Flow cytometric analysis of lectin-oligosaccharide interactions by using fluorescent oligosaccharide probes

Tetraphenylethylene (TPE) derivatives have strong fluorescence in aggregated state. We report here an oligosaccharide binding assay system using tetraphenylethylene derivatives bearing oligosaccharides with aggregation-induced emission (AIE) characteristics. A tetraphenylethylene derivative bearing 6'-sialyllactose (6'SL) bound to microbeads coated with SSA lectin more effectively than RCA120 lectin. Microbeads that bound to fluorescent oligosaccharide probes could be detected by flow cytometric analysis. Tetraphenylethylene derivatives bearing oligosaccharides are useful for flow cytometric analysis of lectin-oligosaccharide interactions.     

The molecular mechanism of membrane proteins probed by evanescent infrared waves

The catalytic action of membrane proteins is vital to many cellular processes. Yet the molecular mechanisms remain poorly understood. We describe here the technique of evanescent infrared difference spectroscopy as a tool to decipher the structural changes associated with the enzymatic action of membrane proteins. Functional changes as minute as the protonation state of individual amino acid side chains can be observed and linked to interactions with a ligand, agonist, effector, or redox partner.     

Identification of Receptor Tyrosine Kinase, Discoidin Domain Receptor 1 (DDR1), as a Potential Biomarker for Serous Ovarian Cancer

Ovarian cancer, one of the most common gynecological malignancies, has an aggressive phenotype. It is necessary to develop novel and more effective treatment strategies against advanced disease. Protein tyrosine kinases (PTKs) play an important role in the signal transduction pathways involved in tumorigenesis, and represent potential targets for anticancer therapies. In this study, we performed cDNA subtraction following polymerase chain reaction (PCR) using degenerate oligonucleotide primers to identify specifically overexpressed PTKs in ovarian cancer. Three PTKs, janus kinase 1, insulin-like growth factor 1 receptor, and discoidin domain receptor 1 (DDR1), were identified and only DDR1 was overexpressed in all ovarian cancer tissues examined for the validation by quantitative real-time PCR. The DDR1 protein was expressed in 63% (42/67) of serous ovarian cancer tissue, whereas it was undetectable in normal ovarian surface epithelium. DDR1 was expressed significantly more frequently in high-grade (79%) and advanced stage (77%) tumors compared to low-grade (50%) and early stage (43%) tumors. The expression of the DDR1 protein significantly correlated with poor disease-free survival. Although its functional role and clinical utility remain to be examined in future studies, our results suggest that the expression of DDR1 may serve as both a potential biomarker and a molecular target for advanced ovarian cancer.     

Photoinduced increase in surfactant solution viscosity using azobenzene dicarboxylate for molecular switching

We provide further insight into the photochemical control of viscoelasticity through the use of azobenzene sodium dicarboxylate for molecular switching. As a photoresponsive molecule, Sodium 3,3'-azobenzene dicarboxylate (3,3'-Azo2Na) was added to a solution of cetyltrimethylammonium bromide (CTAB)/sodium salicylate (NaSal), which is known for inducing the formation of wormlike micelles. This solution maintained a wormlike micellar structure, although a reduction in zero-shear viscosity was observed. When this mixed aqueous solution of CTAB/NaSal/3,3'-Azo2Na (16.7 mM each) was irradiated by ultraviolet light, the 3,3'-Azo2Na exhibited molecular trans-cis photoisomerization. We measured the dynamic viscoelasticity of the sample in the photostationary state and found that the zero-shear viscosity increased approximately sevenfold compared to the preirradiation state. This phenomenon is the opposite of the system wherein viscosity reduced by irradiation, as reported by us. We discuss the mechanism of this viscosity change.     

Viscoelastic behavior of poly(butyl methacrylate) densely grafted on silica nanoparticles

A series of poly(butyl methacrylate)s (PBMAs) with various molar masses (33 000–270 000 g mol^?1), which were densely grafted on fumed silica nanoparticles (PBMA–SiO₂), were synthesized by surface‐initiated atom transfer radical polymerization. The dynamic viscoelastic behavior of PBMA–SiO₂ was systematically investigated in the solid and molten states with oscillatory strains, and compared to that of a conventional nanocomposite (PBMA/SiO₂). The storage moduli of PBMA–SiO₂ and PBMA/SiO₂ are equivalent in the solid state, whereas the storage modulus of PBMA–SiO₂ is lower than that of PBMA/SiO₂ in the molten state, especially at high silica loading. This is because the formation of a network structure composed of the silica nanoparticles in PBMA–SiO₂ is strongly suppressed by the polymer brushes on the particles. In contrast, even at low silica loading, the PBMA–SiO₂ system exhibits a gel‐like behavior resulting from a steric repulsion between the composite particles, because all of the tethered polymers behave as bound polymers. Copyright © 2011 Society of Chemical Industry     

