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Endosomal trafficking plays an integral role in various eukaryotic cell activities and serves as a basis for higher-order functions in multicellular organisms. An understanding of the importance of endosomal trafficking in plants is rapidly developing, but its molecular mechanism is mostly unknown. Several key regulators of endosomal trafficking, including RAB5, which regulates diverse endocytic events in animal cells, are highly conserved. However, the identification of lineage-specific regulators in eukaryotes indicates that endosomal trafficking is diversified according to distinct body plans and lifestyles. In addition to orthologues of metazoan RAB5, land plants possess a unique RAB5 molecule, which is one of the most prominent features of plant RAB GTPase organization. Plants have also evolved a unique repertoire of SNAREs, the most distinctive of which are diverse VAMP7-related longins, including plant-unique VAMP72 derivatives. Here, we demonstrate that a plant-unique RAB5 protein, ARA6, acts in an endosomal trafficking pathway in Arabidopsis thaliana. ARA6 modulates the assembly of a distinct SNARE complex from conventional RAB5, and has a functional role in the salinity stress response. Our results indicate that plants possess a unique endosomal trafficking network and provide the first indication of a functional link between a specific RAB and a specific SNARE complex in plants.

SNAREs (soluble N-ethylmaleimide sensitive factor attachment protein receptors) mediate specific membrane fusion between transport vesicles or organelles and target membranes. VAM3/SYP22 and PEP12/SYP21 are Qa-SNAREs that act in the vacuolar transport pathway of Arabidopsis thaliana, and are localized predominantly on the vacuolar membrane and the pre-vacuolar compartment (PVC), respectively. Previous studies have shown that loss-of-function mutants of VAM3/SYP22 or PEP12/SYP21 showed male gametophytic lethality, suggesting that VAM3/SYP22 and PEP12/SYP21 possess different, non-redundant functions. We have re-evaluated the effects of mutations in these genes using T-DNA insertion mutants in the Columbia accession. We found that a mutation in VAM3/SYP22 (vam3-1) caused pleiotropic abnormalities, including semi-dwarfism and wavy leaves. In contrast, a loss-of-function mutant of PEP12/SYP21 (pep12) showed no apparent abnormal phenotype. We also found that the double vam3-1 pep12 mutant had severely reduced fertilization competence, although male and female gametophytes (vam3-1(-) pep12(-) ) maintained the ability to fertilize. Moreover, promoter swapping analysis revealed that expression of a GFP-PEP12/SYP21 fusion under the control of the VAM3/SYP22 promoter suppressed all phenotypes of the vam3-1 mutant. These results indicate that the functions of VAM3/SYP22 and PEP12/SYP21 were redundant and interchangeable., (© 2010 The Authors. The Plant Journal © 2010 Blackwell Publishing Ltd.)

The SNARE complex is a key regulator of vesicular traffic, executing membrane fusion between transport vesicles or organelles and target membranes. A functional SNARE complex consists of four coiled-coil helical bundles, three of which are supplied by Q-SNAREs and another from an R-SNARE. Arabidopsis thaliana VAMP727 is an R-SNARE, with homologs only in seed plants. We have found that VAMP727 colocalizes with SYP22/ VAM3, a Q-SNARE, on a subpopulation of prevacuolar compartments/endosomes closely associated with the vacuolar membrane. Genetic and biochemical analyses, including examination of a synergistic interaction of vamp727 and syp22 mutations, histological examination of protein localization, and coimmunoprecipitation from Arabidopsis lysates indicate that VAMP727 forms a complex with SYP22, VTI11, and SYP51 and that this complex plays a crucial role in vacuolar transport, seed maturation, and vacuole biogenesis. We suggest that the VAMP727 complex mediates the membrane fusion between the prevacuolar compartment and the vacuole and that this process has evolved as an essential step for seed development.

