Induction by perfluorinated fatty acids with different carbon chain length of peroxisomal beta-oxidation in the liver of rats

The potency of the induction of peroxisomal beta-oxidation was compared between perfluorinated fatty acids (PFCAs) with different carbon chain lengths in the liver of male and female rats. In male rats, perfluoroheptanoic acid (PFHA) has little effect, although perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA) and perfluorodecanoic acid (PFDA) potentially induced the activity. By contrast, PFHA and PFOA did not induce the activity of peroxisomal beta-oxidation in the liver of female rats while PFNA and PFDA effectively induced the activity. The induction of the activity by these PFCAs was in a dose-dependent manner, and there is a highly significant correlation between the induction and hepatic concentrations of PFCAs in the liver regardless of their carbon chain lengths. These results strongly suggest that the difference in their chemical structure is not the cause of the difference in the potency of the induction. Hepatic concentrations of PFOA and PFNA was markedly higher in male compared with female rats. Castration of male rats reduced the concentration of PFNA in the liver and treatment with testosterone entirely restored the reduction. In contrast to the results obtained from the in vivo experiments, the activity of peroxisomal beta-oxidation was induced by PFDA and PFOA to the same extent in cultured hepatocytes prepared from both male and female rats. These results, taken together, indicate that difference in accumulation between PFCAs in the liver was responsible for the different potency of the induction of peroxisomal beta-oxidation between PFCAs with different carbon chain lengths and between sexes.     

Inhibition of 3β- and 17β-hydroxysteroid dehydrogenase activities in rat Leydig cells by perfluorooctane acid

Perfluorooctane acid (PFOA) is classified as a persistent organic pollutant and as an endocrine disruptor. The mechanism by which PFOA causes reduced testosterone production in males is not known. We tested our hypothesis that PFOA interferes with Leydig cell steroidogenic enzymes by measuring its effect on 3β-hydroxysteroid dehydrogenase (3β-HSD) and 17β-hydroxysteroid dehydrogenase 3 (17β-HSD3) activities in rat testis microsomes and Leydig cells. The IC₅₀s of PFOA and mode of inhibition were assayed. PFOA inhibited microsomal 3β-HSD with an IC₅₀ of 53.2 ± 25.9 μM and 17β-HSD3 with an IC₅₀ 17.7 ± 6.8 μM. PFOA inhibited intact Leydig cell 3β-HSD with an IC₅₀ of 146.1 ± 0.9 μM and 17β-HSD3 with an IC₅₀ of 194.8 ± 1.0 μM. The inhibitions of 3β-HSD and 17β-HSD3 by PFOA were competitive for the substrates. In conclusion, PFOA inhibits 3β-HSD and 17β-HSD3 in rat Leydig cells.     

Comparison of the elimination between perfluorinated fatty acids with different carbon chain length in rats

Elimination in urine and feces was compared between four perfluorinated fatty acids (PFCAs) with different carbon chain length. In male rats, perfluoroheptanoic acid (PFHA) was rapidly eliminated in urine with the proportion of 92% of the dose being eliminated within 120 h after an intraperitoneal injection. Perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA) and perfluorodecanoic acid (PFDA) was eliminated in urine with the proportions of 55, 2.0 and 0.2% of the dose, respectively. By contrast, four PFCAs were eliminated in feces with the proportion of less than 5% of the dose within 120 h after an injection. In female rats, the proportions of PFOA and PFNA eliminated in urine within 120 h were 80% and 51% of the dose, respectively, which were significantly higher compared with those in male rats. There was the tendency that PFCA with longer carbon chain length is less eliminated in urine in both male and female rats. Fecal elimination of PFCAs was not different between PFCAs in female rats and comparable to those in male rats. The rates of biliary excretion of PFCAs in male rats were slower than those in female rats. Sex-related difference in urinary elimination of PFOA was abolished when male rats had been castrated. On the contrary, treatment with testosterone suppressed the elimination of PFOA in urine in both castrated male rats and female rats. The effect of testosterone was in a time- and dose-dependent manner. These results suggest that PFCAs are distinguished by their carbon chain length by a renal excretion system, which is regulated by testosterone.     

