Cloning and characterization of deoxymugineic acid synthase genes from graminaceous plants.
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Graminaceous plants have evolved a unique mechanism to acquire iron through the secretion of a family of small molecules, called mugineic acid family phytosiderophores (MAs). All MAs are synthesized from l-Met, sharing the same pathway from l-Met to 2'-deoxymugineic acid (DMA). DMA is synthesized through the reduction of a 3''-keto intermediate by deoxymugineic acid synthase (DMAS). We have isolated DMAS genes from rice (OsDMAS1), barley (HvDMAS1), wheat (TaD-MAS1), and maize (ZmDMAS1). Their nucleotide sequences indicate that OsDMAS1 encodes a predicted polypeptide of 318 amino acids, whereas the other three orthologs all encode predicted polypeptides of 314 amino acids and are highly homologous (82-97.5%) to each other. The DMAS proteins belong to the aldo-keto reductase superfamily 4 (AKR4) but do not fall within the existing subfamilies of AKR4 and appear to constitute a new subfamily within the AKR4 group. All of the proteins showed DMA synthesis activity in vitro. Their enzymatic activities were highest at pH 8-9, consistent with the hypothesis that DMA is synthesized in subcellular vesicles. Northern blot analysis revealed that the expression of each of the above DMAS genes is up-regulated under iron-deficient conditions in root tissue, and that of the genes OsDMAS1 and TaDMAS1 is up-regulated in shoot tissue. OsDMAS1 promoter-GUS analysis in iron-sufficient roots showed that its expression is restricted to cells participating in long distance transport and that it is highly up-regulated in the entire root under iron-deficient conditions. In shoot tissue, OsDMAS1 promoter drove expression in vascular bundles specifically under iron-deficient conditions. (+info)
Calcium channel blocker azelnidipine reduces glucose intolerance in diabetic mice via different mechanism than angiotensin receptor blocker olmesartan.
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The potential combined effect and mechanism of calcium channel blockers (CCB) and angiotensin II type 1 receptor blockers (ARB) to improve insulin resistance were investigated in type 2 diabetic KK-Ay mice, focusing on their antioxidative action. Treatment of KK-Ay mice with a CCB, azelnidipine (3 mg/kg/day), or with an ARB, olmesartan (3 mg/kg/day), for 2 weeks lowered the plasma concentrations of glucose and insulin in the fed state, attenuated the increase in plasma glucose in the oral glucose tolerance test (OGTT), and increased 2-[(3)H]deoxy-d-glucose (2-[(3)H]DG) uptake into skeletal muscle with the increase in translocation of glucose transporter 4 (GLUT4) to the plasma membrane. Both blockers also decreased the in situ superoxide production in skeletal muscle. The decrease in plasma concentrations of glucose and insulin in the fed state and superoxide production in skeletal muscle, as well as GLUT4 translocation to the plasma membrane, after azelnidipine administration was not significantly affected by coadministration of an antioxidant, 2,2,6,6-tetramethyl-1-piperidinyloxy (tempol). However, those changes caused by olmesartan were further improved by tempol. Moreover, olmesartan enhanced the insulin-induced tyrosine phosphorylation of insulin receptor substrate-1 induced in skeletal muscle, whereas azelnidipine did not change it. Coadministration of azelnidipine and olmesartan further decreased the plasma concentrations of glucose and insulin, improved OGTT, and increased 2-[(3)H]DG uptake in skeletal muscle. These results suggest that azelnidipine improved glucose intolerance mainly through inhibition of oxidative stress and enhanced the inhibitory effects of olmesartan on glucose intolerance, as well as the clinical possibility that the combination of CCB and ARB could be more effective than monotherapy in the treatment of insulin resistance. (+info)
The actions of azelnidipine, a dihydropyridine-derivative Ca antagonist, on voltage-dependent Ba2+ currents in guinea-pig vascular smooth muscle.
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BACKGROUND AND PURPOSE: Although azelnidipine is used clinically to treat hypertension its effects on its target cells, Ca2+ channels, in smooth muscle have not been elucidated. Therefore, its effects on spontaneous contractions and voltage-dependent L-type Ca2+ channels were investigated in guinea-pig portal vein. EXPERIMENTAL APPROACH: The inhibitory potency of azelnidipine on spontaneous contractions in guinea-pig portal vein was compared with those of other dihydropyridine (DHP)-derived Ca antagonists (amlodipine and nifedipine) by recording tension. Also its effects on voltage-dependent nifedipine-sensitive inward Ba2+ currents (IBa) in smooth muscle cells dispersed from guinea-pig portal vein were investigated by use of a conventional whole-cell patch-clamp technique. KEY RESULTS: Spontaneous contractions in guinea-pig portal vein were reduced by all of the Ca antagonists (azelnidipine, Ki = 153 nM; amlodipine, Ki = 16 nM; nifedipine, Ki = 7 nM). In the whole-cell experiments, azelnidipine inhibited the peak amplitude of IBa in a concentration- and voltage-dependent manner (-60 mV, Ki = 282 nM; -90 mV, Ki = 2 microM) and shifted the steady-state inactivation curve of IBa to the left at -90 mV by 16 mV. The inhibitory effects of azelnidipine on IBa persisted after 7 min washout at -60 mV. In contrast, IBa gradually recovered after being inhibited by amlodipine, but did not return to control levels. Both azelnidipine and amlodipine caused a resting block of IBa at -90 mV. Only nifedipine appeared to interact competitively with S(-)-Bay K 8644. CONCLUSIONS AND IMPLICATIONS: These results suggest that azelnidipine induces long-lasting vascular relaxation by inhibiting voltage-dependent L-type Ca2+ channels in vascular smooth muscle. (+info)
Azelnidipine down-regulates renal angiotensin-converting enzyme and mineralocorticoid receptor mRNA in diabetic hypertensive rats.
