Biomedical subjects
Y Kamiya
Publications and source records attributed to Y Kamiya.
The tomato DWARF enzyme catalyses C-6 oxidation in brassinosteroid biosynthesis.
Brassinosteroids (BRs) are steroidal plant hormones essential for normal plant growth and development. Mutants in the biosynthesis or perception of BRs are usually dwarf. The tomato Dwarf gene (D), which was predicted to encode a cytochrome P450 enzyme (P450) with homology to other P450s involved in BR biosynthesis, was cloned previously. Here, we show that DWARF catalyses the C-6 oxidation of 6-deoxocastasterone (6-deoxoCS) to castasterone (CS), the immediate precursor of brassinolide. To do this, we first confirmed that the D cDNA complemented the mutant light- and dark-grown phenotypes of the extreme dwarf (dx) allele of tomato. To identify a substrate for the DWARF enzyme, exogenous application of BR intermediates to dx plants was carried out. C-6 oxoBR intermediates enhanced hypocotyl elongation whereas the C-6 deoxoBR, 6-deoxoCS, had little effect. Quantitative analysis of endogenous BR levels in tomato showed mainly the presence of 6-deoxoBRs. Furthermore, dx plants were found to lack CS and had a high level of 6-deoxoCS in comparison to D plants that had CS and a lower level of 6-deoxoCS. Confirmation that DWARF catalyzed the C-6 oxidation of 6-deoxoCS to CS was obtained by functional expression of DWARF in yeast. In these experiments, the intermediate 6alpha-hydroxycastasterone was identified, indicating that DWARF catalyzes two steps in BR biosynthesis. These data show that DWARF is involved in the C-6 oxidation in BR biosynthesis.
Effects of dehydroepiandrosterone on mitogen-activated protein kinase in human aortic smooth muscle cells.
The objective of the present study was to determine whether dehydroepiandrosterone (DHEA) modifies growth factor-induced mitogen-activated protein kinase (MAPK) activation, based on our previous study demonstrating that DHEA attenuates fetal calf serum-induced proliferation in human male aortic smooth muscle cells (human male aortic SMCs). Human male aortic SMCs were used for this study. Platelet-derived growth factor-BB (PDGF-BB), epidermal growth factor (EGF), and basic fibroblast growth factor (bFGF), but not insulin-like growth factor-1 (IGF-1), stimulated MAPK activity. Only MAPK activation induced by PDGF-BB was reduced by pretreatment with DHEA, although DHEA did not affect the MAPK activation induced by EGF or bFGF. The basal and PDGF-stimulated MAPK activity were decreased by two types of cyclic AMP (cAMP) elevating agents and increased by cAMP-dependent protein kinase (PKA) inhibitor in human male aortic SMCs, suggesting that cAMP regulates MAPK negatively. The intracellular cAMP was increased by PDGF-BB. The increase of cAMP by PDGF-BB was augmented by pretreatment with DHEA, although DHEA alone did not affect cAMP. Neither EGF nor bFGF affected cAMP with and without DHEA pretreatment. Secretion of PGE2 induced by PDGF was augmented by pretreatment with DHEA. Stimulatory effects of DHEA on the production of PGE2 and cAMP were partially canceled by aromatase inhibitor and completely canceled by indomethacin or selective inhibitor of cyclooxygenase-2. These results suggest that DHEA inhibited MAPK activation induced by PDGF-BB via PGE2 overproduction and subsequent cAMP-dependent pathway in human male aortic SMCs.
Regulation of gibberellin biosynthesis by light.
Phytochromes regulate transcript levels of gibberellin biosynthesis enzymes, GA 20-oxidases and/or GA 3beta-hydroxylases, in germinating lettuce and Arabidopsis seeds and in de-etiolating pea seedlings. Feedback regulation of GA biosynthesis by active GA is well established, but other mechanisms for regulation of these biosynthetic genes also exist, as this feedback does not operate on a GA 3beta-hydroxylase gene of Arabidopsis during seed germination.