Several Conserved Positively Charged Amino Acids in OATP1B1 are Involved in Binding or Translocation of Different Substrates

OATP1B1 and 1B3 are related transporters mediating uptake of numerous compounds into hepatocytes. A putative model of OATP1B3 with a “positive binding pocket” containing conserved positively charged amino acids was predicted (Meier-Abt et al. J Membr Biol 208:213–227, 2005). Based on this model, we tested the hypothesis that these positive amino acids are important for OATP1B1 function. We made mutants and measured surface expression and uptake of estradiol-17β-glucuronide, estrone-3-sulfate and bromosulfophthalein in HEK293 cells. Two of the mutants had low surface expression levels: R181K at 10% and R580A at 30% of wild-type OATP1B1. A lysine at position 580 (R580K) rescued the expression of R580A. Mutations of several amino acids resulted in substrate-dependent effects. The largest changes were seen for estradiol-17β-glucuronide, while estrone-3-sulfate and bromosulfophthalein transport were less affected. The wild-type OATP1B1 K _m value for estradiol-17β-glucuronide of 5.35 ± 0.54 μM was increased by R57A to 30.5 ± 3.64 μM and decreased by R580K to 0.52 ± 0.18 μM. For estrone-3-sulfate the wild-type high-affinity K _m value of 0.55 ± 0.12 μM was increased by K361R to 1.8 ± 0.47 μM and decreased by R580K to 0.1 ± 0.04 μM. In addition, R580K reduced the V _max values for all three substrates to <25% of wild-type OATP1B1. Mutations at intracellular K90, H92 and R93 mainly affected V _max values for estradiol-17β-glucuronide uptake. In conclusion, the conserved amino acids R57, K361 and R580 seem to be part of the substrate binding sites and/or translocation pathways in OATP1B1.     

Inhibitory Effects of Green Tea and (–)-Epigallocatechin Gallate on Transport by OATP1B1, OATP1B3, OCT1, OCT2, MATE1, MATE2-K and P-Glycoprotein

Green tea catechins inhibit the function of organic anion transporting polypeptides (OATPs) that mediate the uptake of a diverse group of drugs and endogenous compounds into cells. The present study was aimed at investigating the effect of green tea and its most abundant catechin epigallocatechin gallate (EGCG) on the transport activity of several drug transporters expressed in enterocytes, hepatocytes and renal proximal tubular cells such as OATPs, organic cation transporters (OCTs), multidrug and toxin extrusion proteins (MATEs), and P-glycoprotein (P-gp). Uptake of the typical substrates metformin for OCTs and MATEs and bromosulphophthalein (BSP) and atorvastatin for OATPs was measured in the absence and presence of a commercially available green tea and EGCG. Transcellular transport of digoxin, a typical substrate of P-gp, was measured over 4 hours in the absence and presence of green tea or EGCG in Caco-2 cell monolayers. OCT1-, OCT2-, MATE1- and MATE2-K-mediated metformin uptake was significantly reduced in the presence of green tea and EGCG (P < 0.05). BSP net uptake by OATP1B1 and OATP1B3 was inhibited by green tea [IC₅₀ 2.6% (v/v) and 0.39% (v/v), respectively]. Green tea also inhibited OATP1B1- and OATP1B3-mediated atorvastatin net uptake with IC₅₀ values of 1.9% (v/v) and 1.0% (v/v), respectively. Basolateral to apical transport of digoxin was significantly decreased in the presence of green tea and EGCG. These findings indicate that green tea and EGCG inhibit multiple drug transporters in vitro. Further studies are necessary to investigate the effects of green tea on prototoypical substrates of these transporters in humans, in particular on substrates of hepatic uptake transporters (e.g. statins) as well as on P-glycoprotein substrates.     

