Abnormal glycosphingolipid mannosylation triggers salicylic acid-mediated responses in Arabidopsis.

The Arabidopsis thaliana protein GOLGI-LOCALIZED NUCLEOTIDE SUGAR TRANSPORTER (GONST1) has been previously identified as a GDP-d-mannose transporter. It has been hypothesized that GONST1 provides precursors for the synthesis of cell wall polysaccharides, such as glucomannan. Here, we show that in vitro GONST1 can transport all four plant GDP-sugars. However, gonst1 mutants have no reduction in glucomannan quantity and show no detectable alterations in other cell wall polysaccharides. By contrast, we show that a class of glycosylated sphingolipids (glycosylinositol phosphoceramides [GIPCs]) contains Man and that this mannosylation is affected in gonst1. GONST1 therefore is a Golgi GDP-sugar transporter that specifically supplies GDP-Man to the Golgi lumen for GIPC synthesis. gonst1 plants have a dwarfed phenotype and a constitutive hypersensitive response with elevated salicylic acid levels. This suggests an unexpected role for GIPC sugar decorations in sphingolipid function and plant defense signaling. Additionally, we discuss these data in the context of substrate channeling within the Golgi.

Diversity of seed cesium accumulation in soybean mini-core collections.

Radiocesium is an extremely harmful radionuclide because of its long half-life; it is important to reduce its transfer from contaminated soil into crops. Here we surveyed genetic variation for seed cesium (Cs) concentration in soybean mini-core collections representing large genetic diversity. The collections grown over 3 years in rotational paddy fields exhibited varying seed Cs concentrations with significant year-to-year correlations, although the phenotypic stability of Cs concentration was lower than that of the congeners potassium (K) and rubidium (Rb). Although Cs is supposedly accumulated in plants via the K transport system, there was no apparent relationship between Cs and K concentrations, whereas a clear positive correlation was observed between Cs and Rb concentrations. Cs and K concentrations in seed showed slightly positive and negative correlations, respectively, with days to flowering. We selected several high or low Cs accumulator candidates on the basis of the 3 years of seed concentration data. These two groups showed significantly different seed Cs concentrations in another field. The differences could not be explained by flowering time alone. These results suggest that genetic variation for seed Cs concentration is present in soybean germplasm and would be useful for breeding low Cs-accumulating varieties.

Roles of BOR2, a boron exporter, in cross linking of rhamnogalacturonan II and root elongation under boron limitation in Arabidopsis.

Boron (B) is required for cross linking of the pectic polysaccharide rhamnogalacturonan II (RG-II) and is consequently essential for the maintenance of cell wall structure. Arabidopsis (Arabidopsis thaliana) BOR1 is an efflux B transporter for xylem loading of B. Here, we describe the roles of BOR2, the most similar paralog of BOR1. BOR2 encodes an efflux B transporter localized in plasma membrane and is strongly expressed in lateral root caps and epidermis of elongation zones of roots. Transfer DNA insertion of BOR2 reduced root elongation by 68%, whereas the mutation in BOR1 reduced it by 32% under low B availability (0.1 µm), but the reduction in shoot growth was not as obvious as that in the BOR1 mutant. A double mutant of BOR1 and BOR2 exhibited much more severe growth defects in both roots and shoots under B-limited conditions than the corresponding single mutants. All single and double mutants grew normally under B-sufficient conditions. These results suggest that both BOR1 and BOR2 are required under B limitation and that their roles are, at least in part, different. The total B concentrations in roots of BOR2 mutants were not significantly different from those in wild-type plants, but the proportion of cross-linked RG-II was reduced under low B availability. Such a reduction in RG-II cross linking was not evident in roots of the BOR1 mutant. Thus, we propose that under B-limited conditions, transport of boric acid/borate by BOR2 from symplast to apoplast is required for effective cross linking of RG-II in cell wall and root cell elongation.

The Arabidopsis IRX10 and IRX10-LIKE glycosyltransferases are critical for glucuronoxylan biosynthesis during secondary cell wall formation.