Role of Metabolism in Bone Development and Homeostasis

Carbohydrates, fats, and proteins are the underlying energy sources for animals and are catabolized through specific biochemical cascades involving numerous enzymes. The catabolites and metabolites in these metabolic pathways are crucial for many cellular functions; therefore, an imbalance and/or dysregulation of these pathways causes cellular dysfunction, resulting in various metabolic diseases. Bone, a highly mineralized organ that serves as a skeleton of the body, undergoes continuous active turnover, which is required for the maintenance of healthy bony components through the deposition and resorption of bone matrix and minerals. This highly coordinated event is regulated throughout life by bone cells such as osteoblasts, osteoclasts, and osteocytes, and requires synchronized activities from different metabolic pathways. Here, we aim to provide a comprehensive review of the cellular metabolism involved in bone development and homeostasis, as revealed by mouse genetic studies.     

Photolytic crosslinking of poly(p-hydroxystyrene)

Light-induced crosslinking of poly(p-hydroxystyrene) (PPHS) is significantly enhanced by O₂. This was evidenced by molar mass (light scattering measurements) and by gel content determinations which were performed on various polymer samples before and after continuous irradiation at λ_inc = 254 nm. The following mechanism was elucidated with the aid of flash photolysis studies: Crosslinking in the absence or presence of O₂ is mainly due to the combination of phenoxyl type radicals. In the absence of O₂ the latter are exclusively formed by O? H bond cleavage of singlet excited phenolic groups. Triplet excited phenolic groups which are also formed do not deactivate via O? H bond cleavage but react very effectively with O₂. This reaction leads to the formation of HO and additional phenoxyl type radicals. All Commercial and most laboratory-prepared PPHS samples contain chemically bound impurities of quinoid nature. On the basis of results performed with model compounds of low molar mass, it is concluded that triplet excited quinoid groups react effectively with phenolic groups forming phenoxyl type radicals and that they are quite unreactive with respect to the abstraction of alphatic hydrogen atoms. Irradiation of PPHS at λ_inc = 254 nm causes the formation of quinoid groups which absorb strongly at this wavelength. Light absorption by these groups becomes a determining factor with respect to photochemical alteration in the course of further irradiation.     

Gene expression within cell-sized lipid vesicles

Functional protein synthesis was observed in cell-sized lipid vesicles following encapsulation of a gene-expression system. Expression of rsGFP (red-shifted green fluorescent protein) within individual vesicles was observed by fluorescence microscopy. Interestingly, at the early stage of the reaction, the expression efficiency inside the vesicle was remarkably higher than that in the solution outside. The synthesized rsGFP in individual vesicles is safe from attack by proteinase K added to the external aqueous solution. Studies on cell-sized vesicles expressing protein should contribute to a fundamental understanding of certain aspects of living systems and will be useful for practical applications, such as the construction of microreactors.     

Structurally Diverse Polyamines: Solid‐Phase Synthesis and Interaction with DNA

A versatile solid‐phase approach based on peptide chemistry was used to construct four classes of structurally diverse polyamines with modified backbones: linear, partially constrained, branched, and cyclic. Their effects on DNA duplex stability and structure were examined. The polyamines showed distinct activities, thus highlighting the importance of polyamine backbone structure. Interestingly, the rank order of polyamine ability for DNA compaction was different to that for their effects on circular dichroism and melting temperature, thus indicating that these polyamines have distinct effects on secondary and higher‐order structures of DNA.     

CuI and H2O2 Inactivate and FeII Inhibits [Fe]‐Hydrogenase at Very Low Concentrations

[Fe]‐Hydrogenase (Hmd) catalyzes reversible hydride transfer from H₂. It harbors an iron‐guanylylpyridinol as a cofactor with an Fe^II that is ligated to one thiolate, two COs, one acyl‐C, one pyridinol‐N, and solvent. Here, we report that Cu^I and H₂O₂ inactivate Hmd (half‐maximal rates at 1 μM Cu^I and 20 μM H₂O₂) and that Fe^II inhibits the enzyme with very high affinity (K_i=40 nM ). Infrared and EPR studies together with competitive inhibition studies with isocyanide indicated that Cu^I exerts its inhibitory effect most probably by binding to the active site iron‐thiolate ligand. Using the same methods, it was found that H₂O₂ binds to the active‐site iron at the solvent‐binding site and oxidizes Fe^II to Fe^III. Also it was shown that Fe^II reversibly binds away from the active site iron, with binding being competitive to the organic hydride acceptor; this inhibition is specific for Fe^II and is reminiscent of that for the [FeFe]‐hydrogenase second iron, which specifically interacts with H₂.