Biomedical subjects

Ayako Harada

Publications and source records attributed to Ayako Harada.

8 recordsLinked to original sources

PubMed2005

Red blood cells highly express type I platelet-activating factor-acetylhydrolase (PAF-AH) which consists of the alpha1/alpha2 complex.

Although red blood cells account for about 30% of total PAF-AH activity found in the blood, the physiological function of this enzyme is unknown. To understand the role and regulatory mechanism of this enzyme, we purified it from easily obtainable pig red blood cells. PAF-AH activity was mainly found in the soluble fraction of the red blood cells. Two peaks of enzyme activity appeared with increasing concentration of imidazole on column chromatography on nickel-nitroacetic acid (Ni-NTA) resin. We called these peaks of small and large enzyme activities fractions X and Y, respectively, and then further purified the enzymes by sequential chromatofocusing on Mono P and gel filtration on TSK G-3000. In the final preparation from fraction Y, two proteins bands corresponding to 26 kDa and 28 kDa were related to enzyme activity. Determination of the partial amino acid sequences of the proteins of 26 kDa and 28 kDa revealed that these proteins were identical to alpha(1) and alpha(2), respectively, both of which are catalytic subunits of Type I intracellular PAF-AH. On Western analysis, the 26 kDa and 28 kDa protein bands cross-reacted with specific monoclonal antibodies to alpha(1) and alpha(2), respectively. Since the apparent molecular weight of the natural enzyme was estimated to be about 60 kDa, the enzyme activity in fraction Y was thought to be that of a heterodimer consisting of alpha(1) and alpha(2). On the other hand, the enzyme activity in fraction X was thought to be that of a homodimer consisting of alpha(2). Other blood cells such as polymorphonuclear leukocytes and platelets only contained the alpha(2)/alpha(2) homodimer. It has been reported that the alpha(1)/alpha(2) heterodimer is poorly expressed in adult animals except for in the spermatogonium. Taken altogether, these results suggest that high expression of the alpha(1)/alpha(2) heterodimer is important for the physiological function of mature red blood cells.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗

PubMed2005

MafA is a key regulator of glucose-stimulated insulin secretion.

MafA is a transcription factor that binds to the promoter in the insulin gene and has been postulated to regulate insulin transcription in response to serum glucose levels, but there is no current in vivo evidence to support this hypothesis. To analyze the role of MafA in insulin transcription and glucose homeostasis in vivo, we generated MafA-deficient mice. Here we report that MafA mutant mice display intolerance to glucose and develop diabetes mellitus. Detailed analyses revealed that glucose-, arginine-, or KCl-stimulated insulin secretion from pancreatic beta cells is severely impaired, although insulin content per se is not significantly affected. MafA-deficient mice also display age-dependent pancreatic islet abnormalities. Further analysis revealed that insulin 1, insulin 2, Pdx1, Beta2, and Glut-2 transcripts are diminished in MafA-deficient mice. These results show that MafA is a key regulator of glucose-stimulated insulin secretion in vivo.

Animals↗

PubMed2004

High-resolution analysis by nano-electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry for the identification of molecular species of phospholipids and their oxidized metabolites.

Nano-electrospray ionization (ESI) Fourier transform ion cyclotron resonance mass spectrometry (FTICRMS) was applied to identify the molecular species of phosphatidylethanolamine of Caenorhabditis elegans, which has a high concentration of phospholipids with a fatty acyl chain having an odd number of carbon atoms. The molecular species of diacyl phosphatidylethanolamine with one fatty acyl chain having an odd number of carbon atoms and one fatty acyl chain having an even number of carbon atoms was identified separately from alkyl-acyl phosphatidylethanolamine with an alkyl chain having an even number of carbon atoms and a fatty acyl chain having an even number of carbon atoms. Furthermore, nano-ESI-FTICRMS was applied to the direct identification of oxidized phosphatidylcholine from soybean. The mass peaks of individual molecular species of oxidative phosphatidylcholine, such as 34:3 diacyl phosphatidylcholine with peroxide (+2O) (m/z 788.544) or 34:2 diacyl phosphatidylcholine with peroxide (+2O) (m/z 790.560), can be separated from the peaks of the molecular species of the non-oxidative phospholipids. This suggests that the mass peaks with a difference of less than 0.1 mass units in their molecular weight can be separated and that their individual exact molecular compositions can be obtained by the FTICRMS analysis. The high resolution and high accuracy of FTICRMS are very effective in the analysis of molecular species of phospholipids and their derivatives.