Uptake kinetics and storage capacity of dissolved inorganic phosphorus and corresponding dissolved inorganic nitrate uptake in Saccharina latissima and Laminaria digitata (Phaeophyceae)

Uptake rates of dissolved inorganic phosphorus and dissolved inorganic nitrogen under unsaturated and saturated conditions were studied in young sporophytes of the seaweeds Saccharina latissima and Laminaria digitata (Phaeophyceae) using a “pulse‐and‐chase” assay under fully controlled laboratory conditions. In a subsequent second “pulse‐and‐chase” assay, internal storage capacity (ISC) was calculated based on V_M and the parameter for photosynthetic efficiency F_v/F_m. Sporophytes of S. latissima showed a V_S of 0.80 ± 0.03 μmol · cm^?2 · d^?1 and a V_M of 0.30 ± 0.09 μmol · cm^?2 · d^?1 for dissolved inorganic phosphate (DIP), whereas V_S for DIN was 11.26 ± 0.56 μmol · cm^?2 · d^?1 and V_M was 3.94 ± 0.67 μmol · cm^?2 · d^?1. In L. digitata, uptake kinetics for DIP and DIN were substantially lower: V_S for DIP did not exceed 0.38 ± 0.03 μmol · cm^?2 · d^?1 while V_M for DIP was 0.22 ± 0.01 μmol · cm^?2 · d^?1. V_S for DIN was 3.92 ± 0.08 μmol · cm^?2 · d^?1 and the V_M for DIN was 1.81 ± 0.38 μmol · cm^?2 · d^?1. Accordingly, S. latissima exhibited a larger ISC for DIP (27 μmol · cm^?2) than L. digitata (10 μmol · cm^?2), and was able to maintain high growth rates for a longer period under limiting DIP conditions. Our standardized data add to the physiological understanding of S. latissima and L. digitata, thus helping to identify potential locations for their cultivation. This could further contribute to the development and modification of applications in a bio‐based economy, for example, in evaluating the potential for bioremediation in integrated multitrophic aquacultures that produce biomass simultaneously for use in the food, feed, and energy industries.     

M234Glu is a component of the proton sponge in the reaction center from photosynthetic bacteria

Bacterial reaction centers use light energy to couple the uptake of protons to the successive semi-reduction of two quinones, namely Q_A and Q_B. These molecules are situated symmetrically in regard to a non-heme iron atom. Four histidines and one glutamic acid, M234Glu, constitute the five ligands of this atom. By flash-induced absorption spectroscopy and delayed fluorescence we have studied in the M234EH and M234EL variants the role played by this acidic residue on the energetic balance between the two quinones as well as in proton uptake. Delayed fluorescence from the P⁺Q_A^? state (P is the primary electron donor) and temperature dependence of the rate of P⁺Q_A^? charge recombination that are in good agreement show that in the two RC variants, both Q_A^? and Q_B^? are destabilized by about the same free energy amount: respectively ～ 100 ± 5 meV and 90 ± 5 meV for the M234EH and M234EL variants, as compared to the WT. Importantly, in the M234EH and M234EL variants we observe a collapse of the high pH band (present in the wild-type reaction center) of the proton uptake amplitudes associated with formation of Q_A^? and Q_B^?. This band has recently been shown to be a signature of a collective behaviour of an extended, multi-entry, proton uptake network. M234Glu seems to play a central role in the proton sponge-like system formed by the RC protein.     

Nitric oxide interacts with mitochondrial complex III producing antimycin-like effects

