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Shozo Yamamoto - One of the best experts on this subject based on the ideXlab platform.
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Hinokitiol, a Selective Inhibitor of the Platelet-Type Isozyme of Arachidonate 12-Lipoxygenase
Biochemical and biophysical research communications, 2000Co-Authors: Hiroshi Suzuki, Shozo Yamamoto, Tetsuji Ueda, Ivo Juránek, Takahiro Katoh, Manabu Node, Toshiyuki SuzukiAbstract:Abstract Hinokitiol (4-isopropyltropolone), a constituent of Japanese cypress, reversibly inhibited platelet-type 12-Lipoxygenase with an IC50 of 0.1 μM, and the enzyme activity was almost lost at 1 μM. The compound was much less active with other Lipoxygenase enzymes with higher IC50 values (leukocyte-type 12-Lipoxygenase, 50 μM; soybean Lipoxygenase, 17 μM; 15-Lipoxygenase-1, >100 μM; 5-Lipoxygenase, 17 μM). Hinokitiol up to 100 μM had almost no effect on cyclooxygenases-1 and -2. Their structure–activity relationship examined with various tropolone derivatives indicated the requirements of the 2-hydroxyl group and 4-alkyl group for the potent and selective inhibition of platelet-type 12-Lipoxygenase.
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Arachidonate 12-Lipoxygenase isozymes.
Advances in experimental medicine and biology, 1999Co-Authors: Shozo Yamamoto, Michihiro Nakamura, Hiroshi Suzuki, Kazunori IshimuraAbstract:A Lipoxygenase is an enzyme which incorporates one molecule of oxygen into arachidonic acid or other polyunsaturated fatty acids producing hydroperoxy acids with a conjugated diene system. Enzymes oxygenating at the positions 5, 8, 12 and 15 of arachidonic acid and producing the corresponding hydroperoxy eicosatetraenoic acid (HpETE) have been isolated from mammalian tissues (Figure 1). All enzymes except for the 8-Lipoxygenase have been highly purified, and their cDNAs and genomic DNAs have been already cloned (1,2).
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platelet type Arachidonate 12 Lipoxygenase in mouse gastrointestinal tract
Acta Histochemica Et Cytochemica, 1998Co-Authors: Michihiro Nakamura, Shozo Yamamoto, Kazunori IshimuraAbstract:12-Lipoxygenase enzyme oxygenates the position 12 of arachidonic acid, and produces 12S-hydroperoxy-arachidonic acid. When mouse gastrointestinal tract was immunostained with an antiserum against human platelet 12-Lipoxygenase and examined by light microscopy, positively-stained cells were found in the epithelium of stomach and small and large intestines. Electron microscopic immunohistochemistry revealed that the positively-stained cells were open-type enteroendocrine cells. The endocrine cells had many granules of heterogeneous sizes, forms and internal structures, and most of the granules had electron lucent area. 12-Lipoxygenase was localized in the cytoplasm, but not in the nucleus, plasma membranes and other subcellular organelles. Double immunostaining revealed that all 12-Lipoxygenasepositive cells contained serotonin. With an antiserum against 12-Lipoxygenase isozyme of porcine leukocytes, no positively-stained cells were observed in gastrointestinal epithelium.
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Diurnal Change of Arachidonate 12-Lipoxygenase in Rat Pineal Gland
Biochemical and biophysical research communications, 1997Co-Authors: Hiroo Kawajiri, Shozo Yamamoto, Tanihiro Yoshimoto, Daming Zhuang, Na Qiao, Hiroshi Hagiya, Hiroyoshi Sei, Yusuke MoritaAbstract:Abstract Rat pineal gland contains a 12-Lipoxygenase as demonstrated by the enzyme activity, RNA blot analysis and in situ hybridization. Using rats maintained with 12-h dark and light cycles, dynamic changes of the enzyme in pineal gland were examined. When the crude extract of pineal glands was incubated with arachidonic acid and the reaction products were analyzed by reverse-phase HPLC, the glands obtained from rats in the dark showed a higher 12-Lipoxygenase activity than those obtained from rats in the light. The pineal 12-Lipoxygenase activity decreased after the light was on at 7 o'clock and reached the lowest level around 16 o'clock. Upon Western blot analysis the amount of 12-Lipoxygenase protein in pineal glands was high in the dark and lowest around 16 o'clock. A half life of the enzyme protein was estimated to be approximately 2.8 h in organ culture of rat pineal glands. Northern blot analysis also revealed a higher 12-Lipoxygenase mRNA level in pineal glands obtained in the dark than those obtained in the light. Thus, the 12-Lipoxygenase of rat pineal glands shows a diurnal fluctuation that is regulated at the transcription level, and may play a certain role in the regulation of neuroendocrine processes of this gland.
