Monitoring of microbiological water quality by real-time pcr.

A new method was developed to evaluate the microbiological water quality. Deoxyribonucleic acid of water-borne bacteria was extracted and quantified using real-time polymer chain reaction detection system with a selected universal primer set. Quantification of the deoxyribonucleic acid in environmental water samples is independent of the culture condition, nutrient condition, or the bacterial metabolic states and can reflect the relatively small environmental changes which can not be detected through the other parameters. Therefore, this method can well represent the microbiological water quality. Compared with the conventional plate count method, deoxyribonucleic acid quantification has higher sensitivity, precision and reliability. The relationship between concentration of bacterial deoxyribonucleic acid and other water quality parameters was examined. Concentration of deoxyribonucleic acid was not well correlated with the plate count number, turbidity or other physical and chemical parameters.

Cellular thiol status-dependent inhibition of tumor cell growth via modulation of p27(kip1) translocation and retinoblastoma protein phosphorylation by 1'-acetoxychavicol acetate.

1'-Acetoxychavicol acetate (ACA) has been shown to inhibit tumor cell growth, but there is limited information on its effects on cell signaling and the cell cycle control pathway. In this study, we sought to determine how ACA alters cell cycle and its related control factors in its growth inhibitory effect in Ehrlich ascites tumor cells (EATC). ACA caused an accumulation of cells in the G1 phase and an inhibition of DNA synthesis, which were reversed by supplementation with N-acetylcysteine (NAC) or glutathione ethyl ester (GEE). Furthermore, ACA decreased hyperphosphorylated Rb levels and increased hypophosphorylated Rb levels. NAC and GEE also abolished the decease in Rb phosphorylation by ACA. As Rb phosphorylation is regulated by G1 cyclin dependent kinase and CDK inhibitor p27(kip1), which is an important regulator of the mammalian cell cycle, we estimated the amount of p27(kip1) levels by western blotting. Treatment with ACA had virtually no effect on the amount of p27(kip1) levels, but caused a decrease in phosphorylated p27(kip1) and an increase in unphosphorylated p27(kip1) as well as an increase in the nuclear localization of p27(kip1). These events were abolished in the presence of NAC or GEE. These results suggest that in EATC, cell growth inhibition elicited by ACA involves decreases in Rb and p27(kip1) phosphorylation and an increase in nuclear localization of p27(kip1), and these events are dependent on the cellular thiol status.

Protective effect of gamma-aminobutyric acid (GABA) against cytotoxicity of ethanol in isolated rat hepatocytes involves modulations in cellular polyamine levels.

Gamma-aminobutyric acid (GABA) is considered to be a multifunctional molecule with various physiological effects throughout the body. It is also evident that the liver contains GABA and its transporter. However, the functions of GABA in liver have not been well documented. In this study, the cytoprotective effect of GABA against ethanol-induced hepatotoxicity was evaluated in primary cultured rat hepatocytes. Addition of ethanol induced decrease of cell viability in a dose-dependent manner. However, treatment with GABA resulted in a dose-dependent recovery from ethanol (150 mM)-induced cytotoxicity.GABA reversed the ethanol-induced decrease in intracellular polyamine levels. Furthermore, the addition of polyamines also reversed the ethanol-induced decrease of cell viability. These results suggest that GABA is protective against the cytotoxicity of ethanol in isolated rat hepatocytes and this effect may be modulated by the maintenance of intracellular polyamine levels.

Involvement of polyamines in evening primrose extract-induced apoptosis in Ehrlich ascites tumor cells.

We previously demonstrated that evening primrose extract (EPE) induced apoptosis and inhibited the DNA synthesis in Ehrlich ascites tumor cells (EATC) and suggested that EPE-induced inhibition of the growth of EATC are via at least two pathway differentially modulated by reactive oxygen species, notably intracellular peroxides. These are (a) the EPE-induced apoptosis pathway which is dependent on increases in hydrogen peroxide and (b) the EPE-induced inhibition of cell proliferation which is hydrogen peroxide independent. In this study, EPE brought about a significant decrease in intracellular polyamine levels. Furthermore, the addition of polyamines reversed the EPE-induced decrease in cell viability and suppressed the EPE-induced increase in intracellular hydrogen peroxides. However, the addition of polyamines did not reverse EPE-induced decrease in DNA synthesis and phosphorylation of Rb protein, and EPE-induced translocation of AIF. These results suggest the involvement of polyamines in the EPE-induced apoptosis pathway which is dependent on increase in hydrogen peroxide.