The blood concentrations of melatonin are elevated by stress-induced sympathetic nerve excitation and are affected by some anesthetics. Isoflurane has an effect to increase sympathetic nerve activity when compared with sevoflurane. This study was performed to investigate the effects of these two anesthetics on the blood concentrations of melatonin. Female patients were anesthetized with either isoflurane or sevoflurane. We obtained blood samples before and 5 min after 5% isoflurane (ISO group) or 7% sevoflurane (SEV group) anesthesia. The blood melatonin concentrations during anesthesia in the ISO group increased significantly, from 65 +/- 60 to 170 +/- 90 pg x ml(-l); mean +/- SD ( P < 0.05), whereas those in the SEV group decreased, from 60 +/- 50 to 30 +/- 30 pg x ml(-l) ( P < 0.05). In conclusion, isoflurane increases, but sevoflurane decreases blood melatonin concentrations.

Background: Estrogen replacement therapy (ERT) has an antioxidant effect that opposes the oxidation of LDL. Oral ERT-induced increases in plasma triglyceride, however, are associated with decreased LDL size, which may counteract this antioxidant effect. Because lower doses of oral estrogen do not affect plasma triglyceride concentrations, LDL size might not change, and the antioxidant effect of estrogen might be preserved. We investigated whether a lower dose of oral estrogen could eliminate the adverse effects of high-dose oral ERT on the size and oxidative susceptibility of LDL in postmenopausal women., Methods and Results: Postmenopausal women received no treatment or were treated with oral conjugated equine estrogen (CEE) 0.625 or 0.3125 mg/d for 3 months. CEE at a dose of 0.625 mg/d significantly increased plasma triglyceride concentrations and decreased LDL diameter, but the concentrations of LDL-derived thiobarbituric acid reactive substances (TBARS) and lag time for conjugated diene formation did not change. In contrast, 0.3125 mg of CEE did not affect plasma triglyceride concentrations or LDL diameter and significantly decreased LDL-derived TBARS concentrations and significantly prolonged LDL lag time. Estrogen-induced changes in LDL diameter correlated negatively with changes in plasma triglyceride (r=-0.44, P<0.01) and LDL-derived TBARS (r=-0.57, P<0.001) but positively with changes in LDL lag time (r=0.42, P<0.01)., Conclusions: Because oral CEE at a dose of 0.3125 mg/d does not elevate plasma triglyceride, resulting in unchanged size of LDL particles that are resistant to oxidation, the antioxidant effect of estrogen can be preserved.

Melatonin, a pineal secretory product, is a potent scavenger of a variety of free radicals. The aim of this study was to investigate the effect of melatonin on the prevention of mitochondrial injury induced by hepatic ischemia and reperfusion. Rats were subjected to 70 min of hepatic ischemia and 2 h of reperfusion. Fifteen minutes prior to ischemia and at reperfusion, animals received vehicle or melatonin (10 mg/kg body weight) intraperitoneally. In the vehicle-treated animals, the respiratory control index, ADP/O, State 3 respiration and dinitrophenol-induced uncoupled respiration decreased markedly after ischemia/reperfusion and were restored by melatonin administration. Similarly, pH change coupled with mitochondrial energy transfer was suppressed by ischemia/reperfusion with the effects being reduced by melatonin treatment. Mitochondrial lipid peroxidation was elevated in the ischemic/reperfused vehicle-treated livers, but this elevation was attenuated by melatonin. Mitochondrial glutathione peroxidase activity decreased in the vehicle-treated group with this decrease being reduced by melatonin treatment. Electron microscopic studies demonstrated that treatment with melatonin restored to near normal the ischemia/reperfusion-induced disorganization of mitochondrial structure. Melatonin protects against mitochondrial injury which reduces mitochondrial oxidative stress and improves ischemia/reperfusion-induced hepatic energy metabolism.

This study investigated the effects of edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one, MCI-186), a potent free radical scavenger, on the prevention of mitochondrial injury induced by hepatic ischemia and reperfusion. Mature male rats were subjected to 70 min of hepatic ischemia and 2 h of reperfusion. The rats received vehicle or edaravone (10 mg/kg body weight) intravenously prior to ischemia, before reperfusion and 1 h after reperfusion. In the vehicle-treated animals, the respiratory control index, ADP/O, State 3 respiration and dinitrophenol-induced uncoupled respiration decreased markedly after ischemia/reperfusion and were restored by edaravone administration. Mitochondrial lipid peroxidation was elevated in the vehicle-treated group, which was attenuated by edaravone, while mitochondrial glutathione peroxidase activity decreased in the vehicle-treated group, which was similarly abrogated by edaravone treatment. Electron microscopic observation demonstrated that treatment with edaravone restored the ischemia/reperfusion-induced disorganization of mitochondrial structures. Edaravone protects against mitochondrial injury, which prevents mitochondrial oxidative stress and improves ischemia/reperfusion-induced hepatic energy metabolism.