The effect of NO between cytochromes b and c of the mitochondrial respiratory chain were studied using submitochondrial particles (SMP) from bovine heart and GSNO and SPER-NO as NO sources. Succinate-cytochrome c reductase (complex II-III) activity (222±4 nmol/min. mg protein) was inhibited by 51% in the presence of 500 μM GSNO and by 48% in the presence of 30 μM SPER-NO, in both cases at ~1.25 μM NO. Neither GSNO nor SPER-NO were able to inhibit succinate-Q reductase activity (complex II; 220±9 nmol/min. mg protein), showing that NO affects complex III. Complex II-III activity was decreased (36%) when SMP were incubated with l-arginine and mtNOS cofactors, indicating that this effect is also produced by endogenous NO. GSNO (500 μM) reduced cytochrome b₅₆₂ by 71%, in an [O₂] independent manner. Hyperbolic increases in O₂^•- (up to 1.3±0.1 nmol/min. mg protein) and H₂O₂ (up to 0.64±0.05 nmol/min. mg protein) productions were observed with a maximal effect at 500 μM GSNO. The O₂^•-/H₂O₂ ratio was 1.98 in accordance with the stoichiometry of the O₂^•- disproportionation. Moreover, H₂O₂ production was increased by 72–74% when heart coupled mitochondria were exposed to 500 μM GSNO or 30 μM SPER-NO. SMP incubated in the presence of succinate showed an EPR signal (g=1.99) compatible with a stable semiquinone. This EPR signal was increased not only by antimycin but also by GSNO and SPER-NO. These signals were not modified under N₂ atmosphere, indicating that they are not a consequence to the effect of NOx species on complex III area. These results show that NO interacts with ubiquinone-cytochrome b area producing antimycin-like effects. This behaviour comprises the inhibition of electron transfer, the interruption of the oxidation of cytochromes b, and the enhancement of [UQH^•]_ss which, in turn, leads to an increase in O₂^•- and H₂O₂ mitochondrial production rates.     

Contribution of organic anion transporting polypeptide OATP2B1 to amiodarone accumulation in lung epithelial cells

The accumulation mechanisms of amiodarone (AMD) involving transporters in lung alveolar epithelial type II cells were studied. The uptake of AMD was examined using human alveolar epithelial-derived cell line A549 as a model. AMD was transported by the carrier-mediated system, and the apparent K_m and V_max values were 66.8 ± 30.3 μM and 49.7 ± 9.7 nmol/mg protein/5 min, respectively. The uptake of AMD by A549 cells was Na⁺-independent and was inhibited by substrates of human organic anion transporting polypeptide (OATP). The inhibition profiles were similar to the inhibitory effects of several compounds on OATP2B1-mediated E-3-S transport, and RT-PCR analysis showed mRNA expression of OATP2B1 and 1B3 in A549 cells. SiRNAs targeted to the OATP2B1 gene decreased the OATP2B1 mRNA expression level in A549 cells up to about 50% and reduced the uptake of AMD up to about 40%. These results indicate that AMD uptake mediated by carriers, including OATP2B1, might lead to accumulation of AMD in the lung and AMD-induced pulmonary toxicity (AIPT).     

Identification of phalloidin uptake systems of rat and human liver

To determine whether the liver toxin phalloidin is transported into hepatocytes by one of the known bile salt transporters, we expressed the sodium-dependent Na⁺/taurocholate cotransporting polypeptide (Ntcp) and several sodium-independent bile salt transporters of the organic anion transporting polypeptide (OATP/SLCO) superfamily in Xenopus laevis oocytes and measured uptake of the radiolabeled phalloidin derivative [³H]demethylphalloin. We found that rat Oatp1b2 (previously called Oatp4 (Slc21a10)) as well as human OATP1B1 (previously called OATP-C (SLC21A6)) and OATP1B3 (previously called OATP8 (SLC21A8)) mediate uptake of [³H]demethylphalloin when expressed in X. laevis oocytes. Transport of increasing [³H]demethylphalloin concentrations was saturable with apparent K_m values of 5.7 μM (Oatp1b2), 17 μM (OATP1B1) and 7.5 μM (OATP1B3). All other tested Oatps/OATPs as well as the rat liver Ntcp did not transport [³H]demethylphalloin. Therefore, we conclude that rat Oatp1b2 as well as human OATP1B1 and OATP1B3 are responsible for phalloidin uptake into rat and human hepatocytes.     

