Synthetic urea derivatives such as N-phenyl-N′-(4-pyridyl)urea (4PU) and N-(2-chloro-4-pyridyl)-N′-phenylurea (4PU30) have strong cytokinin activities. Using tritiated 4PU30 as a probe, we previously established the presence of a cytokinin-specific binding protein (CSBP) of high affinity (K a for 4PU30 ϭ 4ϫ10 10 M Ϫ1 ) in the soluble fraction of etiolated mung bean seedlings [Nagata, R., Kawachi, E., Hashimoto, Y. & Shudo, K. (1993) Biochem. Biophys. Res. Commun. 191, 543Ϫ549]. In this report, we purified CSBP by the use of 4PU-Sepharose 4B, an affinity gel liganded with 4PU. We determined partial amino acid sequences of CSBP and isolated its cDNA by reverse-transcription (RT) PCR. The cDNA encoded a protein with a calculated molecular mass of 17 kDa. A data base homology search revealed that CSBP is a novel member of a major pollen allergen/pathogenesis-related protein family. Recombinant CSBP was expressed in Escherichia coli and was confirmed to bind specifically to cytokinins.Keywords : cytokinin; binding protein; purification; cDNA cloning ; recombinant protein.Cytokinins are plant growth hormones that control the proliferation and differentiation of plants [1,2]. Natural cytokinins include purine analogues such as zeatin and N 6 -(∆ 2 -isopentenyl)-adenine, which exert potent and specific biological activities at nanomolar concentrations. We have found a series of synthetic urea derivatives with very potent cytokinin activity, such as N-. 4PU30 possesses activity almost equivalent to that of natural purine-type cytokinins, while 4PU1080 possesses much stronger activity. In the tobacco callus growth test, the optimum concentrations of 4PU, 4PU30 and 4PU1080 were 0.5 µM, 4 nM and Ͻ 1 nM, respectively [4]. At first glance, the chemical structures of urea-type and purine-type cytokinins are different. However, both urea-type and purinetype cytokinin antagonists inhibited the biological responses of both types of cytokinins [5], suggesting that both structural types of cytokinins share a common molecular mechanism of action. Lineweaver-Burk analysis of the inhibitory effects of these antagonists and the quantitative structure/activity relationships Note. The novel nucleotide sequence data reported in this paper have been submitted to the DDBJ/EMBL/GenBank nucleotide sequence databases and are available under accession numbers AB012218, AB012219 and AB012220. Y. Fujimoto and R. Nagata contributed equally to this publication.[6] also suggested that urea-type and purine-type cytokinins have a common site of action. However, despite the potent and specific biological activities of these cytokinins, and the accumulated data on the structure/activity relationships of a variety of cytokinin structures, the molecular mechanisms of their biological actions have not been fully elucidated.Various kinds of soluble cytokinin-binding proteins have been found in various plant species using purine-type cytokinins as probes [7Ϫ14]. However, most of these proteins binds potent cytokinins with association constants (K a's) of les...
Anatomic resection may decrease local recurrence and improve the surgical outcomes in solitary HCC measuring 2 to 5 cm in diameter.
SummaryTumor markers [alpha-fetoprotein (AFP) or des-gamma-carboxyprothrombin (DCP)] and neutrophil/lymphocyte ratio (NLR) reportedly correlate with longterm outcomes for hepatocellular carcinoma (HCC). However, no standardized method has been established for evaluating the pretransplant data. One hundred and twenty-four patients who underwent living donor liver transplantation (LDLT) were retrospectively reviewed. The best predictive parameters for tumor recurrence were maximum values for AFP or DCP and 90-day mean values for NLR, respectively, and multivariate analysis confirmed these values were correlated with tumor recurrence. However, receiver operating characteristic analysis revealed that discriminative powers were sufficient only in maximum AFP [area under the curve (AUC) 0.88, P < 0.001] and maximum DCP (AUC 0.76, P < 0.001), while mean NLR was less predictive (AUC 0.62, P = 0.20). When incorporating AFP and DCP to the Tokyo criteria (≤5 tumors with each tumor ≤5 cm), the presence of at least two of the following factors: (i) beyond the Tokyo criteria, (ii) AFP>250 ng/ml, and (iii) DCP > 450 mAu/ml (>450 ng/ml), was correlated with a worse 5-year disease-free survival rate (20.0% vs. 96.8%, P < 0.001) and 5-year overall survival rate (20.0% vs. 84.0%, P < 0.001). The prognosis of patients undergoing LDLT for HCC strongly relies on maximum AFP or DCP values before transplantation, while the prognostic impact of NLR is limited.
The complete nucleotide sequence of the genomic RNA of cucumber green mottle mosaic virus watermelon strain SH (CGMMV-SH) was determined using cloned cDNA. This sequence is 6421 nucleotides long containing at least four open reading frames, which correspond to 186K, 129K, 29K and 17.3K proteins. The 17.3K protein is the coat protein. Sequence analysis shows that CGMMV-SH is very closely related to another watermelon strain, CGMMV-W, although three amino acid substitutions in the 29K protein were found between these strains. The sequence was also compared to those of other tobamoviruses, tobacco mosaic virus (TMV) vulgare, TMV-L (a tomato strain) and tobacco mild green mosaic virus reported by other groups. It shows 55 to 56% identity with these viruses. The size and location of the open reading frames are very similar to those of TMV but the 129K and 186K proteins are composed of 1142 and 1646 amino acids, being larger than those of TMV by 27 and 31 amino acids, respectively. The deduced amino acid sequences of these proteins are highly homologous to those of TMV, especially in the readthrough downstream region of the 186K protein.
BackgroundThe dysbiosis of gut microbiota has been implicated in the pathogenesis of inflammatory bowel diseases; however, the underlying mechanisms have not yet been elucidated. Heavily glycosylated mucin establishes a first-line barrier against pathogens and serves as a niche for microbial growth.MethodsTo elucidate relationships among dysbiosis, abnormal mucin utilisation, and microbial metabolic dysfunction, we analysed short-chain fatty acids (SCFAs) and mucin components in stool samples of 40 healthy subjects, 49 ulcerative colitis (UC) patients, and 44 Crohn's disease (CD) patients from Japan.FindingsLevels of n-butyrate were significantly lower in stools of both CD and UC patients than in stools of healthy subjects. Correlation analysis identified seven bacterial species positively correlated with n-butyrate levels; the major n-butyrate producer, Faecalibacterium prausnitzii, was particularly underrepresented in CD patients, but not in UC patients. In UC patients, there were inverse correlations between mucin O-glycan levels and the production of SCFAs, such as n-butyrate, suggesting that mucin O-glycans serve as an endogenous fermentation substrate for n-butyrate production. Indeed, mucin-fed rodents exhibited enhanced n-butyrate production, leading to the expansion of RORgt+Treg cells and IgA-producing cells in colonic lamina propria. Microbial utilisation of mucin-associated O-glycans was significantly reduced in n-butyrate-deficient UC patients.InterpretationMucin O-glycans facilitate symbiosynthesis of n-butyrate by gut microbiota. Abnormal mucin utilisation may lead to reduced n-butyrate production in UC patients.FundJapan Society for the Promotion of Science, Health Labour Sciences Research Grant, AMED-Crest, AMED, Yakult Foundation, Keio Gijuku Academic Development Funds, The Aashi Grass Foundation, and The Canon Foundation.
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