Damage to mitochondria as a result of the intrinsic generation of free radicals is theoretically involved in the processes of cellular aging. Herein, we investigated whether acutely administered melatonin, due to its free radical scavenging activity, would influence mitochondrial metabolism. Mitochondrial respiratory activity and respiratory chain complex I and IV activities in liver mitochondria from a strain of senescence-accelerated-prone mice (SAMP8) and a strain of senescence-accelerated-resistant mice (SAMR1) were measured when the animals were 12 months of age. Respiratory control index (RCI), ADP/O ratio, State 3 respiration and dinitrophenol (DNP)-dependent uncoupled respiration were significantly lower in SAMP8 than in SAMR1. In contrast, State 4 respiration was significantly higher in SAMP8 than in SAMR1. Activities of complexes I and IV in SAMP8 were significantly lower than in SAMR1. Melatonin administration (10mg/kg body weight, intraperitoneally) 1h prior to sacrifice significantly increased RCI, ADP/O ratio, State 3 respiration and DNP-induced uncoupled respiration in SAMP8 while also significantly reducing State 4 respiration in SAMP8. The injection of melatonin also significantly increased complex I activity in both mouse strains and complex IV activity in the liver of SAMP8 mice. These results document an age-related decrease in hepatic mitochondrial function in SAM which can be modified by an acute pharmacological injection of melatonin; the indole stimulated mitochondrial respiratory chain activity which would likely reduce deteriorative oxidative changes in mitochondria that normally occur in advanced age.

Background: Postmenopausal estrogen replacement therapy (ERT) has an antioxidant effect that opposes the oxidation of LDL particles. Oral ERT-induced increases in plasma triglyceride, however, decrease LDL particle size, which may counteract this antioxidant effect. Because transdermal ERT decreases plasma triglyceride, it may not decrease LDL particle size and may preserve estrogen's antioxidant effect. The present study investigates whether transdermal ERT can eliminate the adverse effects of oral ERT on the size and oxidative susceptibility of LDL in postmenopausal women., Methods and Results: Postmenopausal women received no treatment (n=12) or were treated with either 0.625 mg oral conjugated equine estrogen daily (n=16) or with transdermal estradiol (50 microg/d, n=16) for 3 months. Plasma lipids and the diameter of LDL particles were determined. Susceptibility of LDL to oxidation was analyzed by incubation with CuSO4 and subsequent measurement of thiobarbituric acid reactive substance (TBARS) concentrations. Oral ERT significantly increased plasma triglyceride and decreased LDL diameter but did not affect LDL-derived TBARS concentrations. In contrast, transdermal ERT significantly decreased the concentrations of plasma triglyceride and LDL-derived TBARS and significantly increased LDL diameter. Estrogen-induced changes in LDL diameter correlated negatively with changes in plasma triglyceride (r=-0.51, P<0.001) and LDL-derived TBARS (r=-0.50, P<0.001)., Conclusions: Because transdermal, but not oral ERT, decreases plasma triglyceride and produces larger LDL particles that are resistant to oxidation, the antioxidant effect of estrogen can be preserved.

Mitochondrial oxidative damage from free radicals may be a factor underlying aging. We investigated whether long-term administration of physiological levels of melatonin, a direct free radical scavenger and indirect antioxidant, influences mitochondrial respiratory activity in liver of senescence-accelerated mice (SAM). Liver was obtained in the middle of dark period of the daily light:dark cycle from SAMP8, a strain of mice prone to accelerated senescence, and from SAMR1, a senescence-resistant strain, at 6 and 12 months of age. Respiratory control index (RCI), adenosine-5-diphosphate (ADP)/O ratio, State 3 respiration and dinitophenol (DNP)-dependent uncoupled respiration exhibited significant age-associated decreases in SAMP8. SAMP8 also showed significant age-associated reductions in respiratory chain complex I and IV activities. No age-related effects were found in these parameters in SAMR1. Daily oral melatonin administration (2 microg/mL of drinking fluid) beginning at 7 months of age significantly increased RCI, State 3 respiration, DNP-dependent uncoupled respiration, and complex I and IV activities in both mouse strains when they were 12 months old. These results reveal age-related reductions in mitochondrial function in SAM mice which are modified by melatonin; the most likely explanation for the corrective actions of melatonin relate to its antioxidative actions in mitochondria and other portions of the cell. The implication of the findings is that melatonin may be beneficial during aging as it reduces the deteriorative oxidative changes in mitochondria and other portions of the cell associated with advanced age.