Fluorescence-based assays for the assessment of drug interaction with the human transporters OATP1B1 and OATP1B3

Hepatic disposition plays a significant role in the pharmacokinetics and pharmacodynamics of a variety of drugs. Sinusoidal membrane transporters have been shown to participate in the hepatic disposition of many pharmaceuticals. Two sinusoidal membrane transporters with an established role in hepatic disposition are OATP1B1 and OATP1B3 (organic anion-transporting polypeptides 1B1 and 1B3, respectively). OATP1B1 and OATP1B3 have been implicated in the hepatic uptake of statin drugs, and polymorphisms linked to OATP1B1 have been associated with deleterious patient endpoints. As a result, OATP1B1 and OATP1B3 represent sites for potential drug-drug interactions. Numerous methods exist for identifying potential drug-drug interactions with transporters. However, relatively few offer the convenience and speed of fluorescence-based assays. Here a fluorescence-based assay was developed for measuring the OATP1B1- and OATP1B3-mediated transport of 8-fluorescein-cAMP (8-FcA). The OATP1B1- and OATP1B3-mediated transport of 8-FcA was time dependent and saturable (K_m = 2.9 and 1.8 μM, V_max = 0.20 and 0.33 pmol/min/cm², respectively). Molecules known to interact with OATPs, including cyclosporin A, rifampicin, and glibenclamide, each demonstrated concentration-dependent inhibition of 8-FcA transport by OATP1B1 and OATP1B3. The in vitro fluorescence-based assays described here using 8-FcA as the substrate are convenient and rapid and have utility in screening drug candidates for potential drug-drug interactions with OATP1B1 and OATP1B3.     

Novel fluorine‐containing chiral hydrazide‐hydrazones: Design,synthesis, structural elucidation,antioxidant and anticholinesterase activity,and in silico studies

In this study, a series of fluorine‐containing chiral hydrazide‐hydrazone derivatives [III‐XII] from ?‐cysteine ethyl ester hydrochloride was synthesized as new antioxidant and anticholinesterase agents. The antioxidant activity of these derivatives was evaluated by ABTS^+· and DPPH^· scavenging and CUPRAC assays and the anticholinesterase activity by the Ellman method spectrophotometrically. The results of the antioxidant assay showed that compounds V , IX , and X exhibited higher activity than BHT and α‐tocopherol used as positive standards. Among the synthesized derivatives, compound IX (IC₅₀: 2.3 ± 1.6 μM) exhibited higher acetylcholinesterase inhibitory activity than galantamine (IC₅₀: 4.5 ± 0.8 μM). Compounds XI (IC₅₀: 9.6 ± 1.0 μM), IX (IC₅₀: 12.5 ± 1.6 μM), III (IC₅₀: 16.0 ± 1.6 μM), X (IC₅₀: 17.2 ± 1.8 μM), VI (IC₅₀: 20.2 ± 0.8 μM), XII (IC₅₀: 21.5 ± 1.0 μM), and VII (IC₅₀: 24.6 ± 0.6 μM) displayed better butyrylcholinesterase inhibitory activity than galantamine (IC₅₀: 46.03 ± 0.14 μM). ADME‐Tox analysis was used to probe the drug‐like properties of the compounds. Molecular docking studies were also applied to understand the interactions between compounds and targets. The docking calculations were supported by the experimental data. In particular, compound IX , having better activity than galantamine against acetylcholinesterase and butyrylcholinesterase enzymes, was visualized using molecular docking.     

Design and synthesis of plant cyclopeptide Astin C analogues and investigation of their immunosuppressive activity

To further investigate on the structure-activity relationships of immunosuppressive Astin C, seventeen analogues 1–17 were designed and synthetized via amino acid substitution strategy by the solid-phase peptide synthesis method for the first time. In comparison with Astin C (IC₅₀?=?12.6?±?3.3?μM), only compounds 2 (IC₅₀?=?38.4?±?16.2?μM), 4 (IC₅₀?=?51.8?±?12.7?μM), 5 (IC₅₀?=?65.2?±?15.6?μM), and 8 (IC₅₀?=?61.8?±?12.4?μM) exhibited immunosuppressive activity in the Lymph node cells of mice. These results showed that the Astin C analogues containing _D-amino acid residues, hydrophobic long-chain alkyl substituents, and aryl substituents performed better than those carrying hydrophilic amino acid residues and short-chain alkyl substituents. Moreover compounds 15, 16, and 17 had no immunosuppressive activity, which suggested that cis-3,4-dichlorinated proline played an important role in the immunosuppressive activity of Astin C.     