Activation-induced apoptosis of peripheral lymphocytes treated with 7-hydroxystaurosporine, UCN-01.
7-hydroxystaurosporine (UCN-01) is a new anticancer agent which exerts an inhibitory effect on cell cycle check points and is currently under phase I clinical trials in US and Japan. Preliminary clinical data indicated that UCN-01 remained in plasma at high concentrations for long periods of time. This unavoidable high plasma drug exposure is likely to lead to hematological toxicities in patients. In the present study, cultured human peripheral blood lymphocytes (PBLs) were used to evaluate the possible hematological toxicities of UCN-01 treatment. UCN-01 induces apoptosis, and the induction of apoptosis-related surface markers were also examined to investigate the involvement of these molecules in UCN-01-induced apoptosis in PBLs. In vitro viability of PBLs was decreased by high dose of UCN-01 (25 microM, 3-day exposure). This effect of UCN-01 was significantly suppressed by the presence of human serum, suggesting that some specific inhibitory factor(s) in human serum may antagonize the lympholytic effect of UCN-01. The percentage of annexin V-positive PI-negative cells increased with exposure to UCN-01 in a time- and dose-dependent manner; by up to 30.3% after exposure to 25 microM UCN-01 for 3 days. At the same time, the expression of both interleukin-2 receptor (IL-2R, CD25) and Fas (CD95), analyzed by flow cytometry, was induced. Con A-stimulated PBLs were more sensitive to UCN-01-induced apoptosis than non-stimulated lymphocytes and UCN-01 increased the sFas-L released into culture medium from con A-stimulated PBLs. Therefore, lymphocyte depletion mediated by activation-induced apoptosis is likely to occur in patients treated with UCN-01 at high doses.
Progressive disease rate as a surrogate endpoint of phase II trials for non-small-cell lung cancer.
BACKGROUND: Although the potential activity of anticancer agents has been traditionally assessed by the response rate (RR) in phase II trials, there is an increasing need to identify alternative endpoints to evaluate the efficacy of novel types of antineoplastic agents such as cytostatic agents. However, none of the proposed alternatives have been validated. DESIGN: RR, rate of progressive disease (PD), and median survival time (MST) were obtained from 44 treatment arms in 42 single-agent phase II trials for non-small-cell lung cancer (NSCLC). Correlations between these parameters and their significance in selection of promising drugs were evaluated. RESULTS: The median (range) RR and PD rate per treatment arm were 17% (0%-40%) and 41% (8%-93%), respectively. The PD rate correlated more closely with MST (correlation coefficient (r) = 0.80, P < 0.001) than did the RR (r = 0.62, P < 0.001). The RR of active agents against NSCLC ranged broadly from 7% to 40%, whereas their PD rates were all 50% or less. In addition, all treatment arms with a PD rate over 50% had a poor MST of six months or shorter. CONCLUSIONS: The PD rate was potentially as good an endpoint as RR, and it may be a good candidate for the primary endpoint of phase II trials for novel types of anticancer agents.
Regulation of gibberellin biosynthesis genes during flower and early fruit development of tomato.
