A sulfoglycolipid beta-sulfoquinovosyldiacylglycerol (betaSQDG) binds to Met1-Arg95 region of murine DNA polymerase lambda (Mmpol lambda) and inhibits its nuclear transit.

Beta-sulfoquinovosyldiacylglycerol (betaSQDG) is a synthetic sulfoglycolipid that shows inhibitory activity of DNA polymerase lambda (pol lambda). Here we identified a betaSQDG binding region within murine pol lambda (Mmpol lambda) using T7 phage display technology. We compared the binding intensity of betaSQDG with recombinant phages (phages lambda1-6) that displayed different segments of Mmpol lambda. The binding assay clearly showed that phage lambda1, which displayed the non-structural Met1-Arg95 region including the nuclear localization signal (NLS) and part of the BRCT domain, bound more strongly to betaSQDG than the other recombinant phages. Binding assays using recombinant proteins gave similar results, showing specific betaSQDG binding to Met1-Arg95 with a K(D) value of 9.9 nM. Furthermore, in a cell-based assay, nuclear localization of EGFP-pollambda was inhibited in the presence of betaSQDG possibly due to binding of betaSQDG to NLS. These experiments clearly show that the binding region of betaSQDG within Mmpol lambda could be successfully identified using T7 phage display technology. We suggest that the strategy we describe here will be of value for identifying the binding site within a protein for small ligands, and will provide information that cannot be obtained using other experimental techniques due to their inherent technical limitations.

The entrapment of kojic oleate in bilayer vesicles.

The entrapment of kojic acid and its newly synthesized ester (kojic oleate) has been evaluated. Kojic oleate was synthesized by DCC (N,N'-dicyclohexylcarbodiimide, DCC)/(4-(N,N-dimethylamino)pyridine, DMAP) esterification method and identified by FAB-MS and 1H NMR. The synthesized product was mainly 7-O-kojic oleate with more than 80% yield. It was entrapped in vesicular membrane prepared from 9.5:9.5:1.0 molar ratio of amphiphiles (Span 60, Tween 61 or DPPC), cholesterol and dicetyl phosphate. Kojic acid was encapsulated in the water compartment of these vesicles in order to confirm the vesicle formation. The morphology and particle size of the vesicles were characterized by an optical microscope and transmission electron microscope (TEM). The entrapment efficiencies of kojic acid and kojic oleate in the vesicles were investigated by dialysis and column chromatography, respectively. The contents of the entrapped kojic acid and kojic oleate were assayed by HPLC. The entrapment efficiency of kojic acid was 0.01-0.04 mol, whereas kojic oleate gave higher entrapment efficiency of 0.25-0.35 mol/mol of the total compositions of amphiphile/cholesterol/dicetyl phosphate. Structural modification of kojic acid improved its entrapment in the vesicles. Tween 61 vesicles could entrap kojic oleate more than did Span 60 vesicles. The pi-A isotherms revealed the lower area per molecule of Span 60, which formed a more rigid pack of its molecule on air/water interface than that of Tween 61. This implied the high rigidity of vesicular membrane prepared with Span 60 led to the lower amount of kojic oleate entrapped in the vesicles. From the release study of kojic acid through the dialysis membrane, it indicated that the intercalation of kojic oleate in the vesicular membranes did not significantly affect the release of kojic acid from the vesicles.

Mechanism of the immunosuppressive effect in vivo of novel immunosuppressive drug beta-SQAG9, which inhibits the response of the CD62L+ T-cell subset.

INTRODUCTION: We synthesized sulfo-glycolipid, beta-SQAG9 (designate square beta-SQAG9 liposome, because it efficiently forms a liposome structure) that possessed immunosuppressive effects such as inhibition of T-cell responses in human allogeneic MLR and skin allograft survival in rats, and bound to CD62L (L-selectin) in vitro. In this study, we further investigated the immunosuppressive mechanism in vivo by beta-SQAG9 liposome in a skin-allografted rat model. METHODS: ACI rats (RT1(a)) were grafted skin of LEW rats (RT1(1)) treated with PBS or beta-SQAG9 liposome IV once a day for 7 days. Subsequently, we investigated the population of T cells and CD62L(+) T-cell subset in the spleen, axillary lymph nodes (ALNs), and peripheral blood of skin-allografted rats by two-color flow cytometry. RESULTS: Five of 11 (45.5%) rats that were treated with 50 mg/kg beta-SQAG9 liposome showed graft survival and another showed moderate rejection in graft. The CD62L(+) T-cell subset population in ALNs of beta-SQAG9 liposome-treated rats decreased in a dose-dependent manner. No significant difference in the T-cell population was observed between the beta-SQAG9 and control groups. These data suggest that beta-SQAG9 could bind to the CD62L(+) T-cell subset in vivo as well as in vitro and affect T-cell migration, which might lead to T-cell tolerance in vivo.

