Chemical Constituents of the Bulbs of Scilla peruviana and Their Pancreatic Lipase Inhibitory Activity.

Scilla species are used as medicinal plants and contain lanosterol-type triterpene glycosides. The phytochemical investigation of the bulbs of Scilla peruviana led to the isolation of 17 compounds, including three new rearranged pentacyclic-lanosterol glycosides (1-3) and two new homoisoflavanone glycosides (12 and 13). The structures of the undescribed compounds were determined by extensive spectroscopic analyses, including two-dimensional (2D) NMR. Among the triterpene glycosides, 2, 3, and 6 showed significant pancreatic lipase inhibitory activity in a concentration-dependent manner in vitro. The oral administration of scillascilloside D-2 (6) reduced serum triglyceride levels in a dose-dependent manner in soybean oil-loaded mice.

Daisaikoto Inhibits Pancreatic Lipase Activity and Decreases Serum Triglyceride Levels in Mice.

Daisaikoto Extract, a Kampo medicine listed in the Japanese pharmacopoeia 17th edition, is clinically used to treat obesity and related symptoms. Lipid metabolism is closely related to obesity, and pancreatic lipase inhibitors are therefore regarded as effective for the treatment of obesity. Although Daisaikoto has shown promise in the treatment of obesity, its mechanism of action has yet to be elucidated. In the present study, we found that Daisaikoto extract inhibits pancreatic lipase activity in a dose-dependent manner and decreases serum triglyceride levels in mice. To determine the crude drugs responsible for lipase inhibition, 8 variants of Daisaikoto extract without one crude drug were prepared and evaluated for lipase inhibitory activity. The lipase inhibitory activity of the Daisaikoto extract was reduced by excluding Scutellariae Radix (SR), which was found to inhibit lipase activity with an IC50 value of 1.70 mg/mL. In conclusion, Daisaikoto represents a natural medicine, in particular SR, capable of inhibiting pancreatic lipase and lipid absorption.

Diet-induced hyperhomocysteinemia impairs vasodilation in 5/6-nephrectomized rats.

Plasma homocysteine is elevated in patients with impaired renal function, and markedly so at end-stage renal disease. As chronic kidney disease and hyperhomocysteinemia are also independent risk factors for cardiovascular disease, the latter is hypothesized to accelerate vascular abnormalities following renal failure. This study aimed to investigate the combined effect of impaired renal function and hyperhomocysteinemia on vascular function. We show that in 5/6-nephrectomized rats, a model of chronic kidney disease, a methionine-rich diet for 8 weeks induces moderate hyperhomocysteinemia, exacerbates hypertension, and attenuates the vascular response to acetylcholine, sodium nitroprusside, 8-bromo-cGMP, and isoprenaline. However, plasma nitrate/nitrite and total NOS activity in the thoracic aorta were not affected. Collectively, the data imply that hyperhomocysteinemia and end-stage renal disease synergistically impair endothelium-dependent and endothelium-independent vasodilatation by blocking the cGMP/protein kinase G and/or cAMP/protein kinase A pathways. 5/6-Nephrectomized rat with hyperhomocysteinemia induced by a methionine-rich diet would be a useful model for elucidating the pathogenesis of vascular impairment in patients with end-stage renal disease.

Chronic high-fat feeding impairs adaptive induction of mitochondrial fatty acid combustion-associated proteins in brown adipose tissue of mice.

Since brown adipose tissue (BAT) is involved in thermogenesis using fatty acids as a fuel, BAT activation is a potential strategy for treating obesity and diabetes. However, whether BAT fatty acid combusting capacity is preserved in these conditions has remained unclear. We therefore evaluated expression levels of fatty acid oxidation-associated enzymes and uncoupling protein 1 (Ucp1) in BAT by western blot using a diet-induced obesity C57BL/6J mouse model. In C57BL/6J mice fed a high-fat diet (HFD) over 2-4 weeks, carnitine palmitoyltransferase 2 (Cpt2), acyl-CoA thioesterase (Acot) 2, Acot11 and Ucp1 levels were significantly increased compared with baseline and control low-fat diet (LFD)-fed mice. Similar results were obtained in other mouse strains, including ddY, ICR and KK-Ay, but the magnitudes of the increase in Ucp1 level were much smaller than in C57BL/6J mice, with decreased Acot11 levels after HFD-feeding. In C57BL/6J mice, increased levels of these mitochondrial proteins declined to near baseline levels after a longer-term HFD-feeding (20 weeks), concurrent with the accumulation of unilocular, large lipid droplets in brown adipocytes. Extramitochondrial Acot11 and acyl-CoA oxidase remained elevated. Treatment of mice with Wy-14,643 also increased these proteins, but was less effective than 4 week-HFD, suggesting that mechanisms other than peroxisome proliferator-activated receptor α were also involved in the upregulation. These results suggest that BAT enhances its fatty acid combusting capacity in response to fat overload, however profound obesity deprives BAT of the responsiveness to fat, possibly via mitochondrial alteration.