Gibberellins (GAs) are essential for the development of fertile flowers in tomato, and may also be required immediately after fertilization. In the GA-biosynthetic pathway, the reactions catalyzed by GA 20-oxidases have been implicated as site of regulation. To study the regulation of GA biosynthesis in flower and early fruit development, we isolated three tomato GA 20-oxidase cDNA clones, Le20ox-1, -2 and -3. The three genes showed different organ-specific patterns of mRNA accumulation. Analysis of the transcript levels of the three GA 20-oxidase genes, as well as those of copalyl diphosphate synthase (LeCPS) and GA 3 beta-hydroxylase (Le3OH-2) during flower bud and early fruit development, revealed temporally distinct patterns of mRNA accumulation. Up until anthesis, transcripts were observed for LeCPS, Le20ox-1, -2 and Le3OH-2, with an accumulation of Le20ox-1 mRNA. In contrast to the high level of Le3OH-2 transcripts in the fully open flower, mRNA levels of Le20ox-1, -2 and LeCPS were reduced at this stage. After anthesis, LeCPS and Le20ox-1 transcripts increased again. In addition, Le20ox-3transcripts increased whereas the transcripts of Le3OH-2 decreased to an undetectable level. In situ hybridization results demonstrated that during early stages of bud development, Le20ox-2 transcripts were localized in the tapetum and placenta. The presented results supply novel data about localization of GA biosynthesis gene transcripts, and indicate that transcript levels of GA biosynthesis genes are all highly regulated during flower bud development.
The gene encoding tobacco gibberellin 3beta-hydroxylase is expressed at the site of GA action during stem elongation and flower organ development.
Gibberellin 3beta-hydroxylase catalyzes the final step in the biosynthetic pathway leading to the plant hormone gibberellin (GA) and, therefore, the in vivo localization of this enzyme should give a direct indication of the site of synthesis of bioactive GAs in plants. We have isolated a cDNA clone, Nty (Nicotiana tabacum GA 3beta-hydroxylase), which encodes a putative GA 3beta-hydroxylase, by RT-PCR using RNA from tobacco shoot apices. Functional analysis, using an NTY protein expressed in Escherichia coli, revealed that Nty encoded an active GA 3beta-hydroxylase. A high expression level of Nty was observed in shoot apices, flowers, roots, young internodes but not in leaves or seeds. We performed more detailed expression analyses using in situ hybridization and histochemical analyses of the GUS activity in transgenic tobacco plants carrying an Nty promoter:GUS fusion gene. These studies revealed that expression of Nty was restricted to specific regions, including actively dividing and elongating cells in the various organs; rib meristem and elongation zones of shoot apices, tapetum and pollen grains in developing anthers and root tips, which are consistent with the sites of GA action. It is proposed that GA actions depend on the modulation of endogenous bioactive GA levels through the regulation of GA 3beta-hydroxylase expression in situ.
Therapy-related leukemia and myelodysplastic syndrome in breast cancer patients treated with cyclophosphamide or anthracyclines.
BACKGROUND: Accumulation of data regarding therapy-related leukemia (TRL) or myelodysplastic syndrome (t-MDS) is critical for assessing the risk of developing such diseases and for subsequent decision-making processes for better treatment. METHODS: We evaluated the clinical characteristics of patients with TRL/t-MDS diagnosed at the National Cancer Center Hospital between January 1989 and September 1997. This report is concerned with those patients who initially had been treated with chemotherapeutic agents for breast cancer. RESULTS: Thirteen patients (median age, 55 years) developed TRL (n = 4) or t-MDS (n = 9). The median interval between the development of TRL/t-MDS and initial treatment was 94 months (range 23-190 months). For the primary therapy, all patients had received intense and prolonged treatment with cyclophosphamide (CPA) and/or anthracyclines including doxorubicin (DOX), with a median cumulative dose of 55 g/body (range 16.4-288.5 g) for CPA and 480 mg/m2 (range 395-625.5 mg/m2) for DOX. Seven patients were subsequently treated by chemotherapy and one received an allogeneic bone marrow transplantation. CONCLUSIONS: Clinicians must remain alert to the risks associated with unproven medical practices which include long-term administration of alkylating agents. Selected patients with TRL/t-MDS may respond to intense salvage combination chemotherapy.
Production of homo- and hetero-dimeric isozymes from two aldehyde oxidase genes of Arabidopsis thaliana.
