A novel strategy for identifying differential gene expression: An improved method of differential display analysis.

We propose a novel alternative approach, an advanced method for recently developed strategies, for identifying differentially expressed genes. Firstly, double-stranded cDNAs were digested using Sau3AI and the 3'-end restriction fragments of the cDNA were ligated to a double-stranded adapter. Next, the restriction fragments were directly amplified using several combinations of adapter-specific primers and FITC-labeled oligo dT primers. The selected cDNA fragments were displayed on a polyacrylamide gel. Neither nested PCR nor purification of 3'-end fragments are necessary. We examined the validity of this approach by evaluating gene expression changes during granulocytic differentiation of HL-60 cells. This method can theoretically detect almost all gene expression changes more rapidly and through simpler manipulations than by any other approach.     

Design and synthesis of para-fluorophenylalanine amide derivatives as thrombin receptor antagonists.

An antagonist specific for the thrombin receptor is expected to be a remedy for thrombosis. Structure-activity studies of thrombin receptor-tethered ligand SFLLRNP have revealed the importance of the Phe-2-phenyl group in receptor recognition and the replacement of the Phe-2 by para-fluorophenylalanine [(p-F)Phe] was found to enhance its activity [Nose, T. et al. (1993) Biochem. Biophys. Res. Commun. 193, 694-699]. In order to obtain a small sized antagonist, a series of (p-F)Phe derivatives was designed and synthesized novel structural elements essential for receptor interactions being introduced at both the N and C-termini. beta-Mercaptopropionyl (betaMp) or its derivative activated by S-3-nitro-2-pyridinesulphenyl (Npys) was introduced at the N-terminus, and phenylmethyl amines were coupled to the C-terminus. All compounds were inactive when assayed for human platelet aggregation, indicating that they are not agonists. beta-Mercaptopropionyl derivatives were also inactive as antagonists. However, Npys-containing analogs were found to inhibit the agonist activity of SFLLRNP. In particular, SNpys-betaMp-(p-F)Phe-NH-R [R = -CH(C6H5)2 and -CH2-CH-(C6H5)2] potently suppressed platelet aggregation. The results suggested that (p-F)Phe can be used as a structural core to construct an effective antagonist conformation.     

Cell Surface Galactosylation Is Essential for Nonsexual Flocculation in Schizosaccharomyces pombe

We have isolated fission yeast mutants that constitutively flocculate upon growth in liquid media. One of these mutants, the gsf1 mutant, was found to cause dominant, nonsexual, and calcium-dependent aggregation of cells into flocs. Its flocculation was inhibited by the addition of galactose but was not affected by the addition of mannose or glucose, unlike Saccharomyces cerevisiae FLO mutants. The gsf1 mutant coflocculated with Schizosaccharomyces pombe wild-type cells, while no coflocculation was found with galactose-deficient (gms1Δ) cells. Moreover, flocculation of the gsf1 mutant was also inhibited by addition of cell wall galactomannan from wild-type cells but not from gms1Δ cells. These results suggested that galactose residues in the cell wall glycoproteins may be receptors of gsf1-mediated flocculation, and therefore cell surface galactosylation is required for nonsexual flocculation in S. pombe.     

Serum antibody level against GroEL type heat-shock protein of Campylobacter jejuni in patients with Guillain-Barré syndrome.

Recently there has been an increase in the number of cases reported of Guillain-Barré syndrome (GBS) developed after Campylobacter jejuni infection. To investigate the role of a C.jejuni GroEL-type heat-shock protein (CjHsp60) in the infection and induction of GBS, we examined the antibody level against CjHsp60 in 27 human sera, including GBS and non-GBS patients, by an enzyme-linked immunosorbent assay. Sera from patients with C. jejuni infection, despite the development of GBS, had a higher titer of anti-CjHsp60 antibody than those of patients without the infection and healthy control subjects. The patients with C. jejuni infection followed by GBS had slightly higher levels of this antibody than did the patients with infection who did not develop GBS, but there was no statistical significance. In conclusion, CjHsp60 is found to be one of the major immunogenic antigens in actual C. jejuni infection, but no evidence that supports the direct relationship between this protein and C. jejuni-associated GBS was found in this study.     