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Mammalian Arachidonate 12-Lipoxygenases
Advances in Experimental Medicine and Biology, 1997Co-Authors: Shozo Yamamoto, Toshiya Arakawa, Yoshitaka Takahashi, Michihiro Nakamura, Natsuo Ueda, Takahiko Hada, Hiroshi Suzuki, G R Reddy, Hiroshi Hagiya, Satoshi MatsudaAbstract:Since Arachidonate 12-Lipoxygenase was earlier found in platelets as the first mammalian Lipoxygenase, the enzyme has been found in a variety of tissues of a number of animal species. On the basis of our studies in the last decade, we have demonstrated that there are two isoforms of 12-Lipoxygenase (1).
Tanihiro Yoshimoto - One of the best experts on this subject based on the ideXlab platform.
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expression of Arachidonate 12 Lipoxygenase in rat tissues a possible role in glucagon secretion
Journal of Histochemistry and Cytochemistry, 2000Co-Authors: Hiroo Kawajiri, Tanihiro Yoshimoto, Daming Zhuang, Na Qiao, Miyuki Yamamoto, Shoichi Iseki, Kazuyuki HamaguchiAbstract:There are three isoforms of Arachidonate 12-Lipoxygenase in mammals: platelet, leukocyte, and epidermal types. We found in this study that the leukocyte-type enzyme was present in rat pineal gland, lung, spleen, aorta, adrenal gland, spinal cord, and pancreas, as assessed by RT-PCR. Immunohistochemical analysis showed that the enzyme was localized in macrophages in lung and spleen, alpha-cells of pancreatic islet, zona glomerulosa cells of adrenal cortex, and neuronal cells of spinal cord and superior cervical ganglion. The presence of the 12-Lipoxygenase in pancreatic alpha-cells was confirmed by glucagon staining in a consecutive section. We overexpressed the leukocyte-type 12-Lipoxygenase cDNA in a glucagon-secreting alphaTC clone 6 cell line that had been established from a transgenic mouse. Glucagon secretion was stimulated by approximately twofold in the 12-Lipoxygenase-expressing cells compared to the mock-transfected and original cells. The results suggest that the 12-Lipoxygenase of the leukocyte type augments glucagon secretion from pancreatic islets.
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Expression of Arachidonate 12-Lipoxygenase in Rat Tissues: A Possible Role in Glucagon Secretion
The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society, 2000Co-Authors: Hiroo Kawajiri, Tanihiro Yoshimoto, Daming Zhuang, Na Qiao, Miyuki Yamamoto, Shoichi Iseki, Kazuyuki HamaguchiAbstract:SUMMARY There are three isoforms of Arachidonate 12-Lipoxygenase in mammals: platelet, leukocyte, and epidermal types. We found in this study that the leukocyte-type enzyme was present in rat pineal gland, lung, spleen, aorta, adrenal gland, spinal cord, and pancreas, as assessed by RT-PCR. Immunohistochemical analysis showed that the enzyme was localized in macrophages in lung and spleen, a -cells of pancreatic islet, zona glomerulosa cells of adrenal cortex, and neuronal cells of spinal cord and superior cervical ganglion. The presence of the 12-Lipoxygenase in pancreatic a -cells was confirmed by glucagon staining in a consecutive section. We overexpressed the leukocyte-type 12-Lipoxygenase cDNA in a glucagon-secreting a TC clone 6 cell line that had been established from a transgenic mouse. Glucagon secretion was stimulated by approximately twofold in the 12-Lipoxygenase-expressing cells compared to the mock-transfected and original cells. The results suggest that the 12-Lipoxygenase of the leukocyte type augments glucagon secretion from pancreatic islets. (J Histochem Cytochem 48:1411‐1419, 2000)
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Decreased activity of Arachidonate 12-Lipoxygenase in platelets of Japanese patients with non-insulin-dependent diabetes mellitus.