Predicting pesticide concentrations in river water with a hydrologically calibrated basin-scale runoff model.

Hydrological diffuse pollution models require calibration before they can be used to make accurate long-term predictions for a range of hydrological and meteorological conditions. As such, the applicability of the models to the dispersion of new pesticides is limited due to the lack of calibration data. In this study, the performance of a GIS-based basin-scale runoff model for predicting the concentrations of paddy-farming pesticides in river water was examined when calibrated using hydrological data alone, without optimization based on empirical pesticide concentration data. The prediction accuracy on a daily or hourly scale was somewhat unsatisfactory due to inevitable compromises concerning rice farming schedules. However, the month-averaged pesticide concentrations were satisfactorily accurate; more than 50% of predicted values were between half and twice the observed values, considering the deficiencies of the input data, particularly for pesticide usage, which may include up to 50% error.

High expression of 92 kDa type IV collagenase (matrix metalloproteinase-9) in canine mammary adenocarcinoma.

The 92 kDa type VI collagenase (matrix metalloproteinase-9 (MMP-9)) activities on zymography assay were found to be 1-6 times higher in benign tumor breast tissues of 12 canines and 4-26 times higher in adenocarcinoma breast tissues of nine canines than that of control tissues, respectively. A full-length canine MMP-9 cDNA was cloned from the adenocarcinoma tissue by reverse transcription-PCR and 5'- and 3'-RACE. The isolated cDNA contained an open reading frame coding for a polypeptide of 704 amino acids. The predicted protein sequence displayed extensive similarity to that of known MMP-9s and contained a putative signal sequence, a propeptide, an active site with three zinc-binding histidine residues, a calcium-binding domain, a hemopexin region, and three key cysteine residues. Western blotting using MMP-9-specific antibodies prepared against the peptide corresponding to Arg(642)-Asp(704) of canine MMP-9 and Northern blotting using a MMP-9-specific cDNA fragment as a probe confirmed that MMP-9 (the 92 kDa protein band) was highly expressed in canine mammary adenocarcinoma tissues. Higher levels of MMP-9 activity were found in the sera of canines with mammary adenocarcinoma. The results indicated that MMP-9 plays an important role in the progression of a canine mammary tumor and that assay of serum MMP-9 is helpful for early diagnosis as progress of adenocarcinoma.

Bisphenol a glucuronide, a major metabolite in rat bile after liver perfusion.

The environmental estrogen bisphenol A, orally introduced into the body, passes through the liver and modulates the endocrine system to elicit irreversible changes in the functioning of reproduction. To elucidate the actual and dynamic metabolism of bisphenol A in the liver before its arrival at target organs, this study evaluated the metabolism and disposition of the compound within the passage through the liver in Sprague-Dawley rats. On perfusion of 7.5 micromol of bisphenol A into the liver via the portal vein, approximately 91% of the infused bisphenol A was absorbed by the liver tissue, and about 65% of the absorbed bisphenol A was glucuronidated within 60 min. Roughly 65% of the bisphenol A glucuronide that formed in the liver was excreted into the bile and about 35% into the hepatic vein. On perfusion of 0.01, 0.05, and 0.1 mM bisphenol A solution into the liver, free bisphenol A was excreted only into the vein at 5.6, 9.3, and 14.6%, respectively, of the total bisphenol A. These results suggest that most bisphenol A absorbed by the intestine is probably glucuronidated exclusively in the liver and the conjugate is excreted mainly into the bile.

cDNA cloning and expression of a bovine phenol UDP-glucuronosyltransferase, BovUGT1A6.

A full-length cDNA encoding a phenol UDP-glucuronosyltransferase was isolated by plaque hybridization, RT-PCR and 5'-RACE from a cDNA library prepared from the bovine liver. The deduced amino acid sequence (529 amino acid residues) has A signal sequence (23 amino acid residues) at the amino terminus and a transmembrane-anchoring domain (17 amino acid residues) at the carboxyl terminus. The encoded protein has a potential asparagine-linked glycosylation site (Asn291). The cloned cDNA was named bovUGT1A6 on the basis of the amino acid similarity. BovUGT1A6 cloned in the pAAH5 expression vector was transformed into Saccharomyces cerevisiea AH22 cells to obtain an active 54-kDa bovUGT1A6 enzyme. The expressed enzyme represented UDP-glucuronosyltransferase activities toward 1-naphthol and 4-methylumbelliferone, confirming that the isolated cDNA is an isoform of bovine phenol UDP-glucuronosyltransferase. Microsomal UDP-glucuronosyltransferase activity toward 1-naphthol in the bovine kidney cortex was found to be higher than that in the liver and other organs, and mRNA of bovUGT1A6 was more strongly detected in the kidney on Northern blotting analysis. These results suggest that the bovine kidney, which strongly expresses bovUGT1A6, is a significant organ for xenobiotics glucuronidation.