Aerosols↗

PubMed2003

Plasma platelet activating factor-acetylhydrolase (PAF-AH).

The platelet-activating factor-acetylhydrolase (PAF-AH) is an enzyme which catalyzes the hydrolysis of acetyl ester at the sn-2 position of PAF. The family of PAF-AHs consists of two intracellular isoforms (Ib and II), and one secreted isoform (plasma). These PAF-AHs show different biochemical characteristics and molecular structures. Plasma PAF-AH and intracellular isoform, II degrade not only PAF but also oxidatively fragmented phospholipids with potent biological activities. Among these PAF-AHs, plasma PAF-AH has been the target of many clinical studies in inflammatory diseases, such as asthma, sepsis, and vascular diseases, because the plasma PAF-AH activity in the patients with these diseases is altered when compared with normal individuals. Finding a genetic deficiency in the plasma PAF-AH opened the gate in elucidating the protecting role of this enzyme in inflammatory diseases. The most common loss-of-function mutation, V279F, is found in more than 30% of Japanese subjects (4% homozygous, 27% heterozygous). This single nucleotide polymorphism in plasma PAF-AH and the resulting enzymatic deficiency is thought to be a genetic risk factor in various inflammatory diseases in Japanese subjects. Administration of recombinant plasma PAF-AH or transfer of the plasma PAF-AH gene improves pathology in animal models. Therefore, substitution of plasma PAF-AH would be an effective in the treatment of the patients with the inflammatory diseases and a novel clinical approach. In addition, the detection of polymorphisms in the plasma PAF-AH gene and abnormalities in enzyme activity would be beneficial in the diagnosis of the inflammatory diseases.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗

PubMed2003

Pineal gland (melatonin) affects the parturition time, but not luteal function and fetal growth, in pregnant rats.

The purpose of the present study was to investigate the role of pineal gland (melatonin) on parturition time, luteal function, and fetal growth in pregnant rats. Cycling rats were subjected to pinealectomy or sham operation under ether anesthesia; and pinealectomized rats immediately underwent implantation of a melatonin capsule (PINX + Mel group) or a vehicle-containing capsule (PINX group), and sham operated rats also underwent implantation of a vehicle-containing capsule (control group). All rats were maintained under the same photoperiod conditions (14 L:10 D) and were induced pregnancy. Blood samples were obtained on days 7, 12, 15, 17, 19, and 21 of pregnancy to measure serum progesterone concentrations, and parturition times were recorded on days 22 and 23. In the next experiment, pregnant PINX rats received subcutaneous injection of melatonin (10 microg/body) at 08:00 h (PINX + 8 h group) or at 20:00 h (PINX + 20 h group) from day 15 to the end of pregnancy, and parturition times were recorded. Parturition times of rats in the PINX group, the PINX + Mel group or the PINX + 8 h group, but not the PINX + 20 h group, were significantly different compared with those in the control group. Pinealectomy or melatonin implantation did not affect serum progesterone concentrations during pregnancy or the number and weight of fetuses or corpora lutea. The present results indicate that pineal gland (melatonin rhythm) synchronizing with photoperiodic rhythm is likely to be an important determinant of parturition time, but it does not affect progesterone production or fetal growth in pregnant rats.

Animals↗

PubMed2002

Identification and functional characterization of nadrin variants, a novel family of GTPase activating protein for rho GTPases.

