Fine mapping of a gene for low-tiller number, Ltn, in japonica rice (Oryza sativa L.) variety Aikawa 1

Tillering is one of the most important agronomic traits related to grain production in rice (Oryza sativa L.). A japonica-type variety, Aikawa 1, is known to have low-tiller number. The detailed location of a low-tillering gene, Ltn, which has been localized on chromosome 8 in Aikawa 1, was confirmed by molecular mapping. Using BC₅F₂ individuals derived from a cross between IR64 and Aikawa 1, the low-tillering gene was mapped to an interval defined by SSR markers ssr5816-3 and A4765. This was designated as Ltn because there was no reported gene for tillering in the region of chromosome 8. Through high-resolution linkage analysis, the candidate region of Ltn was located between DNA markers ssr6049-23 and ind6049-1 corresponding to 38.6 kbp on the Nipponbare genome sequence. These DNA markers, which were tightly linked to Ltn, are useful for marker-assisted selection in breeding studies.     

Importance of the anti-interferon capacity of Sendai virus C protein for pathogenicity in mice

The Sendai virus (SeV) C protein blocks signal transduction of interferon (IFN), thereby counteracting the antiviral actions of IFN. Using HeLa cell lines expressing truncated or mutated SeV C proteins, we found that the C-terminal half has anti-IFN capacity, and that K(151)A, E(153)A, and R(154)A substitutions in the C protein eliminated this capacity. Here, we further created the mutant virus SeV Cm*, in which K(151)A, E(153)K, and R(157)L substitutions in the C protein were introduced without changing the amino acid sequence of overlapped P, V, and W proteins. SeV Cm* was found to lack anti-IFN capacity, as expected. While the growth rate and final yield of SeV Cm* were inferior to those of the wild-type SeV in IFN-responsive, STAT1-positive 2fTGH cells, SeV Cm* grew equivalently to the wild-type SeV in IFN-nonresponsive, STAT1-deficient U3A cells. SeV Cm* was thus shown to maintain multiplication capacity, except that it lacked anti-IFN capacity. Intranasally inoculated SeV Cm* could propagate in the lungs of STAT1(-/-) mice but was cleared from those of STAT1(+/+) mice without propagation. It was found that the anti-IFN capacity of the SeV C protein was indispensable for pathogenicity in mice. Conversely, the results show that the innate immunity contributed to elimination of SeV in early stages of infection in the absence of anti-IFN capacity.     

Anti-tumor and anti-invasive effects of diverse delta-alkyllactones: dependence on molecular side-chain length, action period and intracellular uptake

New delta-alkyllactones (DALs) with diverse side-chain lengths (184-254 Da), which are structurally different from the widespread, naturally occurring delta-lactones of higher molecular weight (348-439 Da), such as camptothecin and sultriecin, were chemically synthesized and analyzed for their carcinostatic activity. Of the DALs with 11, 12, 13, 14, or 16 carbon atoms, delta-hexadecalactone (DH16:0) was the most carcinostatic when administered to Ehrlich ascites tumor (EAT) cells at 37 degrees C for 20 h, and measured by the mitochondrial dehydrogenase-based WST-1 assay. Prolongation of the administration period to 72 h enhanced the carcinostatic activity more markedly for DH16:0 than for other DALs. The carcinostatic activity of DALs was unexpectedly augmented by increasing the number of carbon atoms, in contrast to the conventional view that carcinostatic activity is attenuated by the addition of carbon atoms to fatty acids. Intracellular accumulation of DH16:0, as analyzed by gas chromatography, was detected (1.5 Pg/cell), whereas other DALs studied were rarely found. The results indicate a close relationship between carcinostatic activity and intracellular accumulation. Invasion of human fibrosarcoma HT-1080 cells through the reconstituted basement membrane was inhibited by several DALs, even at doses as low as 5-10% of those necessary for carcinostatic activity, suggesting an invasive mechanism different from carcinostasis. The invasion-inhibitory activity was intensified by increasing the number of carbon atoms, in a manner similar to that for the carcinostatic activity. The lifespan of EAT-cell-transplanted mice was markedly prolonged with DH16:0, presumably due to excellent distribution throughout the body and tumor cells. Thus DH16:0 may be a potent anticancer agent, in term of its carcinostatic, anti-invasive, and lifespan-prolonging activities.     