Removal of a synthetic organic chemical by PAC-UF systems--I: Theory and modeling.

Based on the age distributions of powdered activated carbon (PAC) in reactors, the competitive bi-solute isotherm and the pore-surface diffusion mechanism, a model was developed to predict the removal of a trace synthetic organic chemical (SOC) when PAC was applied to an ultrafiltration (UF) membrane system. Independent experiments evaluated the input parameters for the model. The pore diffusion in liquid-filled pore was the dominant internal mass transfer mechanism in a PAC particle and the surface diffusion mechanism was neglected in the model. Model prediction was compared with data from pilot plant experiments treating simazine in natural water. Due to the blending and the increasing PAC in the UF loop, a transient behavior of effluent simazine concentration was observed in the UF effluent. The model predicted successfully this periodical variation of the simazine concentration in the UF loop effluent, as well as the simazine concentration in the effluent from the PAC slurry contactor. The faithful model prediction required the modeling of the phenomenon of adsorption/desorption of an SOC on membrane itself.

Removal of a synthetic organic chemical by PAC-UF systems. II: Model application.

This paper describes several application potentials with a recently developed model for predicting the synthetic organic chemical (SOC) removal by powdered activated carbon (PAC) adsorption during ultrafiltration (UF) and discusses the removal mechanism. The model was successfully applied, without any modification, to dead-end mode operation as well as to cross-flow mode operation, validating the assumption of the internal diffusion control mechanism and the continuously-stirred-tank-reactor (CSTR) concept. Even when UF was operated in a cross-flow mode, PAC added was re-circulating in suspension for only a short time. Then, solute uptake took place mostly by PAC immobilized in membrane tubes not only for dead-end operation but also for cross-flow operation. Therefore, cross-flow operation did not have any advantage regarding the SOC mass transfer on PAC in UF loop over dead-end operation. The model simulation implied that pulse PAC addition at the beginning of filtration cycle resulted better SOC removal than continuous PAC addition. However, for the pulse PAC addition mode, the model predicted somewhat lower effluent SOC concentration than the observed values, and the benefit of pulse PAC application in terms of reducing SOC over its continuous dosage was not confirmed. Longer detention time of PAC dosed in a pulse than continuously dosed PAC could possibly further decrease internal diffusivity.

Pore distribution effect of activated carbon in adsorbing organic micropollutants from natural water.

Adsorption isotherms of organic micropollutants in coexistence with natural organic matter (NOM) were analyzed to evaluate the impacts of pore size distribution of activated carbon (AC) on the competition effects of the NOM. Single solute adsorption experiments and simultaneous adsorption experiments with NOM contained in a coagulation-pretreated surface water were performed for four agricultural chemicals and three coal-based activated carbons (ACs) having different pore distributions. The results showed that, for all the carbons used, the adsorption capacity of the chemicals was reduced distinctly in the presence of NOM. Such a reduction was more apparent for AC with a larger portion of small pores suitable for the adsorption of small organic molecules and for the agricultural chemicals with a more hydrophilic nature. Ideal adsorbed solution theory (IAST) incorporated with the Freundlich isotherm expression (IAST-Freundlich model) could not interpret the impact of NOM on the adsorption capacity of the chemicals unless a pore blockage effect caused by the adsorption of NOM was also considered. By taking into account this effect, the adsorption isotherm of the chemicals in the presence of NOM was well described, and the capacity reduction caused by the NOM was quantitatively assessed from the viewpoints of the site competition and the pore blockage. Analytical results clearly indicated that pore blockage was an important competition mechanism that contributed to 10-99% of the total capacity reductions of the chemicals, the level depended greatly on the ACs, the chemicals and the equilibrium concentrations, and could possibly be alleviated by broadening the pore size distributions of the ACs to provide a large volume percentage for pores with sizes above 30 A.