Identification of acyl-CoA thioesterase in mouse mesenteric lymph nodes.

Acyl-CoA thioesterases (ACOTs) are a group of enzymes that catalyze the hydrolysis of fatty acyl-CoAs to free fatty acids and CoA, with the potential to regulate the intracellular levels of these molecules. In this study, we show that a cytosolic isoform, ACOT7, is expressed at a significant level in the mesenteric lymph nodes (MLNs) of mice. While crude preparations of the mesenteric visceral fat contained significant levels of palmitoyl-CoA thioesterase activity, enzyme activity was concentrated 6.9-fold in MLNs compared with the residual adipose portion after excision of MLNs. When MLN homogenates were centrifuged, 82% of the enzyme activity was recovered in the cytosolic fraction, concomitant with almost exclusive recovery of ACOT7. Immunoprecipitation using anti-ACOT7 antibody estimated that 87% of enzyme activity in the homogenates was accounted for by ACOT7. On MLN sections, the germinal centers of secondary lymphoid follicles were immunostained with the antibody. In MLNs of mice fasted for 16 h, ACOT7 levels were induced 1.8-fold, which reflected a 1.5-fold increase in enzyme activity. These findings suggest that ACOT7 may be involved in dietary intake-associated responses in fatty acid metabolism in MLNs.

Pretreatment prediction of interferon-alfa efficacy in chronic hepatitis C patients.

BACKGROUND & AIMS: Interferon has been used widely to treat patients with chronic hepatitis C infections. Prediction of interferon efficacy before treatment has been performed mainly by using viral information, such as viral load and genotype. This information has allowed the successful prediction of sustained responders (SR) and non-SRs, which includes transient responders (TR) and nonresponders (NR). In the current study we examined whether liver messenger RNA expression profiles also can be used to predict interferon efficacy. METHODS: RNA was isolated from 69 liver biopsy samples from patients receiving interferon monotherapy and was analyzed on a complementary DNA microarray. Of these 69 samples, 31 were used to develop an algorithm for predicting interferon efficacy, and 38 were used to validate the precision of the algorithm. We also applied our methodology to the prediction of the efficacy of interferon/ribavirin combination therapy using an additional 56 biopsy samples. RESULTS: Our microarray analysis combined with the algorithm was 94% successful at predicting SR/TR and NR patients. A validation study confirmed that this algorithm can predict interferon efficacy with 95% accuracy and a P value of less than .00001. Similarly, we obtained a 93% prediction efficacy and a P value of less than .0001 for patients receiving combination therapy. CONCLUSIONS: By using only host data from the complementary DNA microarray we are able to successfully predict SR/TR and NR patients for interferon therapy. Therefore, this technique can help determine the appropriate treatment for hepatitis C patients.

Optimization of liver biopsy RNA sampling and use of reference RNA for cDNA microarray analysis.

In this study, we used the rat liver as a model system to optimize the conditions for extracting RNA from liver biopsies for use in cDNA microarrays. We found that a 5-mm biopsy with a 16-gauge needle and storage in RNA later at 4 degrees C were optimal conditions for RNA extraction. The most important factor for the quantity and quality of RNA extraction was the sample diameter. Using the optimized sampling conditions and a cDNA microarray, we compared the expression of genes in the normal and the fibrotic tissues of the LEC rat liver, a model of liver tumorigenesis, with SD rat liver RNA as a reference. We found 29 genes that were up-regulated and 33 genes that were down-regulated in the fibrotic part of the liver. Furthermore, with the help of the reference RNA, we were able to classify the expression profiles into five groups without complex mathematical analyses; without the reference RNA, the genes could be classified into only two groups. Finally, we found that osteopontin was expressed at a very high level in the fibrotic portion of the LEC rat liver. This cDNA microarray result was validated by immunohistochemistry, which showed an elevated expression of osteopontin in the region of cholangiocarcinoma and a lack of expression in normal tissues. With optimized conditions, we should be able to apply the microarray system for routine practice.