Polyclonal antibodies were raised against synthetic peptides or recombinant polypeptides encoded by Arabidopsis atAO-1 and atAO-2 cDNAs, which have sequences similar to maize and animal aldehyde oxidase (AO) cDNAs. Anti-atAO-1 antibodies recognized AOalpha and AObeta among the three isoforms, AOalpha, AObeta, and AOgamma, detected in Arabidopsis seedlings after native PAGE, while anti-atAO-2 antibodies reacted with AObeta and AOgamma. The polypeptide specifically recognized by each antibody was collected as the Protein-A/IgG/antigen complex. The 150- and 145-kDa polypeptides were purified by SDS-PAGE and digested with Achromobacter Protease I. From the amino acid sequences and molecular masses of the derivative peptides, it was revealed that the 150- and 145-kDa polypeptides were the products of atAO-1 and atAO-2, respectively. Molecular masses of the native forms of AOalpha, AObeta, and AOgamma were estimated as approximately 290-300 kDa. These results suggest that AOalpha and AOgamma are homodimers consisting of atAO-1 and atAO-2 products, respectively, and that AObeta is a heterodimer of the atAO-1 and atAO-2 products.
The effects of ketamine and propofol on neuronal nicotinic acetylcholine receptors and P2x purinoceptors in PC12 cells.
UNLABELLED: We studied the effects of ketamine and propofol on two ligand-gated ion channels mediating fast synaptic transmission through sympathetic ganglia, neuronal nicotinic acetylcholine receptors (nAchRs), and P2X purinoceptors in a rat pheochromocytoma cell line PC12 using whole cell voltage clamp recording. Ketamine and propofol similarly inhibited the nicotine-induced inward current reversibly and dose-dependently at the membrane potential of -60 mV but had no effects on the adenosine triphosphate-induced current. Both anesthetics accelerated the current decay during agonist application, resulting in greater inhibition on the steady current than the peak current. The 50% inhibition concentration values for the steady current were lower than the clinically relevant concentrations for ketamine (2.8+/-0.6 microM) and higher than those for propofol (5.4+/-0.6 microM). Both anesthetics induced an addition of the fast component to the decay phase and an acceleration of the slow component, which suggests an open channel blockade or an enhancement of desensitization as a mechanism. The effects on closed channels seemed to be small because preincubation with the anesthetics did not significantly augment the block. Inhibition was voltage-independent at membrane potentials between -20 and -70 mV and was consistent with a noncompetitive block. Inhibition of the neuronal nAchR-mediated current may lead to the suppression of synaptic transmission in sympathetic ganglia by ketamine, but not by propofol, at the clinically relevant concentrations. However, these results are not consistent with changes in sympathetic nerve activities reported for animals or humans anesthetized with ketamine or propofol, which suggests effects from other systems, such as the central nervous system in vivo. IMPLICATIONS: Ketamine (at smaller than clinically relevant concentrations) and propofol (at larger than clinically relevant concentrations) inhibited neuronal nicotinic acetylcholine receptor-mediated current in PC12 cells, which possess the receptors that resemble those in postganglionic sympathetic neurons. These findings are not consistent with in vivo experiments, which suggests that effects from other systems, such as the central nervous system, are of importance.
Depressant and convulsant barbiturates both inhibit neuronal nicotinic acetylcholine receptors.