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Effects of a new lipophilic L-type calcium channel blocker, azelnidipine, on the expression of molecular components of the renin-angiotensin-aldosterone system (RAAS) were assessed. Male Otsuka Long-Evans Tokushima Fatty (OLETF) rats, a model of diabetes with hypertension, and their lean littermates, Long-Evans Tokushima Otsuka (LETO) rats, were treated with azelnidipine for 2 weeks. The renal cortical mineralocorticoid receptor mRNA in OLETF was higher than in LETO, but was suppressed (P<0.05) by azelnidipine. Renal cortical angiotensin-converting enzyme mRNA of OLETF was lower than that of LETO rats, and it was further suppressed by azelnidipine (P<0.05). Azelnidipine can down-regulate the gene expression of molecular components of RAAS. (+info)
Root-to-shoot long-distance circulation of nicotianamine and nicotianamine-nickel chelates in the metal hyperaccumulator Thlaspi caerulescens.
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Plant metal hyperaccumulator species are widely used as models to unravel the heavy metal tolerance and hyperaccumulation mechanisms. Thlaspi caerulescens is capable of tolerating and hyperaccumulating Zn, Cd, and Ni. A search for factors involved in the cellular tolerance to Ni, based on yeast screens, led to isolation of a cDNA encoding a functional nicotianamine (NA) synthase (NAS). The T. caerulescens genome appears to contain a single copy of the NAS gene named TcNAS whose expression is restricted to the leaves. The analysis of dose-response and time-course Ni treatments have revealed that the exposure to Ni triggers the accumulation of NA in the roots. Because neither TcNAS expression nor NAS activity were detected in the roots, the NA accumulation in roots is most probably the result of its translocation from the leaves. Once in the roots, NA, together with Ni, is subsequently found in the xylem, for redirection to the aerial parts. Using liquid chromatography coupled to inductively coupled plasma or electrospray ionization mass spectrometry, it has been shown that part of the Ni is translocated as a stable Ni-NA complex in the xylem sap. This circulation of NA, Ni, and NA-Ni chelates is absent in the non-tolerant non-hyperaccumulator related species T. arvense. Taken together, the results provide direct physiological and chemical evidence for NA and NA-heavy metal complex translocation in a hyperaccumulator species. (+info)
Highly sensitive quantitative analysis of nicotianamine using LC/ESI-TOF-MS with an internal standard.
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A highly sensitive quantitative method for analyzing nicotianamine (NA) by liquid chromatography/electrospray ionization time-of-flight mass spectrometry (LC/ESI-TOF-MS) is reported. Fluorenylmethoxycarbonylation of nicotianamine reduced its polarity and enabled its retention in a reversed-phase column. The adoption of N(epsilon)-nicotyllysine (NL) as an internal standard ensured reliable quantification by giving a linear calibration curve drawn between the NA/NL molar ratios of standard solutions injected and the NA/NL area ratios in mass chromatograms. The high sensitivity of this analytical method allowed us to measure the amount of NA. This analytical method has applications to all research concerning NA. (+info)
The long-acting Ca2+-channel blocker azelnidipine prevents left ventricular remodeling after myocardial infarction.
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Long-acting Ca(2+)-channel blockers have been reported to be effective in treating ischemic heart disease. However, their effects on cardiac remodeling after myocardial infarction (MI) are still unclear. We performed this study to examine the effect of azelnidipine on left ventricular (LV) remodeling, including systolic and diastolic dysfunction, in rats with MI. MI was induced by ligation of the left anterior descending artery. The rats were then separated into 3 groups: a sham-operated group (n = 9), untreated MI group (n = 10), and azelnidipine-treated MI group (n = 10). Four weeks after MI, hemodynamic measurements and Doppler echocardiographic assessment were performed. LV weight and LV end-diastolic dimension were significantly higher in the untreated MI group than in the sham-operated group. Azelnidipine significantly prevented the increases in these parameters. Azelnidipine also improved the ejection fraction (42 +/- 3%, P<0.05) and the E wave to A wave ratio (3.2 +/- 0.5, P<0.05), compared with the untreated MI group (31 +/- 3% and 5.3 +/- 0.8, respectively). In conclusion, azelnidipine can prevent LV remodeling and improve systolic and diastolic function after MI. Administration of long-acting Ca(2+)-channel blockers after MI is an effective strategy for treating MI. (+info)
Effect of benidipine on simvastatin metabolism in human liver microsomes.
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Benidipine, which is a calcium channel blocker that has clinical advantages in the treatment of hypertension, is metabolized by CYP3A4 in humans. The effect of benidipine on the metabolism of simvastatin by human liver microsomes was investigated in order to predict the potential of in vivo drug-drug interactions between benidipine and other substrates of CYP3A4. The results were compared with data generated with azelnidipine, which is also metabolized by CYP3A4. Both benidipine and azelnidipine inhibited simvastatin metabolism in vitro in a concentration-dependent manner. Assuming competitive inhibition, the K(i) values based on the unbound concentrations, were calculated to be 0.846 and 0.0181 microM for benidipine and azelnidipine, respectively. If simvastatin (10 mg) and benidipine (8 mg, the clinically recommended highest dose) were to be administered concomitantly, the ratio of the areas under the concentration-time curves of simvastatin with and without benidipine (AUC((+I))/AUC) was predicted to be 1.01. On the other hand, if simvastatin (10 mg) and azelnidipine (8 mg) were co-administered, the AUC((+I))/AUC for simvastatin was predicted to be 1.72, which is close to the observed value (1.9) in healthy volunteers. These data suggest that benidipine is unlikely to cause a drug interaction by inhibiting CYP3A4 activity in the liver. (+info)