Functional characterization of human and cynomolgus monkey UDP-glucuronosyltransferase 1A6 enzymes

UDP-glucuronosyltransferase 1A6 (UGT1A6) is a major isoform in the human liver that glucuronidates numerous drugs, environmental chemicals and endogenous substrates. In this study, human and cynomolgus monkey UGT1A6 cDNAs (humUGT1A6 and monUGT1A6, respectively) were cloned, and the corresponding proteins were heterologously expressed in yeast cells to identify the functions of primate UGT1A6s. The enzymatic properties of UGT1A6 proteins were characterized by the kinetic analysis of serotonin (5-hydroxytryptamine, 5-HT) and 4-methylumbelliferone (4-MU) glucuronidation. humUGT1A6 and monUGT1A6 showed 96% identity in their nucleotide and amino acid sequences. Immunoblotting analysis using an antibody raised against human UGT1A6 showed that protein staining intensities were different between human and cynomolgus monkey UGT1A6 enzymes in microsomal fractions from livers and yeast cells, although both enzymes were detectable. The apparent K(m) value (15 mM) for 5-HT glucuronidation of cynomolgus monkey liver microsomes was significantly higher than that (8.6mM) of human liver microsomes, whereas V(max) values were lower in cynomolgus monkeys (2.8 nmol/min/mg protein) than in humans (8.6 nmol/min/mg protein). No significant species difference was observed in K(m) (approximately 90 microM) or V(max) (approximately 25 nmol/min/mg protein) values for liver microsomal 4-MU glucuronidation. In yeast cell microsomes, K(m) values (approximately 6mM) for 5-HT glucuronidation by recombinant UGT1A6s were similar, while a V(max) value (0.1nmol/min/mg protein) of monUGT1A6 was significantly lower than that (0.7 nmol/min/mg protein) of humUGT1A6. In 4-MU glucuronidation, both K(m) (210 microM) and V(max) (3.5 nmol/min/mg protein) values of monUGT1A6 were significantly higher than those of humUGT1A6 (K(m), 110 microM; V(max), 1.5nmol/min/mg protein). These findings suggest that the enzymatic properties of UGT1A6 were extensively different between humans and cynomolgus monkeys, although humUGT1A6 and monUGT1A6 showed high homology at the amino acid level. The information gained in this study should help with in vivo extrapolation and to assess the toxicity of xenobiotics.     

Interactions of environmental factors influencing pupal coloration in swallowtail butterfly Papilio xuthus

The swallowtail butterfly Papilio xuthus Linné [Lepidoptera: Papilionidae] exhibits pupal protective color polyphenism. Interactions of various environmental factors on pupal coloration were analyzed in non-diapausing individuals. Under sufficient light (200lux), most pupating larvae became green pupae when the surface of the pupation site was smooth, while they became brown when the surface was rough. Tactile signals are the positive environmental factors causing induction of the brown pupal coloration. In dark boxes, the induction of the brown pupal coloration was easily induced even on a smooth surface, suggesting that light suppresses induction of brown coloration. Different colors of pupation sites did not affect pupal coloration under sufficient light. Environmental factors received during a critical period both before girdling and after girdling affected pupal coloration. When tactile signals received from rough surfaces reach threshold levels during pupation, brown pupal coloration is determined. Larvae reared under a daily periodicity of natural light formed a girdle at midnight, subsequently, the prepupae received strong daylight the following day. Under natural light most larvae produced brown pupae on rough surfaces and green pupae on smooth surfaces.     

UGT75L6 and UGT94E5 mediate sequential glucosylation of crocetin to crocin in Gardenia jasminoides

Crocin is an apocarotenoid glycosyl ester accumulating in fruits of Gardenia jasminoides and used as a food coloring and nutraceutical. For the first time, the two glucosyltransferases UGT75L6 and UGT94E5 that sequentially mediate the final glucosylation steps in crocin biosynthesis in G. jasminoides have been identified and functionally characterized. UGT75L6 preferentially glucosylates the carboxyl group of crocetin yielding crocetin glucosyl esters, while UGT94E5 glucosylates the 6' hydroxyl group of the glucose moiety of crocetin glucosyl esters. The expression pattern of neither UGT75L6 nor UGT94E5 correlated with the pattern of crocin accumulation in G. jasminoides.     

