Volatile Components of Quince Fruit (Cydonia oblonga Mill.)

Steam distilled oil of quince fruit (Cydonia oblonga Mill.=C. vulgaris Pers., marmelo in Japanese) was analyzed by gas chromatography and gas chromatography-mass spectrometry. Sixty-two compounds, 2 hydrocarbons, 13 esters, 11 alcohols, 11 aldehydes, 11 ketones, 5 lactones and 9 miscellaneous compounds, were identified. Of them, the chemical structures of two new oxide compounds, trans- and cis-3-methyl-5-[(E)-3′-methyl-13′-butadien-1′-yl]tetrahydrofuran, were elucidated by instrumental analyses.     

Rat gastric mucins recognized by monoclonal antibodies RGM21 and HIK1083: isolation of mucin species characteristic of the surface and glandular mucosa.

Whole mucins and reduced subunits were extracted from the corpus of the rat stomach. After purification by Sepharose CL-4B chromatography followed by cesium trifluoroacetate equilibrium centrifugation, they were analyzed by Sepharose CL-2B chromatography, rate-zonal sedimentation centrifugation, and Q-Sepharose chromatography. Monoclonal antibodies RGM21 and HIK1083, which histochemically stained mucins in the surface and glandular mucosa of the rat stomach, respectively, were used to detect the site-specific mucins. Although RGM21- and HIK1083-reactive mucins both had a multimerized structure, the density and size of both the whole mucins and reduced subunits differed, thus indicating the presence of distinct mucin species in the surface and glandular mucosa. The mucin subunits were separated into four fractions, UB, B1, B2a, and B2b, by Q-Sepharose chromatography. HIK1083 reacted mainly with UB, while RGM21 reacted with B1, B2a, and B2b. These results, combined with dot-blot, amino acid, and carbohydrate composition analyses, showed that the surface mucins may consist of three kinds of subunits. In contrast, the glandular mucins may consist of one kind of subunit which differs from that of surface mucins.     

Calyculin A and okadaic acid: inhibitors of protein phosphatase activity

Calyculin A and okadaic acid induce contraction in smooth muscle fibers. Okadaic acid is an inhibitor of phosphatase activity and the aims of this study were to determine if calyculin A also inhibits phosphatase and to screen effects of both compounds on various phosphatases. Neither compound inhibited acid or alkaline phosphatases, nor the phosphotyrosine protein phosphatase. Both compounds were potent inhibitors of the catalytic subunit of type-2A phosphatase, with IC50 values of 0.5 to 1 nM. With the catalytic subunit of protein phosphatase type-1, calyculin A was a more effective inhibitor than okadaic acid, IC50 values for calyculin A were about 2 nM and for okadaic acid between 60 and 500 nM. The endogenous phosphatase of smooth muscle myosin B was inhibited by both compounds with IC50 values of 0.3 to 0.7 nM and 15 to 70 nM, for calyculin A and okadaic acid, respectively. The partially purified catalytic subunit from myosin B had IC50 values of 0.7 and 200 nM for calyculin A and okadaic acid, respectively. The pattern of inhibition for the phosphatase in myosin B therefore is similar to that of the type-1 enzyme.     

Two distinct Vps34 phosphatidylinositol 3-kinase complexes function in autophagy and carboxypeptidase Y sorting in Saccharomyces cerevisiae

Vps30p/Apg6p is required for both autophagy and sorting of carboxypeptidase Y (CPY). Although Vps30p is known to interact with Apg14p, its precise role remains unclear. We found that two proteins copurify with Vps30p. They were identified by mass spectrometry to be Vps38p and Vps34p, a phosphatidylinositol (PtdIns) 3-kinase. Vps34p, Vps38p, Apg14p, and Vps15p, an activator of Vps34p, were coimmunoprecipitated with Vps30p. These results indicate that Vps30p functions as a subunit of a Vps34 PtdIns 3-kinase complex(es). Phenotypic analyses indicated that Apg14p and Vps38p are each required for autophagy and CPY sorting, respectively, whereas Vps30p, Vps34p, and Vps15p are required for both processes. Coimmunoprecipitation using anti-Apg14p and anti-Vps38p antibodies and pull-down experiments showed that two distinct Vps34 PtdIns 3-kinase complexes exist: one, containing Vps15p, Vps30p, and Apg14p, functions in autophagy and the other containing Vps15p, Vps30p, and Vps38p functions in CPY sorting. The vps34 and vps15 mutants displayed additional phenotypes such as defects in transport of proteinase A and proteinase B, implying the existence of another PtdIns 3-kinase complex(es). We propose that multiple Vps34p-Vps15p complexes associated with specific regulatory proteins might fulfill their membrane trafficking events at different sites.     

