Functional Differentiation of the Glycosyltransferases That Contribute to the Chemical Diversity of Bioactive Flavonol Glycosides in Grapevines (Vitis vinifera)

We identified two glycosyltransferases that contribute to the structural diversification of flavonol glycosides in grapevine (Vitis vinifera): glycosyltransferase 5 (Vv GT5) and Vv GT6. Biochemical analyses showed that Vv GT5 is a UDP-glucuronic acid:flavonol-3-O-glucuronosyltransferase (GAT), and Vv GT6 is a bifunctional UDP-glucose/UDP-galactose:flavonol-3-O-glucosyltransferase/galactosyltransferase. The Vv GT5 and Vv GT6 genes have very high sequence similarity (91%) and are located in tandem on chromosome 11, suggesting that one of these genes arose from the other by gene duplication. Both of these enzymes were expressed in accordance with flavonol synthase gene expression and flavonoid distribution patterns in this plant, corroborating their significance in flavonol glycoside biosynthesis. The determinant of the specificity of Vv GT5 for UDP-glucuronic acid was found to be Arg-140, which corresponded to none of the determinants previously identified for other plant GATs in primary structures, providing another example of convergent evolution of plant GAT. We also analyzed the determinants of the sugar donor specificity of Vv GT6. Gln-373 and Pro-19 were found to play important roles in the bifunctional specificity of the enzyme. The results presented here suggest that the sugar donor specificities of these Vv GTs could be determined by a limited number of amino acid substitutions in the primary structures of protein duplicates, illustrating the plasticity of plant glycosyltransferases in acquiring new sugar donor specificities.     

A Highlights from MBoC Selection: Initial Polarized Bud Growth by Endocytic Recycling in the Absence of Actin Cable–dependent Vesicle Transport in Yeast

The assembly of filamentous actin is essential for polarized bud growth in budding yeast. Actin cables, which are assembled by the formins Bni1p and Bnr1p, are thought to be the only actin structures that are essential for budding. However, we found that formin or tropomyosin mutants, which lack actin cables, are still able to form a small bud. Additional mutations in components for cortical actin patches, which are assembled by the Arp2/3 complex to play a pivotal role in endocytic vesicle formation, inhibited this budding. Genes involved in endocytic recycling were also required for small-bud formation in actin cable-less mutants. These results suggest that budding yeast possesses a mechanism that promotes polarized growth by local recycling of endocytic vesicles. Interestingly, the type V myosin Myo2p, which was thought to use only actin cables to track, also contributed to budding in the absence of actin cables. These results suggest that some actin network may serve as the track for Myo2p-driven vesicle transport in the absence of actin cables or that Myo2p can function independent of actin filaments. Our results also show that polarity regulators including Cdc42p were still polarized in mutants defective in both actin cables and cortical actin patches, suggesting that the actin cytoskeleton does not play a major role in cortical assembly of polarity regulators in budding yeast.     

Cultured human corneal epithelial stem/progenitor cells derived from the corneal limbus

Stem/progenitor cells of the human corneal epithelium are present in the human corneal limbus, and several corneal epithelial stem/progenitor cell markers have been reported. Recently, the neurotrophin family receptors were reported to be useful markers of corneal epithelial stem/progenitor cells. Therefore, we examined an enzymatic separation method for obtaining corneal epithelial stem/progenitor cells and measuring the change in the expression of low-affinity neurotrophin receptor p75 (p75^NTR), a receptor belonging to the neurotrophin family. As a result, it was found that our separation method preserved cell viability. Furthermore, p75^NTR was mainly observed in epithelial basal cells as were the corneal epithelial stem/progenitor markers p63 and integrin β1. p75^NTR was also observed in the cultured cells, but its frequency decreased with passage. In conclusion, we propose that our culture method will enable the culture of corneal stem cells and that it is a useful tool for elucidating the molecular basis of the niche that is necessary for the maintenance of epithelial stem cells in the corneal limbus. Furthermore, we conclude that p75^NTR is a useful cell marker for evaluating the characteristics of stem/progenitor cells in culture.     

Structural basis for oligosaccharide recognition of misfolded glycoproteins by OS-9 in ER-associated degradation

Misfolded glycoproteins are translocated from endoplasmic reticulum (ER) into the cytosol for proteasome-mediated degradation. A mannose-6-phosphate receptor homology (MRH) domain is commonly identified in a variety of proteins and, in the case of OS-9 and XTP3-B, is involved in glycoprotein ER-associated degradation (ERAD). Trimming of outermost α1,2-linked mannose on C-arm of high-mannose-type glycan and binding of processed α1,6-linked mannosyl residues by the MRH domain are critical steps in guiding misfolded glycoproteins to enter ERAD. Here we report the crystal structure of a human OS-9 MRH domain (OS-9(MRH)) complexed with α3,α6-mannopentaose. The OS-9(MRH) has a flattened β-barrel structure with a characteristic P-type lectin fold and possesses distinctive double tryptophan residues in the oligosaccharide-binding site. Our crystallographic result in conjunction with nuclear magnetic resonance (NMR) spectroscopic and biochemical results provides structural insights into the mechanism whereby OS-9 specifically recognizes Manα1,6Manα1,6Man residues on the processed C-arm through the continuous double tryptophan (WW) motif.     

