X-ray single-crystal diffraction (SCD) analysis has the intrinsic limitation that the target molecules must be obtained as single crystals. Here we report a protocol for SCD analysis that does not require the crystallization of the sample. In our method, tiny crystals of porous complexes are soaked in a solution of the target, such that the complexes can absorb the target molecules. Crystallographic analysis clearly determines the absorbed guest structures along with the host frameworks. Because the SCD analysis is carried out on only one tiny crystal of the complex, the required sample mass is of the nanogram-microgram order. We demonstrate that as little as about 80 nanograms of a sample is enough for the SCD analysis. In combination with high-performance liquid chromatography, our protocol allows the direct characterization of multiple fractions, establishing a prototypical means of liquid chromatography SCD analysis. Furthermore, we unambiguously determined the structure of a scarce marine natural product using only 5 micrograms of the compound.

Abnormal activation the WNT/β-catenin signaling pathway has been associated with ovarian carcinomas, but a specific WNT ligand and pertinent downstream mechanisms are not fully understood. In this study, we found abundant WNT7A in the epithelium of serous ovarian carcinomas, but not detected in borderline and benign tumors, normal ovary or endometrioid carcinomas. To characterize the role of WNT7A in ovarian tumor growth and progression, nude mice were injected either intraperitoneally (i.p.) or subcutaneously (s.c.) with WNT7A knocked down SKOV3.ip1 and overexpressed SKOV3 cells. In the i.p. group, mice receiving SKOV3.ip1 cells with reduced WNT7A expression developed significantly fewer tumor lesions. Gross and histological examination revealed greatly reduced invasion of WNT7A knockdown cells into intestinal mesentery and serosa compared to the control cells. Tumor growth was regulated by loss or overexpression of WNT7A in mice receiving s.c. injection as well. In vitro analysis of cell function revealed that cell proliferation, adhesion, and invasion were regulated by WNT7A. The activity of the TCF/LEF reporter was stimulated by overexpression of WNT7A in ovarian cancer cells. Co-transfection with WNT7A and FZD5 receptor further increased activity, and this effect was inhibited by co-transfection with SFRP2, or dominant-negative TCF4. Overexpression of WNT7A stimulated MMP7 promoter, and mutation of TCF binding sites in MMP7 promoter confirmed that activation of MMP7 promoter by WNT7A was mediated by β-catenin/TCF signaling. Collectively, these results suggest that re-expression of WNT7A during malignant transformation of ovarian epithelial cells plays a critical role in ovarian cancer progression mediated by WNT/β-catenin signaling pathway.

We recently reported a new method for single-crystal X-ray diffraction (SCD) analysis that does not require the crystallization of the target compound. In this 'crystal-free' crystallography, a tiny crystal of a porous complex is soaked in the solution of the target guest. The guest molecules are absorbed and oriented in the crystal pores and can be analyzed by X-ray diffraction. We describe here a detailed synthetic protocol for the preparation of uniform single crystals of the porous host complex and for the subsequent guest uptake. The protocol describes our most versatile porous complex, which is prepared from commercially available ZnI2 and 2,4,6-tri(4-pyridyl)-1,3,5-triazine. The host complex has large pores with a cross-section of 8 × 5 Å(2). Single crystals of the complex are grown from layered solutions of the two components. The pores of the as-synthesized complex are filled with nitrobenzene, which is replaced with the inert solvent cyclohexane. This solvent exchange is essential for the rapid and effective inclusion of target compounds. The most crucial and delicate step is the selection of high-quality single crystals from the mixture of crystals of various shapes and sizes. We suggest using the facial indices of the single crystals as a criterion for crystal selection. Single-crystal samples for X-ray analysis can be prepared by immersing the selected crystals in a cyclohexane/dichloromethane solution of target compound. After a very slow evaporation of the solvent, typically over 2 d, the final crystal can be picked and directly subjected to SCD analysis. The protocol can be completed within ∼16 d.

The WNTs are secreted proteins that control essential developmental processes, such as embryonic patterning, cell growth, migration, and differentiation. In mice, three members of the Wnt gene family (Wnt4, Wnt5a, and Wnt7a) have been studied extensively in the female reproductive tract. The present study determined effects of postnatal day and exposure to diethylstilbestrol (DES) on Wnt and Fzd gene expression in the mouse uterus as well as the biological role of Wnt11 in postnatal mouse uterine development and function. Wnt4, Wnt5a, Wnt7a, Wnt7b, Wnt11, Wnt16, Fzd6, and Fzd10 were detected by in situ hybridization in the neonatal mouse uterus. In situ hybridization analyses revealed that Wnt4, Wnt5a, and Wnt16 were localized in the endometrial stroma, whereas Wnt7a, Wnt7b, Wnt11, Fzd6, and Fzd10 were in the uterine epithelia of neonatal mice. Exposure of mice to estrogen or estrogen receptor agonists during critical development periods inhibits endometrial adenogenesis. In the present study, DES-induced disruption of endometrial gland development was associated with reduction or suppression of Wnt4, Wnt5a, Wnt7a, Wnt11, Wnt16, and Fzd10. Ablation of Wnt11, an epithelial-expressed, DES-regulated gene, in the neonatal uterus did not affect endometrial adenogenesis or expression of other Wnt genes. Interestingly, Wnt11-deleted uteri had more endometrial glands on Postnatal Day 10. Although CTNNB1 expression was not affected by ablation of Wnt11, Vangl2 was inhibited in the uteri of Wnt11(d/d) mice. These results support the idea that a number of different Wnt genes are potential regulators for uterine morphogenesis; however, Wnt11 does not have a direct effect on uterine development.

