A novel cytoplasmic GTPase XAB1 interacts with DNA repair protein XPA

The xeroderma pigmentosum group A protein (XPA) plays a central role in nucleotide excision repair (NER). To identify proteins that bind to XPA, we screened a HeLa cDNA library using the yeast two-hybrid system. Here we report a novel cytoplasmic GTP-binding protein, designated XPA binding protein 1 (XAB1). The deduced amino acid sequence of XAB1 consisted of 374 residues with a molecular weight of 41 kDa and an isoelectric point of 4.65. Sequence analysis revealed that XAB1 has four sequence motifs G1–G4 of the GTP-binding protein family in the N-terminal half. XAB1 also contains an acidic region in the C-terminal portion. Northern blot analysis showed that XAB1 mRNA is expressed ubiquitously, and immunofluorescence analysis revealed that XAB1 is localized mainly in the cytoplasm. Consistent with the GTP-binding motif, purified recombinant XAB1 protein has intrinsic GTPase activity. Using the yeast two-hybrid system, we elucidated that XAB1 binds to the N-terminal region of XPA. The deletion of five amino acids, residues 30–34 of XPA, required for nuclear localization of XPA abolished the interaction with XAB1. These results suggest that XAB1 is a novel cytoplasmic GTPase involved in nuclear localization of XPA.     

Genetic linkage relationships between two enzyme loci,Est-2 and acph,in the mosquito Aedes (Finlaya) togoi

An esterase locus (Est-2), coding for carboxylesterase, and an acid phosphatase locus (Acph) were genetically studied by agar gel electrophoresis in the mosquito Aedes (Finlaya) togoi. The Est-2 and Acph variants occur as a monomer and a dimer, respectively. Both enzyme loci are linked to the sex locus (M) and s (straw-colored larva); the gene arrangement and recombination distances were Est-2—12.6%—s—31.7%—M—2.9%—Acph—3.2%—Est-3. The Est-3 locus was previously shown to code for carboxylesterase.This work was supported by Grant AI 16983-02 from the National Institutes of Health, Bethesda, Md.     

The Rab27a/granuphilin complex regulates the exocytosis of insulin-containing dense-core granules

Recently, we identified and characterized a novel protein, granuphilin, whose domain structure is similar to that of the Rab3 effector protein rabphilin3 (J. Wang, T. Takeuchi, H. Yokota, and T. Izumi, J. Biol. Chem. 274:28542-28548, 1999). Screening its possible Rab partner by a yeast two-hybrid system revealed that an amino-terminal zinc-finger domain of granuphilin interacts with Rab27a. Granuphilin preferentially bound to the GTP form of Rab27a. Formation of the Rab27a/granuphilin complex was readily detected in the pancreatic beta cell line MIN6. Moreover, the tissue distributions of Rab27a and granuphilin are remarkably similar: both had significant and specific expression in pancreatic islets and in pituitary tissue, but no expression was noted in the brain. Analyses by immunofluorescence, immunoelectron microscopy, and sucrose density gradient subcellular fractionation showed that Rab27a and granuphilin are localized on the membrane of insulin granules. These findings suggest that granuphilin functions as a Rab27a effector protein in beta cells. Overexpression of wild-type Rab27a and its GTPase-deficient mutant significantly enhanced high K(+)-induced insulin secretion without affecting basal insulin release. Although Rab3a, another exocytotic Rab protein, has some similarities with Rab27a in primary sequence, intracellular distribution, and affinity toward granuphilin, overexpression of Rab3a caused different effects on insulin secretion. These results indicate that Rab27a is involved in the regulated exocytosis of conventional dense-core granules possibly through the interaction with granuphilin, in addition to its recently identified role in lysosome-related organelles.     

