Phosphorylation of plasma membrane aquaporin regulates temperature-dependent opening of tulip petals

The opening and closing of tulip petals was reproduced in the dark by changing the temperature from 5 degrees C to 20 degrees C for opening and 20 degrees C to 5 degrees C for closing. The opening process was accompanied by (3)H(2)O transport through the stem from the incubation medium to the petals. A Ca(2+)-channel blocker and a Ca(2+)-chelator inhibited petal opening and (3)H(2)O transport. Several proteins in the isolated plasma membrane fraction were phosphorylated in the presence of 25 micro M Ca(2+) at 20 degrees C. The 31-kDa protein that was phosphorylated, was suggested immunologically as the putative plasma membrane aquaporin (PM-AQP). This phosphorylated PM-AQP clearly reacted with the anti-phospho-Ser. In-gel assay revealed the presence of a 45-kDa Ca(2+)-dependent protein kinase in the isolated plasma membrane. Phosphorylation of the putative PM-AQP was thought to activate the water channel composed of PM-AQP. Dephosphorylation of the phosphorylated PM-AQP was also observed during petal closing at 5 degrees C, suggesting the inactivation of the water channel.     

Plant-specific microtubule-associated protein SPIRAL2 is required for anisotropic growth in Arabidopsis

In diffusely growing plant cells, cortical microtubules play an important role in regulating the direction of cell expansion. Arabidopsis (Arabidopsis thaliana) spiral2 (spr2) mutant is defective in directional cell elongation and exhibits right-handed helical growth in longitudinally expanding organs such as root, hypocotyl, stem, petiole, and petal. The growth of spr2 roots is more sensitive to microtubule-interacting drugs than is wild-type root growth. The SPR2 gene encodes a plant-specific 94-kD protein containing HEAT-repeat motifs that are implicated in protein-protein interaction. When expressed constitutively, SPR2-green fluorescent protein fusion protein complemented the spr2 mutant phenotype and was localized to cortical microtubules as well as other mitotic microtubule arrays in transgenic plants. Recombinant SPR2 protein directly bound to taxol-stabilized microtubules in vitro. Furthermore, SPR2-specific antibody and mass spectrometry identified a tobacco (Nicotiana tabacum) SPR2 homolog in highly purified microtubule-associated protein fractions from tobacco BY-2 cell cultures. These results suggest that SPR2 is a novel microtubule-associated protein and is required for proper microtubule function involved in anisotropic growth.     

Quantitative PCR with 16S rRNA-gene-targeted species-specific primers for analysis of human intestinal bifidobacteria

A highly sensitive quantitative PCR detection method has been developed and applied to the distribution analysis of human intestinal bifidobacteria by combining real-time PCR with Bifidobacterium genus- and species-specific primers. Real-time PCR detection of serially diluted DNA extracted from cultured bifidobacteria was linear for cell counts ranging from 10(6) to 10 cells per PCR assay. It was also found that the method was applicable to the detection of Bifidobacterium in feces when it was present at concentrations of >10(6) cells per g of feces. Concerning the distribution of Bifidobacterium species in intestinal flora, the Bifidobacterium adolescentis group, the Bifidobacterium catenulatum group, and Bifidobacterium longum were found to be the three predominant species by examination of DNA extracted from the feces of 46 healthy adults. We also examined changes in the population and composition of Bifidobacterium species in human intestinal flora of six healthy adults over an 8-month period. The results showed that the composition of bifidobacterial flora was basically stable throughout the test period.     

Genetic variations at urotensin II and urotensin II receptor genes and risk of type 2 diabetes mellitus in Japanese

Urotensin II is among the most potent vasoactive hormones known and the urotensin II (UTS2) gene is localized to 1p36-p32, one of the regions reported to show possible linkage with type 2 diabetes in Japanese. When we surveyed genetic polymorphisms in the UTS2 and urotensin II receptor (GPR14) gene, we identified two SNPs with amino acid substitutions (designated T21M and S89N and an SNP in the promotor region (-605G>A) of the UTS2 gene, and two SNPs in the non-coding region of the GPR14 gene. We then studied these three SNPs in the UTS2 gene and two SNPs in the GPR14 gene in 152 Japanese subjects with type 2 diabetes mellitus and two control Japanese populations. The allele frequency of 89N was significantly higher in type 2 diabetic patients than in both elderly normal subjects (P = 0.0018) and subjects with normal glucose tolerance (P = 0.0011), whereas the allele frequency of T21M and -605G>A in the UTS2 gene and those of two SNPs in the GPR14 gene were essentially identical in these three groups. Furthermore, in the subjects with normal glucose tolerance, 89N was associated with significantly higher insulin levels on oral glucose tolerance test, suggesting reduced insulin sensitivity in subjects with 89N. These results strongly suggest that subjects with S89N in the UTS2 gene are more insulin-resistant and thus more susceptible to type 2 diabetes mellitus development.     