A novel beta1,3-N-acetylglucosaminyltransferase (beta3Gn-T8), which synthesizes poly-N-acetyllactosamine, is dramatically upregulated in colon cancer.

A new member of the UDP-N-acetylglucosamine: beta-galactose beta1,3-N-acetylglucosaminyltransferase (beta3Gn-T) family having the beta3-glycosyltransferase motifs was identified using an in silico method. This novel beta3Gn-T was cloned from a human colon cancer cell line and named beta3Gn-T8 based on its position in a phylogenetic tree and enzymatic activity. Beta3Gn-T8 transfers GlcNAc to the non-reducing terminus of the Galbeta1-4GlcNAc of tetraantennary N-glycan in vitro. HCT15 cells transfected with beta3Gn-T8 cDNA showed an increase in reactivity to both LEA and PHA-L4 in a flow cytometric analysis. These results indicated that beta3Gn-T8 is involved in the biosynthesis of poly-N-acetyllactosamine chains on tetraantennary (beta1,6-branched) N-glycan. In most of the colorectal cancer tissues examined, the level of beta3Gn-T8 transcript was significantly higher than in normal tissue. Beta3Gn-T8 could be an enzyme involved in the synthesis of poly-N-acetyllactosamine on beta1-6 branched N-glycans in colon cancer.

A low-density cDNA microarray with a unique reference RNA: pattern recognition analysis for IFN efficacy prediction to HCV as a model.

We have designed and established a low-density (295 genes) cDNA microarray for the prediction of IFN efficacy in hepatitis C patients. To obtain a precise and consistent microarray data, we collected a data set from three spots for each gene (mRNA) and using three different scanning conditions. We also established an artificial reference RNA representing pseudo-inflammatory conditions from established hepatocyte cell lines supplemented with synthetic RNAs to 48 inflammatory genes. We also developed a novel algorithm that replaces the standard hierarchical-clustering method and allows handling of the large data set with ease. This algorithm utilizes a standard space database (SSDB) as a key scale to calculate the Mahalanobis distance (MD) from the center of gravity in the SSDB. We further utilized sMD (divided by parameter k: MD/k) to reduce MD number as a predictive value. The efficacy prediction of conventional IFN mono-therapy was 100% for non-responder (NR) vs. transient responder (TR)/sustained responder (SR) (P < 0.0005). Finally, we show that this method is acceptable for clinical application.

Characterization of a heparan sulfate 3-O-sulfotransferase-5, an enzyme synthesizing a tetrasulfated disaccharide.

Heparan sulfate d-glucosaminyl 3-O-sulfotransferases (3-OSTs) catalyze the transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to position 3 of the glucosamine residue of heparan sulfate and heparin. A sixth member of the human 3-OST family, named 3-OST-5, was recently reported (Xia, G., Chen, J., Tiwari, V., Ju, W., Li, J.-P., Malmstrom, A., Shukla, D., and Liu, J. (2002) J. Biol. Chem. 277, 37912-37919). In the present study, we cloned putative catalytic domain of the human 3-OST-5 and expressed it in insect cells as a soluble enzyme. Recombinant 3-OST-5 only exhibited sulfotransferase activity toward heparan sulfate and heparin. When incubated heparan sulfate with [35S]PAPS, the highest incorporation of35S was observed, and digestion of the product with a mixture of heparin lyases yielded two major35S-labeled disaccharides, which were determined as DeltaHexA-GlcN(NS,3S,6S) and DeltaHexA(2S)-GlcN(NS,3S) by further digestion with 2-sulfatase and degradation with mercuric acetate. However, when used heparin as acceptor, we identified a highly sulfated disaccharide unit as a major product. This had a structure of DeltaHexA(2S)-GlcN(NS,3S,6S). Quantitative real-time PCR analysis revealed that 3-OST-5 was highly expressed in fetal brain, followed by adult brain and spinal cord, and at very low or undetectable levels in the other tissues. Finally, we detected a tetrasulfated disaccharide unit in bovine intestinal heparan sulfate. To our knowledge, this is the first report to describe not only the natural occurrence of tetrasulfated disaccharide unit but also the enzymatic formation of this novel structure.

