Base-pairing requirement for RNA silencing by a bacterial small RNA and acceleration of duplex formation by Hfq

SgrS is an Hfq-binding small antisense RNA that is induced upon phosphosugar stress. It forms a ribonucleoprotein complex with RNase E through Hfq to mediate silencing of the target ptsG mRNA encoding the membrane component of the glucose-specific phosphoenolpyruvate phosphotransferase system. Although SgrS is believed to act on ptsG mRNA through base pairing between complementary regions, this was not previously tested experimentally. We addressed the question of whether SgrS indeed forms an RNA-RNA duplex with ptsG mRNA to exert its regulatory function. Specific single nucleotide substitutions around the Shine-Dalgarno (SD) sequence of ptsG completely eliminated SgrS action while compensatory mutations in SgrS restored it. A systematic mutational analysis of both ptsG and SgrS RNAs revealed that six base pairs around SD sequence of ptsG are particularly important for SgrS action. We also showed in vitro that SgrS forms a stable duplex with the ptsG mRNA, and that Hfq markedly facilitates the rate of duplex formation.     

Complex genetic nature of sex-independent transmission ratio distortion in Asian rice species: the involvement of unlinked modifiers and sex-specific mechanisms

Transmission ratio distortion (TRD), in which one allele is transmitted more frequently than the opposite allele, is presumed to act as a driving force in the emergence of a reproductive barrier. TRD acting in a sex-specific manner has been frequently observed in interspecific and intraspecific hybrids across a broad range of organisms. In contrast, sex-independent TRD (siTRD), which results from preferential transmission of one of the two alleles in the heterozygote through both sexes, has been detected in only a few plant species. We previously reported an S(6) locus-mediated siTRD, in which the S(6) allele from an Asian wild rice strain (Oryza rufipogon) was transmitted more frequently than the S(6)(a) allele from an Asian cultivated rice strain (O. sativa) through both male and female gametes in heterozygous plants. Here, we report on the effect of a difference in genetic background on S(6) locus-mediated siTRD, based on the analysis using near-isogenic lines and the original wild strain as a parental strain for crossing. We found that the degree of TRD through the male gametes varied depending on the genetic background of the female (pistil) plants. Despite the occurrence of TRD through both male and female gametes, abnormality was detected in ovules, but not in pollen grains, in the heterozygote. These results suggest the involvement of unlinked modifiers and developmentally distinct, sex-specific genetic mechanisms in S(6) locus-mediated siTRD, raising the possibility that siTRD driven by a single locus may be affected by multiple genetic factors harbored in natural populations.     

A Lectin from the Mussel Mytilus galloprovincialis Has a Highly Novel Primary Structure and Induces Glycan-mediated Cytotoxicity of Globotriaosylceramide-expressing Lymphoma Cells

A novel lectin structure was found for a 17-kDa α-d-galactose-binding lectin (termed “MytiLec”) isolated from the Mediterranean mussel, Mytilus galloprovincialis. The complete primary structure of the lectin was determined by Edman degradation and mass spectrometric analysis. MytiLec was found to consist of 149 amino acids with a total molecular mass of 16,812.59 Da by Fourier transform-ion cyclotron resonance mass spectrometry, in good agreement with the calculated value of 16,823.22 Da. MytiLec had an N terminus of acetylthreonine and a primary structure that was highly novel in comparison with those of all known lectins in the structure database. The polypeptide structure consisted of three tandem-repeat domains of ∼50 amino acids each having 45–52% homology with each other. Frontal affinity chromatography technology indicated that MytiLec bound specifically to globotriose (Gb3; Galα1–4Galβ1–4Glc), the epitope of globotriaosylceramide. MytiLec showed a dose-dependent cytotoxic effect on human Burkitt lymphoma Raji cells (which have high surface expression of Gb3) but had no such effect on erythroleukemia K562 cells (which do not express Gb3). The cytotoxic effect of MytiLec was specifically blocked by the co-presence of an α-galactoside. MytiLec treatment of Raji cells caused increased binding of anti-annexin V antibody and incorporation of propidium iodide, which are indicators of cell membrane inversion and perforation. MytiLec is the first reported lectin having a primary structure with the highly novel triple tandem-repeat domain and showing transduction of apoptotic signaling against Burkitt lymphoma cells by interaction with a glycosphingolipid-enriched microdomain containing Gb3.     

