[Spectroscopic and Molecular Characterization of Water Soluble Organic Matter from Sediments in the Macrophyte-dominated and Algae-dominated Zones of Taihu Lake].

Macrophyte- and algae-dominated lakes (zones) are the two typical states of shallow lakes, where the source and composition of organic matter are distinct. The burial of organic matter (OM) in the sediment supports the role of lakes as carbon sinks. However, organic matter in the sediments could be further processed, influencing the carbon cycle. The post-burial metabolism of the sedimentary OM relates closely to its composition. However, information on the differences in composition remains limited, especially the molecular composition of organic matter from sediments in the macrophyte-dominated and algae-dominated lakes. In this study, sediments were collected from the macrophyte-dominated and algae-dominated zones of Taihu Lake (East Taihu Lake and Meiliang Bay, respectively), and the active pool of sedimentary OM (water soluble organic matter, WSOM) was extracted and purified. The composition of the WSOM was characterized in detail via absorption spectroscopy, fluorescent spectroscopy, infrared spectroscopy, and Fourier transform-ion cyclotron resonance mass spectrometry (FT-ICR MS). The optical index of E2:E3 showed that the molecular size of WSOM in the macrophyte-dominated zone (M-WSOM) was slightly larger than that in the algae-dominated zone (A-WSOM). Consistently, the intensity-weighted molecular weights were identified as 388.9 and 379.9, respectively, via FT-ICR MS analysis. M-WSOM was more humified than A-WSOM, as evidenced by the SUVA254 and HIX values. The FT-ICR MS results showed that the relative abundance of the condensed aromatic substance and the aromatics were 6.3% (intensity-weighted) and 7.7% for M-WSOM and 1.1% and 4.4% for A-WSOM, respectively. The excitation-emission matrix fluorescence-parallel factor analysis (EEM-PARAFAC) suggested that the protein-like component was more in A-WSOM than that in M-WSOM, and the FT-ICR MS results showed that the intensity-weighted relative abundances of peptides were 35.6% and 15.6% for A-WSOM and M-WSOM, respectively. The FT-ICR MS results further showed that the heteroatom-containing molecules were abundant in the sedimentary WSOM, i.e., 82.9% and 91.7% for M-WSOM and A-WSOM, respectively. The nitrogen-containing molecules dominated, contributing to 53.5% and 78.5% of M-WSOM and A-WSOM, respectively. There were 30.4% and 41.4% phosphorus-containing molecules in M-WSOM and A-WSOM, respectively. The phosphorus-containing molecules in M-WSOM were mainly aliphatics and highly unsaturated structures with low oxygen, whereas those in A-WSOM were mainly peptides. This study elucidated the detailed molecular composition of WSOM in the macrophyte-dominated and algae-dominated zones of Taihu Lake, which aids understanding of the carbon, nitrogen, and phosphorus biogeochemical cycles in lakes.

Human organic cation transporter 3 mediates the transport of antiarrhythmic drugs.

Antiarrhythmic drugs have been considered to be transported by the organic cation transport system. The purpose of this study was to elucidate the molecular mechanism underlying the transport of antiarrhythmic drugs using cells from the second segment of the proximal tubule (S2) cells of mice expressing human-organic cation transporter 3 (S2 human-OCT3). The antiarrhythmic drugs tested were cibenzoline, disopyramide, lidocaine, mexiletine, phenytoin, pilsicanide, procainamide and quinidine. Human-OCT3 mediated a time- and dose-dependent uptake of quinidine and lidocaine, with Km values of 216 and 139 microM, respectively. Human-OCT3 also mediated the uptake of disopyramide and procainamide but not that of phenytoin. All antiarrhythmic drugs tested inhibited histamine uptake mediated by human-OCT3 in a dose-dependent manner. The IC50 values of antiarrhythmic drugs for human-OCT3 ranged between 0.75 and 656 microM. Kinetic analysis revealed that disopyramide, lidocaine, procainamide and quinidine inhibited histamine uptake mediated by human-OCT3 in a competitive manner. In conclusion, these results suggest that human-OCT3 mediates the transport of antiarrhythmic drugs, which may be the mechanism underlying the distribution and the elimination of these drugs.

Characterization of the renal tubular transport of zonampanel, a novel alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor antagonist, by human organic anion transporters.