The purpose of the present study was to investigate the effects of tamoxifen on the size and oxidative susceptibility of low-density lipoprotein (LDL) particles in breast cancer patients with tamoxifen-induced fatty liver. We investigated the following breast cancer patients: 13 receiving no tamoxifen (group A), 13 receiving tamoxifen 40 mg daily but without fatty liver (group B), and 13 receiving tamoxifen 40 mg daily with fatty liver (group C). Plasma lipids and diameter of LDL particles were measured. Susceptibility of LDL to oxidation was analyzed by incubation with CuSO(4) while monitoring conjugated diene formation and assaying thiobarbituric acid reactive substances (TBARS). Plasma total and LDL cholesterol concentrations in groups B and C were significantly lower than those in group A. In group C, concentrations of plasma triglyceride (TG) and TBARS were significantly greater, but LDL particle diameter and lag time for LDL oxidation were significantly smaller than those in groups A and B. Plasma TG concentrations correlated negatively with computed tomography ratio of liver to spleen (r = -0.76; P < 0.001). LDL particle diameter correlated negatively with plasma TG (r = -0.62; P < 0.001) and TBARS (r = -0.44; P < 0.01), but positively with LDL lag time (r = 0.47; P < 0.01). Tamoxifen-induced fatty liver in breast cancer patients may be atherogenic, via increased TG and consequent small, easily oxidized LDL particles.

We investigated whether long-term melatonin administration in the drinking water influences oxidative modification of lipids and proteins and antioxidative enzyme activity in brain of senescence-accelerated mice (SAM). Cerebral cortex was obtained in the middle of the dark period of the daily light cycle from SAMP8, a strain of mice prone to accelerated senescence, and from SAMR1, a senescence-resistant strain, at 3, 6, and 12 months of age. Thiobarbituric acid-reactive substances (TBARS) and protein carbonyls exhibited significant age-related increases in both strains. Glutathione peroxidase (GPx) activity decreased significantly at 12 months of age in SAMP8. No age effect was found in GPx activity in SAMR1, or in superoxide dismutase (SOD) activity in either strain. Melatonin administration (2 microg/mL) via the drinking fluid beginning at 7 months significantly decreased neural TBARS content (over 30%) in both strains and lowered the protein carbonyl content in the brain of SAMP8 mice. Furthermore, melatonin significantly augmented GPx activity (over 20%) in both strains. Melatonin had no effect on SOD activity. These results suggest an age-related increase in cerebral tissue vulnerability to oxidation in SAM that can be modified by melatonin, most likely through the ability of melatonin to scavenge oxygen free radicals and to stimulate antioxidant enzyme activity.

Mitochondrial oxidative damage from free radicals may be a factor underlying aging, and melatonin, a powerful free radical scavenger, may participate in mitochondrial metabolism. We measured respiratory chain complex I and IV activities in liver mitochondria from a strain of senescence-accelerated prone mice (SAMP8) and a strain of senescence-accelerated resistant mice (SAMR1) at age 3, 6, and 12 months. No age-associated effects were found in either complex I and IV activities, thiobarbituric acid-reactive substances (TBARS), or glutathione peroxidase (GPx) activity in SAMRI. In contrast, SAMP8 showed significant age-associated decreases in complex I and IV activities. While no age effect was found in TBARS in SAMP8, TBARS levels in SAMP8 were significantly more abundant than in SAMRI. GPx activity in SAMP8 decreased significantly by 12 months. Daily oral melatonin administration (2 microg/mL of drinking fluid) beginning when the mice were 7 months old significantly increased complex I and IV activity, decreased TBARS, and increased GPx activities in both SAMRI and SAMP8 at 12 months. The implication of the findings is that melatonin may be beneficial during aging as it reduced the deteriorative oxidative changes in mitochondria and other portions of the cell associated with advanced age.