Metabolism: clinical and experimental, 1998Co-Authors: Toshio Tohjima, Michihiro Nakamura, Naoko Honda, Kentaro Mochizuki, Junichiro Kinoshita, Kenji Watanabe, Tomoyuki Arisaka, Ryuzo Kawamori, Yuko Kurahashi, Tanihiro YoshimotoAbstract:Abstract To study the metabolism of the platelet 12-Lipoxygenase pathway in diabetes, we evaluated the correlation between the activity and amount of Arachidonate 12-Lipoxygenase in the platelets of patients with non—insulin-dependent-diabetes mellitus (NIDDM). There were four parts in this investigation: (1) examination of abnormalities in platelet 12-Lipoxygenase in patients with NIDDM recruited from the Hospital of Juntendo University School of Medicine; (2) comparison of 12-Lipoxygenase in the platelets of non-obese NIDDM patients without angiopathy versus normal subjects matched for age, sex, and body mass index (BMI); (3) evaluation of gender differences; and (4) assessment of the potential influence of glycemic control. The activity of 12-Lipoxygenase was assayed by incubation of [1-14C]arachidonic acid with the platelet cytosol. The reaction mixture was extracted and separated by thin-layer chromatography, and the radioactive end products were detected. The activity of 12-Lipoxygenase in the platelets of patients with NIDDM was significantly less than in normal subjects (P
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Diurnal Change of Arachidonate 12-Lipoxygenase in Rat Pineal Gland
Biochemical and biophysical research communications, 1997Co-Authors: Hiroo Kawajiri, Shozo Yamamoto, Tanihiro Yoshimoto, Daming Zhuang, Na Qiao, Hiroshi Hagiya, Hiroyoshi Sei, Yusuke MoritaAbstract:Abstract Rat pineal gland contains a 12-Lipoxygenase as demonstrated by the enzyme activity, RNA blot analysis and in situ hybridization. Using rats maintained with 12-h dark and light cycles, dynamic changes of the enzyme in pineal gland were examined. When the crude extract of pineal glands was incubated with arachidonic acid and the reaction products were analyzed by reverse-phase HPLC, the glands obtained from rats in the dark showed a higher 12-Lipoxygenase activity than those obtained from rats in the light. The pineal 12-Lipoxygenase activity decreased after the light was on at 7 o'clock and reached the lowest level around 16 o'clock. Upon Western blot analysis the amount of 12-Lipoxygenase protein in pineal glands was high in the dark and lowest around 16 o'clock. A half life of the enzyme protein was estimated to be approximately 2.8 h in organ culture of rat pineal glands. Northern blot analysis also revealed a higher 12-Lipoxygenase mRNA level in pineal glands obtained in the dark than those obtained in the light. Thus, the 12-Lipoxygenase of rat pineal glands shows a diurnal fluctuation that is regulated at the transcription level, and may play a certain role in the regulation of neuroendocrine processes of this gland.
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Epidermal growth factor enhances transcription of human Arachidonate 12-Lipoxygenase in A431 cells.