Molecular cloning and characterization of a gene encoding glutaminase from Aspergillus oryzae.

A glutaminase from Aspergillus oryzae was purified and its molecular weight was determined to be 82,091 by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Purified glutaminase catalysed the hydrolysis not only of L-glutamine but also of D-glutamine. Both the molecular weight and the substrate specificity of this glutaminase were different from those reported previously [Yano et al. (1998) J Ferment Technol 66: 137-143]. On the basis of its internal amino acid sequences, we have isolated and characterized the glutaminase gene (gtaA) from A. oryzae. The gtaA gene had an open reading frame coding for 690 amino acid residues, including a signal peptide of 20 amino acid residues and a mature protein of 670 amino acid residues. In the 5'-flanking region of the gene, there were three putative CreAp binding sequences and one putative AreAp binding sequence. The gtaA structural gene was introduced into A. oryzae NS4 and a marked increase in activity was detected in comparison with the control strain. The gtaA gene was also isolated from Aspergillus nidulans on the basis of the determined nucleotide sequence of the gtaA gene from A. oryzae.

Smooth muscle layer-dependent distribution of 5-hydroxytryptamine(7) receptor in the porcine myometrium.

1. To analyse the mechanisms of muscle layer-dependent inhibition of porcine myometrial contractility by 5-hydroxytryptamine (5-HT), the effects of 5-HT, 5-carboxamidotryptamine(5-CT), 5-methoxytryptamine (5-MeOT), forskolin and cyclic adenosine 3', 5'-monophosphate (cyclic AMP) analogues on spontaneous and stimulant-induced contractions were examined in longitudinal (LM) and circular muscles (CM). In addition, accumulation of cyclic AMP by 5-HT and distribution of 5-HT(7) receptors in LM and CM layers were compared using biochemical and molecular approaches. 2. 5-HT receptor agonists inhibited the spontaneous contractions of LM and CM (5-CT>5-HT>5-MeOT), but CM was more sensitive than was LM. The inhibition by the agonists was antagonized by methiothepin (100 nM). 3. Carbachol-, high-K(+)-, histamine- and Ca(2+)-induced contractions were inhibited by 5-HT with different responses (CM>LM). Even in the presence of 3-isobutyl-1-methylxanthine (IBMX), the inhibition by 5-HT in the CM was still more conspicuous than that in the LM. 4. Compared with the CM, the inhibition of spontaneous contraction by forskolin, dibutyryl-cyclic AMP and 8-bromo-cyclic AMP was marked in the LM. 5. 5-HT (1 nM - 1 microM) increased the cyclic AMP in both muscle layers, but the increment in the CM was higher than that in the LM whether IBMX was present or not. 6. LM and CM layers contained a single class of [(3)H]-5-CT binding sites with a similar K(d) value (0.21 - 0.24 nM). However, B(max) (5-HT(7) receptor concentration) in the CM (120.6 fmol mg(-1) protein) was higher than that in the LM (30.4 fmol mg(-1) protein). 7. The molecular study (reverse transcription polymerase chain reaction) demonstrated the expression of 5-HT(7) receptor mRNA in the CM was higher than that in the LM. 8. These results suggest that the muscle layer-dependent difference in inhibition by 5-HT is not restricted to spontaneous contraction but applies to various contractions in the porcine myometrium. Different inhibition of the contractility by 5-HT is caused by muscle layer-related accumulation of cyclic AMP (CM>LM), due to smooth muscle-layer dependent distribution (CM>LM) of 5-HT(7) receptors.

1-Naphthol beta-D-glucuronide formed intraluminally in rat small intestine mucosa and absorbed into the colon.

UDP-glucuronosyltransferase expressed in the rat intestinal epithelial cells is important as the first barrier against chemicals. The distribution of 1-naphthol and its glucuronide formed in rat intestine was estimated by using everted intestine. Roughly 60% of the 1-naphthol added to the mucosal fluid was absorbed into the mucosa of the small intestine and colon within 30 min. Approximately 66% of the 1-naphthol absorbed in the proximal intestine was secreted intraluminally as a glucuronide, and a minimal 9% was transported into the serosal fluid as a glucuronide. In the distal intestine, approximately 34% was secreted intraluminally and 30% was transported into the serosal fluid as a glucuronide. The greatest amount of the glucuronide (37% of the absorbed 1-naphthol) was transported into the serosal fluid, whereas a minimal 7% was secreted intraluminally in the colon. In marked contrast, the colon was found to transport 1-naphthol-glucuronide from the mucosal fluid into the serosal fluid at an approximately 8-fold higher rate than that of the small intestine. These results suggest that, in the small intestine, phenolic xenobiotics are mostly glucuronidated and secreted intraluminally and that the resulting glucuronide is absorbed and transported into the serosal side of the colon.

