Accelerated biodegradation of poly(vinyl alcohol) by glycosidations of the hydroxyl groups or addition of sugars

Biodegradabilities of N-acetyl-d-glucosamine (GlcNAc)- (1) and chitobiose-substituted (2) poly(vinyl alcohol)s (PVA)s in a soil suspension (pH 6.5) were investigated at 25 degrees C for 40 days. Biochemical oxygen demand of 1 with a degree of substitution of 0.2-0.3 (DP = 430-480) was higher than that of PVA under the degradation condition. Size exclusion chromatography, (1)H NMR, and Fourier-transform infrared measurements of the recovered sample indicated that biodegradation of the PVA main chain was accelerated by partial glycosidation of hydroxyl groups in PVA. Similar acceleration was observed in a PVA/GlcNAc (50:50, w/w) mixture. Microbes which relate with degradation of the glycosidated polymers were grown in a culture medium including the soil suspension and the polymer as the carbon source. Polyacrylamide gel electrophoresis (SDS-PAGE) and IR measurements indicated that a cell-free extract derived from GlcNAc-substituted PVA was different from that in the PVA/GlcNAc mixture. The results suggested that the PVA main chain in GlcNAc-substituted PVA was cleaved by a different microorganism or via a mechanism different from that in the mixture. Chitobiose-substituted PVA 2 showed more enhanced acceleration, indicating that the sugar length influenced the degradability.     

Recognition of virus infected cells by NK cells

NK cells show cytotoxicity against virus-infected cells and tumor cells and play an important role in host defense. Although mecheanism of target cell recognition by NK cells have been unclear for a long time, it has recently been elucidated that certain NK cell receptors specifically recognize virus products. Furthermore, expression pattern of NK cell receptors, which consist of activating and inhibitory receptors, determines susceptibility to virus-infection. Here, we review recent progress of mechanism of recognition of virus-infected by NK cells.     

A novel approach and protocol for discovering extremely low-abundance proteins in serum

The proteomic analysis of serum (plasma) has been a major approach to determining biomarkers essential for early disease diagnoses and drug discoveries. The determination of these biomarkers, however, is analytically challenging since the dynamic concentration range of serum proteins/peptides is extremely wide (more than 10 orders of magnitude). Thus, the reduction in sample complexity prior to proteomic analyses is essential, particularly in analyzing low-abundance protein biomarkers. Here, we demonstrate a novel approach to the proteomic analyses of human serum that uses an originally developed serum protein separation device and a sequentially linked 3-D-LC-MS/MS system. Our hollow-fiber-membrane-based serum pretreatment device can efficiently deplete high-molecular weight proteins and concentrate low-molecular weight proteins/peptides automatically within 1 h. Four independent analyses of healthy human sera pretreated using this unique device, followed by the 3-D-LC-MS/MS successfully produced 12 000-13 000 MS/MS spectra and hit around 1800 proteins (>95% reliability) and 2300 proteins (>80% reliability). We believe that the unique serum pretreatment device and proteomic analysis protocol reported here could be a powerful tool for searching physiological biomarkers by its high throughput (3.7 days per one sample analysis) and high performance of finding low abundant proteins from serum or plasma samples.     

Binding of FAD to cytochrome b558 is facilitated during activation of the phagocyte NADPH oxidase, leading to superoxide production

The superoxide-producing phagocyte NADPH oxidase can be reconstituted in a cell-free system. The activity of NADPH oxidase is dependent on FAD, but the physiological status of FAD in the oxidase is not fully elucidated. To clarify the role of FAD in NADPH oxidase, FAD-free full-length recombinant p47(phox), p67(phox), p40(phox), and Rac were prepared, and the activity was reconstituted with these proteins and purified cytochrome b(558) (cyt b(558)) with different amounts of FAD. A remarkably high activity, over 100 micromol/s/micromol heme, was obtained in the oxidase with purified cyt b(558), ternary complex (p47-p67-p40(phox)), and Rac. From titration with FAD of the activity of NADPH oxidase reconstituted with purified FAD-devoid cyt b, the dissociation constant K(d) of FAD in cyt b(558) of reconstituted oxidase was estimated as nearly 1 nm. We also examined addition of FAD on the assembly process in reconstituted oxidase. The activity was remarkably enhanced when FAD was present during assembly process, and the efficacy of incorporating FAD into the vacant FAD site in purified cyt b(558) increased, compared when FAD was added after assembly processes. The absorption spectra of reconstituted oxidase under anaerobiosis showed that incorporation of FAD into cyt b(558) recovered electron flow from NADPH to heme. From both K(d) values of FAD and the amount of incorporated FAD in cyt b(558) of reconstituted oxidase, in combination with spectra, we propose the model in which the K(d) values of FAD in cyt b(558) is changeable after activation and FAD binding works as a switch to regulate electron transfer in NADPH oxidase.     