Identification of the functional expression of adenosine A<Subscript>3</Subscript> receptor in pancreas using transgenic mice expressing jellyfish apoaequorin

To investigate the functional expression of adenosine A₃ receptor (A3AR) in mammalian living tissues, we generated an apoaequorin-transgenic mouse that expresses jellyfish apoaequorin throughout its body. The expression of apoaequorin under the control of a strong CAG promoter was detected in various tissues, including the abdominal skin, adipose, ear, brain, esophagus, heart, inferior vena cava vessel, kidney, lens, liver, lung, pancreas, skeletal muscle, spleen, tail, testis, and thymus. The transgene was mapped to the C1–2 region of chromosome 16 by Fluorescence in situ hybridization analysis. Among these transgenic mouse tissues, we succeeded in detecting elevated responses of intracellular Ca²⁺ as a light emission of aequorin induced by the A3AR agonist in the pancreas, brain, and testis, the last two of which are known to be main tissues abundantly expressing A3AR. The A3AR agonist led to the phosphorylation of both extracellular signal-regulated kinase 1/2 and protein kinase B in mouse pancreas, and all the intracellular responses via A3AR were antagonized by the A3AR-specific antagonist. In addition, the mRNA expression of A3AR and the A3AR-induced intracellular responses were also found in the rat pancreatic acinar cell line AR42J. These results suggest that pancreas is one of the main tissues functionally expressing A3AR in mammalians in vivo, and that the present approach using transgenic mice that express apoaequorin throughout their bodies will facilitate the functional analysis of proteins of interest. Kazuya Yamano and Katsuhiro Mori contributed equally to this work     

Comparison of biological activity among nonfucosylated therapeutic IgG1 antibodies with three different N-linked Fc oligosaccharides: the high-mannose, hybrid, and complex types

The structure of asparagine-linked oligosaccharides attached to the antibody constant region (Fc) of human immunoglobulin G1 (IgG1) has been shown to affect the pharmacokinetics and antibody effector functions of antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). However, it is still unclear how differences in the N-linked oligosaccharide structures impact the biological activities of antibodies, especially those lacking core fucose. Here, we succeeded in generating core fucose-lacking human IgG1 antibodies with three different N-linked Fc oligosaccharides, namely, a high-mannose, hybrid, and complex type, using the same producing clone, and compared their activities. Cultivation of an alpha-1,6-fucosyltransferase (FUT8) knockout Chinese hamster ovary cell line in the presence or absence of a glycosidase inhibitor (either swainsonine or kifunensine) yielded antibody production of each of the three types without contamination by the others. Two of three types of nonnaturally occurring atypical oligosaccharide IgG1, except the complex type, reduced the affinity for both human lymphocyte receptor IIIa (FcgammaRIIIa) and the C1q component of the complement, resulting in reduction of ADCC and CDC. The bulky structure of the nonreducing end of N-linked Fc oligosaccharides is considered to contribute the CDC change, whereas the structural change in the reducing end, i.e. the removal of core fucose, causes ADCC enhancement through improved FcgammaRIIIa binding. In the pharmacokinetic profile, although no significant difference of human neonatal Fc receptor (FcRn)-binding affinity was observed among the three types, the complex type showed longer serum half-lives than the other types irrespective of core fucosylation in mice, which also suggests the contribution of the nonreducing end structure. The present study provides basic information on the effects of core fucose-lacking N-linked Fc oligosaccharides on antibody biological activities.     

Lack of adenomatous polyposis coli protein correlates with a decrease in cell migration and overall changes in microtubule stability

Most sporadic colorectal tumors carry truncation mutations in the adenomatous polyposis coli (APC) gene. The APC protein is involved in many processes that govern gut tissue. In addition to its involvement in the regulation of beta-catenin, APC is a cytoskeletal regulator with direct and indirect effects on microtubules. Cancer-related truncation mutations lack direct and indirect binding sites for microtubules in APC, suggesting that loss of this function contributes to defects in APC-mutant cells. In this study, we show that loss of APC results in disappearance of cellular protrusions and decreased cell migration. These changes are accompanied by a decrease in overall microtubule stability and also by a decrease in posttranslationally modified microtubules in the cell periphery particularly the migrating edge. Consistent with the ability of APC to affect cell shape, the overexpression of APC in cells can induce cellular protrusions. These data demonstrate that cell migration and microtubule stability are linked to APC status, thereby revealing a weakness in APC-deficient cells with potential therapeutic implications.     