Glucuronidation of the environmental oestrogen bisphenol A by an isoform of UDP-glucuronosyltransferase, UGT2B1, in the rat liver.

Bisphenol A, an environmental oestrogenic chemical, was found to conjugate highly with glucuronic acid in male rat liver microsomes studied in vitro. In the various isoforms tested (1A1, 1A3, 1A5, 1A6, 1A7 and 2B1), glucuronidation of bisphenol A and of diethylstilboestrol, a synthetic crystalline compound possessing oestrogenic activity and known to be glucuronidated by liver microsomes, was catalysed by an isoform of UDP-glucuronosyltransferase (UGT), namely UGT2B1, which glucuronidates some endogenous androgens. UGT activity towards bisphenol A in liver microsomes and in UGT2B1 expressed in yeast AH22 cells (22.9 and 0.58 nmol/min per mg of microsomal proteins respectively) was higher than that towards diethylstilboestrol (75.0 and 4.66 pmol/min per mg of microsomal proteins respectively). UGT activities towards both bisphenol A and diethylstilboestrol were distributed mainly in the liver but were also observed at substantial levels in the kidney and testis. Northern blot analysis disclosed the presence of UGT2B1 solely in the liver, and about 65% of the male rat liver microsomal UGT activities towards bisphenol A were absorbed by the anti-UGT2B1 antibody. These results indicate that bisphenol A, in male rat liver, is glucuronidated by UGT2B1, an isoform of UGT.

Small intestinal UDP-glucuronosyltransferase sheUGT1A07: partial purification and cDNA cloning from sheep small intestine.

A phenol UDPglucuronosyltransferase (UGT) was partially purified, and the cDNA encoding the isoform was cloned and sequenced from sheep small intestine. The purified preparation containing a one major band (57 kDa) and one minor band (50 kDa) revealed high activities toward xenobiotics such as 1-naphthol (1-NA), 4-nitrophenol, and 4-methylumbelliferone. The preparation, however, had only little activity toward 4-hydroxybiphenyl and no activity toward bilirubin, suggesting that the preparation contains UGT1 isoforms. The NH2-terminal amino acid sequence of the major band was determined to be Gly-Lys-Leu-Leu-Val-Val-Pro-Met-Asp-Gly-Ser. A full-length UGT cDNA was obtained by reverse transcription-polymerase chain reaction with the degenerated 5'-primer from the NH2-terminal amino acid sequence of the purified major one and rapid amplification of cDNA ends from sheep small intestine. The cloned cDNA named sheUGT1A07 by amino acid similarity has a NH2-terminus sequence identical to that of the purified major one. Another phenol UGT cDNA named sheUGT1A6 was also cloned from sheep liver. sheUGT1A6 was expressed mainly in the liver, whereas sheUGT1A07 mRNA was expressed almost only in the alimentary organs, suggesting that sheUGT1A6 plays a role as a general drug metabolizing UGT isoform in the liver and sheUGT1A07 plays important role in the xenobiotics glucuronidation in the sheep small intestine.

The significance of amino acid residue Asp446 for enzymatic stability of rat UDP-glucuronosyltransferase UGT1A6.

Asp446 in rat UDP-glucuronosyltransferase (UGT), UGT1A6, is an essential amino acid residue for its enzymatic activity (H. Iwano et al. Biochem. J. 325, 587-591, 1997). The role of Asp446 in UGT1A6 was investigated by comparing some properties of UGT mutant proteins that have a single mutation (D446K, D446E, D446N, D446Q, D446A, and D446T). These mutants, except D446K, had catalytic activities toward 1-naphthol and 4-methylumbelliferone. The UGT activities of D446E and D446N were about half of that of the wild type, and the activities of the other mutants were only about 1/5-1/10 of that of the wild type. The Km values for 1-naphthol of these mutants were similar to that of the wild type, while the values for UDP-glucuronic acid were slightly higher. The mutants were unstable in a low-pH buffer solution and were dramatically inactivated by heat treatments. Interestingly, after 30 min of treatment at 37 degrees C in the presence of UDP-glucuronic acid, the UGT activities of all functional mutants were elevated. These results suggest that Asp446 is an indispensable residue for folding a functional conformation of rat UGT1A6 by cooperation with UDP-glucuronic acid.

