to versicolorin B (VB). In a competition experiment using the cytosol fraction of Aspergillus parasiticus, a high concentration of VHOH inhibited the enzyme reaction from OAVN to AVR, and instead VB was newly formed. The recombinant Vbs protein, which was expressed in Pichia pastoris, showed OAVN cyclase activity, as well as VHOH cyclase activity. A mutant of A. parasiticus SYS-4 (= NRRL 2999) with vbs deleted accumulated large amounts of OAVN, 5'-hydroxyaverantin, averantin, AVR, and averufanin in the mycelium. These results indicated that the cyclase encoded by the vbs gene is also involved in the reaction from OAVN to AVR in aflatoxin biosynthesis. Small amounts of VHOH, VB, and aflatoxins also accumulated in the same mutant, and this accumulation may have been due to an unknown enzyme(s) not involved in aflatoxin biosynthesis. This is the first report of one enzyme catalyzing two different reactions in a pathway of secondary metabolism.     

Rapid proteasomal degradation of translocation-deficient UDP-glucuronosyltransferase 1A1 proteins in patients with Crigler-Najjar type II

UDP-glucuronosyltransferase form 1A1 (UGT1A1) is the only bilirubin-glucuronidating isoform of this protein, and genetic deficiencies of UGT1A1 cause Crigler-Najjar syndrome, a disorder resulting from nonhemolytic unconjugated hyperbilirubinemia. Here we have focused on the instability of a translocation-deficient UGT1A1 protein, which has been found in patients with Crigler-Najjar type II, to elucidate the molecular basis underlying the deficiency in glucuronidation of bilirubin. A substitution of leucine to arginine at position 15 (L15R/1A1) is predicted to disrupt the hydrophobic core of the signal peptide of UGT1A1. L15R/1A1 was synthesized in similar amounts to wild-type UGT1A1 protein (WT/1A1) in transfected COS cells. However, L15R/1A1 did not translocate across the endoplasmic reticulum membrane and was degraded rapidly with a half-life of about 50min, in contrast to the much longer half-life of about 12.8h for WT/1A1. Our findings demonstrate that L15R/1A1 was rapidly degraded by the proteasome owing to its mislocalization in the cell.