Epigenetic mark sequence of the H19 gene in human sperm

We have investigated the epigenetic mark in the human H19 gene. The H19 promoter is methylation-free in human sperm, but it is methylated in the paternally derived allele of most adult tissues. Consequently, the H19 gene is exclusively transcribed from the maternal allele. It was demonstrated that the differentially methylated region (DMR) located 2 kb upstream from mouse H19 is essential for the imprinting of H19. A 39 bp sequence in DMR has a high degree of similarity between humans, mice and rats. The highly conserved 15 bp core region of the consensus sequence contains four methylatable sites, and thus has been proposed as a potential imprinting mark region. In this study, fine epigenetic sequencing analysis was performed on the sperm DNA in comparison with other adult organs. Interestingly, the conserved sequence of the potential mark region was methylated in almost all the sperm genomes analyzed. Furthermore, the single dinucleotide CpG, whose methylation affects the accessibility of the element to CTCF, was methylated in the conserved core in the human sperm. These results suggest that the human core sequences may act as an imprinting center in the reciprocal monoallelic expression of H19.     

Characterization of expression, and cloning, of beta-D-xylosidase and alpha-L-arabinofuranosidase in developing and ripening tomato (Lycopersicon esculentum Mill.) fruit

Modifications to the cell wall of developing and ripening tomato fruit are mediated by cell wall-degrading enzymes, including a beta-d-xylosidase or alpha-l-arabinofuranosidase, which participate in the breakdown of xylans and/or arabinoxylans. The activity of both enzymes was highest during early fruit growth, before decreasing during later development and ripening. Two beta-d-xylosidase cDNAs, designated LeXYL1 and LeXYL2, and an alpha-l-arabinofuranosidase cDNA, designated LeARF1, were obtained. Accumulation of mRNAs for beta-d-xylosidase and alpha-l-arabinofuranosidase was examined during fruit development and ripening. LeARF1 and LeXYL2 genes were relatively highly expressed during fruit development and decreased after the onset of ripening. By contrast, LeXYL1 was not expressed during fruit development, but was expressed later, particularly during over-ripening. The expression of all three genes was also followed in ripening-impaired mutants, Nr, Nr2, nor, and rin of cv. Ailsa Craig fruit. LeXYL2 mRNA was detected in the ripe fruits of all the mutants and its abundance was similar to that in mature green wild-type fruit. By contrast, LEXYL1 mRNA was expressed only in the ripe fruits of the Nr mutant, suggesting that the two beta-d-xylosidase genes are subject to distinct regulatory control during fruit development and ripening. LeARF1 mRNA was detected in ripe fruits of Nr2, nor and rin, and not in ripe fruit of the Nr mutant. The accumulation of LeARF1 in ripe fruit was restored by 1-methylcyclopropene (1-MCP), an inhibitor of ethylene action, while 1-MCP had no effect on the expression of LeXYL1 or LeXYL2. This suggests that LeARF1 expression is subject to negative regulation by ethylene and that the two beta-d-xylosidase genes are independent of ethylene action.     

Cell-penetrating peptide-conjugated lipid nanoparticles for siRNA delivery

Lipid nanoparticles (LNP) modified with cell-penetrating peptides (CPP) were prepared for the delivery of small interfering RNA (siRNA) into cells. Lipid derivatives of CPP derived from protamine were newly synthesized and used to prepare CPP-decorated LNP (CPP-LNP). Encapsulation of siRNA into CPP-LNP improved the stability of the siRNA in serum. Fluorescence-labeled siRNA formulated in CPP-LNP was efficiently internalized into B16F10 murine melanoma cells in a time-dependent manner, although that in LNP without CPP was hardly internalized into these cells. In cells transfected with siRNA in CPP-LNP, most of the siRNA was distributed in the cytoplasm of these cells and did not localize in the lysosomes. Analysis of the endocytotic pathway indicated that CPP-LNP were mainly internalized via macropinocytosis and heparan sulfate-mediated endocytosis. CPP-LNP encapsulating siRNA effectively induced RNA interference-mediated silencing of reporter genes in B16F10 cells expressing luciferase and in HT1080 human fibrosarcoma cells expressing enhanced green fluorescent protein. These data suggest that modification of LNP with the protamine-derived CPP was effective to facilitate internalization of siRNA in the cytoplasm and thereby to enhance gene silencing.     