Inhibitory effects of uridine diphosphate on UDP-glucuronosyltransferase.

Inhibitory effects of uridine diphosphate on the enzymatic activity of UDP-glucuronosyltransferase (UGT) were investigated. Pyrimidine nucleotides such as UDP, UTP and cytidine diphosphate reduced the activity of rat purified UGT (phenol UGT) to about 10%, 48% and 46% of the control, respectively, at the same concentration as a donor substrate, UDP-glucuronic acid. Purine nucleotides, uridine monophosphate, glucuronic acid and some UDP-sugars were only slightly inhibitory toward the transferase. Similar effects were observed in the expressed UGT (UGT1A6; corresponding to phenol UGT) in yeast cells and rat liver microsomal membrane-binding UGT, indicating that uracil and diphosphate residues are essential for the UDP inhibition. Interestingly, 2'-deoxy UDP was found to be a less effective inhibitor (about 50% inhibition) than UDP on the purified, the expressed (UGT1A6 and UGT2B1) and microsomal membrane-binding UGTs. These results indicate that not only uracil and diphosphate residues but also 2'-hydroxyl residue of UDP ribose participates in the interactions between UDP and UDP-glucuronosyltransferase.

UDP-glucuronosyltransferase UGT1A7 induced in rat small intestinal mucosa by oral administration of 2-naphthoflavone.

In the rat intestine, UDP-glucuronosyltransferase (UGT) isoforms were highly induced by oral administration of 2-naphthoflavone, as shown by intestinal UGT activity toward 1-naphthol (1-NA). The greatest increase in UGT activity occurred in the duodenum. Using UGT1A6 cDNA as a probe, we obtained three types of clones corresponding to UGT1A2, UGT1A6 and UGT1A7, in a ratio of 1:1:8, from a cDNA library constructed from the 2-naphthoflavone-treated rat intestine. The induction of each isoform was evaluated by means of Northern blotting with isoform-specific probes. The mRNAs of UGT1A6 (glucuronizing various phenolic xenobiotics) and the mRNAs of UGT1A7 (glucuronizing the ultimate carcinogenic metabolite of benzo(a)pyrene) were expressed constitutively and were highly induced in the duodenum and proximal jejunum. S1 mapping showed that induction of the isoforms of the UGT1 family was more pronounced in the liver than in the small intestine and that UGT1A7 was the major UGT1 isoform in the small intestine of vehicle-treated rats and in that of 2-naphthoflavone-treated rats. These results indicate that, in rats, UGT1A7 is expressed constitutively and is particularly inducible in the small intestine. In the light of these results, we believe that the UGT1A7 isoform would play an important role in glucuronidation in the small intestinal mucosa of rats.

A critical amino acid residue, asp446, in UDP-glucuronosyltransferase.

An amino acid residue, Asp446, was found to be essential for the enzymic activity of UDP-glucuronosyltransferase (UGT). We obtained a rat phenol UGT (UGT1*06) cDNA (named Ysh) from male rat liver by reverse-transcription (RT)-PCR using pfu polymerase. A mutant Ysh having two different bases, A1337G and G1384A (named Ysh A1337GC1384A), that result in two amino acid substitutions, D446G and V462M, was obtained by RT-PCR using Taq polymerase. Ysh was expressed functionally in microsomes of Saccharomyces cerevisiae strain AH22. However, the expressed protein from YshA1337GG1384A had no transferase activity. Two other mutant cDNAs with YshA1337G having one changed base, A1337G, resulting in one amino acid substitution, D446G, and YshG1384A having a changed base, G1384A, resulting in an amino acid substitution, V462M, were constructed and expressed in the yeast. The expressed protein from YshG1384A (named YshV462M) exhibited enzymic activity, but the one from YshA1337G (named YshD446G) did not show any activity at all. Asp446 was conserved in all UGTs and UDP-galactose:ceramide galactosyltransferases reported, suggesting that Asp446 plays a critical role in each enzyme.