Cloning and characterization of a new human UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase, designated pp-GalNAc-T13, that is specifically expressed in neurons and synthesizes GalNAc alpha-serine/threonine antigen.

To date, 10 members of the UDP-N-acetyl-alpha-d-galactosamine:polypeptide N-acetylgalactosaminyltransferase (pp-GalNAc-T) family have been cloned and analyzed in human. In this study, we cloned and analyzed a novel human pp-GalNAc-T from an NT2 cell cDNA library, and we named it pp-GalNAc-T13. In amino acid sequences, pp-GalNAc-T13 was highly homologous, showing 84.3% identity, to pp-GalNAc-T1. Real time PCR analysis revealed pp-GalNAc-T13 to be highly and restrictively expressed in the brain and present at very low or undetectable levels in other tissues, in contrast to the ubiquitous expression of pp-GalNAc-T1. pp-GalNAc-T13 was abundantly expressed in all neuroblastoma cells examined and primary cultured neurons but not in glioblastoma cells and primary cultured astrocytes. pp-GalNAc-T13 exhibited much stronger activity to transfer GalNAc to mucin peptides, such as Muc5Ac and MUC7, than did pp-GalNAc-T1. In addition, pp-GalNAc-T13 differed in substrate specificity to pp-GalNAc-T1. pp-GalNAc-T13 was able to form a triplet Tn epitope, three consecutive GalNAc-Ser/Thr structures, on peptides encoded in syndecan-3, a proteoglycan expressed in neurons. pp-GalNAc-T13-deficient mice have been established in a previous work. Immunohistochemical study showed a remarkable decrease in Tn antigen expression in the cerebellum of the pp-GalNAc-T13 knockout mouse. pp-GalNAc-T13 would be a major enzyme responsible for the synthesis of O-glycan and specifically the Tn antigen epitope in neurons.

Differential roles of two N-acetylgalactosaminyltransferases, CSGalNAcT-1, and a novel enzyme, CSGalNAcT-2. Initiation and elongation in synthesis of chondroitin sulfate.

By a tblastn search with beta 1,4-galactosyltransferases as query sequences, we found an expressed sequence tag that showed similarity in beta 1,4-glycosyltransferase motifs. The full-length complementary DNA was obtained by a method of 5'-rapid amplification of complementary DNA ends. The predicted open reading frame encodes a typical type II membrane protein comprising 543 amino acids, the sequence of which was highly homologous to chondroitin sulfate N-acetylgalactosaminyltransferase (CSGalNAcT-1), and we designated this novel enzyme CSGalNAcT-2. CSGalNAcT-2 showed much stronger N-acetylgalactosaminyltransferase activity toward glucuronic acid of chondroitin poly- and oligosaccharides, and chondroitin sulfate poly- and oligosaccharides with a beta 1-4 linkage, i.e. elongation activity for chondroitin and chondroitin sulfate, but showed much weaker activity toward a tetrasaccharide of the glycosaminoglycan linkage structure (GlcA-Gal-Gal-Xyl-O-methoxyphenyl), i.e. initiation activity, than CSGalNAcT-1. Transfection of the CSGalNAcT-1 gene into Chinese hamster ovary cells yielded a change of glycosaminoglycan composition, i.e. the replacement of heparan sulfate on a syndecan-4/fibroblast growth factor-1 chimera protein by chondroitin sulfate, however, transfection of the CSGalNAcT-2 gene did not. The above results indicated that CSGalNAcT-1 is involved in the initiation of chondroitin sulfate synthesis, whereas CSGalNAcT-2 participates mainly in the elongation, not initiation. Quantitative real-time PCR analysis revealed that CSGalNAcT-2 transcripts were highly expressed in the small intestine, leukocytes, and spleen, however, both CSGalNAcTs were ubiquitously expressed in various tissues.

A novel I-branching beta-1,6-N-acetylglucosaminyltransferase involved in human blood group I antigen expression.

