Requirement of Skp1-Bub1 interaction for kinetochore-mediated activation of the spindle checkpoint

The spindle checkpoint transiently prevents cell cycle progression of cells that have incurred errors or failed to complete steps during mitosis, including those involving kinetochore function. The molecular nature of the primary signal transmitted from defective kinetochores and how it is detected by the spindle checkpoint are unknown. We report biochemical evidence that Bub1, a component of the spindle checkpoint, associates with centromere (CEN) DNA via Skp1, a core kinetochore component in budding yeast. The Skp1's interaction with Bub1 is required for the mitotic delay induced by kinetochore tension defects, but not for the arrest induced by spindle depolymerization, kinetochore assembly defects, or Mps1 overexpression. We propose that the Skp1-Bub1 interaction is important for transmitting a signal to the spindle checkpoint pathway when insufficient tension is present at kinetochores.     

Thalictrum minus cell cultures and ABC-like transporter

Cultured Thalictrum minus cells produce a benzylisoquinoline alkaloid, berberine, in the presence of benzyladenine, and excrete it into the culture medium. T. minus cells excluded berberine, even if berberine was exogenously added to the medium, without benzyladenine treatment. Similarly, T. minus cells excluded a heterocyclic dye (neutral red) and calcein AM, which is used as a fluorescent probe to detect the drug efflux pump activity by ABC transporters. The addition of several inhibitors of P-glycoprotein, a representative ABC transporter, induced the accumulation in of both berberine and calcein AM ATP-dependent manner. The expression of P-glycoprotein-like ABC transporter genes was also demonstrated. The involvement of ABC transporter in the secretion of berberine in T. minus cells is discussed.     

Glycosides of 2-C-methyl-D-erythritol from the fruits of anise,coriander and cumin

Eight glycosides of 2-C-methyl-D-erythritol (1) were isolated from the fruit of anise, and their structures were clarified as 1-O-beta-D-glucopyranoside, 3-O-beta-D-glucopyranoside, 4-O-beta-D-glucopyranoside, 1-O-beta-D-fructofuranoside, 3-O-beta-D-fructofuranoside, 4-O-beta-D-fructofuranoside, 1-O-beta-D-(6-O-4-hydroxybenzoyl)-glucopyranoside and 1-O-beta-D-(6-O-4-methoxybenzoyl)-glucopyranoside of 2-C-methyl-D-erythritol (2-9), respectively. Furthermore, 2 and 4 were isolated from the fruit of coriander, and 2, 3 and 4 were isolated from the fruit of cumin. Though the phosphate of 1 was known to be one of the first precursors of isoprenoids in the non-mevalonate pathway, and 1 is considered to be a common constituent in Umbelliferous plants, the glycosides of 1 are found for the first time.     

Three nicotianamine synthase genes isolated from maize are differentially regulated by iron nutritional status

Nicotianamine synthase (NAS) is an enzyme that is critical for the biosynthesis of the mugineic acid family of phytosiderophores in graminaceous plants, and for the homeostasis of metal ions in nongraminaceous plants. We isolated one genomic NAS clone, ZmNAS3, and two cDNA NAS clones, ZmNAS1 and ZmNAS2, from maize (Zea mays cv Alice). In agreement with the increased secretion of phytosiderophores with Fe deficiency, ZmNAS1 and ZmNAS2 were positively expressed only in Fe-deficient roots. In contrast, ZmNAS3 was expressed under Fe-sufficient conditions, and was negatively regulated by Fe deficiency. This is the first report describing down-regulation of NAS gene expression in response to Fe deficiency in plants, shedding light on the role of nicotianamine in graminaceous plants, other than as a precursor in phytosiderophore production. ZmNAS1-green fluorescent protein (sGFP) and ZmNAS2-sGFP were localized at spots in the cytoplasm of onion (Allium cepa) epidermal cells, whereas ZmNAS3-sGFP was distributed throughout the cytoplasm of these cells. ZmNAS1 and ZmNAS3 showed NAS activity in vitro, whereas ZmNAS2 showed none. Due to its duplicated structure, ZmNAS2 was much larger (65.8 kD) than ZmNAS1, ZmNAS3, and previously characterized NAS proteins (30-38 kD) from other plant species. We reveal that maize has two types of NAS proteins based on their expression pattern and subcellular localization.     

Enzymatic synthesis of hydrophilic undecylenic acid sugar esters and their biodegradability

To enhance water solubility of 10-undecylenic acid, which has anti-fungus, anti-bacterial and anti-virus activity, d-glucose, trehalose and sucrose were regioselectively esterified with vinyl 10-undecylenic acid ester in dimethyl formamide by a commercial protease, Bioprase conc., from Bacillus subtilis. 6-O-(10-Undecylenoyl) d-glucose, 6-O-(10-undecylenoyl) trehalose and 1-O-(10-undecylenoyl) sucrose were obtained. The influence of structural variation by changing the sugar moiety was analyzed the surface tension and biodegradability.     

SOCS-3 regulates onset and maintenance of T(H)2-mediated allergic responses

Members of the suppressor of cytokine signaling (SOCS) family are involved in the pathogenesis of many inflammatory diseases. SOCS-3 is predominantly expressed in T-helper type 2 (T(H)2) cells, but its role in T(H)2-related allergic diseases remains to be investigated. In this study we provide a strong correlation between SOCS-3 expression and the pathology of asthma and atopic dermatitis, as well as serum IgE levels in allergic human patients. SOCS-3 transgenic mice showed increased T(H)2 responses and multiple pathological features characteristic of asthma in an airway hypersensitivity model system. In contrast, dominant-negative mutant SOCS-3 transgenic mice, as well as mice with a heterozygous deletion of Socs3, had decreased T(H)2 development. These data indicate that SOCS-3 has an important role in regulating the onset and maintenance of T(H)2-mediated allergic immune disease, and suggest that SOCS-3 may be a new therapeutic target for the development of antiallergic drugs.     

Site-specific PEGylation of a lysine-deficient TNF-alpha with full bioactivity

Addition of polyethylene glycol to protein (PEGylation) to improve stability and other characteristics is mostly nonspecific and may occur at all lysine residues, some of which may be within or near an active site. Resultant PEGylated proteins are heterogeneous and can show markedly lower bioactivity. We attempted to develop a strategy for site-specific mono-PEGylation using tumor necrosis factor-alpha (TNF-alpha). We prepared phage libraries expressing TNF-alpha mutants in which all the lysine residues were replaced with other amino acids. A fully bioactive lysine-deficient mutant TNF-alpha (mTNF-alpha-Lys(-)) was isolated by panning against TNF-alpha-neutralizing antibody despite reports that some lysine residues were essential for its bioactivity. mTNF-alpha-Lys(-) was site-specifically mono-PEGylated at its N terminus. This mono-PEGylated mTNF-alpha-Lys(-), with superior molecular uniformity, showed higher bioactivity in vitro and greater antitumor therapeutic potency than randomly mono-PEGylated wild-type TNF-alpha. These results suggest the usefulness of the phage display system for creating functional mutant proteins and of our site-specific PEGylation approach.     

Enzymatic synthesis of trehalose esters having lipophilicity

To improve trehalose lipophilicity, trehalose was regioselectively esterified with vinyl fatty acid esters in dimethyl formamide by protease from Bacillus subtilis to give 6-O-lauroyltrehalose, 6-O-myristoyltrehalose, 6-O-palmitoyltrehalose, 6-O-stearoyltrehalose, 6-O-oleoyltrehalose and 6-O-linoleoyltrehalose. The influence of structural variation by changing fatty acid substitute was examined by measurement of the surface tension and biodegradability.