Background: Estrogen increases C-reactive protein (CRP) in postmenopausal women. Estrogen also decreases cell adhesion molecules, whereas elevated CRP stimulates the expression of cell adhesion molecules. Because androgens have antiinflammatory effects, androgenic progestins such as medroxyprogesterone acetate (MPA) may inhibit proinflammatory effects of estrogen. We investigated the effects of MPA on estrogen-induced changes in acute inflammatory proteins and cell adhesion molecules in postmenopausal women., Methods and Results: Postmenopausal women were treated daily with conjugated equine estrogen (CEE, 0.625 mg), CEE plus MPA 2.5 mg, or CEE plus MPA 5.0 mg for 3 months. CEE significantly increased CRP concentrations by 320.1+/-210.2% (P<0.05). The addition of MPA to CEE, however, inhibited the increase in CRP in a concentration-dependent manner (MPA 2.5 mg, 169.8+/-66.9%, P<0.05; MPA 5 mg, 55.0+/-30.4%, not significant). Similarly, CEE increased amyloid A protein concentrations, whereas MPA reversed this effect. Interleukin-6 concentration did not change significantly in any treatment group. CEE alone significantly decreased the concentration of E-selectin, but the concentrations of intercellular adhesion molecule and vascular cellular adhesion molecule did not change significantly. The addition of MPA tended to decrease the levels of cell adhesion molecules, and use of 5.0 mg MPA showed significant decreases in all cell-adhesion molecule concentrations., Conclusions: Concurrent MPA administration may attenuate estrogen's proinflammatory effect. Because MPA in combination with CEE decreased cell adhesion molecule concentrations, the anti-inflammatory effect of MPA may actually be responsible for the favorable effect of estrogen-progestogen combinations on cell adhesion molecules in postmenopausal women.

Background: Estrogen increases endothelium-dependent vasodilation in postmenopausal women. However, use of progestins in combination with estrogen may counter beneficial effects of estrogen on endothelium. We investigated the effect of medroxyprogesterone acetate (MPA) on estrogen-induced increase in endothelium-dependent vasodilation in postmenopausal women., Methods and Results: Postmenopausal women were treated daily with conjugated equine estrogen (CEE) 0.625 mg (n=14), CEE 0.625 mg and MPA 2.5 mg (n=15) or CEE 0.625 mg and MPA 5.0 mg (n=16) for 3 months. Plasma lipids and hormones were measured before and after treatment. Vasodilatory responses of the brachial artery were evaluated by measuring flow-mediated vasodilation (FMD) and nitroglycerin-induced vasodilation by use of high-resolution ultrasonography. Susceptibility of LDL to oxidation was analyzed by incubation with CuSO(4) while kinetics of conjugated diene formation was monitored. Plasma total and LDL cholesterol concentrations were decreased significantly in all groups. CEE increased FMD significantly, from 4.5+/-1.7% to 8.5+/-2.8% (P<0.001). Addition of MPA reversed this effect in a concentration-dependent manner (for MPA 2.5 mg, from 5.0+/-3.2% to 6.2+/-3.1%; for MPA 5.0 mg, from 4.9+/-3.4% to 3.6+/-3.7%; P=NS for each). No treatment significantly altered nitroglycerin-induced dilation. Lag time for conjugated diene formation was prolonged significantly in all groups, and the oxidation rate was significantly reduced., Conclusions: Concurrent MPA administration may offset favorable effects of estrogen on endothelial function in postmenopausal women. Because MPA did not diminish LDL-lowering and antioxidant effects of estrogen, MPA-induced inhibition of endothelium-dependent vasodilation may be independent of changes in oxidative susceptibility and plasma concentration of LDL.