Nadrin is a GTPase-activating protein (GAP) for the rho family of GTPases that controls Ca2+-dependent exocytosis in nerve endings. In this study, three novel splice variants of nadrin were identified and the variants were designated as nadrin-102, -104, -116 and -126 according to their relative molecular masses. All nadrin variants share the GAP domain, coiled-coil domain, serine/threonine/proline-rich domain, SH3-binding motif, and a successive repeat of 29 glutamines. Tissue distribution analyses using polyclonal antibodies that can discriminate each variant showed that the expression of nadrin-102, -104 and -116 was dominant in neuronal tissues and correlates well with the differentiation of neurons while nadrin-126 was strongly expressed in embryonic brain. Expression of nadrin-116 in PC12 cells strongly inhibited NGF-dependent neurite outgrowth and this effect was dependent on its GAP activity. In contrast, no significant effect on either cell morphology or neurite outgrowth was observed with other variants. All variants showed punctate appearance throughout the cytoplasm, while the 66-kDa carboxyl-terminal fragment of nadrin-102 and/or nadrin-116 was localized to the nucleus and its nuclear translocation was accelerated by NGF-induced differentiation of the cells. These results suggested that nadrin variants are different in their ability to regulate rho-mediated signaling and that, in addition to being a GTPase-activating protein, nadrin-102 and -116 have other distinct functions in the nucleus of the cell, implying a possible role in the cross-talk between the cytoskeleton and the nucleus.

Alternative Splicing↗

PubMed2002

Alterations in nitrate/nitrite and nitric oxide synthase in preovulatory follicles in gonadotropin-primed immature rat.

Nitric oxide synthase (NOS) and nitric oxide (NO) play important roles in ovulation. The purpose of this study was to investigate the changes of intrafollicular nitrate/nitrite concentration and NOS mRNA expression in preovulatory follicles during equine CG (eCG) and human CG (hCG) induced ovulation in immature rats. Immature Sprague-Dawley rats received 15 IU eCG and then 15 IU hCG 48 h later. Rats were killed immediately before, 5 h after or 10 h after hCG injection, and their preovulatory follicles were dissected. Follicular fluid, granulosa cell, and theca cell layers were collected from preovulatory follicles and assayed for NO or NOS mRNA or for in vitro incubation study. Nitrate/nitrite concentration in the follicular fluid decreased significantly 5 and 10 h after hCG injection. Inducible NOS (iNOS) mRNA expression, which was greater in granulosa cell than in the theca cell layer, decreased significantly 5 and 10 h after hCG injection. However, endothelial NOS (eNOS) mRNA expression was detected mainly in the theca cell layer and further increased 5 and 10 h after hCG injection but remained low in granulosa cells. In vitro treatment of granulosa cells with 10(-4) or 5x10(-4) M S-nitroso-L-acetyl penicillamine (NO donor) decreased progesterone production and increased DNA fragmentation. We concluded that the decrease in nitrate/nitrite concentration in preovulatory follicles after hCG injection was due mainly to decreased iNOS expression in granulosa cells. These changes in nitrate/nitrite concentration may prevent apoptosis in preovulatory follicles.

Animals↗

PubMed2002

Changes in telomerase activity in experimentally induced atretic follicles of immature rats.

Follicular atresia is characterized by apoptosis of granulosa cells, and telomerase plays an important role in the apoptotic process. To study the relationship between follicular atresia and telomerase activity, we investigated changes in telomerase activity and localization in experimentally induced atretic follicles of immature rats. Immature female Sprague-Dawley rats received 15 IU equine CG (eCG) by subcutaneous injection. Rats were killed under ether anesthesia at 2, 3, 4, or 5 days after eCG injection. Telomerase activity in granulosa cells was measured by telomeric repeat amplification protocol (TRAP) assay, and telomerase localization in the ovary was examined by in situ TRAP assay. Telomerase activity was detected at high levels in granulosa cells on day 2 after eCG injection regardless of follicle size, and levels were significantly decreased in large follicles, with atretic changes, on days 4 and 5. No such decrease was observed in granulosa cells of small follicles. In the next experiment, rats received subcutaneous injections of estradiol (1, 10, or 50 microg/rat) to prevent follicular atresia or sesame oil as a control from day 2 to day 4 after eCG injection and were killed under ether anesthesia on day 5 after eCG injection. The changes observed in the large follicles on days 4 and 5 in oil treated rats were not observed with estradiol treatment. These findings suggest that the telomerase in granulosa cells is likely to play an important role for healthy follicle life and that loss of its activity may be associated with follicular atresia.

Animals↗