Hydrotropic response and expression pattern of auxin-inducible gene,CS-IAA1, in the primary roots of clinorotated cucumber seedlings

Primary roots of cucumber seedlings showed positive hydrotropism when exposed to a moisture gradient and rotated on a two-axis clinostat. To examine the role of auxin in the differential growth of the hydrotropically responding roots, we first examined the expression of auxin-inducible genes, CS-AUX/IAAs, in cucumber roots. After auxin starvation, mRNA levels of CS-IAA1 and CS-IAA3 decreased in the roots. Applying auxin to the auxin-starved roots resulted in accumulation of CS-IAA1 and CS-IAA3 mRNA. The level of expression of these genes increased when the auxin concentration was increased. CS-IAA1 mRNA accumulated in response to 10(-8) M auxin, and the level increased further, depending on the dose. Auxin starvation did not result in a decrease in the level of CS-IAA2 mRNA; however, adding exogenous auxin at concentrations higher than 10(-7) M increased its accumulation. In the primary roots responding hydrotropically or gravitropically, CS-IAA1 expression was greater on the concave side of the curving roots than on the convex side. The difference could be detected 30 min following stimulation by gravity or a moisture gradient, and that difference increased with time. These results support the idea that asymmetry of localization of auxin is associated with differential growth in hydrotropically responding roots.     

<Emphasis Type="Italic">HrNodal</Emphasis>, the ascidian <Emphasis Type="Italic">nodal</Emphasis>-related gene,is expressed in the left side of the epidermis,and lies upstream of <Emphasis Type="Italic">HrPitx</Emphasis>

The nodal-related genes are well known for their fundamental roles during vertebrate development, including mesoderm induction, neural induction, and left-right axis formation, as several nodal-related genes show left-sided expression in mesodermal lineages. We have isolated the first non-vertebrate nodal-related gene, HrNodal, from the ascidian Halocynthia roretzi. During the late cleavage and gastrula stages, HrNodal is transiently and bilaterally expressed in several different cell lineages. Expression at the tailbud stage is observed asymmetrically in the left side, but unexpectedly only in the epidermis of the embryo. We also demonstrate the relationship of HrNodal with HrPitx, a Halocynthia homologue of the Pitx2 gene. HrNodal overexpression results in the disturbance of left-sided HrPitx expression. Our results demonstrate that left-right specification during ascidian embryogenesis involves the HrNodal gene, and that the left-sidedness of the expression is evolutionarily conserved throughout the chordate clade.     

Preparation of chiral 4-benzyloxymethyldihydrofuran-2-one using lipase-catalyzed kinetic resolution: synthesis of (-)-virginiae butanolide C (VB C)

Lipase-catalyzed kinetic resolution of the N,N-dialkyl-3-benzyloxymethyl-4-hydroxybutanamide 10a,b afforded the acetate 11a,b with (R) configuration, whereas the N-monoalkyl-3-benzyloxymethyl-4-hydroxybutanamide 10c-e gave the acetate 11c-e with (S) configuration. The butanamide 10 smoothly cyclized to give chiral 4-benzyloxymethyldihydrofuran-2-one 9 without racemization, which was effectively transformed into highly stereocontrolled virginiae butanolide C (VB C).     

Preparation of poly(L-lactide)-based microspheres having a cationic or anionic surface using biodegradable surfactants

Poly(L-lactide)-based microspheres having cationic or anionic surfaces were prepared using polydepsipeptide-block-poly(L-lactide)s as surfactants. Polydepsipeptide-block-poly(L-lactide)s having amino or carboxylic acid groups on their side chains were synthesized through anionic ring-opening polymerizations of L-lactide using the corresponding protected polydepsipeptides as macroinitiators and consequent deprotections. Since these amphiphilic copolymers consisting of hydrophobic segments and hydrophilic segments with amino or carboxylic acid groups could be converted to cationic or anionic block copolymers, they could act as surfactants preparing poly(L-lactide)-based microspheres by an oil-in-water emulsion method. The amount of ionic groups located on the surfaces of the obtained microspheres was found to increase with increasing the feed of charged polydepsipeptide-block-poly(L-lactide)s in the blend of poly(L-lactide) and block copolymers. The average diameters of the dried microspheres estimated by scanning electron microscopy were found to decrease with an increase in feed of block copolymers in polymer blends.     