UNLABELLED: Neuronal nicotinic acetylcholine receptors (neuronal nAchRs) are sensitive to many anesthetics, including barbiturates, which suggests that these receptors are potential sites for anesthetic action. Subtle changes in molecular structures of the anesthetic barbiturates can produce compounds with potent convulsant activity. Whereas R(-) isomer of 1-methyl-5-phenyl-5-propyl barbituric acid (MPPB) exerts anesthetic action, S(+)MPPB exhibits pure excitatory effects, including convulsion. 5-(2-cyclohexilidene-ethyl)-5-ethyl barbituric acid is another example of a convulsant barbiturate. We compared the effects of depressant and convulsant barbiturates on the neuronal nAchR-mediated current to determine whether inhibition of neuronal nAchRs contributes to the anesthetic action of barbiturates. Whole cell nicotine-induced currents were recorded in PC12 derived from rat pheochromocytoma, using the conventional whole cell patch clamp technique in the presence and absence of barbiturates. Both depressant and convulsant barbiturates inhibited the nicotine-induced inward current reversibly and in a dose-dependent manner when co-applied with nicotine. All barbiturates accelerated the current decay. There was no significant difference between the concentrations for 50% inhibition for MPPB isomers. There was no correlation between inhibition of ganglionic nAchRs and anesthetic effects of the barbiturates. These results strongly oppose the idea that inhibition of neuronal nAchRs contributes to the anesthetic action of barbiturates. IMPLICATIONS: We found that both convulsant and depressant barbiturates inhibit the current mediated through ganglionic nicotinic acetylcholine receptors in PC12 cells. This finding suggests that the inhibition of neuronal nicotinic acetylcholine receptors does not contribute to the anesthetic action of barbiturates.
Comparison of the effects of convulsant and depressant barbiturate stereoisomers on AMPA-type glutamate receptors.
BACKGROUND: Alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-type glutamate receptors mediate fast excitatory synaptic transmission in the central nervous system. Although barbiturates have been shown to suppress the AMPA receptor-mediated responses, it is unclear whether this effect contributes to the anesthetic action of barbiturates. The authors compared the effects of depressant [R(-)] and convulsant [S(+)] stereoisomers of 1-methyl-5-phenyl-5-propyl barbituric acid (MPPB) on the AMPA and gamma-aminobutyric acid type A (GABA(A)) receptor-mediated currents to determine if the inhibitory effects on AMPA receptors correlate to the in vivo effects of the isomers. METHOD: The authors measured whole-cell currents in the rat cultured cortical neuron at holding potential of -60 mV. Kainate 500 microM was applied as the agonist for AMPA receptors. Thiopental (3-300 microM), R(-)-MPPB or S(+)-MPPB (100-1,000 microM) was coapplied with kainate under the condition in which the GABA(A) receptor-mediated current was blocked. Effects of MPPB isomers on the current elicited by GABA 1 microM were studied in the separate experiments. RESULTS: Thiopental inhibited the kainate-induced current reversibly and in a dose-dependent manner, with a concentration for 50% inhibition of 49.3 microM. Both R(-)-MPPB and S(+)-MPPB inhibited the kainate-induced current with a little stereoselectivity. R(-)-MPPB was slightly but significantly more potent than S(+)-MPPB. In contrast, R(-)-MPPB enhanced but S(+)-MPPB reduced the GABA-induced current. CONCLUSIONS: Both convulsant and depressant stereoisomers of the barbiturate inhibited the AMPA receptor-mediated current despite of their opposite effects on the central nervous system in vivo. Although thiopental exhibited a considerable inhibition of AMPA receptors, the results suggest that the inhibition of AMPA receptors contributes little to the hypnotic action of the barbiturates.
Cloning and molecular analyses of a gibberellin 20-oxidase gene expressed specifically in developing seeds of watermelon.
To understand the biosynthesis and functional role of gibberellins (GAs) in developing seeds, we isolated Cv20ox, a cDNA clone from watermelon (Citrullus lanatus) that shows significant amino acid homology with GA 20-oxidases. The complementary DNA clone was expressed in Escherichia coli as a fusion protein, which oxidized GA(12) at C-20 to the C(19) compound GA(9), a precursor of bioactive GAs. RNA-blot analysis showed that the Cv20ox gene was expressed specifically in developing seeds. The gene was strongly expressed in the integument tissues, and it was also expressed weakly in inner seed tissues. In parthenocarpic fruits induced by 1-(2-chloro-4-pyridyl)-3-phenylurea treatment, the expression pattern of Cv20ox did not change, indicating that the GA 20-oxidase gene is expressed primarily in the maternal cells of developing seeds. The promoter of Cv20ox was isolated and fused to the beta-glucuronidase (GUS) gene. In a transient expression system, beta-glucuronidase staining was detectable only in the integument tissues of developing watermelon seeds.
