A Nitrate Reductase Inactivator Protein from Spinach. Purification, Molecular Weight and Subunit Composition

A nitrate reductase-inactivator protein has been purified 16,000-foldfrom spinach leaves by pH 5 treatment, chromatography on SE53,Con A-Sepharose, and chromatofocusing. The yield was 12%, thespecific activity was 115 units mg^–1. Polyacrylamide gelelectrophoresis of the final purified inactivator fraction yielded2 major protein bands and both bands exhibited nitrate reductase-inactivatoractivity. Analysis of this inactivator protein by gel filtrationand SDS-gel electrophoresis revealed protein stainable materialonly in a molecular weight range of 110,000–115,000. SDSgel electrophoresis under reducing conditions yielded 2 proteinbands corresponding to molecular weights of 51,000 and 53,000.The proteolytic mapping for the two separated subunits appearedsimilar and possibly identical. (Received October 28, 1991; Accepted February 24, 1992)     

Femtoliter compartment in liposomes for in vitro selection of proteins

The aqueous compartment in liposomes provides a reaction resembling the cell and therefore is used as a microcompartment in which to study enzymatic reactions. However, regardless of their method of preparation, the heterogeneity in size of cell-size liposomes limits their potential uses. We established a strategy to estimate the internal aqueous volume of cell-size liposomes using a fluorescence-activated cell sorter (FACS). Reactions inside individual liposomes can be measured in a high-throughput format provided that the encapsulated proteins give rise to a fluorescent signal such as by exhibiting fluorescence themselves or by catalyzing production of a fluorescent compound. The strategy of volume estimation was applied to in vitro selection experiments. The green fluorescent protein (GFP) gene was encapsulated into liposomes together with an in vitro translation system. Here liposomes carrying a single copy of the gene were identified using the internal aqueous volume information of individual liposomes, and those exhibiting higher green fluorescence intensity were sorted by the FACS machine. This system was able to enrich those encoding GFP with higher fluorescence intensity over those with lower intensity. These results suggest the possibility of performing evolutionary experiments in an environment that mimics the cell.     

ROOT GROWTH INHIBITING, a Rice Endo-1,4-β-d-Glucanase, Regulates Cell Wall Loosening and is Essential for Root Elongation

The molecular mechanism involved in cell wall dynamics has not been well clarified, although it is quite important for organ growth. We characterized a rice mutant, root growth inhibiting (rt), which is defective in root elongation. The rt mutant showed a severe defect in cell elongation at the root-elongating zone with additional collapse of epidermal and cortex cells at the root tip caused by the defect in the smooth exfoliation of root cap cells. Consistent with these phenotypes, expression of the RT gene, which encodes a member of the membrane-anchored endo-1,4-??-d-glucanase, was specifically localized in the root-elongating zone and at the junction between epidermal and root cap cells. The enzymatic analysis of root extracts from the wild-type and rt mutant indicated that RT hydrolyzes noncrystalline amorphous cellulose. The cellulose content was slightly increased but the crystallinity of cellulose was decreased in the rt root. In addition, the hemicellulose composition was different between wild-type and rt roots. The total extensibility was significantly lower in the rt root explants. Based on these results, we concluded that RT is involved in the disassembly of the cell wall for cell elongation in roots as well as for root cap exfoliation from the epidermal cell layer by hydrolyzing the noncrystalline amorphous cellulose fibers of cellulose microfibrils resulting in loosening of the hemicellulose and cellulose interaction.     

Annexin A4 is a possible biomarker for cisplatin susceptibility of malignant mesothelioma cells

Mesothelioma is a highly malignant tumor with a poor prognosis and limited treatment options. Although cisplatin (CDDP) is an effective anticancer drug, its response rate is only 20%. Therefore, discovery of biomarkers is desirable to distinguish the CDDP-susceptible versus resistant cases. To this end, differential proteome analysis was performed to distinguish between mesothelioma cells of different CDDP susceptibilities, and this revealed that expression of annexin A4 (ANXA4) protein was higher in CDDP-resistant cells than in CDDP-susceptible cells. Furthermore, ANXA4 expression levels were higher in human clinical malignant mesothelioma tissues than in benign mesothelioma and normal mesothelial tissues. Finally, increased susceptibility was observed following gene knockdown of ANXA4 in mesothelioma cells, whereas the opposite effect was observed following transfection of an ANXA4 plasmid. These results suggest that ANXA4 has a regulatory function related to the cisplatin susceptibility of mesothelioma cells and that it could be a biomarker for CDDP susceptibility in pathological diagnoses.     

Novel biotin linker with alkyne and amino groups for chemical labelling of a target protein of a bioactive small molecule

We synthesized a novel linker (1) with biotin, alkyne and amino groups for the identification of target proteins using a small molecule that contains an azide group (azide probe). The alkyne in the linker bound the azide probe via an azide-alkyne Huisgen cycloaddition. A protein cross-linker effectively bound the conjugate of the linker and an azide probe with a target protein. The covalently bound complex was detected by western blotting. Linker 1 was applied to a model system using an abscisic acid receptor, RCAR/PYR/PYL (PYL). Cross-linked complexes of linker 1, the azide probes and the target proteins were successfully visualized by western blotting. This method of target protein identification was more effective than a previously developed method that uses a second linker with biotin, alkyne, and benzophenone (linker 2) that acts to photo-crosslink target proteins. The system developed in this study is a method for identifying the target proteins of small bioactive molecules and is different from photo-affinity labelling.     

Antiproliferative activity of root extract from gentian plant (Gentiana triflora) on cultured and implanted tumor cells

We describe a novel pharmacological activity of the gentian root, an ingredient of Chinese medicines. Root extract from Gentiana triflora triggered cell death of human Daudi cells in culture. In addition, daily administration of the extract to mice inhibited growth of implanted solid tumors. Extract treatment of cultured cells resulted in the appearance of shranken, fragmented, or condensed cell and nuclear morphologies, and in chromosomal DNA degradation. But, the extract-treated cells did not show DNA fragmentation, which exhibits a nucleosome ladder, suggesting that extract-triggered cell death is not mediated through a typical apoptotic pathway.