The absolute structure determination of compounds with axial and planar chirality obtained by recently developed asymmetric syntheses was achieved using the crystalline sponge method without using any reference compounds or synthetic modifications.

Superior manual dexterity in higher primates emerged together with the appearance of cortico-motoneuronal (CM) connections during the evolution of the mammalian corticospinal (CS) system. Previously thought to be unique to higher primates, we identified transient CM connections in early postnatal mice, which are eventually eliminated by Sema6D-PlexA1 signaling. PlexA1 mutant mice maintain CM connections into adulthood and exhibit superior manual dexterity compared to controls. Finally, differing PlexA1 expression in layer 5 of the motor cortex, which is strong in wild-type mice but weak in humans, may be explained by FEZF2-mediated cis-regulatory elements that are found only in higher primates. Thus, species-dependent regulation of PlexA1 expression may have been crucial in the evolution of mammalian CS systems that improved fine motor control in higher primates.

The CRISPR/Cas9 system has been used for spatio-temporal gene modification through the ubiquitous expression of gRNA by an RNA polymerase III promoter and the controlled expression of Cas9 using a tissue-specific or inducible promoter. However, unexpected gene disruptions indicate the necessity of a tissue-specific or inducible expression of not only Cas9 but also gRNA. In the present study, we attempted to develop a CRISPR/Cas9 system that could express functional gRNAs and Cas9 by a single RNA polymerase II promoter and induce multi-loci disruptions in specific cells. To this end, we designed vectors expressing ribozyme-flanked gRNAs (RGRs) and Cas9 mRNAs simultaneously. We showed that the mono-promoter-driven vector induces gene disruptions at the target loci in HEK 293 cells after transfection. In addition, two target loci were disrupted simultaneously by the transfection of a mono-promoter-driven vector expressing two RGRs and Cas9 mRNA. Finally, we constructed a universal vector for use in the construction of plasmids to be applied to the present mono-promoter-driven CRISPR/Cas9 system. We have thus provided a versatile tool for generating gene disruptions by the CRISPR/Cas9 system; this system should contribute to a wide range of investigations, including studies on spatio-temporal gene functions.

j-Glucosidase was purified from a crude cellulase preparation from Aspergillus niger by affinity chromatography on a methacrylamide-N-methylene-his-methacrylamide copolymer bearing cellobiamine. The purified enzyme was a dimer with an isoelectric point of 4.0. The molecular mass of the enzyme was estimated to be 240 kDa by gel-permeation chromatography. The enzyme hydrolyzed specificically P-glucosidic bonds and catalyzed transglucosylation of the p-glucosyl group of cellobiose to yield 4-0-j-gentiobiosylglucose in the presence of organic solvents or under neutral conditions. The fungus Aspergillus niger excretes a wide range of carbohydrate-hydrolyzing enzymes. Various techniques, including size-exclusion, hydroxyapatite and ion-exchange chromatography, have been applied to the fractionation and purification of each enzyme from the multicomponent enzyme system. However, only limited success has been achieved with the purification of /Y-glucosidase from culture filtrates [l -71. So far, it has been thought that the substrate specificity of A . niger j-glucosidase was not strict for the fl-D-gluco configuration. For Instance, Tavobilov reported that p-glucosidase from A . niger hydrolyzed not only P-D-glucosides, but also j-D-xylosides, P-D-galactosides and r-L-arabinosides [l]. Later, McCleary et al. purified A . niger j-glucosidase 66.7-fold by gel-filtration and ion-exchange chromatography and reported that the enzyme hydrolyzed P-glucosidic bonds almost specifically but contaminating 8-xylosidase and /Igalactosidase activities still remained in the enzyme preparation [2]. Thus, the substrate specificity of A. niger p-glucosidase has differed depending on the extent of purification. Regarding the transglycosylation of this enzyme, Barker et al. reported that culture filtrates of A . niger catalyzed transglycosylation of the /I-glucosyl group of cellobiose to another cellobiose to form trisacchandes, and the principal linkages synthesized were Pl-6. However, Pl-2, j l -3 and pl-4 linkages were also formed, suggesting that j-glucosidase catalyzed the transglucosylation of p-glucosyl group [8]. However, catalytic activities of the purified enzyme have not been reported. In this study, we aimed at characterizing the substrate specificity and transglycosilation of A . niger P-glucosidase using a highly purified enzyme. We have, therefore, developed an affinity chromatography on a methacrylamide-N-methylene-bismethacrylamide copolymer bearing cellobiamine which interacts with subsites of P-glucosidases. Purification and properties of the P-glucosidase are reported here.Correspondence to T. Watanabe, Wood Research Institute, Kyoto University, Gokasho, Uji, Kyoto, Japan, 611Ahhreviutions. pNpp, p-nitrophenyl; CH,CN, acetonitrile; MeOH, methyl alcohol; EtOH, ethyl alcohol; Me2S0, dimethglsulroxide; DMF, N,N-dimethylformamide.Enzyme. &glucosidase (EC 3.2.1.21). MATERIALS AND METHODS MaterialsSephadex G-75 and PBE-94 were obtained from Pharmacia. SP-Toyopearl was obtained from Toso. Cellulosin AC-40 was obtained from Ued...