Biochimica et biophysica acta, 1997Co-Authors: Yi Wen Liu, Shozo Yamamoto, Toshiya Arakawa, Tanihiro Yoshimoto, Ben Kuen Chen, Ching Jiunn Chen, Wen Chang ChangAbstract:Abstract Epidermal growth factor (EGF), determined by immunoprecipitation and Western blot analysis, increased both enzyme activity and protein level of 12-Lipoxygenase in the solubilized microsomes of human epidermoid carcinoma A431 cells, respectively. The EGF-induced expression of 12-Lipoxygenase mRNA was inhibited by transcription inhibitors such as actinomycin D and 5,6-dichlorobenzimidazole riboside. Promoters of different lengths for human 12-Lipoxygenase gene were used to prepare the luciferase fusion vectors. These construct plasmids were transiently transfected into A431 cells, and the induction of luciferase expression by EGF was examined. A 4- to 6-fold increase in luciferase reporter activity stimulated by EGF for 18 h treatment was observed in plasmids with the 5′-flanking region length of −951 bp and that of −224 bp upstream from translation starting site. The time-dependent induction of luciferase activity by EGF paralleled the EGF-induced enzyme activity and expression of 12-Lipoxygenase protein. Taken together, the results of this study indicate that EGF enhanced the transcription of the human 12-Lipoxygenase gene, resulting in an increase in the amount and activity of 12-Lipoxygenase.
Michihiro Nakamura - One of the best experts on this subject based on the ideXlab platform.
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Arachidonate 12-Lipoxygenase isozymes.
Advances in experimental medicine and biology, 1999Co-Authors: Shozo Yamamoto, Michihiro Nakamura, Hiroshi Suzuki, Kazunori IshimuraAbstract:A Lipoxygenase is an enzyme which incorporates one molecule of oxygen into arachidonic acid or other polyunsaturated fatty acids producing hydroperoxy acids with a conjugated diene system. Enzymes oxygenating at the positions 5, 8, 12 and 15 of arachidonic acid and producing the corresponding hydroperoxy eicosatetraenoic acid (HpETE) have been isolated from mammalian tissues (Figure 1). All enzymes except for the 8-Lipoxygenase have been highly purified, and their cDNAs and genomic DNAs have been already cloned (1,2).
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Decreased activity of Arachidonate 12-Lipoxygenase in platelets of Japanese patients with non-insulin-dependent diabetes mellitus.
Metabolism: clinical and experimental, 1998Co-Authors: Toshio Tohjima, Michihiro Nakamura, Naoko Honda, Kentaro Mochizuki, Junichiro Kinoshita, Kenji Watanabe, Tomoyuki Arisaka, Ryuzo Kawamori, Yuko Kurahashi, Tanihiro YoshimotoAbstract:Abstract To study the metabolism of the platelet 12-Lipoxygenase pathway in diabetes, we evaluated the correlation between the activity and amount of Arachidonate 12-Lipoxygenase in the platelets of patients with non—insulin-dependent-diabetes mellitus (NIDDM). There were four parts in this investigation: (1) examination of abnormalities in platelet 12-Lipoxygenase in patients with NIDDM recruited from the Hospital of Juntendo University School of Medicine; (2) comparison of 12-Lipoxygenase in the platelets of non-obese NIDDM patients without angiopathy versus normal subjects matched for age, sex, and body mass index (BMI); (3) evaluation of gender differences; and (4) assessment of the potential influence of glycemic control. The activity of 12-Lipoxygenase was assayed by incubation of [1-14C]arachidonic acid with the platelet cytosol. The reaction mixture was extracted and separated by thin-layer chromatography, and the radioactive end products were detected. The activity of 12-Lipoxygenase in the platelets of patients with NIDDM was significantly less than in normal subjects (P
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platelet type Arachidonate 12 Lipoxygenase in mouse gastrointestinal tract
Acta Histochemica Et Cytochemica, 1998Co-Authors: Michihiro Nakamura, Shozo Yamamoto, Kazunori IshimuraAbstract:12-Lipoxygenase enzyme oxygenates the position 12 of arachidonic acid, and produces 12S-hydroperoxy-arachidonic acid. When mouse gastrointestinal tract was immunostained with an antiserum against human platelet 12-Lipoxygenase and examined by light microscopy, positively-stained cells were found in the epithelium of stomach and small and large intestines. Electron microscopic immunohistochemistry revealed that the positively-stained cells were open-type enteroendocrine cells. The endocrine cells had many granules of heterogeneous sizes, forms and internal structures, and most of the granules had electron lucent area. 12-Lipoxygenase was localized in the cytoplasm, but not in the nucleus, plasma membranes and other subcellular organelles. Double immunostaining revealed that all 12-Lipoxygenasepositive cells contained serotonin. With an antiserum against 12-Lipoxygenase isozyme of porcine leukocytes, no positively-stained cells were observed in gastrointestinal epithelium.