Synthesis of acetone glycerol acyl esters by immobilized lipase ofMucor miehei

Summary The applications of immobilized lipase ofMucor miehei for the synthesis of acetone glycerol acyl ester from acetone glycerol and fatty acid, which is the first step for monoglyceride production was investigated. With a high oleic acid to acetone glycerol ratio (O/A, mol/mol), a high catalytic activity was observed under low water content in the reaction mixture. By the combination of high O/A ratio (>3) and removal of water which was produced during the reaction, the conversion degree was increased to almost 100%. With the O/A ratio of 3, the approximate half-life of the immobilized lipase and productivity of ester was estimated to be 20 days and 869 g product/g immobilized enzyme per 2 half-lives, respectively.     

Mycobacteria Exploit Host Hyaluronan for Efficient Extracellular Replication

In spite of the importance of hyaluronan in host protection against infectious organisms in the alveolar spaces, its role in mycobacterial infection is unknown. In a previous study, we found that mycobacteria interact with hyaluronan on lung epithelial cells. Here, we have analyzed the role of hyaluronan after mycobacterial infection was established and found that pathogenic mycobacteria can grow by utilizing hyaluronan as a carbon source. Both mouse and human possess 3 kinds of hyaluronan synthases (HAS), designated HAS1, HAS2, and HAS3. Utilizing individual HAS-transfected cells, we show that HAS1 and HAS3 but not HAS2 support growth of mycobacteria. We found that the major hyaluronan synthase expressed in the lung is HAS1, and that its expression was increased after infection with Mycobacterium tuberculosis. Histochemical analysis demonstrated that hyaluronan profoundly accumulated in the granulomatous legion of the lungs in M. tuberculosis-infected mice and rhesus monkeys that died from tuberculosis. We detected hyaluronidase activity in the lysate of mycobacteria and showed that it was critical for hyaluronan-dependent extracellular growth. Finally, we showed that L-Ascorbic acid 6-hexadecanoate, a hyaluronidase inhibitor, suppressed growth of mycobacteria in vivo. Taken together, our data show that pathogenic mycobacteria exploit an intrinsic host-protective molecule, hyaluronan, to grow in the respiratory tract and demonstrate the potential usefulness of hyaluronidase inhibitors against mycobacterial diseases.     

Discovery and biological profile of isoindolinone derivatives as novel metabotropic glutamate receptor 1 antagonists: A potential treatment for psychotic disorders

We describe here the discovery and biological profile of a series of isoindolinone derivatives as developed mGluR1 antagonists. Our combined strategy of rapid parallel synthesis and conventional medicinal optimization successfully led to N-cyclopropyl 22 and N-isopropyl isoindolinone analogs 21 and 23 with improved in vivo DMPK profiles. Moreover the most advanced analog 23 showed an oral antipsychotic-like effect at a dose of 1 mg/kg in an animal model.     

Identification of MK-1925: A selective,orally active and brain-penetrable opioid receptor-like 1 (ORL1) antagonist

Structure–activity relationship studies directed toward improving the metabolic stability of compound 1 resulted in the identification of 3-[5-(3,5-difluorophenyl)-3-({[(1S,3R)-3-fluorocyclopentyl]amino}methyl)-4-methyl-1H-pyrazol-1-yl]propanenitrile 39 (MK-1925) as a selective, orally available and brain-penetrable opioid receptor-like 1 (ORL1) antagonist. The compound also showed in vivo efficacy after oral dosing. Therefore, compound 39 was selected to undergo further studies as a clinical candidate.