Arabidopsis IRX10 and IRX10-LIKE (IRX10-L) proteins are closely related members of the GT47 glycosyltransferase family. Single gene knock-outs of IRX10 or IRX10-L result in plants with either a weak or no mutant phenotype. However irx10 irx10-L double mutants are severely affected in their development, with a reduced rosette size and infrequent formation of a small infertile inflorescence. Plants homozygous for irx10 and heterozygous for irx10-L have an intermediate phenotype exhibiting a short inflorescence compared with the wild type, and an almost complete loss of fertility. Stem sections of the irx10 homozygous irx10-L heterozygous or irx10 irx10-L double mutants show decreased secondary cell-wall formation. NMR analysis shows that signals derived from the reducing end structure of glucuronoxylan were detected in the irx10 single mutant, and in the irx10 homozygous irx10-L heterozygous combination, but that the degree of polymerization of the xylan backbone was reduced compared with the wild type. Additionally, xylans from irx10 stem tissues have an almost complete loss of the GlcUA side chain, whereas the level of 4-O-Me-GlcUA was similar to that in wild type. Deletion of the predicted signal peptide from the N terminus of IRX10 or IRX10-L results in an inability to rescue the irx10 irx10-L double mutant phenotype. These findings demonstrate that IRX10 and IRX10-L perform a critical function in the synthesis of glucuronoxylan during secondary cell-wall formation, and that this activity is associated with the formation of the xylan backbone structure. This contrasts with the proposed function of the tobacco NpGUT1, which is closely related to the Arabidopsis IRX10 and IRX10-L proteins, in rhamnogalacturonan II biosynthesis.

The glycerophosphoryl diester phosphodiesterase-like proteins SHV3 and its homologs play important roles in cell wall organization.

Despite the importance of extracellular events in cell wall organization and biogenesis, the mechanisms and related factors are largely unknown. We isolated an allele of the shaven3 (shv3) mutant of Arabidopsis thaliana, which exhibits ruptured root hair cells during tip growth. SHV3 encodes a novel protein with two tandemly repeated glycerophosphoryl diester phosphodiesterase-like domains and a glycosylphosphatidylinositol anchor, and several of its paralogs are found in Arabidopsis. Here, we report the detailed characterization of mutants of SHV3 and one of its paralogs, SVL1. The shv3 and svl1 double mutant exhibited additional defects, including swollen guard cells, aberrant expansion of the hypocotyl epidermis and ectopic lignin deposits, suggesting decreased rigidity of the cell wall. Fourier-transform infrared spectroscopy and measurement of the cell wall components indicated an altered cellulose content and pectin modification with cross-linking in the double mutant. Furthermore, we found that the ruptured root hair phenotype of shv3 was suppressed by increasing the amount of borate, which is supposed to be involved in pectic polysaccharide cross-linking, in the medium. These findings indicate that SHV3 and its paralogs are novel important factors involved in primary cell wall organization.

C-reactive protein-induced upregulation of extracellular matrix metalloproteinase inducer in macrophages: inhibitory effect of fluvastatin.

OBJECTIVE: Extracellular matrix metalloproteinase inducer (EMMPRIN) and matrix metalloproteinase (MMP)-9 were reported to be expressed at the macrophage-rich area in human coronary atherosclerotic plaque. We examined whether C-reactive protein (CRP) activates macrophages to express EMMPRIN and MMP-9 in vitro and whether statins inhibit it. METHODS AND RESULTS: Rat peritoneal macrophages were collected by peritoneal lavage, and were incubated in the presence or absence of CRP. CRP at 5 microg/ml increased the gene expression of EMMPRIN relative to GAPDH, measured by RT-PCR, by 1.67+/-0.07 fold at 24 h and by 1.85+/-0.49 fold at 48 h (both p<0.05). The gene expression of MMP-9 in the presence of CRP at 5 microg/ml was followed by 1.36+/-0.11 fold increase at 24 h and by 3.95+/-0.81 fold at 48 h (both p<0.05). CRP at 5 microg/ml for 48 h increased by 6 fold MMP-9 activity, measured by zymography, without affecting tissue inhibitor of metalloproteinases-1. Boiled CRP at 5 mug/ml for 48 h unaffected MMP-9 activity. Fluvastatin blocked the CRP-induced increases in EMMPRIN and MMP-9 expression and activity. Diphenylene iodonium, an inhibitor of NADPH oxidase, had a similar effect on MMP-9 activity. Fluvastatin suppressed the CRP-induced increases in 8-epi-prostaglandin F(2alpha) levels in the condition media. CONCLUSIONS: CRP is an activator for macrophages to enhance EMMPRIN and MMP-9 expression. Fluvastatin inhibits them presumably through its antioxidant effect.