Large scale in vitro experiment system for 2 GHz exposure

A beam formed radiofrequency (RF) exposure-incubator employing a horn antenna, a dielectric lens, and a culture case in an anechoic chamber is developed for large scale in vitro studies. The combination of an open type RF exposure source and a culture case through which RF is transmitted realizes a uniform electric field (+/-1.5 dB) in a 300 x 300 mm area that accommodates 49 35 mm diameter culture dishes. This large culture dish area enables simultaneous RF exposure of a large number of cells or various cell lines. The RF exposure source operates at 2142.5 MHz corresponding to the middle frequency of the downlink band of the International Mobile Telecommunication 2000 (IMT-2000) cellular system. The dielectric lens, which has a gain of 7 dB, focuses RF energy in the direction of the culture case and provides a uniform electric field. The culture case is sealed and connected to the main unit for environmental control, located outside the anechoic chamber, via ducts. The temperature at the center of the tray, which contains the culture dishes in the culture room, is maintained at 37.0 +/- 0.2 degrees C by air circulation. In addition, the appropriate CO2 density and humidity supplied to the culture case realizes stable long-term culture conditions. Specific absorption rate (SAR) dosimetry is performed using an electric field measurement technique and the Finite Difference Time Domain (FDTD) calculation method. The results indicate that the mean SAR of the culture fluid at the bottom of the 49 (7 x 7 array) culture dishes used in the in vitro experiments is 0.175 W/kg for an antenna input power of 1 W and the standard deviation of the SAR distribution is 59%. When only 25 culture dishes (5 x 5 array) are evaluated, the mean SAR is 0.139 W/kg for the same antenna input power and the standard deviation of the SAR distribution is 47%. The proliferation of the H4 cell line in 72 h in a pair of RF exposure-incubators reveals that the culture conditions are equivalent to those of a common CO2 incubator.     

Prediction of the coding sequences of mouse homologues of FLJ genes: the complete nucleotide sequences of 110 mouse FLJ-homologous cDnas identified by screening of terminal sequences of cDNA clones randomly sampled from size-fractionated libraries.

We have been conducting a mouse cDNA project to predict protein-coding sequences of mouse KIAA-homologous genes since 2001. As an extension of this project, we also started to accumulate mouse cDNA clones homologous to the human FLJ cDNA clones which are another long cDNA resource produced in our institute. We have isolated the cDNA clones from size-fractionated cDNA libraries derived from five different mouse tissues and natural killer T-cells. Although the human FLJ cDNA clones were originally derived from human spleen libraries, one-third of their mouse homologues were obtained from the brain library. We designated these homologues "mFLJ" plus a 5-digit number and herein characterized 110 mFLJ cDNA clones. We assigned an integrity of the CDSs from the comparison of the 110 cDNA clones with the corresponding human FLJ cDNA clones. The average size of the 110 mouse cDNA sequences was 3.8 kb and that of the deduced amino acid sequences from their longest CDS in each cDNA was 663 amino acid residues. Homology and/or motif search against public databases revealed new domains and/or motifs in 26 mFLJ gene products which provide additional speculation regarding the function of FLJ genes.     

Solution structure of the RWD domain of the mouse GCN2 protein

GCN2 is the alpha-subunit of the only translation initiation factor (eIF2alpha) kinase that appears in all eukaryotes. Its function requires an interaction with GCN1 via the domain at its N-terminus, which is termed the RWD domain after three major RWD-containing proteins: RING finger-containing proteins, WD-repeat-containing proteins, and yeast DEAD (DEXD)-like helicases. In this study, we determined the solution structure of the mouse GCN2 RWD domain using NMR spectroscopy. The structure forms an alpha + beta sandwich fold consisting of two layers: a four-stranded antiparallel beta-sheet, and three side-by-side alpha-helices, with an alphabetabetabetabetaalphaalpha topology. A characteristic YPXXXP motif, which always occurs in RWD domains, forms a stable loop including three consecutive beta-turns that overlap with each other by two residues (triple beta-turn). As putative binding sites with GCN1, a structure-based alignment allowed the identification of several surface residues in alpha-helix 3 that are characteristic of the GCN2 RWD domains. Despite the apparent absence of sequence similarity, the RWD structure significantly resembles that of ubiquitin-conjugating enzymes (E2s), with most of the structural differences in the region connecting beta-strand 4 and alpha-helix 3. The structural architecture, including the triple beta-turn, is fundamentally common among various RWD domains and E2s, but most of the surface residues on the structure vary. Thus, it appears that the RWD domain is a novel structural domain for protein-binding that plays specific roles in individual RWD-containing proteins.     

