Biomedical subjects

Takahiro Hasegawa

Publications and source records attributed to Takahiro Hasegawa.

8 recordsLinked to original sources

PubMed2007

Differential localization of aquaporin-2 and glucose transporter 4 in polarized MDCK cells.

Membrane water channel aquaporin-2 (AQP2) and glucose transporter 4 (GLUT4) exhibit a common feature in that they are stored in intracellular storage compartments and undergo translocation to the plasma membrane upon hormonal stimulation. We compared the intracellular localization and trafficking of AQP2 and GLUT4 in polarized Madin-Darby canine kidney cells stably transfected with human AQP2 (MDCK-hAQP2) by immunofluorescence microscopy. When expressed in MDCK-hAQP2 cells, GLUT4 and GLUT4-EGFP were predominantly localized in the perinuclear region close to and within the Golgi apparatus, similar to endogenous GLUT4 in adipocytes and myocytes. In addition, GLUT4 was occasionally seen in EEA1-positive early endosomes. AQP2, on the other hand, was sequestered in subapical Rab11-positive vesicles. In the basal state, the intracellular storage site of GLUT4 was distinct from that of AQP2. Forskolin induced translocation of AQP2 from the subapical storage vesicles to the apical plasma membrane, which did not affect GLUT4 localization. When forskolin was washed out, AQP2 was first retrieved to early endosomes from the apical plasma membrane, where it was partly colocalized with GLUT4. AQP2 was then transferred to Rab11-positive storage vesicles. These results show that AQP2 and GLUT4 share a common compartment after retrieval from the plasma membrane, but their storage compartments are distinct from each other in polarized MDCK-hAQP2 cells.

Animals↗

PubMed2006

A starvation-specific serine protease gene, isp6+, is involved in both autophagy and sexual development in Schizosaccharomyces pombe.

Schizosaccharomyces pombe isp6(+) gene encodes a vacuolar serine protease, which is specifically induced during nitrogen starvation. An isp6-disruption mutant, isp6Delta, grew normally under normal conditions but was defective in large-scale protein degradation during nitrogen starvation, a hallmark of autophagy. Vacuoles are the organelles for such drastic protein degradation but those of isp6Delta were apparently aberrant. isp6Delta was infertile under nitrogen source-free conditions with poor expression of ste11(+), a gene critical for sexual development. A protein kinase A-disruption mutant, pka1Delta, is prone to sexual development because expression of ste11(+) is derepressed. However, isp6Deltapka1Delta still showed defects in ste11(+) expression and sexual development under nitrogen source-free conditions. isp6Delta and isp6Deltapka1Delta were able to initiate sexual development to produce spores when only a small amount of a nitrogen source was present. Pat1 protein kinase negatively controls meiosis, and a temperature-sensitive mutant of pat1, pat1-114, initiates meiosis irrespective of ploidy at the restrictive temperature. However, isp6Deltapat1-114 did not start meiosis under nitrogen source-free conditions even at the restrictive temperature. These observations suggest that isp6(+) contributes to sexual development by providing a nitrogen source through autophagy.

Autophagy↗

PubMed2006

An effective method to increase solvability in biochemical systems using S-system.

In this paper we propose an effective method to estimate the intrinsic values in an immobilized enzyme system, i.e., Michaelis constant K(m) and the maximum reaction rate V(m). We combine three techniques: (1) the non-linear least square method for estimating the kinetic values, (2) orthogonal collocation with the Gauss integration method, and (3) Newton-Raphson method (NRM) or S-system method (SSM) as Newton-like method. We build a procedure to combine the first two methods to estimate the unknown kinetic values in a system. We apply this procedure to solve the intrinsic kinetic parameters determination problem in an immobilized enzyme systems following Michaelis-Menten reaction. To demonstrate the effectiveness of the current method, we test their convergence performance in detail. The results show that the basin of attraction in the current method is extremely enlarged compared with that of the S-system alone. We suggest that the current method is one of the most effective ways to solve fairly complicated biochemical reaction systems in general.

Enzymes, Immobilized↗

PubMed2006

Molecular and cellular characterization of a new aquaporin, AQP-x5, specifically expressed in the small granular glands of Xenopus skin.

A new toad aquaporin (AQP) cDNA was cloned from a cDNA library constructed from the ventral skin of Xenopus laevis. This AQP (Xenopus AQP-x5) consisted of 273 amino acid residues with a high sequence homology to mammalian AQP5. The predicted amino acid sequence contained the two conserved Asn-Pro-Ala motifs found in all major intrinsic protein (MIP) family members and six putative transmembrane domains. The sequence also contained a mercurial-sensitive cysteine and a putative phosphorylation motif site for protein kinase A at Ser-257. The swelling assay using Xenopus oocytes revealed that AQP-x5 facilitated water permeability. Expression of AQP-x5 mRNA was restricted to the skin, brain, lungs and testes. Immunofluorescence and immunoelectron microscopical studies using an anti-peptide antibody (ST-156) against the C-terminal region of the AQP-x5 protein revealed the presence of immunopositive cells in the skin, with the label predominately localized in the apical plasma membrane of the secretory cells of the small granular glands. These glands are unique both in being close to the epidermal layer of the skin and in containing mitochondria-rich cells with vacuolar H+-ATPase dispersed among its secretory cells. Results from immunohistochemical experiments on the mucous or seromucous glands of several other anurans verified this result. We conclude that the presence of AQP-x5 in the apical plasma membrane of the small granular glands suggests its involvement in water secretion from the skins. The physiological roles of the AQP-x5 protein in the small or mucous glands are discussed.