Borate cross-linked/total rhamnogalacturonan II ratio in cell walls for the biochemical diagnosis of boron deficiency in hydroponically grown pumpkin.

Boron (B) is an essential micronutrient for vascular plants. The function of B has been demonstrated to cross-link monomeric rhamnogalacturonan II (mRG-II) to form dimeric RG-II-borate (dRG-II-B), and thus to stabilize plant cell walls. The dRG-II-B to total RG-II ratio in the cell walls of pumpkin hydroponically grown under various low-B conditions was analyzed to evaluate its applicability to the diagnosis of plant B deficiency. The dRG-II-B ratio in cell walls ranged between approximately 0.9 in B-sufficient tissues and approximately 0.15 in severe B-deficient tissues, reflecting the B nutritional status of tissues. This result indicates that the degree of B shortage in plant tissues is very likely to be diagnosed by the dRG-II-B ratio in cell walls.

11B solid-state NMR investigation of the rhamnogalacturonan II-borate complex in plant cell walls.

The boron in plant cell walls, which is water-insoluble and in the solid state, is solubilized by pectinase digestion to give a dimeric rhamnogalacturonan II-borate (dRG-II-B) complex. To clarify the nondestructive structure of boron present in plant cell walls (as represented by sugar beet fiber), we performed 192- and 96-MHz 11B solid state NMR measurements. The use of a high field magnet frequency of 192-MHz enabled us to observe 11B isotropic chemical shifts at -9.7 and -9.6 ppm for dRG-II-B and sugar beet fiber in the solid state, respectively, demonstrating that the boron in isolated dRG-II-B and in plant cell walls is present as a borate-diol ester (1:2). The observation of the magnetic field dependence of the chemical shift and lineshape for the borate-diol ester (1:2) by quadrupolar interaction suggested that the borate complex had a distorted tetrahedral boron structure.

Angiostatin inhibits coronary angiogenesis during impaired production of nitric oxide.

BACKGROUND: The in vivo mechanism by which inhibition of NO synthase impairs ischemia-induced coronary vascular growth is unknown. We hypothesized that production of the growth inhibitor angiostatin increases during decreased NO production, blunting angiogenesis and collateral growth. METHODS AND RESULTS: Measurements were made in myocardial tissue or interstitial fluid (MIF) from dogs undergoing repetitive coronary occlusions under control conditions or during antagonism of NO synthase (N(G)-nitro-L-arginine methyl ester [L-NAME]) for 7, 14, or 21 days. A sham group was instrumented identically but received no occlusions. In controls, capillary density in the ischemic zone increased initially but returned to baseline at the later times. In the L-NAME group, capillary density was lower at 7 days compared with that of controls. MIF from control dogs induced in vitro endothelial tube formation and cell proliferation, significantly greater than that from the L-NAME group. MIF from shams did not stimulate tube formation. In controls or shams, tube formation or cell proliferation did not change after administration of antiangiostatin, but this antibody restored the responses to control levels in the L-NAME group. Angiostatin expression in MIF was increased in the L-NAME group compared with controls and shams. The activities of tissue matrix metalloproteinases (MMPs) MMP-2 and MMP-9, which generate angiostatin, were increased in the L-NAME group. CONCLUSIONS: Inhibition of NO synthase increased expression of angiostatin and activities of MMP-2 and MMP-9. Our findings indicate that angiostatin inhibits coronary angiogenesis during compromised NO production and may underscore the impairment of coronary angiogenesis during endothelial dysfunction.