Competitive ability of two Brassica varieties in relation to biomass allocation and morphological plasticity under varying nutrient availability

Green cabbage (Brassica campestris, leafy variety) and turnip (Brassica campestris var. rapifera, rooty variety) were grown in both monocultures and mixtures at three nutrient levels to investigate their responses to nutrient availability with respect to biomass allocation, morphological plasticity and competitive ability. Their allocation parameters and leaf morphological traits were affected by both nutrient availability and developmental stage. Both of the varieties had a smaller biomass allocation to leaf blades, but a greater allocation to petioles at high nutrient levels. Root:shoot ratio (RSR) of green cabbage decreased with increasing nutrient availability, whereas that of turnip increased. Turnip had a smaller leaf blade weight ratio (LBWR) than cabbage, being compensated for by larger leaf area ratio (LAR) and specific leaf area (SLA). Leaf area ratio and SLA of both the varieties increased with increasing nutrient availability as did their mean dry weights. The mean dry weight of turnip was slightly greater than that of green cabbage in their respective monocultures, while that of green cabbage was greater than that of turnip in their 1:1 mixture. Therefore, green cabbage, having inherently greater biomass allocation to leaves, was generally more competitive than turnip with more biomass allocation to roots, especially at higher nutrient levels. However, within a variety, morphological plasticity (variation in LAR and SLA) was more important than the plasticity in biomass allocation (e.g. variation in RSR and LBWR) in determining competitive ability. The implication of our results is that competition models based on biomass allocation pattern alone may fail to predict competitive outcomes and that such models should also take morphological plasticity into full account.     

Phosphate metabolism during diel vertical migration in the raphidophycean alga, Chattonella antiqua

The ecological advantage of diel vertical migration on the nutrition and accumulation of Chattonella antiqua, which is one of the dominant red-tide forming phytoplankton species in the Seto Inland Sea of Japan, was examined using a large axenic culture tank, in which vertical stratification of salinity, temperature and nutrients was maintained, analogous to natural conditions observed when red tides occur. C. antiqua was capable of migrating through very sharp salinity and temperature gradients. At night the species migrated to the deep nutrient-rich water and assimilated nutrients. During the daytime it migrated to the nutrient-depleted surface water and used the accumulated nutrients for photosynthesis. Nitrogen uptake was synchronized with phosphate uptake. ³¹P-NMR spectroscopy during the migration experiment revealed that C. antiqua has the capability of nocturnal phosphate uptake in the deep nutrient-rich water, but no capability of synthesizing polyphosphate, which was considered to be the intracellular phosphate pool. These findings were compared with those reported for another raphidophycean, Heterosigma akashiwo. Although both species carry out vertical migration and nocturnal nutrient uptake, only H. akashiwo has the capability of making an intracellular polyphosphate pool. This revised version was published online in July 2006 with corrections to the Cover Date.     

Brain-Derived Neurotrophic Factor Stimulates Interactions of Shp2 with Phosphatidylinositol 3-Kinase and Grb2 in Cultured Cerebral Cortical Neurons

Shp2, a protein tyrosine phosphatase possessing SH2 domains, is utilized in the intracellular signaling of various growth factors. Shp2 is highly expressed in the CNS. Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, which also shows high levels of expression in the CNS, exerts neurotrophic and neuromodulatory effects in CNS neurons. We examined how BDNF utilizes Shp2 in its signaling pathway in cultured cerebral cortical neurons. We found that BDNF stimulated coprecipitation of several tyrosine-phosphorylated proteins with anti-Shp2 antibody and that Grb2 and phosphatidylinositol 3-kinase (PI3-K) were coprecipitated with anti-Shp2 antibody in response to BDNF. In addition, both anti-Grb2 and anti-PI3-K antibodies coprecipitated Shp2 in response to BDNF. The BDNF-stimulated coprecipitation of the tyrosine-phosphorylated proteins, Grb2, and PI3-K with anti-Shp2 antibody was completely inhibited by K252a, an inhibitor of TrkB receptor tyrosine kinase. This BDNF-stimulated Shp2 signaling was markedly sustained as well as BDNF-induced phosphorylation of TrkB and mitogen-activated protein kinases. In PC12 cells stably expressing TrkB, both BDNF and nerve growth factor stimulated Shp2 signaling similarly to that by BDNF in cultured cortical neurons. These results indicated that Shp2 shows cross-talk with various signaling molecules including Grb2 and PI3-K in BDNF-induced signaling and that Shp2 may be involved in the regulation of various actions of BDNF in CNS neurons.