Zonampanel monohydrate (YM872; [2,3-dioxo-7-(1H-imidazol-1-yl)-6-nitro-1,2,3,4-tetrahydro-1-quinoxalinyl]acetic acid monohydrate) is a novel alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor antagonist. The major elimination route for zonampanel has been reported to be by urine via the kidneys. The purpose of this study is to elucidate the molecular mechanism of the renal excretion of zonampanel using cells stably expressing human organic anion transporters (hOAT) 1, hOAT2, hOAT3, and hOAT4, as well as human organic cation transporters (hOCT) 1 and hOCT2. Another AMPA receptor antagonist, YM90K [6-(1H-imidazol-1-yl)-7-nitro-2,3(1H,4H)-quinoxalinedione monohydrochloride], a decarboxymethylated form of zonampanel, was also used for comparing the substrate specificity. Zonampanel inhibited the uptake of prototypical organic anion substrates, [14C]para-aminohippurate in hOAT1 and [3H]estrone sulfate in hOAT3 and hOAT4, in a competitive manner. A time- and concentration-dependent increase in [14C]zonampanel uptake was observed in cells expressing hOAT1, hOAT3, and hOAT4. The Km values of zonampanel uptake by hOAT1, hOAT3, and hOAT4 were 1.4, 7.7, and 215 microM, respectively. Considering the localization of each transporter, results suggest that zonampanel is taken up via hOAT1 and hOAT3 from the blood into proximal tubular cells and then effluxed into the lumen via hOAT4. Probenecid and cimetidine competitively inhibited [14C]zonampanel uptake by the hOATs (hOAT1, hOAT3, and hOAT4 for probenecid; hOAT3 for cimetidine). YM90K inhibited the uptake of the prototypical substrate via hOAT3 competitively, but the uptake via hOAT1 noncompetitively. These findings suggest that the prototypical organic anion substrates (para-aminohippurate and estrone sulfate), cimetidine, probenecid, and zonampanel share binding specificity in each hOAT, whereas YM90K does not in hOAT1, possibly due to it being the decarboxymethylated form.

Interactions of human- and rat-organic anion transporters with pravastatin and cimetidine.

We have elucidated the interactions of human and rat organic anion transporters (hOATs and rOATs) with pravastatin and cimetidine. Pravastatin inhibited hOAT1/rOAT1, hOAT2/rOAT2, hOAT3/rOAT3, and hOAT4. The mode of inhibition was noncompetitive for hOAT1 and hOAT2, whereas it was competitive for hOAT3 and hOAT4. Cimetidine also inhibited hOAT1/rOAT1, hOAT3/rOAT3, and hOAT4. The mode of inhibition was a combination of competitive and noncompetitive manners for hOAT1, whereas it was competitive for hOAT3. The effects of OAT inhibitors on OAT1, OAT2, and OAT3 exhibited some but not so remarkable interspecies differences between humans and rats. In conclusion, we have characterized pravastatin and cimetidine as OAT inhibitors.

Evidence for a role of human organic anion transporters in the muscular side effects of HMG-CoA reductase inhibitors.

The purpose of this study was to elucidate the role of human organic anion transporters (human OATs) in the induction of drug-induced skeletal muscle abnormalities. 3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors have been clinically used for lowering plasma cholesterol levels, and are known to induce various forms of skeletal muscle abnormalities including myopathy and rhabdomyolysis. Immunohistochemical analysis revealed that human OAT1 and human OAT3 are localized in the cytoplasmic membrane of the human skeletal muscles. The activities of human OATs were measured using mouse cell lines from renal proximal tubules stably expressing human OATs. Human OAT3, but not human OAT1, mediates the transport of pravastatin. Fluvastatin inhibited organic anion uptake mediated by human OAT1 in a mixture of competitive and noncompetitive manner, whereas simvastatin and fluvastatin noncompetitively inhibited the organic anion uptake mediated by human OAT3. In conclusion, the organic anion transporters OAT1 and OAT3 are localized in the cytoplasmic membrane of human skeletal muscles. Pravastatin, simvasatin, and fluvasatin inhibit human OATs activity. These results suggest that muscle organic anion transporters play a role in the muscular side effects of HMG-CoA reductase inhibitors.

Interactions of human organic anion transporters with diuretics.

The tubular secretion of diuretics in the proximal tubule has been shown to be critical for the action of drugs. To elucidate the molecular mechanisms for the tubular excretion of diuretics, we have elucidated the interactions of human organic anion transporters (hOATs) with diuretics using cells stably expressing hOATs. Diuretics tested were thiazides, including chlorothiazide, cyclothiazide, hydrochlorothiazide, and trichlormethiazide; loop diuretics, including bumetanide, ethacrynic acid, and furosemide; and carbonic anhydrase inhibitors, including acetazolamide and methazolamide. These diuretics inhibited organic anion uptake mediated by hOAT1, hOAT2, hOAT3, and hOAT4 in a competitive manner. hOAT1 exhibited the highest affinity interactions for thiazides, whereas hOAT3 did those for loop diuretics. hOAT1, hOAT3, and hOAT4 but not hOAT2, mediated the uptake of bumetanide. hOAT3 and hOAT4, but not hOAT1 mediated the efflux of bumetanide. hOAT1 and hOAT3, but not hOAT2 and hOAT4 mediated the uptake of furosemide. In conclusion, it was suggested that hOAT1 may play an important role in the basolateral uptake of thiazides, and hOAT3 in the uptake of loop diuretics. In addition, it was also suggested that bumetanide taken up by hOAT3 and/or hOAT1 is excreted into the urine by hOAT4.