Selection for reluctance to avoid humans during the domestication of mice

Many animal species have been domesticated over the course of human history and became tame as a result of domestication. Tameness is a behavioral characteristic with 2 potential components: (1) reluctance to avoid humans and (2) motivation to approach humans. However, the specific behavioral characteristics selected during domestication processes remain to be clarified for many species. To quantify these 2 different components of tameness separately, we established 3 behavioral tests: the ‘active tame’, ‘passive tame’ and ‘stay‐on‐hand’ tests. We subjected genetically diverse mouse strains to these tests, including 10 wild strains (BFM/2Ms, PGN2/Ms, HMI/Ms, BLG2/Ms, NJL/Ms, KJR/Ms, SWN/Ms, CHD/Ms, MSM/Ms and CAST/Ei), a fancy strain (JF1/Ms) and 6 standard laboratory strains (C3H/HeNJcl, CBA/J, BALB/cAnNCrlCrlj, DBA/2JJcl, 129^+Ter/SvJcl and C57BL/6JJcl). To analyze the effects of domestication, these 17 strains were divided into 2 groups: domesticated strains (fancy and laboratory strains) and wild strains. Significant differences between strains were observed in all traits, and the calculated estimates of broad‐sense heritability were 0.15–0.72. These results illustrate that tameness in mice is significantly influenced by genetic background. In addition, they clearly show the differences in the features of tameness in domesticated and wild strains. Most of the domesticated strains showed significantly greater reluctance to avoid humans than wild strains, whereas there was no significant difference in the level of motivation to approach humans between these 2 groups. These results might help to clarify the genetic basis of tameness in mice .     

Synthesis and biological evaluation of C-glucosides with azulene rings as selective SGLT2 inhibitors for the treatment of type 2 diabetes mellitus: Discovery of YM543

Here, a series of C-glucosides with azulene rings in the aglycon moiety was synthesized and the inhibitory activities toward hSGLT1 and hSGLT2 were evaluated. Starting from the azulene derivative 7 which had relatively good SGLT2 inhibitory activity, compound 8a which has a 3-[(azulen-2-yl)methyl]phenyl group was identified as a lead compound for further optimization. Introduction of a phenolic hydroxyl group onto the central benzene ring afforded a potent and selective SGLT2 inhibitor 8e, which reduced blood glucose levels in a dose-dependent manner in rodent diabetic models. A mono choline salt of 8e (YM543) was selected as a clinical candidate for use in treating type 2 diabetes mellitus.     

Detection of novel blast resistance genes, Pi58(t) and Pi59(t), in a Myanmar rice landrace based on a standard differential system

The use of broad-spectrum R genes is an effective way to achieve durable resistance against rice blast (Magnaporthe oryzae Couch, anamorph: Pyricularia oryzae Cavara) in rice (Oryza sativa L.). We previously surveyed the diversity of blast resistance in 948 rice varieties and found a Myanmar rice landrace, Haoru (International Rice Research Institute genebank acc. no. IRGC33090), with broad-spectrum resistance against the standard differential blast isolates. Here, we examined the genetic basis of Haoru’s broad-spectrum resistance by using the standard blast differential system consisting of the standard isolates and differential varieties. For genetic analysis, we used a BC1F1 population and BC1F2 lines derived from crosses of Haoru with a susceptible variety, US-2. Co-segregation analysis of the reaction pattern in the BC1F1 population against the 20 standard isolates suggested that Haoru harbors three R genes. By using bulk-segregant and linkage analysis, we mapped two of the three R genes on chromosomes 12 and 6, and designated them as Pi58(t) and Pi59(t), respectively. Pi58(t) and Pi59(t) were differentiated from other reported R genes using the standard differential system. The estimated resistance spectrum of Pi58(t) corresponded with that of Haoru, suggesting that Pi58(t) is primarily responsible for Haoru’s broad-spectrum resistance. In addition, Pi59(t) and the third gene were also proven to be new and useful genetic resources for studying and improving blast resistance in rice.     

Pigment epithelium-derived factor (PEDF) shares binding sites in collagen with heparin/heparan sulfate proteoglycans

Pigment epithelium-derived factor (PEDF) is a collagen-binding protein that is abundantly distributed in various tissues, including the eye. It exhibits various biological functions, such as anti-angiogenic, neurotrophic, and neuroprotective activities. PEDF also interacts with extracellular matrix components such as collagen, heparan sulfate proteoglycans (HSPGs), and hyaluronan. The collagen-binding property has been elucidated to be important for the anti-angiogenic activity in vivo (Hosomichi, J., Yasui, N., Koide, T., Soma, K., and Morita, I. (2005) Biochem. Biophys. Res. Commun. 335, 756-761). Here, we investigated the collagen recognition mechanism by PEDF. We first narrowed down candidate PEDF-binding sequences by taking advantage of previously reported structural requirements in collagen. Subsequent searches for PEDF-binding sequences employing synthetic collagen-like peptides resulted in the identification of one of the critical binding sites for PEDF, human α1(I)(929-938) (IKGHRGFSGL). Further analysis revealed that the collagen recognition by PEDF is sequence- and conformation-specific, and the high affinity binding motif is KGXRGFXGL in the triple helix. The PEDF-binding motif significantly overlapped with the heparin/HSPG-binding motif, KGHRG(F/Y). The interaction of PEDF with collagen I was specifically competed with by heparin but not by chondroitin sulfate-C or hyaluronan. The binding sequences for PEDF and heparin/HSPG also overlapped with the covalent cross-linking sites between collagen molecules. These findings imply a functional relationship between PEDF and HSPGs during angiogenesis, and the interaction of these molecules is regulated by collagen modifications.