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Mammalian Arachidonate 12-Lipoxygenases
Advances in Experimental Medicine and Biology, 1997Co-Authors: Shozo Yamamoto, Toshiya Arakawa, Yoshitaka Takahashi, Michihiro Nakamura, Natsuo Ueda, Takahiko Hada, Hiroshi Suzuki, G R Reddy, Hiroshi Hagiya, Satoshi MatsudaAbstract:Since Arachidonate 12-Lipoxygenase was earlier found in platelets as the first mammalian Lipoxygenase, the enzyme has been found in a variety of tissues of a number of animal species. On the basis of our studies in the last decade, we have demonstrated that there are two isoforms of 12-Lipoxygenase (1).
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Subcellular localization of Arachidonate 12-Lipoxygenase and morphological effect of its overexpression on murine keratinocytes.
Cell and tissue research, 1997Co-Authors: Michihiro Nakamura, Shozo Yamamoto, Kazunori IshimuraAbstract:Arachidonate 12-Lipoxygenase enzyme oxygenates the position 12 of arachidonic acid and produces 12-hydroperoxy-arachidonic acid. Mouse keratinocytes were transiently transfected with an expression vector of human platelet 12-Lipoxygenase cDNA. The cells were homogenized, and the subcellular localization of the enzyme was examined by differential centrifugation. The 12-Lipoxygenase activity was detected predominantly in the particulate fractions. In contrast, immunoelectron microscopy detected the enzyme mainly in the cytoplasm of the transfected cell, but not in the nucleus, subcellular organelles or plasma membrane. To explain the discrepancy between these findings, we performed an electron-microscopic examination of the 176000 g pellet of the keratinocyte homogenate. The pellet contained mainly insoluble proteins such as keratin but not membrane structures such as the plasma membrane. Thus, it is possible that the enzyme was localized originally in the cytoplasm of the keratinocyte, and found in the particulate fractions due to its association with insoluble proteins during fractionation procedures. Unique structural changes were observed in the transfected keratinocytes. The nucleus had very scant karyoplasm and coarse fibrillary structures. When the keratinocytes were transfected with a mutant 12-Lipoxygenase cDNA or a vector without 12-Lipoxygenase cDNA, these structural changes were not observed.
Hiroshi Suzuki - One of the best experts on this subject based on the ideXlab platform.
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Hinokitiol, a Selective Inhibitor of the Platelet-Type Isozyme of Arachidonate 12-Lipoxygenase
Biochemical and biophysical research communications, 2000Co-Authors: Hiroshi Suzuki, Shozo Yamamoto, Tetsuji Ueda, Ivo Juránek, Takahiro Katoh, Manabu Node, Toshiyuki SuzukiAbstract:Abstract Hinokitiol (4-isopropyltropolone), a constituent of Japanese cypress, reversibly inhibited platelet-type 12-Lipoxygenase with an IC50 of 0.1 μM, and the enzyme activity was almost lost at 1 μM. The compound was much less active with other Lipoxygenase enzymes with higher IC50 values (leukocyte-type 12-Lipoxygenase, 50 μM; soybean Lipoxygenase, 17 μM; 15-Lipoxygenase-1, >100 μM; 5-Lipoxygenase, 17 μM). Hinokitiol up to 100 μM had almost no effect on cyclooxygenases-1 and -2. Their structure–activity relationship examined with various tropolone derivatives indicated the requirements of the 2-hydroxyl group and 4-alkyl group for the potent and selective inhibition of platelet-type 12-Lipoxygenase.
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Arachidonate 12-Lipoxygenase isozymes.