Effect of neurotrophins on differentiation, calcification and proliferation in cultures of human pulp cells

Neurotrophins (NTs) are expressed during tooth development. However, little is known about a role of NTs in differentiation of pulp cells into mineralizing cells. In this study, mRNA expressions of hard tissue-related proteins, calcification and proliferation are examined in cultures of human pulp (HP) cells. Nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), neurotrophin (NT)-3 and NT-4/5 increased the mRNA levels of dentin sialophsphoprotein, alkaline phosphatase, osteopontin, type I collagen and bone morphogenetic protein-2 and mineral deposition in cultures of HP cells. The increased levels and manners varied, depending on the concentrations of NTs and hard-tissue related protein tested. On the other hand, only NGF significantly stimulated DNA synthesis in cultures of HP cells. These findings suggest that NTs characteristically regulate hard-tissue related protein expression, calcification and proliferation in pulp cells. NTs may accelerate pulp cell differentiation.     

Non-fucosylated therapeutic antibodies: the next generation of therapeutic antibodies

Therapeutic antibody IgG1 has two N-linked oligosaccharide chains bound to the Fc region. The oligosaccharides are of the complex biantennary type, composed of a trimannosyl core structure with the presence or absence of core fucose, bisecting N-acetylglucosamine (GlcNAc), galactose, and terminal sialic acid, which gives rise to structural heterogeneity. Both human serum IgG and therapeutic antibodies are well known to be heavily fucosylated. Recently, antibody-dependent cellular cytotoxicity (ADCC), a lytic attack on antibody-targeted cells, has been found to be one of the critical effector functions responsible for the clinical efficacy of therapeutic antibodies such as anti-CD20 IgG1 rituximab (Rituxan^®) and anti-Her2/neu IgG1 trastuzumab (Herceptin^®). ADCC is triggered upon the binding of lymphocyte receptors (FcγRs) to the antibody Fc region. The activity is dependent on the amount of fucose attached to the innermost GlcNAc of N-linked Fc oligosaccharide via an α-1,6-linkage, and is dramatically enhanced by a reduction in fucose. Non-fucosylated therapeutic antibodies show more potent efficacy than their fucosylated counterparts both in vitro and in vivo, and are not likely to be immunogenic because their carbohydrate structures are a normal component of natural human serum IgG. Thus, the application of non-fucosylated antibodies is expected to be a powerful and elegant approach to the design of the next generation therapeutic antibodies with improved efficacy. In this review, we discuss the importance of the oligosaccharides attached to the Fc region of therapeutic antibodies, especially regarding the inhibitory effect of fucosylated therapeutic antibodies on the efficacy of non-fucosylated counterparts in one medical agent. The impact of completely non-fucosylated therapeutic antibodies on therapeutic fields will be also discussed.     

Double knockdown of α1,6-fucosyltransferase (<Emphasis Type="Italic">FUT8</Emphasis>) and GDP-mannose 4,6-dehydratase (<Emphasis Type="Italic">GMD</Emphasis>) in antibody-producing cells: a new strategy for generating fully non-fucosylated therapeutic antibodies with enhanced ADCC

Background  

Antibody-dependent cellular cytotoxicity (ADCC) is greatly enhanced by the absence of the core fucose of oligosaccharides attached to the Fc, and is closely related to the clinical efficacy of anticancer activity in humans in vivo. Unfortunately, all licensed therapeutic antibodies and almost all currently-developed therapeutic antibodies are heavily fucosylated and fail to optimize ADCC, which leads to a large dose requirement at a very high cost for the administration of antibody therapy to cancer patients. In this study, we explored the possibility of converting already-established antibody-producing cells to cells that produce antibodies fully lacking core fucosylation in order to facilitate the rapid development of next-generation therapeutic antibodies.