Transgenic pigs expressing human decay-accelerating factor regulated by porcine MCP gene promoter

Porcine membrane cofactor protein (pMCP) is abundantly expressed throughout the body with particularly strong expression on the vascular endothelia. Previous studies demonstrated that the promoter of the pMCP gene induced efficient expression of a human complement regulatory protein, decay-accelerating factor (DAF; CD55), in transgenic mice. In the present study, we tried to produce transgenic pigs with two hybrid genes, 0.9/hDAF and 5.4/hDAF, which were composed of human DAF (hDAF) gene regulated under pMCP promoters of different lengths (0.9 and 5.4 kb). Five live founder transgenic pigs were obtained only with the 0.9/hDAF construct. Although, four founder pigs transmitted the transgene to the second generation, the transmission rates varied among founders. We examined the expression of hDAF in tissues of descendants of two lines (Dm1 and Dm4). Human DAF specific RNAs were confirmed by an RT-PCR analysis in all organs examined. Levels of hDAF protein in the organs from the descendants of Dm1 line were higher than those in the corresponding human organs as determined by enzyme-linked immunosorbent assay. Immunohistochemical studies showed that the tissue distribution of hDAF in the descendants of both lines was similar to that of endogenous pMCP. The expression level of hDAF on the vascular endothelial cells in Dm1 line was twice that on the corresponding human cells. We tested whether proinflammatory cytokines upregulate an efficiency of pMCP promoter on hDAF expression in transgenic pigs. Although the expression of hDAF on the human endothelial cells increased with a combination of cytokines, tumor necrosis factor alpha and interferon-gamma, no cytokine-induced upregulation was seen in the cells of transgenic pigs. The endothelial cells from transgenic pigs exhibited high resistance to the human serum-mediated cytolysis.     

Rapid Screening of a Novel Arrayed Medaka (Oryzias latipes) Cosmid Library

Medaka (Oryzias latipes) has many advantages for genetic and developmental studies. With recent advances in the genome analyses of other species, rapid accumulation of resources for medaka genomics is expected. In this study, we generated an arrayed medaka cosmid library from the HNI inbred strain, carrying a 40-kb insert on average. The library consists of approximately 120,000 clones with a 6-fold genomic coverage. Cosmid clones can be screened within 2 days using standard polymerase chain reaction. Considering the advantage of the cosmid insert size and the compact genome size of the medaka, this library provides a powerful tool for future genome analyses.     

Thymidine phosphorylase suppresses Fas-induced apoptotic signal transduction independent of its enzymatic activity

Thymidine phosphorylase (TP) has chemotactic and angiogenic activities resulting from its enzymatic activity in vitro, and it also promotes tumor growth and inhibits apoptosis in vivo. Recently, we have reported that TP plays an important role in Fas-induced apoptosis. Caspase-8 cleavage, subsequent cytochrome c release, and caspase-3 cleavage were prevented in KB cells transfected with a TP cDNA (KB/TP cells). In this study, treatment with thymidine phosphorylase inhibitor (TPI) or thymidine did not affect cell survival of KB/TP cells during Fas-induced apoptosis. Moreover, treatment with thymine or 2-deoxy-D-ribose (degradation products of thymidine generated by TP) also did not affect cell survival of control transfectant (KB/CV) cells during Fas-induced apoptosis. These findings indicate that TP suppresses Fas-induced apoptotic signal transduction independent of its enzymatic activity.     

High-potential iron-sulfur protein (HiPIP) is the major electron donor to the reaction center complex in photosynthetically growing cells of the purple bacterium Rubrivivax gelatinosus

A gene encoding the high-potential iron-sulfur protein (HiPIP) was cloned from the purple photosynthetic bacterium Rubrivivax gelatinosus. An insertional disruption of this gene by a kanamycin resistance cartridge resulted in a significant decrease in the growth rate under photosynthetic growth conditions. Flash-induced kinetic measurements showed that the rate of reduction of the photooxidized reaction center is greatly diminished in the mutant depleted in the HiPIP. On the other hand, mutants depleted in the low- and high-potential cytochromes c(8), the two other soluble electron carriers, which have been shown to donate an electron to the reaction center in Rvi. gelatinosus, showed growth rates similar to those of the wild type under both photosynthetic and respiratory growth conditions. It was concluded that HiPIP is the major physiological electron donor to the reaction center in Rvi. gelatinosus cells grown under photosynthetic conditions.     

Phosphorylation of Fanconi anemia protein, FANCA, is regulated by Akt kinase

Phosphorylation of the Fanconi anemia complementation group A (FANCA) protein is thought to be important for the function of the FA pathway. However, the kinase for FANCA (so-called FANCA-PK) remains to be identified. FANCA has a consensus sequence for Akt kinase near serine 1149 (Ser1149), suggesting that Akt can phosphorylate FANCA. We performed in vitro kinase assays using as substrate either a GST-fusion wild-type (WT) FANCA fragment or a GST-fusion FANCA fragment containing a mutation from serine to alanine at 1149 (FANCA-S1149A). These experiments confirmed that FANCA is phosphorylated at Ser 1149, in vitro. However, (32)P-orthophosphate labeling experiments revealed that FANCA-S1149A was more efficiently phosphorylated than WT-FANCA. Furthermore, phosphorylation of wild-type FANCA was blocked by coexpression of a constitutively active (CA)-Akt and enhanced by a dominant-negative (DN) Akt. Our results suggest that Akt is a negative regulator of FANCA phosphorylation.