Advances in experimental medicine and biology, 1999Co-Authors: Shozo Yamamoto, Michihiro Nakamura, Hiroshi Suzuki, Kazunori IshimuraAbstract:A Lipoxygenase is an enzyme which incorporates one molecule of oxygen into arachidonic acid or other polyunsaturated fatty acids producing hydroperoxy acids with a conjugated diene system. Enzymes oxygenating at the positions 5, 8, 12 and 15 of arachidonic acid and producing the corresponding hydroperoxy eicosatetraenoic acid (HpETE) have been isolated from mammalian tissues (Figure 1). All enzymes except for the 8-Lipoxygenase have been highly purified, and their cDNAs and genomic DNAs have been already cloned (1,2).
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Mammalian Arachidonate 12-Lipoxygenases
Advances in Experimental Medicine and Biology, 1997Co-Authors: Shozo Yamamoto, Toshiya Arakawa, Yoshitaka Takahashi, Michihiro Nakamura, Natsuo Ueda, Takahiko Hada, Hiroshi Suzuki, G R Reddy, Hiroshi Hagiya, Satoshi MatsudaAbstract:Since Arachidonate 12-Lipoxygenase was earlier found in platelets as the first mammalian Lipoxygenase, the enzyme has been found in a variety of tissues of a number of animal species. On the basis of our studies in the last decade, we have demonstrated that there are two isoforms of 12-Lipoxygenase (1).
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Suicide inactivation of porcine leukocyte 12-Lipoxygenase associated with its incorporation of 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid derivative.
Biochimica et biophysica acta, 1996Co-Authors: Koji Kishimoto, Shozo Yamamoto, Tanihiro Yoshimoto, Michihiro Nakamura, Hiroshi Suzuki, Toshifumi Takao, Yasutsugu Shimonishi, Ta-i TanabeAbstract:Abstract Two isozymes of Arachidonate 12-Lipoxygenase, platelet-type and leukocyte-type, which were distinguished by their substrate specificities and primary structures, were investigated with reference to ‘suicide’ inactivation. Upon reaction with arachidonic acid the leukocyte-type enzyme was inactivated rapidly during the catalysis, whereas the platelet-type enzyme did not show such a rapid inactivation. The two 12-Lipoxygenase isozymes were incubated with various hydroperoxy and hydroxy products from arachidonic acid. (15S)-Hydroperoxy-5,8,11,13-eicosatetraenoic acid (15-HPETE) was found to be a unique substrate of the leukocyte-type 12-Lipoxygenase as follows. (1) 15-HPETE was an active substrate for porcine leukocyte 12-Lipoxygenase, and converted anaerobically to a 14,15-epoxy compound (14,15-leukotriene A4). (2) A rapid inactivation of the enzyme was observed within 2 min upon aerobic and anaerobic incubations with 15-HPETE. (3) 15-HPETE was rapidly incorporated into the enzyme in a nearly equimolar amount under both aerobic and anaerobic conditions. (4) Several findings suggested a covalent binding of 15-HPETE or its derivative to the enzyme. (5) Such a rapid and stoichiometric incorporation of 15-HPETE was not observed with the platelet-type 12-Lipoxygenase. On the basis of these findings we presumed that 15-HPETE was transformed to 14,15-leukotriene A4, which was covalently bound to the leukocyte-type 12-Lipoxygenase leading to the suicide inactivation of the enzyme.