Novel anti-carbohydrate antibodies reveal the cooperative function of sulfated N- and O-glycans in lymphocyte homing

Cell surface glycans play pivotal roles in immune cell trafficking and immunity. Here we present an efficient method for generating anti-carbohydrate monoclonal antibodies (mAbs) using gene-targeted mice and describe critical glycans in lymphocyte homing. We immunized sulfotransferase GlcNAc6ST-1 and GlcNAc6ST-2 doubly deficient mice with sulfotransferase-overexpressing Chinese hamster ovary cells and generated two mAbs, termed S1 and S2. Both S1 and S2 bound high endothelial venules (HEVs) in the lymphoid organs of humans and wild-type mice, but not in those of doubly deficient mice. Glycan array analysis indicated that both S1 and S2 specifically bound 6-sulfo sialyl Lewis X and its defucosylated structure. Interestingly, S2 inhibited lymphocyte homing to peripheral lymph nodes by 95%, whereas S1 inhibited it by only 25%. S2 also significantly inhibited contact hypersensitivity responses and L-selectin-dependent leukocyte adhesion to HEVs. Immunohistochemical and Western blot analyses indicated that S1 preferentially bound sulfated O-glycans, whereas S2 bound both sulfated N- and O-glycans in HEVs. Furthermore, S2 strongly inhibited the N-glycan-dependent residual lymphocyte homing in mutant mice lacking sulfated O-glycans, indicating the importance of both sulfated N- and O-glycans in lymphocyte homing. Thus, the two mAbs generated by a novel method revealed the cooperative function of sulfated N- and O-glycans in lymphocyte homing and immune surveillance.     

Temperature-sensitive phenotype caused by natural mutation in Capsicum latescent in two tropical regions

Plants in tropical regions experience temperature fluctuation only in non-extreme ambient temperatures. Thus, moderate changes in temperatures, which they never experience in their local environments, might be sufficient to manifest the locally hidden phenotype caused by natural mutation. To validate this hypothesis, temperature-treating experiments were performed on Capsicum accessions collected from tropical regions. Thirty-six Capsicum accessions, collected from Caribbean countries, were screened for temperature sensitivity. Similarities in their temperature sensitivities were compared with Sy-2 (C. chinense) from Seychelles, which was previously found to be a temperature-sensitive accession. Tr-13 from Trinidad & Tobago exhibited developmental abnormalities at temperatures below 24 °C. Expression of defense-related genes was induced, and salicylic acid, which is a key molecule in the plant’s defense response, accumulated in Tr-13 at temperatures below 24 °C. Tr-13 and Sy-2 appeared normal when they were grown at temperatures simulating those in Trinidad and Seychelles, respectively. Crossing Tr-13 with No. 3341 or Sy-2 revealed that the temperature-sensitive phenotype of Tr-13 was caused by a genetic mutation in the same locus as Sy-2. Plants having a temperature-sensitive phenotype that is caused by natural mutations evade artificial selection and exist as crops in specific environments, such as tropical regions.     

Regio- and stereoselective oxidation of propranolol enantiomers by human CYP2D6, cynomolgus monkey CYP2D17 and marmoset CYP2D19

Toxic and pharmacokinetic profiles of drug candidates are evaluated in vivo often using monkeys as experimental animals, and the data obtained are extrapolated to humans. Well understanding physiological properties, including drug-metabolizing enzymes, of monkeys should increase the accuracy of the extrapolation. The present study was performed to compare regio- and stereoselectivity in the oxidation of propranolol (PL), a chiral substrate, by cytochrome P450 2D (CYP2D) enzymes among humans, cynomolgus monkeys and marmosets. Complimentary DNAs encoding human CYP2D6, cynomolgus monkey CYP2D17 and marmoset CYP2D19 were cloned, and their proteins expressed in a yeast cell expression system. The regio- and stereoselective oxidation of PL enantiomers by yeast cell microsomal fractions were compared. In terms of efficiency of expression in the system, the holo-proteins ranked CYP2D6 ≒ CYP2D17 ? CYP2D19. This may be caused by the bulky side chain of the amino acid residue at position 119 (leucine for CYP2D19 vs. valine for CYP2D6 and CYP2D17), which can disturb the incorporation of the heme moiety into the active-site cavity. PL enantiomers were oxidized by all of the enzymes mainly into 4-hydroxyproranolol (4-OH-PL), followed by 5-OH-PL and N-desisopropylpropranolol (NDP). In the kinetic analysis, apparent K_m values were commonly in the μM range and substrate enantioselectivity of R-PL < S-PL was observed in both K_m and V_max values for the formation of the three metabolites from PL enantiomers. The activity to produce NDP tended to be higher for the monkey enzymes, particularly CYP2D17, than for the human enzyme. These results indicate that in the oxidation of PL enantiomers by CYP2D enzymes, stereoselectivity is similar but regioselectivity is different between humans and monkeys.     