We evaluated the antioxidative effect of melatonin on the oxidized low-density lipoprotein (LDL)-induced impairment of nitric oxide (NO) production in human umbilical artery, which may be the prime cause of endothelial dysfunction in pre-eclampsia. Umbilical artery sections with intact endothelium were obtained from healthy pregnant women who were delivered between 37 and 40 wk of gestation. The production of NO in the umbilical arteries was stimulated by adding L-arginine followed by incubation for 60 min. NO concentrations were estimated by measuring nitrite ions (NO(2) using high-performance liquid chromatography. LDL was oxidized by incubation with 5 microM CuSO(4) at 37 degrees C for 4 hr, followed by dialysis at 4 degrees C for 24 hr. Prior to the addition of L-arginine, the segments were treated with native or oxidized LDL (0, 50, 100, 200, 400 microg/mL), or were pre-treated with either mannitol (50 mM) or melatonin (20, 100, 500 microM) before adding oxidized LDL. Changes in L-arginine-induced NO(2)(-) production were expressed as a percentage of NO(2)(-) production at the end of pre-incubation. Treatment with oxidized LDL significantly reduced L-arginine-induced NO(2)(-) production (P<0.05), while NO(2)(-) production did not change by incubation with native LDL. Pre-treatment with melatonin significantly increased NO(2)(-) production that had been decreased by oxidized LDL (P<0.05). Similarly, pre-treatment with mannitol reversed the oxidized LDL-induced reduction in NO(2)(-) production (P<0.05). These results indicate that melatonin protects against oxidized LDL-induced inhibition of NO production in the endothelium of human umbilical arteries, most likely through its ability to scavenge hydroxyl radicals.

Hyperhomocysteinemia is a major and independent risk factor for vascular disease. Oxidative stress is a possible mechanism for homocysteine (Hcy)-induced endothelial dysfunction. Herein, we evaluated the antioxidant property of melatonin as related to the vasospastic effect of Hcy on the human umbilical artery. Helical strips of human umbilical arteries with intact endothelium were obtained at elective Caesarean delivery between 37 and 39 wks of gestation. Changes in 5-hydroxytryptamine (5-HT)-induced vasoconstriction were measured. Arterial strips were treated with FeSO4 (10 microM) and Hcy (10 or 100 microM) or pre-treated with a hydroxyl radical (.OH) scavenger (mannitol, 20 mM), a cyclooxygenase inhibitor (indomethacin, 20 microM), nitric oxide (NO) synthesis inhibitor (L-NG-monomethylarginine, LNMA, 200 microM), or melatonin (1 or 10 microM). Hcy potentiated 5-HT-induced constriction in a concentration-dependent manner. Pre-treatment with mannitol significantly suppressed the vasospastic effect of Hcy. LNMA augmented the vasospastic effect of Hcy, but indomethacin did not. Melatonin significantly suppressed the vasospastic effect of Hcy in a concentration-dependent manner. These findings suggest that Hcy potentiates 5-HT-induced vasoconstriction in the human umbilical artery, possibly by suppressing bioavailable NO. Melatonin protects against the vasospastic effect of Hcy, most likely by scavenging.OH arising from Hcy autooxidation.

We investigated the effects of melatonin on ischemia/reperfusion-induced oxidative damage to mitochondria in fetal rat brain. The utero-ovarian arteries were occluded bilaterally for 20 min in female Wistar rats on day 19 of pregnancy to induce fetal ischemia. Reperfusion was achieved by releasing the occlusion and restoring circulation for 30 min. A sham operation was performed in control rats. Melatonin (10 mg/kg) or vehicle was injected intraperitoneally 60 min prior to occlusion. We measured the respiratory control index (RCI) and the adenosine 5-diphosphate (ADP)/oxygen ratio as indicators of mitochondrial respiratory activity, as well as the concentration of thiobarbituric acid-reactive substances (TBARS) in the mitochondria of fetal brain. Ischemia/reperfusion significantly elevated the concentration of TBARS and significantly reduced the RCI as well as the ADP/oxygen ratio. Melatonin treatment reversed the ischemia/reperfusion-induced reductions in the RCI (2.29 +/- 0.06-2.64 +/- 0.09, P < 0.05) and in the ADP/oxygen ratio (1.48 +/- 0.03-1.57 +/- 0.02, P < 0.05), and also reduced the elevation in concentration of TBARS (11.00 +/- 0.34-7.57 +/- 0.74 nM/mg protein, P < 0.01), resulting in values similar to those in untreated, sham-ischemic animals. The results indicate that administration of melatonin to pregnant rats may prevent ischemia/reperfusion-induced oxidative mitochondrial damage in fetal rat brain.