The human blood group i and I antigens are determined by linear and branched poly-N-acetyllactosamine structures, respectively. In erythrocytes, the fetal i antigen is converted to the adult I antigen by I-branching beta-1,6-N-acetylglucosaminyltransferase (IGnT) during development. Dysfunction of the I-branching enzyme may result in the adult i phenotype in erythrocytes. However, the I gene responsible for blood group I antigen has not been fully confirmed. We report here a novel human I-branching enzyme, designated IGnT3. The genes for IGnT1 (reported in 1993), IGnT2 (also presented in this study), and IGnT3 consist of 3 exons and share the second and third exons. Bone marrow cells preferentially expressed IGnT3 transcript. During erythroid differentiation using CD34(+) cells, IGnT3 was markedly up-regulated with concomitant decrease in IGnT1/2. Moreover, reticulocytes expressed the IGnT3 transcript, but IGnT1/2 was below detectable levels. By molecular genetic analyses of an adult i pedigree, individuals with the adult i phenotype were revealed to have heterozygous alleles with mutations in exon 2 (1006G>A; Gly336Arg) and exon 3 (1049G>A; Gly350Glu), respectively, of the IGnT3 gene. Chinese hamster ovary (CHO) cells transfected with each mutated IGnT3 cDNA failed to express I antigen. These findings indicate that the expression of the blood group I antigen in erythrocytes is determined by a novel IGnT3, not by IGnT1 or IGnT2.

Molecular cloning and characterization of a novel UDP-Gal:GalNAc(alpha) peptide beta 1,3-galactosyltransferase (C1Gal-T2), an enzyme synthesizing a core 1 structure of O-glycan.

Recently, a UDP-Gal:GalNAc(alpha) peptide beta1,3-galactosyltransferase (core 1 synthase 1; C1Gal-T1) has been purified from rat liver and its complementary DNA cloned from several species. We isolated a second candidate for core 1 synthase from a Colo205 cDNA library and named it C1Gal-T2. The deduced amino acid sequence of C1Gal-T2, having 26% homology to C1Gal-T1, showed a topology typical of a type II membrane protein. Real time PCR analysis revealed that the expression of C1Gal-T2 transcripts was widespread in many tissues and of relatively high level in salivary gland, stomach, small intestine, kidney, testis, thymus, and spleen. LSC cells, having no core 1 synthase activity, were transfected stably with the C1Gal-T2 gene. Their microsome fraction showed beta1,3-galactosyltransferase activity toward GalNAc-alpha-para-nitrophenyl and GalNAc(alpha)1 peptides resulting in the synthesis of the core 1 structure. The core 1 synthesizing activity of C1Gal-T2 was also determined by flow cytometry and lectin blotting using the LSC cells stably expressing C1Gal-T2. Finally, LSC cells, and Jurkat cells that also lack the core 1 synthase activity, were found to have null alleles of C1Gal-T2. These results indicated that C1Gal-T2 is the second candidate for core 1 synthase that plays an important role in synthesizing O-glycans in digestive organs.

Enzymatic synthesis of chondroitin with a novel chondroitin sulfate N-acetylgalactosaminyltransferase that transfers N-acetylgalactosamine to glucuronic acid in initiation and elongation of chondroitin sulfate synthesis.

We found a novel glycosyltransferase gene having a hypothetical beta 1,4-galactosyltransferase motif (GenBank accession number ) by a BLAST search and cloned its full-length open reading frame using the 5'-rapid amplification of cDNA ends method. The truncated form was expressed in insect cells as a soluble enzyme. It transferred N-acetylgalactosamine, not galactose, to para-nitrophenyl-beta-glucuronic acid. The N-acetylgalactosamine-glucuronic acid linkage has been identified only in chondroitin sulfate; therefore, we examined its chondroitin elongation and initiation activities. N-Acetylgalactosaminyltransferase activity was observed toward chondroitin poly- and oligosaccharides, chondroitin sulfate oligosaccharides, and linkage tetrasaccharide (GlcA-Gal-Gal-Xyl-O-methoxyphenyl), and the chondroitin polysaccharide and linkage tetrasaccharide were better acceptor substrates than the others. Northern blot analysis and quantitative real-time PCR analysis revealed that its 4-kb transcripts were highly expressed in thyroid and placenta, although they were ubiquitously expressed in various tissues and cells. These results suggest that this enzyme has N-acetylgalactosaminyltransferase activity in both the elongation and initiation of chondroitin sulfate synthesis. Furthermore, we performed enzymatic synthesis of chondroitin pentasaccharide in vitro. In one tube reaction with four enzymes, beta 1,4-galactosyltransferase-VII, beta 1,3-galactosyltransferase-VI, glucuronyltransferase-I, and this enzyme, and a synthetic xylose-peptide acceptor, the structure GalNAc-GlcA-Gal-Gal-Xyl-peptide was constructed. This is the first report of a chondroitin pentasaccharide constructed with recombinant glycosyltransferases in vitro.