Effects of Divalent Cations,Trypsin, and Phospholipases on the Passive Permeability to Sodium of Inside-Out Vesicles From Human Red Cells

Inside-out vesicles (IOV) were prepared from human red blood cells. Steady-state uptake of ²²Na was observed to generally follow an exponential time course with a rate constant of 1.57 ± 0.09 h^?1 (SE). One week of cold storage (0–4°C) increased the rate constant to 2.50 ± 0.12 h ^?1 (SE). Mg²⁺, Ca²⁺, or Sr²⁺ decreased the rate of ²²Na uptake with no observable differences between the three divalent cations when tested at concentrations of 50 μM. Mg²⁺ was shown to decrease the rate of ²²Na uptake at concentrations as low as 5 μM with maximal effect at 50 to 100 μM. The decrease in rate of ²²Na uptake induced by Mg²⁺ could be enhanced by exposure of IOV to Mg²⁺ for longer periods of time. Trypsin treatment of IOV increased the rate of uptake of ²²Na and was dependent on the concentration of trypsin added between 5 to 25 μg/ml (treated for 5 min at 25°C). The ability of Mg²⁺ (50 μM) to decrease the rate of ²²Na uptake was still observed after maximal trypsin treatment. Phospholipase A₂ or phospholipase C treatment of IOV increased the rate of ²²Na uptake and was dependent on the amount of phospholipase A₂ (0.1 to 1.0 units/ml) or phospholipase C (0.25 to 2.5 units/ml) added (treated for 5 min at 25°C). After phospholipase A₂ treatment, the observed decrease in the rate of ²²Na uptake induced by Mg²⁺ (50 μM) was generally greater than controls. After phospholipase C treatment, the observed decrease in rate of ²²Na uptake induced by Mg²⁺ (50 μM) was less or absent when compared with controls. Phospholipase C treatment was less effective in preventing the Mg²⁺ effect the longer IOV were exposed to Mg²⁺. The results suggest that Mg²⁺ binds to phospholipid head-groups to reduce Na permeability perhaps by inducing a change in bilayer structure or phospholipid association.     

Inhibition of human and rat 3β-hydroxysteroid dehydrogenase and 17β-hydroxysteroid dehydrogenase 3 activities by perfluoroalkylated substances

Perfluoroalkylated substances (PFASs) including perfluorooctane acid (PFOA) and perfluorooctane sulfonate (PFOS) have been classified as persistent organic pollutants and are known to cause reduced testosterone production in human males. The objective of the present study was to compare the potencies of five different PFASs including PFOA, PFOS, potassium perfluorooctane sulfonate (PFOSK), potassium perfluorohexane sulfonate (PFHxSK) and potassium perfluorobutane sulfonate (PFBSK) in the inhibition of 3β-hydroxysteroid dehydrogenase (3β-HSD) and 17β-hydroxysteroid dehydrogenase 3 (17β-HSD3) activities in the human and rat testes. Human and rat microsomal enzymes were exposed to various PFASs. PFOS and PFOSK inhibited rat 3β-HSD activity with IC₅₀ of 1.35 ± 0.05 and 1.77 ± 0.04 μM, respectively, whereas PFHxSK and PFBSK had no effect at concentrations up to 250 μM. All chemicals tested weakly inhibited human 3β-HSD activity with IC₅₀s over 250 μM. On the other hand, PFOS, PFOSK and PFOA inhibited human 17β-HSD3 activity with IC₅₀s of 6.02 ± 1.02, 4.39 ± 0.46 and 127.60 ± 28.52 μM, respectively. The potencies for inhibition of 17β-HSD3 activity were determined to be PFOSK > PFOS > PFOA > PFHxSK = PFBSK for human 17β-HSD3 activity. There appears to be a species-dependent sensitivity to PFAS-mediated inhibition of enzyme activity because the IC₅₀s of PFOS(K) for inhibition of rat 17β-HSD3 activity was greater than 250 μM. In conclusion, the present study shows that PFOS and PFOSK are potent inhibitors of rat 3β-HSD and human 17β-HSD3 activity, and implies that inhibition of steroidogenic enzyme activity may be a contributing factor to the effects that PFASs exert on androgen secretion in the testis.     