Distribution of chitin/chitosan-like bioflocculant-producing potential in the genus Citrobacter

Some strains belonging to the genera Citrobacter and Enterobacter have been reported to produce chitin/chitosan-like bioflocculants (BFs) from acetate. In this study, to investigate the distribution of the BF-producing potential in the genus Citrobacter and to screen stably and highly BF-producing strains, we obtained 36 Citrobacter strains from different culture collection centers, which were distributed among seven species in the genus, and tested for the flocculating activities of their culture supernatants using a kaolin suspension method. As a result, 21 strains belonging to C. freundii (17 strains in 23 strains tested), C. braakii (two in two), C. youngae (one in one), and C. werkmanii (one in two) showed flocculating activity, but this ability was limited to cells grown on acetate. Gas chromatography/mass spectrometry (GC/MS) analysis of the hydrolysates from the BFs of five selected strains indicated that they consisted of glucosamine and/or N-acetylglucosamine, such as the chitin/chitosan-like BF (BF04) produced by Citrobacter sp. TKF04 (Fujita et al. J Biosci Bioeng 89: 40–46, 2000). Gel filtration chromatography using a high-performance liquid chromatography system revealed that the molecular weight ranges of these BFs varied, but the average sizes were all above 1.66?×?10⁶?Da.     

Design and synthesis of novel DFG-out RAF/vascular endothelial growth factor receptor 2 (VEGFR2) inhibitors: 3. Evaluation of 5-amino-linked thiazolo[5,4-d]pyrimidine and thiazolo[5,4-b]pyridine derivatives

Our aim was to discover RAF/vascular endothelial growth factor receptor 2 (VEGFR2) inhibitors that possess strong activity and sufficient oral absorption, and thus, we selected a 5-amino-linked thiazolo[5,4-d]pyrimidine derivative as the lead compound because of its potential kinase inhibitory activities and its desired solubility. The novel tertiary 1-cyano-1-methylethoxy substituent was designed to occupy the hydrophobic region of 'back pocket' of BRAF on the basis of the X-ray co-crystal structure data of BRAF. In addition, we found that N-methylation of the amine linker could control the twisted molecular conformation leading to improved solubility. These approaches produced N-methyl thiazolo[5,4-b]pyridine-5-amine derivative 5. To maximize the in vivo efficacy, we attempted salt formation of 5. Our result indicated that the besylate monohydrate salt form (5c) showed significant improvement of both solubility and oral absorption. Owing to the improved physicochemical properties, compound 5c demonstrated regressive antitumor efficacy in a HT-29 xenograft model.     

Correction to: An update and reassessment of fern and lycophyte diversity data in the Japanese Archipelago

The fern and lycophyte flora of Japan comprising 721 native taxa (including subspecies and varieties) plus 371 interspecific hybrids was reassessed using a nearly comprehensively sampled distribution map at 10 km resolution vouchered by 216,687 specimens, up-to-date cytotaxonomic information covering 74% of the taxa, and an rbcL sequence dataset covering 97.9% of the taxa. Spatial distribution of species richness and phylogenetic diversity was visualized. Apomixis was observed in 11.0% of the native taxa whose reproductive modes are known. The number of sexually reproducing polyploid taxa (n = 199) is less than sexual diploids (n = 241), and 30 of them are evidently allopolyploid, in contrast with the low number of possible autopolyploids (n = 4). Apomictic taxa were found to have smaller latitudinal ranges than sexual taxa or taxa with multiple reproductive modes. A morphological character dataset in Lucid format is provided for taxonomic identification of the native taxa.