Age-specific mortality rates of reproducing and non-reproducing males of Drosophila melanogaster

The proximate and evolutionary causes of the levelling of mortality rates at late ages, observed in several species, remain obscure. To investigate the causes of mortality levelling late in life in Drosophila melanogaster, we examined the effect of reproduction on mortality patterns, by conducting population cage experiments with a total of more than 45,000 individuals. Several different genotypes of reproducing and non-reproducing males from F(1) crosses among isogenic lines were studied. Our results suggest that significant mortality levelling can occur even in non-reproducing males, but that reproduction also significantly affects mortality patterns. The results show that mortality levelling is strongly affected by the Gompertz initial mortality rate and exponential rate of increase parameters, probably through the effects of heterogeneity in mortality risks.     

Additional effect of epidermal growth factor during in vitro maturation for individual bovine oocytes using a chemically defined medium

This study was performed to establish an individual bovine oocyte-IVP system using a chemically defined simple medium (mSOFaa containing 1 mg/ml polyvinyl alcohol: PVA) and to investigate the effects of epidermal growth factor (EGF) during oocyte maturation on in vitro maturation, fertilization and embryonic development. Cumulus-oocyte complexes were collected from bovine ovaries and were matured in mSOFaa containing PVA (control medium) supplemented with 0, 1, 10 or 50 ng/ml of EGF. Two further groups (TCM199 and mSOFaa, supplemented with 10% fetal calf serum were also included. In this study, mSOFaa containing PVA were used as a basic medium for fertilization and embryo development in vitro. Experiments were conducted in both group- and individual-IVP systems. In the group-IVP system, the proportion of matured oocytes (MII) in the control medium (62.7% +/- 5.0%) was significantly (p < 0.05) lower than in all other treatments, and in the individual-IVP system, the addition of 1 ng/ml EGF significantly (p < 0.05) increased the maturation rate (1 ng/ml EGF vs control: 76.2% +/- 5.4% vs 57.1% +/- 14.4%). The addition of EGF did not affect the proportions of penetrated and normally fertilized oocytes in either individual- or group-culture systems. In the group-IVP system, no significant difference among treatments was found in the rate of blastocyst formation, whereas in the individual-IVP system the control medium supplemented with 10 ng/ml EGF resulted in a significantly (p < 0.05) higher the rate of blastocyst formation (20.0 +/- 5.2%) than that in the control medium (6.2% +/- 3.5%). These results indicate that bovine oocytes can successfully develop to blastocysts in an individual-IVP system using a single chemically defined medium, and that the group-IVP system also resulted in a similar level of blastocyst formation to that in a standard multiple-media system in our laboratory. The effect of EGF during oocyte maturation medium differed depending on whether embryos were cultured individually or in groups.     

Binding activity of H-Ras is necessary for in vivo inhibition of ASK1 activity

H-Ras is well known as one of the essential components of Ras/Raf/MEK/ERK cascade, which is a critical prosurvival signaling mechanism in most eukaryotic cells. Ras targets Raf/MEK/ERK cascade by integrating and transmitting extracellular signals from growth factor receptors to Raf, leading to the propagation of signals to modulate a serious of cellular survival events. Apoptosis signal-regulating kinasel (ASK1) serves as a general mediator of cell death because it is responsive to a variety of death signals. In this study, we found that H-Ras interacted with ASK1 to cause the inhibition of both ASK1 activity and ASKl-induced apoptosis in vivo, which was reversed only partially by addition of RafS621 A, an antagonist of Raf, whereas MEK inhibitor, PD98059, and PI3K inhibitor, LY294002, did not disturb the inhibitory effect of H-Ras on ASK-1-induced apoptosis. Furthermore, by means of immunoprecipitate and kinase assays, we demonstrated that the interaction between H-Ras and ASK1 as well as the inhibition of ASKI activity were dependent on the binding activity of H-Ras. These results suggest that a novel mechanism may be involved in H-Rasmediated cell survival in addition to the well established MEK/ERK and PI3K/Akt kinase-dependent enhancement of cell survival.