Molecular cloning and characterization of a novel chondroitin sulfate glucuronyltransferase that transfers glucuronic acid to N-acetylgalactosamine.

We found a novel human gene (GenBank accession number, Kazusa DNA Research Institute KIAA1402) that possesses homology with chondroitin synthase. The full-length open reading frame consists of 772 amino acids and encodes a typical type II membrane protein. This enzyme had a domain containing beta 3-glycosyltransferase motifs, which might be a beta3-glucuronyltransferase domain, but no domain with beta 4-glycosyltransferase motifs, although both are found in chondroitin synthase. The putative catalytic domain was expressed in COS-7 cells as a soluble enzyme. Its glucuronyltransferase activity was observed when chondroitin and chondroitin sulfate polysaccharides and oligosaccharides were used as acceptor substrates. However, it was not detected when dermatan sulfate, hyaluronan, heparan sulfate, heparin, N-acetylheparosan, lactosamine tetrasaccharide, and linkage tri- and tetrasaccharide acceptors were employed. The reaction product, which was speculated to exhibit a GlcA beta 1-3GalNAc linkage structure at its non-reducing terminus, showed the following characteristics. 1) It was catabolized by beta-glucuronidase. 2) It was an acceptor for Escherichia coli K4 chondroitin polymerase (K4 chondroitin polymerase). 3) The product of K4 chondroitin polymerase was cleaved by chondroitinase ACII. On the other hand, no N-acetylgalactosaminyltransferase activity was detected toward any acceptors. Quantitative real time PCR analysis revealed that its transcripts were highly expressed in the placenta, small intestine, and pancreas, although they were ubiquitously expressed in various tissues and cell lines. This enzyme could play a role in the synthesis of chondroitin sulfate as a glucuronyltransferase.

Molecular cloning and characterization of a novel UDP-GlcNAc:GalNAc-peptide beta1,3-N-acetylglucosaminyltransferase (beta 3Gn-T6), an enzyme synthesizing the core 3 structure of O-glycans.

The core 3 structure of the O-glycan, GlcNAcbeta1-3GalNAcalpha1-serine/threonine, an important precursor in the biosynthesis of mucin-type glycoproteins, is synthesized by UDP-N-acetylglucosamine:GalNAc-peptide beta1,3-N- acetylglucosaminyltransferase (beta3Gn-T; core 3 synthase). The core 3 structure is restricted in its occurrence to mucins from specific tissues such as the stomach, small intestine, and colon. A partial sequence encoding a novel member of the human beta3Gn-T family was found in one of the data bases. We cloned a complementary DNA of this gene and named it beta3Gn-T6. The putative amino acid sequence of beta3Gn-T6 retains the beta3Gn-T motifs and is predicted to comprise a typical type II membrane protein. The soluble form of beta3Gn-T6 expressed in insect cells showed beta3Gn-T activity toward GalNAcalpha-p-nitrophenyl and GalNAcalpha1-serine/threonine. The beta1,3-linkage between GlcNAc and GalNAc of the enzyme reaction product was confirmed by high performance liquid chromatography and NMR analyses. beta3Gn-T6 effectively transferred a GlcNAc to the GalNAc residue on MUC1 mucin, resulting in the synthesis of a core 3 structure. Real time PCR analysis revealed that the beta3Gn-T6 transcript was restricted in its distribution, mainly to the stomach, colon, and small intestine. We concluded that beta3Gn-T6 is the most logical candidate for the core 3 synthase, which plays an important role in the synthesis of mucin-type O-glycans in digestive organs.