Angiotensin receptor blockers shift the circadian rhythm of blood pressure by suppressing tubular sodium reabsorption

Recently, we found that an angiotensin II receptor blocker (ARB) restored the circadian rhythm of the blood pressure (BP) from a nondipper to a dipper pattern, similar to that achieved with sodium intake restriction and diuretics (Fukuda M, Yamanaka T, Mizuno M, Motokawa M, Shirasawa Y, Miyagi S, Nishio T, Yoshida A, Kimura G. J Hypertens 26: 583-588, 2008). ARB enhanced natriuresis during the day, while BP was markedly lower during the night, resulting in the dipper pattern. In the present study, we examined whether the suppression of tubular sodium reabsorption, similar to the action of diuretics, was the mechanism by which ARB normalized the circadian BP rhythm. BP and glomerulotubular balance were compared in 41 patients with chronic kidney disease before and during ARB treatment with olmesartan once a day in the morning for 8 wk. ARB increased natriuresis (sodium excretion rate; U(Na)V) during the day (4.5 ± 2.2 to 5.5 ± 2.1 mmol/h, P = 0.002), while it had no effect during the night (4.3 ± 2.0 to 3.8 ± 1.6 mmol/h, P = 0.1). The night/day ratios of both BP and U(Na)V were decreased. The decrease in the night/day ratio of BP correlated with the increase in the daytime U(Na)V (r = 0.42, P = 0.006). Throughout the whole day, the glomerular filtration rate (P = 0.0006) and tubular sodium reabsorption (P = 0.0005) were both reduced significantly by ARB, although U(Na)V remained constant (107 ± 45 vs. 118 ± 36 mmol/day, P = 0.07). These findings indicate that the suppression of tubular sodium reabsorption, showing a resemblance to the action of diuretics, is the primary mechanism by which ARB can shift the circadian BP rhythm into a dipper pattern.     

Parallel allochronic divergence in a winter moth due to disruption of reproductive period by winter harshness

The disruption of reproductive timing by climatic harshness may result in the temporal isolation of conspecific populations and, ultimately, in speciation. However, whether temporal isolation alone can act as the force initiating speciation and how often the same type of climatic disruption results in the divergence of allochronic populations in a lineage are largely unknown. The reproductive period of the winter geometrid moth Inurois punctigera is separated into early and late winter in habitats with severe winters, but not in habitats with mild winters, suggesting that the reproductive season is disrupted by the harshness of the mid-winter period. Here, we show that sympatric pairs of early- and late-winter populations that differ in origin exist in different regions, suggesting a parallel divergence of reproductive timing. In each region, significant genetic differentiation exists between these early- and late-winter populations, suggesting that the temporal reproductive isolation has persisted. Moreover, we demonstrate that the temporal isolation, in comparison with geographic isolation, contributes greatly to the genetic differentiation among geographic and temporal populations by an analysis of molecular variance and by a comparison of genetic differentiations (F(ST) ) between geographic populations with and without difference in reproductive season. Our results suggest that adaptive divergence of allochronically reproducing populations has occurred independently in different regions, implying the generality of the role of temporal isolation in initiating speciation in a winter moth lineage.     

Discrimination of genotoxic and non-genotoxic hepatocarcinogens by statistical analysis based on gene expression profiling in the mouse liver as determined by quantitative real-time PCR

The general aim of the present study is to discriminate between mouse genotoxic and non-genotoxic hepatocarcinogens via selected gene expression patterns in the liver as analyzed by quantitative real-time PCR (qPCR) and statistical analysis. qPCR was conducted on liver samples from groups of 5 male, 9-week-old B6C3F(1) mice, at 4 and 48h following a single intraperitoneal administration of chemicals. We quantified 35 genes selected from our previous DNA microarray studies using 12 different chemicals: 8 genotoxic hepatocarcinogens (2-acetylaminofluorene, 2,4-diaminotoluene, diisopropanolnitrosamine, 4-dimethylaminoazobenzene, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, N-nitrosomorpholine, quinoline and urethane) and 4 non-genotoxic hepatocarcinogens (1,4-dichlorobenzene, dichlorodiphenyltrichloroethane, di(2-ethylhexyl)phthalate and furan). A considerable number of genes exhibited significant changes in their gene expression ratios (experimental group/control group) analyzed statistically by the Dunnett's test and Welch's t-test. Finally, we distinguished between the genotoxic and non-genotoxic hepatocarcinogens by statistical analysis using principal component analysis (PCA) of the gene expression profiles for 7 genes (Btg2, Ccnf, Ccng1, Lpr1, Mbd1, Phlda3 and Tubb2c) at 4h and for 12 genes (Aen, Bax, Btg2, Ccnf, Ccng1, Cdkn1a, Gdf15, Lrp1, Mbd1, Phlda3, Plk2 and Tubb2c) at 48h. Seven major biological processes were extracted from the gene ontology analysis: apoptosis, the cell cycle, cell proliferation, DNA damage, DNA repair, oncogenes and tumor suppression. The major, biologically relevant gene pathway suggested was the DNA damage response pathway, resulting from signal transduction by a p53-class mediator leading to the induction of apoptosis. Eight genes (Aen, Bax, Btg2, Ccng1, Cdkn1a, Gdf15, Phlda3 and Plk2) that are directly associated with Trp53 contributed to the PCA. The current findings demonstrate a successful discrimination between genotoxic and non-genotoxic hepatocarcinogens, using qPCR and PCA, on 12 genes associated with a Trp53-mediated signaling pathway for DNA damage response at 4 and 48 h after a single administration of chemicals.