Haploid allele mapping of Y-chromosome minisatellite, MSY1 (DYF155S1), to a Japanese population.

The present study analyses the human Y-chromosome minisatellite locus, MSY1 (DYF155S1), in 205 Japanese males of 191 pedigrees using the minisatellite variant repeat (MVR) mapping system. The internal haploid structures of the detected alleles considerably varied and consisted of three major repeat units: types 2, 3 and 4. A comparison of the haploid profiles of the MVR codes identified 185 distinct alleles, of which only five were shared. We did not detect a type 1 repeat unit, and variations were frequent at the 5' end of the minisatellite locus. Within an analysis of 24 paternally linked DNA samples donated by ten families, no mutational events were identified even over two generation gaps. Furthermore, we applied this mapping system to a paternity test in which the alleged father was missing.

Interaction of human and rat organic anion transporter 2 with various cephalosporin antibiotics.

Cephalosporin antibiotics are thought to be excreted into the urine via organic anion transporters (OATs) and OAT can mediate nephrotoxicity by cephalosporins, particularly by cephaloridine. The purpose of this study was to elucidate the interaction of human-OAT2 and rat-OAT2 with cephalosporin antibiotics using proximal tubule cells stably expressing human-OAT2 and rat-OAT2. Human-OAT2 is localized to the basolateral side of the proximal tubule, whereas rat-OAT2 is localized to the apical side of the proximal tubule. Cephalosporins tested were cephalothin, cefoperazone, cefazolin, ceftriaxone, cephaloridine, cefotaxime, cefadroxil and cefamandole. These cephalosporins dose-dependently inhibited organic anion uptake mediated by human-OAT2 and rat-OAT2. There was no species difference observed for the effects of OAT2 with cephalosporins between human and rat transporters. Kinetic analysis revealed that the inhibitory effects for human-OAT2 were competitive. Cephaloridine significantly decreased the viability of cells stably expressing human-OAT2, human-OAT1, human-OAT3 and human-OAT4. The decreased viability of cells stably expressing human-OAT1, human-OAT3 and human-OAT4 but not human-OAT2 was reversed by probenecid. In conclusion, human-OAT2 interacts with cephalosporins, and thus, human-OAT2 may mediate the uptake of cephalosporins on the basolateral side of the proximal tubule. The interaction of human-OAT2 with cephalosporins was the weakest among the basolateral human-OATs tested. In addition, it is suggested that human-OATs mediate cephaloridine-induced nephrotoxicity.

Expression of human organic anion transporters in the choroid plexus and their interactions with neurotransmitter metabolites.

The purpose of the present study was to elucidate the expression of human organic anion transporter 1 (hOAT1) and hOAT3 in the choroid plexus of the human brain and their interactions with neurotransmitter metabolites using stable cell lines. Immunohistochemical analysis revealed that hOAT1 and hOAT3 are expressed in the cytoplasmic membrane and cytoplasm of human choroid plexus. Neurotransmitter metabolites, namely, 5-methoxyindole-3-acetic acid (5-MI-3-AA), homovanillic acid (HVA), vanilmandelic acid (VMA), 3,4-dihydroxyphenylacetic acid (DOPAC), 5-hydroxyindole-3-acetic acid (5-HI-3-AA), N-acetyl-5-hydroxytryptamine (NA-5-HTT), melatonin, 5-methoxytryptamine (5-MTT), 3,4-dihidroxymandelic acid (DHMA), 5-hydroxytryptophol, and 5-methoxytryptophol (5-MTP), but not methanephrine (MN), normethanephrine (NMN), and 3-methyltyramine (3-MT), at 2 mM, inhibited para-aminohippuric acid uptake mediated by hOAT1. On the other hand, melatonin, 5-MI-3-AA, NA-5-HTT, 5-MTT, 5-MTP, HVA, 5-HI-3-AA, VMA, DOPAC, 5-hydroxytryptophol, and MN, but not 3-MT, DHMA, and NMN, at 2 mM, inhibited estrone sulfate uptake mediated by hOAT3. Differences in the IC(50) values between hOAT1 and hOAT3 were observed for DHMA, DOPAC, HVA, 5-HI-3-AA, melatonin, 5-MI-3-AA, 5-MTP, 5-MTT, and VMA. HOAT1 and hOAT3 mediated the transport of VMA but not HVA and melatonin. These results suggest that hOAT1 and hOAT3 are involved in the efflux of various neurotransmitter metabolites from the cerebrospinal fluid to the blood across the choroid plexus.