Synthetic sulfonolipids deduced from sulfonoquinovosyl diacylglycerols of sea urchin reduces hepatic ischemia-reperfusion injury in rats.

BACKGROUND: In hepatic surgery and liver transplantation, ischemia-reperfusion (I/R) is an unavoidable process, and protection against hepatic I/R injury is a major unresolved problem. In this study, we investigated whether 3-O-(6-deoxy-6-sulfono-beta-D-glucopyranosyl)-1,2-di-O-acylglycerol bound to saturated C18 fatty acids (beta-SQAG9), which was derived from sea urchin intestines, could reduce this injury. This agent was recently reported to have immunosuppressive effects in allogeneic rat skin grafts. MATERIALS & METHODS: Male Lewis rats were divided into two experimental groups. Group 1 rats were injected with SQAG9 (50 mg/kg) into the penile vein 15 minutes before the induction of ischemia and into the portal vein just reperfusion. The same amounts of normal saline were injected into rats in the control group (group 2). Each experimental groups included six rats. Seventy percent hepatic ischemia (20 minutes) was induced by occluding the blood vessels and bile duct with a vascular clamp. For examination of hepatic function, serum levels of aspartate aminotransferase, (AST) alanine transaminase (ALT), and lactic dehydrogenase (LDH) were measured. In addition, histological examination was also assessed. RESULTS: Three hours after reperfusion, the mean plasma concentration of AST, ALT, LDH in group 1 was suppressed compared with group 2. Six hours after reperfusion, the hepatic damage in group 1 was mild in comparison with that in group 2. CONCLUSIONS: Our data demonstrated that SQAG-9 reduced the warm hepatic I/R injury.

[The optimal procedure for chylothorax after operation for thoracic esophageal cancer; reasonable approaches to the thoracic duct from the point of view of routes for esophageal replacement].

In general, chylothorax after esophagectomy with lymph node dissection under thoracotomy is a rare postoperative complication. We report a 71-year-old man who developed chylothorax following esophagectomy and 3-field lymph node dissection together with reconstruction using stomach through the posterior mediastinum, and discuss ideal approaches that are less invasive and make it possible to provide better exposure of the thoracic duct. In selecting the ideal approach, the most important thing is differences in routes for esophageal replacement. The anatomical relation between the location of a conduit adopted for reconstruction of the resected esophagus and the thoracic duct should be considered in each case. In the case of the retrosternal or antesternal route, a video-assisted thoracoscopic approach allows for easy detection of the thoracic duct while reducing surgical invasiveness, because there is no conduit in the posterior mediastinum. On the other hand, a conduit interrupts the visual field of thoracoscopy in the case of the posterior mediastinal or intrathoracic route. Drawing up of a conduit to gain a good operative field involves some risks in protection of the vascular pedicle. Therefore, a transabdominomediastinal approach is an optimal option. With this approach, we could easily find the thoracic duct and directly ligate it just cranial to the hiatus, resulting in a remarkable decrease in discharge through the thoracic drainage tube. In addition, we present an intelligible intraoperative photograph.

Three-dimensional structural model analysis of the binding site of lithocholic acid, an inhibitor of DNA polymerase beta and DNA topoisomerase II.

The molecular action of lithocholic acid (LCA), a selective inhibitor of mammalian DNA polymerase beta (pol beta), was investigated. We found that LCA could also strongly inhibit the activity of human DNA topoisomerase II (topo II). No other DNA metabolic enzymes tested were affected by LCA. Therefore, LCA should be classified as an inhibitor of both pol beta and topo II. Here, we report the molecular interaction of LCA with pol beta and topo II. By three-dimensional structural model analysis and by comparison with the spatial positioning of specific amino acids binding to LCA on pol beta (Lys60, Leu77, and Thr79), we obtained supplementary information that allowed us to build a structural model of topo II. Modeling analysis revealed that the LCA-interaction interface in both enzymes has a pocket comprised of three amino acids in common, which binds to the LCA molecule. In topo II, the three amino acid residues were Lys720, Leu760, and Thr791. These results suggested that the LCA binding domains of pol beta and topo II are three-dimensionally very similar.

Human TSC-22 gene: no association with type 2 diabetes.

OBJECTIVE: In order to elucidate whether or not genetic variations of TSC-22 (TGF [transforming growth factor]-beta-stimulated clone 22), which was originally identified as a TGF-beta-responsive leucine zipper protein in murine osteoblastic cells, are associated with type 2 diabetes, the genomic organization of the human TSC-22 gene was determined and the association between its polymorphisms and type 2 diabetes was examined. RESULTS: The human TSC-22 gene spans approximately 5 kilobase pairs and is encoded in three exons. Two single nucleotide polymorphisms (SNPs) were identified in the coding region of the first exon, two other SNPs in the first intron, and one SNP in the putative promoter region. There were, however, no significant differences in the frequency of these polymorphisms between patients with type 2 diabetes and non-diabetic control subjects. CONCLUSION: It is unlikely that the TSC-22 gene is a locus responsible for type 2 diabetes.