The effect of nutrient concentrations,nutrient ratios and temperature on photosynthesis and nutrient uptake by Ulva prolifera: implications for the explosion in green tides

The green-tide macroalga, Ulva prolifera, was tested in the laboratory to determine its nutrient uptake and photosynthesis under different conditions. In the nutrient concentration experiments U. prolifera showed a saturated uptake for nitrate but an escalating uptake in the tested range for phosphorus. Both N/P and NO₃ ^?/NH₄ ⁺ ratios influenced nutrient uptake significantly (p?<?0.05) while the PSII quantum yield [Y(II)] (p?>?0.05) remained unaffected. The maximum N uptake rate (33.9?±?0.8 μmol g^?1 DW h^?1) and P uptake rate (11.1?±?4.7) was detected at N/P ratios of 7.5 and 2.2, respectively. U. prolifera preferred NH₄ ⁺-N to NO₃ ^?-N when the NO₃ ^?-N/NH₄ ⁺-N ratio was less than 2.2 (p?<?0.05). But between ratios of 2.2 and 12.9, the uptake of NO₃ ^?-N surpassed that of NH₄ ⁺-N. In the temperature experiments, the highest N uptake rate and [Y(II)] were observed at 20 °C, while the lowest rates were detected at 5 °C. P uptake rates were correlated with increasing temperature.     

Characterization of phosphate absorption in maize root cortex segments

Uptake of phosphate ions by 1 mm segments of isolated maize root cortex layers was studied. Cortex segments (from roots of 8 days old maize plants) absorb phosphate ions from 1 mM KH₂PO₄ in 0.2 mM CaSCO₄ at the average rate of 34.3 ±3.2 μg P_i g^?1 (fr. m.) h^?1,i.e. 0.35± 0.02 μmol P_i g^?1 (fr. m.) h^?1. Phosphate uptake considerably increases after a certain period of “augmentation”,i.e. washing in aerated 0.2 mM CaSO₄. This increase is completely blocked by the presence of 10 μg ml^?1 cycloheximide. The relation of uptake rate to phosphate concentration in the medium was shown to have 3 phases in the concentration range of 0.02 - 40 mM. Transition points were found between 0.8–1 mM and 10–20 mM. Following K_m and V_max values were found: K_m[mM] : 0.37 - 3.82 - 27.67 V_max[μg P_i g^?1 (fr. m.) h^?1] : 3.33 - 39.40 - 66.67 We have found no sharp pH optimum for phosphate uptake. It proceeds at almost constant rate till pH 6.0 and then the uptake rate drops with increasing pH. At low phosphate concentrations (1 mM) the lowest uptake rate was found at 5 and 13 °C, while the uptake is higher at 5 °C than at 13 °C at phosphate concentrations higher than 1 mM. At these concentrations uptake rate at 35 °C is lower than at 25 °C. Phosphate uptake considerably decreased in anaerobic conditions. DNP and iodoacetate (0.1 mM) completely blocked phosphate uptake from 1 mM KH₂PO₄, while uptake from 5 and 10 mM KH₂PO₄ was left unaffected by these substances. The inhibitors of active - SH groups NEM and PCMB inhibited phosphate uptake: 10^?3 M NEM by 81.6%, 10⁴ M NEM by 42% and 10^?4 M PCMB by 42%.     