Comparison of conventional and liquid-based cytology, and human papillomavirus testing using SurePath preparation in Japan

We compared the detection rate of cervical neoplasias between a liquid-based cytology (LBC) method using SurePath and the conventional method. We also studied the feasibility of human papillomavirus (HPV) typing by linear array assay. Cytological specimens from 1551 Japanese women were prepared using the conventional and SurePath methods; the cytological and histological results from biopsy samples were compared. HPV typing using an HPV linear array assay was carried out on residual specimens using the SurePath method. The cytodiagnostic results showed a concordance rate of 85.3% (Κ= 0.46) between the two methods. The sensitivity of lesions histopathologically diagnosed as CIN1 or above was not significantly different between the two methods (P = 0.575-1.000). The receiver operating characteristic curve analysis of the detectability in CIN2 or above revealed no significant difference between the two methods (P = 0.096). Among the 44 patients who underwent HPV typing using a linear array assay, 33 samples were eligible for HPV testing and were stored at ambient temperature. In conclusion, the SurePath and conventional methods have equivalent abilities for detecting cervical lesions. After preparation for cytological diagnosis, use of the remaining cells from the SurePath specimens to perform HPV typing using the linear array method could be feasible.     

High-rate nitrogen removal from livestock manure digester liquor by combined partial nitritation–anammox process

In this study, combination of a partial nitritation reactor, using immobilized polyethylene glycol (PEG) gel carriers, and a continuous stirred granular anammox reactor was investigated for nitrogen removal from livestock manure digester liquor. Successful nitrite accumulation in the partial nitritation reactor was observed as the nitrite production rate reached 2.1 kg-N/m³/day under aerobic nitrogen loading rate of 3.8 kg-N/m³/day. Simultaneously, relatively high free ammonia concentrations (average 50 mg-NH₃/l) depressed the activity of nitrite oxidizing bacteria with nitrate concentration never exceeding 3% of TN concentration in the effluent of the partial nitritation reactor (maximum 35.2 mg/l). High nitrogen removal rates were achieved in the granular anammox reactor with the highest removal rate being 3.12 kg-N/m³/day under anaerobic nitrogen loading rate of 4.1 kg-N/m³/day. Recalcitrant organic compounds in the digester liquor did not impair anammox reaction and the SS accumulation in the granular anammox reactor was minimal. The results of this study demonstrated that partial nitritation–anammox combination has the potential to successfully remove nitrogen from livestock manure digester liquor.     

Immunohistochemical localization of mitochondrial fatty acid β-oxidation enzymes in Müller cells of the retina

The presence of a mitochondrial fatty acid β-oxidation system in the retina was shown by immunohistochemistry. Fatty acids are considered to serve as a major energy source metabolized by fatty acid β-oxidation together with glucose metabolized by glycolysis in the organs of the entire body, but almost nothing is known about this metabolic system in the retina. Adult rat retinae were subjected to immunofluorescence and immuno-electron microscopy for the localization of fatty acid β-oxidation enzymes, together with western blot analysis for quantitation of the amount of enzyme proteins and DNA microarray analysis for gene expression. All the enzymes examined were shown to be present in the retina, but in small amounts, with the amount of protein and gene expression in the retina being about 1/10 of those in the liver. Immunohistochemistry at light and electron microscopic levels revealed the enzymes to be more preferentially localized to the mitochondria of Müller cells than the retinal neurons. The Müller cells were isolated from the retina and confirmed for the presence of mitochondrial fatty acid β-oxidation enzymes. A mitochondrial fatty acid β-oxidation system was thus shown to be present in the retina heterogeneously.     

Design and synthesis of disubstituted (4-piperidinyl)-piperazine derivatives as potent acetyl-CoA carboxylase inhibitors

Acetyl-CoA carboxylases (ACCs), the rate limiting enzymes in de novo lipid synthesis, play important roles in modulating energy metabolism. The inhibition of ACC has demonstrated promising therapeutic potential for treating obesity and type 2 diabetes mellitus in transgenic mice and preclinical animal models. We describe herein the structure-based design and synthesis of a novel series of disubstituted (4-piperidinyl)-piperazine derivatives as ACC inhibitors. Our structure-based approach led to the discovery of the indole derivatives 13i and 13j, which exhibited potent in vitro ACC inhibitory activity.