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Arachidonate 12-Lipoxygenase of rat pineal glands: Catalytic properties and primary structure deduced from its cDNA
Biochimica et biophysica acta, 1994Co-Authors: Takahiko Hada, Shozo Yamamoto, Satoshi Matsuda, Toshiya Arakawa, Tanihiro Yoshimoto, Michihiro Nakamura, Hiroshi Suzuki, Hiroshi Hagiya, Takaharu Azekawa, Yusuke MoritaAbstract:Abstract When a crude extract of rat pineal glands (the 1000 × g supernatant of a homogenate) was incubated with arachidonic acid, 12-hydroxy-5,8,10,14-eicosatetraenoic acid was found as a major product. The 12-Lipoxygenase of rat pineal gland also reacted with linoleic and α-linolenic acids at 35% and 101% the rate of Arachidonate 12-oxygenation, respectively. Upon Western blot analysis using polyclonal antibody against porcine leukocyte 12-Lipoxygenase, the cytosol fraction of rat pineal gland showed a positive band with a molecular weight of approx. 74 kDa. A full-length cDNA for this enzyme was cloned from a cDNA library of rat pineal gland and the identity of the 12-Lipoxygenase cDNA was confirmed by its expression in E. coli . The amino acid sequence of the enzyme was deduced from the nucleotide sequence of the cDNA, encoding 663 amino acids with a calculated molecular weight of 75 305. The enzyme showed 72% identity of amino acid sequence with porcine leukocyte 12-Lipoxygenase and 73% with bovine tracheal 12-Lipoxygenase, but only 59% with human platelet 12-Lipoxygenase. Taken together, the high reactivity with C-18 fatty acids, the immunoreactivity and the amino acid homology data indicate that the rat pineal 12-Lipoxygenase is more closely related to leukocyte 12-Lipoxygenase than to platelet 12-Lipoxygenase. Upon RNA blot analysis, by far the highest content of 12-Lipoxygenase mRNA was observed in the pineal gland and negligible amounts of mRNA were detected in other parts of the brain. The predominant presence of 12-Lipoxygenase mRNA in pineal gland was confirmed by in situ hybridization of rat brain. Significant amounts of 12-Lipoxygenase mRNA were also detected in rat spleen, aorta, lung and leukocytes.
Kazunori Ishimura - One of the best experts on this subject based on the ideXlab platform.
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Arachidonate 12-Lipoxygenase isozymes.
Advances in experimental medicine and biology, 1999Co-Authors: Shozo Yamamoto, Michihiro Nakamura, Hiroshi Suzuki, Kazunori IshimuraAbstract:A Lipoxygenase is an enzyme which incorporates one molecule of oxygen into arachidonic acid or other polyunsaturated fatty acids producing hydroperoxy acids with a conjugated diene system. Enzymes oxygenating at the positions 5, 8, 12 and 15 of arachidonic acid and producing the corresponding hydroperoxy eicosatetraenoic acid (HpETE) have been isolated from mammalian tissues (Figure 1). All enzymes except for the 8-Lipoxygenase have been highly purified, and their cDNAs and genomic DNAs have been already cloned (1,2).
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platelet type Arachidonate 12 Lipoxygenase in mouse gastrointestinal tract
Acta Histochemica Et Cytochemica, 1998Co-Authors: Michihiro Nakamura, Shozo Yamamoto, Kazunori IshimuraAbstract:12-Lipoxygenase enzyme oxygenates the position 12 of arachidonic acid, and produces 12S-hydroperoxy-arachidonic acid. When mouse gastrointestinal tract was immunostained with an antiserum against human platelet 12-Lipoxygenase and examined by light microscopy, positively-stained cells were found in the epithelium of stomach and small and large intestines. Electron microscopic immunohistochemistry revealed that the positively-stained cells were open-type enteroendocrine cells. The endocrine cells had many granules of heterogeneous sizes, forms and internal structures, and most of the granules had electron lucent area. 12-Lipoxygenase was localized in the cytoplasm, but not in the nucleus, plasma membranes and other subcellular organelles. Double immunostaining revealed that all 12-Lipoxygenasepositive cells contained serotonin. With an antiserum against 12-Lipoxygenase isozyme of porcine leukocytes, no positively-stained cells were observed in gastrointestinal epithelium.
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Subcellular localization of Arachidonate 12-Lipoxygenase and morphological effect of its overexpression on murine keratinocytes.
Cell and tissue research, 1997Co-Authors: Michihiro Nakamura, Shozo Yamamoto, Kazunori IshimuraAbstract:Arachidonate 12-Lipoxygenase enzyme oxygenates the position 12 of arachidonic acid and produces 12-hydroperoxy-arachidonic acid. Mouse keratinocytes were transiently transfected with an expression vector of human platelet 12-Lipoxygenase cDNA. The cells were homogenized, and the subcellular localization of the enzyme was examined by differential centrifugation. The 12-Lipoxygenase activity was detected predominantly in the particulate fractions. In contrast, immunoelectron microscopy detected the enzyme mainly in the cytoplasm of the transfected cell, but not in the nucleus, subcellular organelles or plasma membrane. To explain the discrepancy between these findings, we performed an electron-microscopic examination of the 176000 g pellet of the keratinocyte homogenate. The pellet contained mainly insoluble proteins such as keratin but not membrane structures such as the plasma membrane. Thus, it is possible that the enzyme was localized originally in the cytoplasm of the keratinocyte, and found in the particulate fractions due to its association with insoluble proteins during fractionation procedures. Unique structural changes were observed in the transfected keratinocytes. The nucleus had very scant karyoplasm and coarse fibrillary structures. When the keratinocytes were transfected with a mutant 12-Lipoxygenase cDNA or a vector without 12-Lipoxygenase cDNA, these structural changes were not observed.