Effects of co-administration of estrogen and androgen on induction of sex reversal in the medaka Oryzias latipes

To investigate how estrogen and androgen affect each other in inducing sex reversal in the medaka, O. Iatipes, 17β-estradiol (E2) and 17α-methyldihydrotestosterone (MDHT) were co-administered by a convenient method for hormonal treatment, in which freshly fertilized eggs were immersed for 24 h in saline containing either or both of the two sex steroids in different concentrations and/or ratios. The minimal concentrations of E2 and MDHT sufficient to induce the maximal rate of sex reversal from male to female and from female to male were 500 ng/ml and 2.5 ng/ml, respectively, both of which were referred to as the most efficacious dose (MED), and each equivalent for the inducing potency in sex reversal. E2 and MDHT, when simultaneously administered at MED, greatly suppressed each other to induce each corresponding sex reversal. Thus, the present experimental results indicate that E2 and DMHT are antagonists that induce corresponding sex reversal, and suggest that genotypic sex in the medaka might be modified through an unknown factor of common affinity to both sex steroids, by which the pathway of differentiation of either sex could be switched at the early stages of development far before gonadal sex differentiation.     

Community-wide assessment of protein-interface modeling suggests improvements to design methodology

The CAPRI (Critical Assessment of Predicted Interactions) and CASP (Critical Assessment of protein Structure Prediction) experiments have demonstrated the power of community-wide tests of methodology in assessing the current state of the art and spurring progress in the very challenging areas of protein docking and structure prediction. We sought to bring the power of community-wide experiments to bear on a very challenging protein design problem that provides a complementary but equally fundamental test of current understanding of protein-binding thermodynamics. We have generated a number of designed protein-protein interfaces with very favorable computed binding energies but which do not appear to be formed in experiments, suggesting that there may be important physical chemistry missing in the energy calculations. A total of 28 research groups took up the challenge of determining what is missing: we provided structures of 87 designed complexes and 120 naturally occurring complexes and asked participants to identify energetic contributions and/or structural features that distinguish between the two sets. The community found that electrostatics and solvation terms partially distinguish the designs from the natural complexes, largely due to the nonpolar character of the designed interactions. Beyond this polarity difference, the community found that the designed binding surfaces were, on average, structurally less embedded in the designed monomers, suggesting that backbone conformational rigidity at the designed surface is important for realization of the designed function. These results can be used to improve computational design strategies, but there is still much to be learned; for example, one designed complex, which does form in experiments, was classified by all metrics as a nonbinder.     

Adoption of alternative migratory tactics: a view from the ultimate mechanism and threshold trait changes in a salmonid fish

Partial migration, in which a portion of the population migrates while the rest of the population remains as residents, is a common form of migration. Alternative migratory tactics (AMTs) of partial migration are often determined by polygenic threshold traits. However, the ultimate mechanisms that drive inter‐population variations in threshold traits are not well understood. We present a simple schematic model to explain how the threshold trait changes with fitness consequences under opposing natural and artificial selection forces. We conducted a field test to evaluate the effects of migration difficulty (as a natural selective force) and selective captive breeding (as an artificial selective force) on threshold traits of a partially migratory fish. Male masu salmon Oncorhynchus masou in the Shari River system have AMTs divided into three population categories of hatchery, wild/above the waterfall, and wild/below the waterfall (control). The wild/above the waterfall salmon live in a high‐migration‐cost situation, and the threshold trait changed in a direction that promoted residency. In hatchery salmon, which are produced by migrant‐selective captive breeding, the threshold trait changed in a direction that promoted migration. In contrast, Dolly Varden charr Salvelinus malma displayed only resident tactics, and the threshold trait did not differ between the populations above and below the waterfall, indicating that environment did not explain the variation in the threshold trait. Our results support the model and suggest that opposing natural and artificial selection forces drive variations in the threshold traits and migratory patterns in the studied species. Our conceptual framework for the ultimate mechanism may help to better understand adoption of AMTs and production of diverse intraspecific traits in migratory animals.