Angiostatin: a negative regulator of endothelial-dependent vasodilation.

BACKGROUND: Angiostatin is known to inhibit certain aspects of endothelial function, eg, angiogenesis. Here we investigated the effects of angiostatin on another aspect of endothelial function, vasodilation, and examined mechanisms of inhibition--namely, association of heat-shock protein 90 (hsp90) with endothelial nitric oxide synthase (eNOS) and endothelial generation of nitric oxide (*NO) and superoxide anion (O2-). This avenue of investigation was based on recent reports suggesting that hsp90 modulates NOS production of *NO and O2-. METHODS AND RESULTS: Effects of angiostatin on vasodilation were determined in arterioles with the use of videomicroscopy in response to endothelium- and *NO-dependent vasodilators, acetylcholine (ACh) and vascular endothelial growth factor (VEGF), and an endothelium-independent agonist, papaverine. Association of hsp90 with eNOS was determined in rat aortas and bovine aortic endothelial cells (BAECs). Effects of angiostatin on *NO and O2- generation by BAECs were determined by ozone chemiluminescence and superoxide dismutase (SOD)--inhibitable ferricytochrome c reduction, respectively. Angiostatin impaired vasodilation mediated by ACh and VEGF but not papaverine. Pretreating arterioles with polyethylene glycolated--SOD (PEG-SOD) improved vasodilation to ACh and VEGF. Angiostatin decreased the association of hsp90 with eNOS in aortas and BAEC cultures and increased O2- generation in stimulated BAECs by an Lgamma-nitroargininemethylester (L-NAME)--inhibitable mechanism. CONCLUSIONS: These data indicate angiostatin alters endothelial function by allowing eNOS to generate O2- on activation. Such changes in enzyme function begin to explain, in part, why angiostatin is antiangiogenic and impairs endothelium-dependent vasodilation.

Enalapril suppresses ventricular remodeling more effectively than losartan in patients with acute myocardial infarction.

BACKGROUND: Ventricular remodeling after acute myocardial infarction (AMI) is associated with increased morbidity and mortality. ELITE II study showed that losartan, an angiotensin receptor blocker, shows a survival benefit to the same degree as captopril, an angiotensin-converting enzyme inhibitor, does in patients with heart failure. However, recent OPTIMAAL study showed that clinical outcomes after losartan are not superior to those after captopril in patients with AMI. We examined the effect of losartan on ventricular remodeling after AMI comparatively with that of enalapril. METHODS: We enrolled 203 consecutive patients with AMI (mean age 62 +/- 11 years). All patients underwent primary percutaneous coronary intervention and were randomly assigned to losartan (25-50 mg, n = 101) or enalapril (2.5-10 mg, n = 102) treatment. Biplane left ventriculography was performed just after primary percutaneous transluminal coronary angioplasty (acute phase) and 6 months after the onset of AMI. RESULTS: Any of the maximal creatine kinase level, left ventricular end-diastolic volume index, end-systolic volume index, and ejection fraction measured at acute phase was not different between losartan and enalapril groups. However, changes in left ventricular end-diastolic index (18 +/- 25 vs 8 +/- 24 mL/m2) and left ventricular end-systolic volume index (10 +/- 20 vs 2 +/- 18 mL/m2) from acute phase to 6 months were significantly greater in losartan than in enalapril group. Change in left ventricular ejection fraction (0.2% +/- 10.3% vs 3.4% +/- 11.6%) from acute phase to 6 months was significantly smaller in losartan than in enalapril group. The plasma level of brain natriuretic peptide at 6 months was significantly higher in losartan than in enalapril group (all P < .05). CONCLUSION: These indicate that enalapril suppresses ventricular remodeling after AMI more effectively than losartan.

Circulating level of gelatinase activity predicts ventricular remodeling in patients with acute myocardial infarction.