Regulation of gibberellin 20-oxidase and gibberellin 3beta-hydroxylase transcript accumulation during De-etiolation of pea seedlings.
Gibberellin (GA) 20-oxidase (GA 20-ox) and GA 3beta-hydroxylase (GA 3beta-hy) are enzymes that catalyze the late steps in the formation of active GAs, and are potential control points in the regulation of GA biosynthesis by light. We have investigated the photoregulation of the GA 20-ox and GA 3beta-hy transcript levels in pea (Pisum sativum L.). The GA 20-ox transcript level was higher in light-grown seedlings than in etiolated seedlings, whereas GA 3beta-hy mRNA accumulation was higher in etiolated seedlings. However, transfer of etiolated seedlings to light led to a 5-fold increase in the expression of both transcripts 4 h after transfer. GA 20-ox mRNA accumulation is regulated by both phytochromes A and B. Transfer to light also resulted in a 6-fold decrease in GA(1) levels within 2 h. These results suggest that the light-induced drop in GA(1) level is not achieved through regulation of GA 20-ox and GA 3beta-hy mRNA accumulation. The application of exogenous GA(1) to apical buds of etiolated seedlings prior to light treatments inhibited the light-induced accumulation of both GA 20-ox and GA 3beta-hy mRNA, suggesting that negative feedback regulation is an important mechanism in the regulation of GA 20-ox and GA 3beta-hy mRNA accumulation during de-etiolation of pea seedlings.
Assignment of type II iodothyronine deiodinase gene (DIO2) to human chromosome band 14q24.2-->q24.3 by in situ hybridization.
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Pretranslational regulation of rhythmic type II iodothyronine deiodinase expression by beta-adrenergic mechanism in the rat pineal gland.
It has been demonstrated that type II iodothyronine deiodinase is present in rat pineal gland, and the deiodinase activity markedly increases during the hours of darkness, primarily through beta-adrenergic mechanism. We have studied the relationship between pineal type II iodothyronine deiodinase messenger RNA (mRNA) and the deiodinase activity to elucidate the mechanisms involved in the nocturnal rise in pineal deiodinase activity. Northern analysis has demonstrated that type II iodothyronine deiodinase mRNA is expressed in rat pineal gland, and the mRNA markedly increases during the hours of darkness. The nocturnal increase in pineal type II iodothyronine deiodinase activity is preceded by the increase in its mRNA. Daytime isoproterenol administration resulted in a rapid increase in pineal type II iodothyronine deiodinase mRNA followed by the increase in deiodinase activity. Propranolol treatment, bilateral superior cervical ganglionectomy, or constant light exposure significantly suppressed the nocturnal rise in type II iodothyronine deiodinase mRNA as well as the deiodinase activity. Moreover, isoproterenol or (Bu)2AMP stimulated type II iodothyronine deiodinase mRNA and the deiodinase activity in cultured rat pineal glands. These results suggest that the rhythmic change in pineal type II iodothyronine deiodinase activity is regulated at least in part at the pretranslational level by a beta-adrenergic mechanism transmitted through superior cervical ganglia.
Primary hypomagnesemia caused by isolated magnesium malabsorption: atypical case in adult.
Isolated magnesium malabsorption is a rare disorder, which bas been described in no more than 30 patients worldwide. Patients with this disorder typically present with convulsion and diarrhea in early infancy. Hypomagnesemia and hypocalcemia were found in a 35-year-old man with muscle cramps, who bad been diagnosed as primary hypoparathyroidism. Oral magnesium therapy corrected the low serum calcium, magnesium and parathyroid hormone levels. We report an atypical case of isolated magnesium malabsorption in an adult.