Amino Acid Sequence↗

PubMed2005

Immunocytochemical studies on translocation of phosphorylated aquaporin-h2 protein in granular cells of the frog urinary bladder before and after stimulation with vasotocin.

We have generated a specific antibody against phosphorylated aquaporin-h2 (pAQP-h2) protein to investigate the role of phosphorylation in the translocation of AQP-h2 protein within the granule cells of the urinary bladder of the frog (Hyla japonica). The antibody was generated against a synthetic peptide (ST-160) corresponding to amino acids 255-268, with a phosphorylated Ser-262, a residue that is putatively phosphorylated by protein A kinase. Using this antibody, we found, by Western blot analysis, that phosphorylation of the AQP-h2 protein rapidly increased within 2 min after vasotocin (AVT) stimulation and remained at a higher than normal level for 15 min. Moreover, quantitative immunoelectron microscopy indicated that the location of the AQP-h2 protein dramatically changed after AVT stimulation. Before stimulation, pAQP-h2 protein was localized in only a small number of intracellular vesicles near the nucleus of the granular cells, whereas the labeling density of the intracellular vesicles and the apical membrane rapidly increased after stimulation. This finding was also confirmed by the results of an immunofluorescence study. Thus, phosphorylation of AQP-h2 protein seems to be essential for translocation of the protein from the cytoplasmic pool to the apical plasma membrane of the granular cells in frog urinary bladder.

Amino Acid Sequence↗

PubMed2003

Chloroplasts in seeds and dark-grown seedlings of lotus.

In most higher plants, mature dry seeds have no chloroplasts but etioplasts. Here we show that in a hydrophyte, lotus (Nelumbo nucifera), young chloroplasts already exist in shoots of mature dry seeds and that they give rise to mature chloroplasts during germination, even in darkness. These shoots contain chlorophyll and chlorophyll-binding proteins CP1 and LHCP. The unique features of chloroplast formation in N. nucifera suggest a unique adaptive strategy for seedling development correlated with the plant's habitat.

Adaptation, Physiological↗

PubMed2003

Regulation of water absorption in the frog skins by two vasotocin-dependent water-channel aquaporins, AQP-h2 and AQP-h3.

A new frog aquaporin (AQP) cDNA was cloned from a cDNA library constructed from the ventral skin of the tree frog Hyla japonica. This AQP (Hyla AQP-h2) consisted of 268 amino acid residues with a high homology to mammalian AQP2. The predicted amino acid sequence contained the two conserved Asn-Pro-Ala motifs found in all the major intrinsic protein family members and the putative six transmembrane domains. The sequence also contained a mercurial compound: cysteine, one potential N-glycosylation site at Asn-124, and a putative phosphorylation site recognized by protein kinase A at Ser-262. In a swelling assay using Xenopus oocytes, AQP-h2 facilitated water permeability, especially in response to cAMP. Expression of AQP-h2 mRNA was restricted to several tissues including the ventral skin, kidney, and urinary bladder; but with immunofluorescence staining using an antipeptide antibody (ST-140) against the AQP-h2 protein, immunopositive cells were found only in the ventral skin and urinary bladder. In the ventral pelvic skin, the label for AQP-h2 was localized in the entire plasma membrane of the granular cells beneath the outmost layer of the skin and in the basolateral membrane of the granular cells in this layer. In response to vasotocin, however, the label for AQP-h2 became more intense in the apical membrane in the granular cells of the outermost layer, similar to the case for the earlier studied AQP-h3, which was specifically expressed in the ventral skin. Taken together, these findings suggest that not only AQP-h3, but also AQP-h2 acts as a regulator of the water balance in this frog.

Amino Acid Sequence↗

PubMed2003

Actual state of radiation exposure during coronary angioplasty: a multicenter study in the nationwide database for cost analysis of percutaneous transluminal coronary angioplasty in Japan.

Percutaneous coronary intervention (PCI) has been associated with excessive radiation exposure, so the present study was designed to investigate the determinants of fluoroscopic time during PCI among characteristics inherent to the patient, procedure and hospital in 388 consecutive patients between November 1996 and March 1999 in 11 hospitals included in the Nationwide Database for Cost Analysis of percutaneous transluminal coronary angioplasty in Japan. Fluoroscopic time, which reflects radiation exposure, was used as the dependent variable in a multiple linear regression analysis. The mean fluoroscopic time was 27+/-15 min. The negative value of partial correlation of the hospital code (r=-0.374) and acute myocardial infarction (-0.163) indicated that these were explanatory variables that decreased fluoroscopic time. The positive value of the number of intraaortic balloon pump catheters (r=0.144), the number of balloons (r=0.304) and hospital stay (r=0.147) indicated these were explanatory variables that increased fluoroscopic time. The absolute value of r, (r=0.374), of the hospital code was the highest. In the final R(2) (=0.304) of the model, which is the partial R-square that determined the dependent variable, the hospital code showed the highest value. In conclusion, there was an inter-hospital difference in fluoroscopic time. Although the hospital code was found to be the most powerful determinant of fluoroscopic time, the R(2) (=0.304) of the model showed so lower value that other hospital characteristics that were not included in the model may influence the fluoroscopic time.

Aged↗