     

Postural control adaptability to floor oscillation in the elderly

We established a method to evaluate postural control adaptability, applying it to 341 subjects, aged 18-29 years (young subjects) and 50-79 years, in order to investigate the influences of age and gender on adaptability. Subjects stood with eyes closed on a force plate fixed to a floor oscillator, which was sinusoidally oscillated in the anteroposterior direction with 0.5 Hz frequency and 2.5 cm amplitude. Five trials of 1-minute oscillation were conducted, with a short rest between trials. The mean speed of fluctuation of the center of foot pressure (CFP), as detected by the force plate, was calculated as an index of postural steadiness. Mean CFP speed decreased significantly in all age groups with trial repetition. The adaptability capability of elderly subjects was categorized as "good," "moderate," or "poor," as evaluated against a standard value, based on the variation of the regression of mean CFP speed between the 1st and 5th trials in young subjects. Results showed that the magnitude of reduction in the mean speed, with practice, was linearly related to the initial mean speed. We found a general decline in adaptability, and increase in initial mean speed, in subjects aged 60 years and older, with no gender difference detected in any age group. The proportion of subjects exhibiting moderate and poor adaptability increased gradually with age. In conclusion, age, but not gender, appears to affect adaptation of postural sway with short-term practice, although some elderly subjects maintain postural sway velocity and adaptability capabilities similar to those of young subjects.     

Seawater-resistant, non-spherical protoplasts from seagrass leaves

Two distinct types occurred among enzymatically isolated protoplasts from leaves of eelgrasses ( Zostera marina L., Z. japonica Ascherson and Phyllospadix iwatensis Makino). Spherical protoplasts with a smooth cell membrane were obtained only from young leaf tissues at the basal portions of blades protected from seawater by tightly enclosing sheaths. Non-spherical protoplasts had a highly invaginated cell membrane and were obtained from mature leaf blades, where the cells also in situ have this type of membrane. The protoplasts from mature leaves were rather rigid in shape and resistant to wide ranges of osmotic potential and salinity without change in their non-spherical shape, while the spherical protoplasts were rapidly destroyed in seawater. Detergents lysed the spherical protoplasts but not the non-spherical ones, suggesting that the highly invaginated enclosing structures of the non-spherical protoplasts contained detergent-resistant materials. Thus, the seagrass leaf cells develop seawater resistance, and this change alters the nature of the enclosing structures during the growth of the leaf blades. The non-membranous enclosing structures and their characteristic materials in the mature leaf cells remain to be defined.     

Extraction and preliminary characterization of maturation-promoting factor from starfish oocytes

Cytoplasm of maturing starfish oocytes possesses a factor which induces maturation upon injection into immature oocytes. Such maturation-promoting factor (MPF) was extracted from maturing oocytes of Asterina pectinifera and characterized preliminarily. After 1-methyladenine (1-MeAde) treatment, maturing oocytes were packed in a centrifuge tube to remove jelly and excess medium, and then crushed by centrifugation. The turbid supernatant was homogenized with a buffer containing NaF, Na-beta-glycerophosphate, ATP, EGTA and leupeptin, followed by centrifugation. MPF extracted in the supernatant was purified partially by ammonium sulfate precipitation, hydrophobic chromatography on pentyl-agarose and gel filtration on Sephacryl S-300. The final material induced maturation in the recipient starfish oocytes when 0.5 ng of protein was injected in a volume of 400 pl. The maturation response included germinal vesicle breakdown, and formation of polar bodies and egg pronucleus. Such MPF preparation induced maturation in oocytes of Xenopus laevis as well. Further, starfish MPF was found to be a heat-labile protein; its molecular weight (MW) was estimated as 300 X 10(3) D by gel filtration and its sedimentation coefficient value as 5S by centrifugation on sucrose density gradients.     

Resistance of the neck plasma membrane between the mother and the bud of Saccharomyces cerevisiae and Candida albicans to amphotericin B-induced deformation

Abstract The polyene antibiotic, amphotericin B, at high concentrations (5–20 μg/ml) induced particle-free smooth areas in the plasma membranes of Saccharomyces cerevisiae and Candida albicans . These areas occured more or less over the entire plasma membrane of unbudded cells. In budded cells, however, the neck between the mother and bud did not undergo deformation. This suggests the strong interaction between the filamentous ring, which is firmly attached to the neck plasma membrane, and plasma membrane particles in the neck regions.