BACKGROUND: Matrix metalloproteinase (MMP) plays an important role in the development of ventricular remodeling in an animal model of acute myocardial infarction (AMI). We examined whether circulating MMP activity can predict left ventricular (LV) remodeling after AMI in humans. METHODS: We measured the circulating level of MMP-2 and MMP-9 activities (gelatinase activity) at 14 days after the onset of AMI by gelatin zymography in 52 consecutive patients (age 62+/-2). All patients underwent direct PTCA and stenting at an acute stage, and were treated subsequently with losartan or enalapril. Biplane left ventriculography was performed at admission, and 2 weeks and 6 months after the onset of AMI. RESULTS: We expressed gelatinolysis activity as the ratio to MMP-2 standard. Mean gelatinase activity was 0.721+/-0.013. We divided patients into two groups, groups with gelatinolysis activity <0.72 (low group, n=27) and >0.72 (high group, n=25). Either change in LV end-diastolic volume index (LVEDVI, ml/m(2)) or end-systolic volume index (LVESVI, ml/m(2)) from admission to 2 weeks was not different between the two groups. Changes in both LVEDVI and LVESVI from 2 weeks to 6 months were greater in high gelatinolysis activity group than those in low activity group. Moreover, circulating level of gelatinolysis activity was positively correlated with changes in LVEDVI and LVESVI from 2 weeks to 6 months. CONCLUSION: These results demonstrate that circulating level of gelatinase activity can predict LV remodeling after AMI. Inhibition of gelatinase activity at the acute phase may be a therapeutic strategy for the prevention of remodeling after AMI.

Tumor necrosis factor alpha as an endogenous stimulator for circulating coupling factor 6.

OBJECTIVE: We recently showed that mitochondrial coupling factor 6 (CF6) is present as a pressor substance and a prostacyclin inhibitor in systemic circulation. However, the regulation mechanism for circulating CF6 is unknown. We investigated the role of tumor necrosis factor-alpha (TNF-alpha) in the generation and release of CF6. METHODS AND RESULTS: We used two kinds of cells, human umbilical vein endothelial cells (HUVEC) and ECV-304. The concentration of CF6 in the medium increased with time in both ECV-304 and HUVEC. Treatment of ECV-304 and HUVEC with TNF-alpha enhanced the release of CF6 in a dose-dependent manner concomitantly with the decrease in CF6 content in the mitochondria at 24 h. The released CF6 was characterized to be an active full-length peptide by Western blot. The ratio of CF6 to GAPDH mRNA, measured by real-time polymerase chain reaction, was 1.7 fold increased at 1 h after exposure to TNF-alpha in ECV-304 and HUVEC. This enhanced gene expression and release was blocked or suppressed by 70% by stable transfection of dominant negative mutant I kappa B kinase alpha whose efficacy was confirmed by blockade of translocation of NF-kappa B p65 protein and of degradation of I kappa B alpha protein. Flow cytometry analysis revealed that the cell surface-associated CF6 was significantly increased at 24 h after TNF-alpha in a dose-dependent manner. CONCLUSIONS: TNF-alpha stimulates the gene expression of CF6 via activation of NF-kappa B signaling pathway, and promotes the release of CF6 from ECV-304 and HUVEC.

Nicorandil improves cardiac function and clinical outcome in patients with acute myocardial infarction undergoing primary percutaneous coronary intervention: role of inhibitory effect on reactive oxygen species formation.

BACKGROUND: Early reperfusion therapy improves the clinical outcomes of patients with acute myocardial infarction (AMI), but benefits are limited by reperfusion injury in some patients. We examined the effect of nicorandil, a hybrid of K(ATP) channel opener and nicotinamide nitrate, on reactive oxygen species (ROS) formation and clinical outcomes after primary percutaneous coronary intervention (PCI) for AMI. METHODS: Fifty-eight patients with AMI were randomized into control (n = 25) and nicorandil pretreatment groups (n = 33). In the nicorandil group, nicorandil (4 mg as a bolus injection followed by constant infusion at 8 mg/hour for 24 hours) was administered just after admission. ROS formation was assessed by measuring urinary excretion of 8-epi-prostaglandin F2alpha (PGF2alpha) and compared between the 2 groups. Cardiac function and the incidence of reperfusion injury and cardiac events were also compared. RESULTS: Urinary 8-epi-PGF2alpha excretion was increased 2-fold at 60 to 90 minutes after PCI in the control group, whereas it was unchanged after PCI in the nicorandil group (P <.0001 between the 2 groups). The incidence of no-reflow phenomenon was lower in the nicorandil group than in the control group. Left ventricular ejection fraction and cardiac index at 6 months were greater in the nicorandil group than in controls. Plasma brain natriuretic peptide level at 6 months was lower in the nicorandil group. Incidences of inhospital cardiac events and rehospitalization were lower in the nicorandil group than in controls. CONCLUSIONS: Nicorandil improves cardiac function and clinical outcomes in patients with AMI. Suppression of ROS formation may be involved in the mechanism.