Marmoset glutathione transferases with ketosteroid isomerase activity

The common marmoset Callithrix jacchus encodes two glutathione transferase (GST) enzymes with ketosteroid double-bond isomerase activity. The most active enzyme is CjaGST A3-3 showing a specific activity with 5-androsten-3,17-dione (Δ⁵-AD) of 62.1 ± 1.8 μmol min^-1 mg^-1, and a k_cat value of 261 ± 49 s^-1. The second ketosteroid isomerase CjaGST A1-1 has a 30-fold lower specific activity with Δ⁵-AD and a 37-fold lower k_cat value. Thus, the marmoset CjaGST A3-3 would be the main contributor to the biosynthesis of the steroid hormones testosterone and progesterone, like the human ortholog HsaGST A3-3. Two residues differ in the H-site of the 91.4% sequence identical CjaGST A1-1 and CjaGST A3-3, and modeling of the structures suggests that the bulky phenyl ring of Phe111 in CjaGST A1-1 causes steric hindrance in the binding of the steroid substrate. Tributyltin acetate (IC₅₀=0.16 ± 0.004 μM) and ethacrynic acid (IC₅₀=3.3 ± 0.2 μM) were found to be potent inhibitors of CjaGST A3-3, as previously demonstrated with the human and equine orthologs.     

Cloning/characterization of the canine organic anion transporting polypeptide 1b4 (Oatp1b4) and classification of the canine OATP/SLCO members

The human liver-specific organic anion transporting polypeptides (OATPs) 1B1 and 1B3 are involved in the elimination of numerous xenobiotics and drugs. Although dogs are frequently used for toxicologic and pharmacokinetic characterization of novel drugs, nothing is known about their OATP1B1/1B3 ortholog. Therefore, we cloned and characterized the first canine organic anion transporting polypeptide from dog liver, termed Oatp1b4. The isolated Oatp1b4 cDNA comprises 3661 base pairs (bp) with an open reading frame of 2076 bp, encoding a 692-amino acid protein with a molecular mass of ∼ 85 kDa. The Oatp1b4 gene is approximately 61 kb long and has a similar organization as the human OATP1B1 and OATP1B3 with 13 exons identical in length. Northern blot analysis shows that Oatp1b4 is predominantly expressed in the liver. Oatp1b4 mediates sodium-independent transport of typical organic anions including bromosulfophthalein (BSP), [D-penicillamine^2,5]enkephalin (DPDPE), estradiol-17β-glucuronide (E17βG), estrone-3-sulfate and taurocholate. In addition, Oatp1b4 transports the OATP1B3-specific substrate cholecystokinin octapeptide (CCK-8). Kinetic studies showed that Oatp1b4-mediated E17βG and estrone-3-sulfate transports were monophasic with K_m values of 5 ± 1 µM and 33 ± 4 µM, respectively. In conclusion, the cloned canine Oatp1b4 will provide additional molecular basis to further characterize the species difference of the OATP1B family members.     

Interaction of the Bioactive Flavonol,Icariin, with the Essential Human Solute Carrier Transporters

Solute carrier transporters (SLCs), in particular the organic anion transporting polypeptides (OATPs) and organic anion/cation transporters (OATs/OCTs), are responsible for the cellular entry of many clinically important drugs in body. They largely influence drug safety and efficacy. Icariin is a flavonol widely present in many herbal preparations, which is used to improve sexual function and prevent osteogenesis. However, precautions are necessary in therapies containing icariin due to its involvement in drug–drug/herb interactions, possibly mediated through competing drug uptake via membrane‐transporter proteins. This study is the first to comprehensively evaluate the interactions between icariin and a range of essential SLCs. Our data demonstrated that icariin can significantly inhibit OATP1B3‐ and OATP2B1‐mediated cellular uptake of specific substrates (IC₅₀ of 3.0 ± 1.3 and 6.4 ± 1.9 μM, respectively). Our study revealed that icariin can potentially compete with coadministrated drugs for particular SLCs, which may impact the therapeutic outcome of regimens.