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Tissue Distribution and Subcellular Localization of Platelet-Type Arachidonate 12-Lipoxygenase
Advances in experimental medicine and biology, 1997Co-Authors: Michihiro Nakamura, Shozo Yamamoto, Natsuo Ueda, Kazunori Ishimura, N. Uchida, S. AraseAbstract:Arachidonate 12-Lipoxygenase introduces one oxygen molecule regiospecifically and stereoselectively into the carbon 12 of arachidonic acid. Our group has demonstrated the occurrence of two isoforms of 12-Lipoxygenase (platelet-type and leukocyte-type) which were distinguished in terms of substrate specificity, the cross-reactivity of their antibodies and the homology of their amino acid sequences.1,2 The leukocyte-type 12-lipoxy-genases have been detected not only in leukocytes, but also in several non-hematopoietic cell types, including bovine tracheas,3 porcine anterior pituitary glands,4 canine cerebra5 and rat pineal glands.6 The platelet-type 12-Lipoxygenases were found in platelets of many animal species and also in human and mouse skin cells.7–9 For a better understanding of the physiological role of the platelet-type 12-Lipoxygenase, we attempted to further investigate the expression and the distribution of the enzyme by immunohistochemical study. However, the purification of platelet-type 12-Lipoxygenase as antigen was difficult, and no polyclonal antibody with a high affinity for platelet-type enzyme was available until recently. Therefore, in this study we attempted to purify a recombinant enzyme as an antigen.
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Arachidonate 12-Lipoxygenase in porcine anterior pituitary cells: its localization and possible function in gonadotrophs
The Journal of endocrinology, 1996Co-Authors: H. Ikawa, Kazunori Ishimura, Kei Yamamoto, Y Takahashi, N Ueda, Y Hayashi, S Yamamoto, Minoru Irahara, Toshihiro AonoAbstract:Arachidonate 12-Lipoxygenase, which oxygenates positions 12 and 13 of arachidonic and linoleic acids, is present in porcine anterior pituitary cells. Colocalization of the 12-Lipoxygenase with various pituitary hormones was examined by immunohistochemical double-staining using antibodies against 12-Lipoxygenase and various anterior pituitary hormones. Under light microscopy, approximately 7% of the cells producing luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were positive for 12-Lipoxygenase, whereas the enzyme was detected in less than 2% of the cells producing thyrotrophin, prolactin, growth hormone (GH), and adrenocorticotrophin. In an attempt to examine the participation of 12-Lipoxygenase metabolites in pituitary hormone release, we incubated the primary culture of porcine anterior pituitary cells with 12-hydroperoxy-arachidonic acid or 13-hydroperoxy-linoleic acid. Significant stimulation of LH and FSH release by these hydroperoxides was observed at 10 microM in a time-dependent manner. At doses around 10 microM these compounds produced responses of similar magnitude to 1 nM gonadotrophin-releasing hormone (GnRH), but higher concentrations (30 microM) of the compounds were required for GH release. In contrast, 12-hydroxy-arachidonic and 13-hydroxy-linoleic acids were almost ineffective. Furthermore, the gonadotrophin release by 1 nM GnRH was inhibited by nordihydroguaiaretic acid (a Lipoxygenase inhibitor) with an IC50 of about 5 microM. Thus, the hydroperoxy (but not hydroxy) products of 12-Lipoxygenase may be involved in the release of pituitary hormones especially LH and FSH.