Circulating coupling factor 6 in human hypertension: role of reactive oxygen species.

OBJECTIVE: Coupling factor 6 is an endogenous inhibitor of prostacyclin synthesis and might function as an endogenous vasoconstrictor in the fashion of a circulating hormone in rats. We investigated the role of coupling factor 6 in human hypertension. METHODS AND RESULTS: The patients with essential hypertension (EH) (n = 30) received a series of normal salt diet (12 g salt/day) for 3 days, low salt diet (2 g salt/day) for 7 days, and high salt diet (20-23 g salt/day) for 7 days. Normotensive control subjects (n = 27) received normal and low salt diets. The plasma level of coupling factor 6, measured by radioimmunoassay, during normal salt diet was higher in patients with EH than in normotensive subjects (17.6 +/- 1.7 versus 12.8 +/- 0.5 ng/ml, P < 0.01). Whereas the plasma level of coupling factor 6 was unchanged after salt restriction in normotensive subjects, it was decreased after salt restriction (from 12 g/day to 2 g/day) and was increased after salt loading (from 2 g/day to 20-23 g/day) in patients with EH. This increase in plasma level of coupling factor 6 was abolished by oral administration of ascorbic acid, but the level of blood pressure was unaffected. The percentage changes in plasma coupling factor 6 level after salt restriction and loading were positively correlated with those in mean blood pressure (r = 0.57, P < 0.01), and negatively correlated with those in plasma nitric oxide level (r = -0.51, P < 0.05). CONCLUSION: These indicate that circulating coupling factor 6 is elevated in human hypertension and modulated by salt intake presumably via reactive oxygen species.

Separation of human serum transferrins with different iron-binding states by high-performance liquid chromatography using a pyridinium polymer column.

Four molecular forms of transferrins with different iron-binding states were separated by HPLC using a pyridinium polymer column. The elution order was monoferric transferrin bound to the C-site, holotransferrin, apotransferrin and monoferric transferrin bound to the N-site. Human sera were also analyzed with the column, and ICP-MS combined with HPLC was used to detect iron in each peak. Transferrin peaks separated by HPLC were also confirmed by an immunological method. The percentages of iron saturation in transferrins obtained by the HPLC method were compared with the values calculated from clinical data.

Characterization of metal binding properties of rhamnogalacturonan II from plant cell walls by size-exclusion HPLC/ICP-MS.

The binding properties of metal ions to a pectic polysaccharide, rhamnogalacturonan II (RG-II), from plant cell walls were analyzed by size-exclusion HPLC/ICP-MS. The dimeric RG-II borate complex (dRG-II-B) from sugar beet and red wine contained 0.8 - 1.1 mol/mol of B, 0.8 - 1.1 mol/mol of Ca, 0.1 - 0.3 mol/mol of Sr, and 0.03 - 0.07 mol/mol of Ba. The treatment of dRG-II-B with Sr2+, Ba2+, Pb2+ or La3+ exchanged the originally present Ca, Sr and Ba. In contrast, monomeric RG-II (mRG-II), which contained approximately 0.1 mol/mol of Ca, formed complexes with La3+, Eu3+, and Lu3+ added to the solution, but did not do so with Ca2+, Sr2+, Ba2+ and Pb2+. The HPLC/ICP-MS and HPLC/RI (refractive index detector) analysis of the partially hydrolyzed mRG-II that was treated with La3+ indicated that the side chains and backbone of mRG-II together form a lanthanoid binding site.

Molecular characterization of organic nitrogen in cattle manure compost by size-exclusion HPLC with chemiluminescent nitrogen detection.

To characterize organic nitrogen present in cattle manure compost, a hot-water extract of the compost along with an acid-insoluble (humic acid-like) fraction either treated with alkali or laccase or untreated were separated by size-exclusion high-performance liquid chromatography (SE-HPLC). Nitrogen was then detected by chemiluminescent nitrogen detection (CLND), and humic acid-like substances were detected by measuring the absorbance at 420 nm. The acid-insoluble fraction comprised the higher molecular-weight region in the chromatogram of the whole extract, and its chromatogram pattern of CLND was similar to that of the absorbance at 420 nm. Changes in the molecular-weight distribution of the acid-insoluble fraction detected by CLND due to alkaline degradation and laccase polymerization were in agreement with those observed by measuring the absorbance at 420 nm. These results, together with other data, demonstrate that protein and humic acid-like moieties are present in the same molecule in compost organic matter.

Characterization of protein-like fluorophores released from lake phytoplankton on the basis of fractionation and electrophoresis.

Three kinds of lake plankton were cultivated, and the properties of protein-like fluorophores released from the plankton were characterized using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The results were compared with those by gel chromatography with a fluorescence detector and three-dimensional excitation-emission matrix (3-DEEM). The concentrated protein-like fluorophores of algal dissolved organic matter (DOM) were successfully separated from the fulvic-like fluorophores, and analyzed using SDS-PAGE. SDS-PAGE analysis revealed that the protein-like fluorescence DOM released from Microcystis aeruginosa consisted of proteins with molecular weights of 17, 37, 50, 75, 150 kDa, and greater than 250 kDa. The results of SDS-PAGE were consistent with those of gel chromatography. Those substances with molecular weights greater than 250 kDa may be a polysaccharide-peptide complex, called peptidoglycan, which is a component of bacterial cell walls. The molecular weights of protein-like fluorescence DOM from Staurastrum dorsidentiferum were determined to be 37 and 50 kDa. For Cryptomonas ovata, its DOM was found to be composed of substances with molecular weights of between 10 and 150 kDa. The results by high-performance size exclusion chromatography with chemiluminescent nitrogen detection (HPSEC/CLND) analysis suggest that the protein-like fluorophores from the plankton might be composed of substances containing organic nitrogen.

Increased serum anandamide level at ruptured plaque site in patients with acute myocardial infarction.

Inflammation caused by activated macrophages and T lymphocytes may trigger plaque rapture in acute coronary syndrome (ACS). Anandamide and 2-arachidonylglycerol (2-AG) are macrophage-derived signal lipids and may be involved in the pathogenesis of ACS, but no clinical relevant data have been reported. In 43 acute myocardial infarction (AMI) patients (66 +/- 2 years), blood samples were obtained from the aortic root and the infarct-related coronary artery (IRA) using a PercuSurge system during primary percutaneous coronary intervention (PCI). In six patients with stable effort angina (SEA) (56 +/- 6 years), blood samples were obtained from the site of stenosis during elective PCI. In 25 of the 43 AMI patients, anandamide was detected in the serum. Serum anandamide level was 35 +/- 20 pmol/mL in the aorta and was significantly increased to 401 +/- 134 pmol/mL in the IRA (P < 0.01). 2-AG was undetectable in most of the patients. In patients with SEA, neither anandamide nor 2-AG was detected in the serum at the plaque site. In AMI patients with anandamide detected, left ventricular ejection fraction at 2 weeks after PCI was increased by 3.7 +/- 2.1% compared with that at the acute phase, while it was decreased by 3.0 +/- 1.8% in those without anandamide detected (P < 0.05). The serum anandamide level at the culprit lesion was elevated compared with the systemic level in a significant number of AMI patients, indicating the synthesis of anandamide at the IRA. Anandamide was suggested to be derived from ruptured plaque and may exert beneficial effects in humans.