Specific interaction between an oligosaccharide on the tumor cell surface and the novel antitumor agents, sulfoquinovosylacylglycerols.

Some sulfoquinovosylacylglycerols (SQAG) have been shown to be potent DNA polymerase inhibitors, and to have strong antitumor activity in vivo. In this study, we investigated the mode of action of SQAG with regard to the interaction with the tumor cell surface. Of the SQAG used, the monoacyl forms (SQMG) with C18-, C18:1- or C16-fatty acids (SQMG-alphaC18, -alphaC18:1 or -alphaC16) effectively inhibited cell proliferation of a human adenocarcinoma cell line, DLD-1, but SQMG-alphaC14 and the diacyl forms (SQDG) did not. Analysis of the interaction of SQMG-alphaC18 and -alphaC18:1 on three oligosaccharides of cell surface, sLe(A), Le(X), and SM3, by flow cytometry demonstrated that the most effective interaction was observed on sLe(A). DLD-1 cells bound to SQMG-alphaC18:1-coated plates, and this binding was inhibited by monoclonal antibody against sLe(A) or SM3. However, these cells did not bind to SQMG-alphaC14-coated plates. Moreover the cytotoxic effects of SQMG-alphaC18, -alphaC18:1 on DLD-1 cells was inhibited by monoclonal antibodies against sLe(A) or SM3. Our results suggested that the interaction of SQMGs and sLe(A) plays an important role in suppression of the DLD-1 cell proliferation.

Galactosyldiacylglycerol, a mammalian DNA polymerase alpha-specific inhibitor from a sea alga, Petalonia bingbamiae.

The glycolipid galactosyldiacylglycerol (GDG), containing C16:0 and C18:1 fatty acids, was isolated from the sea alga Petalonia bingbamiae as a potent inhibitor of the activities of mammalian DNA polymerase alpha (pol. alpha). GDG, however, had no effect on pol. alpha from a fish or a higher plant. The inhibition of pol. alpha by GDG was dose-dependent with an IC50 value of 54 microM. The compound did not influence the activities of other replicative DNA polymerases such as mammalian pol. delta, or repair-related enzymes such as mammalian pol. beta. GDG also did not influence the activities of prokaryotic DNA polymerases such as the Klenow fragment of DNA polymerase I, T4 DNA polymerase, Taq DNA polymerase, DNA polymerases from the higher plant, cauliflower, or DNA metabolic enzymes such as calf thymus terminal deoxynucleotidyl transferase, human immunodeficiency virus type 1 reverse transcriptase and deoxyribonuclease 1. Kinetic analysis of the compound showed that pol. alpha was non-competitively inhibited with respect to both the DNA template and the nucleotide substrate. In this study, we demonstrated the structure-function relationship in the selective inhibition of pol. alpha by the glycolipid group.

Structural homology between DNA binding sites of DNA polymerase beta and DNA topoisomerase II.

Unsaturated long-chain fatty acids selectively bind to the DNA binding sites of DNA polymerase beta and DNA topoisomerase II, and inhibit their activities, although the amino acid sequences of these enzymes are markedly different from each other. Computer modeling analysis revealed that the fatty acid interaction interface in both enzymes has a group of four amino acid residues in common, forming a pocket which binds to the fatty acid molecule. The four amino acid residues were Thr596, His735, Leu741 and Lys983 for yeast DNA topoisomerase II, corresponding to Thr79, His51, Leu11 and Lys35 for rat DNA polymerase beta. Using three-dimensional structure model analysis, we determined the spatial positioning of specific amino acid residues binding to the fatty acids in DNA topoisomerase II, and subsequently obtained supplementary information to build the structural model.

Structure of lithocholic acid binding to the N-terminal 8-kDa domain of DNA polymerase beta.

The purpose of this study was to investigate the molecular action of lithocholic acid (LCA), known as a selective inhibitor of DNA polymerase beta (pol beta). The 39-kDa pol beta was separated proteolytically into two fragments of the template-primer binding domain (8 kDa) and the catalytic domain (31 kDa). LCA bound tightly to the 8-kDa fragment but not to the 31-kDa fragment. We examined the structural interaction with the 8-kDa domain using LCA. On (1)H-(15)N HMQC NMR analysis of pol beta with LCA, the 8-kDa domain bound to LCA as a 1:1 complex with a dissociation constant (K(D)) of 1.56 mM. The chemical shifts were observed only in residues mainly in helix-3, helix-4, and the 79-87 turn of the same face. No significant shifts were observed for helix-1, helix-2, and other loops of the 8-kDa domain. This region was composed mainly of three amino acid residues (Lys60, Leu77, and Thr79) of pol beta on the LCA interaction interface. The inhibition mechanism and the structure-function relationship between pol beta and LCA is discussed.

Structure-activity relationship of a novel group of mammalian DNA polymerase inhibitors, synthetic sulfoquinovosylacylglycerols.

We reported previously that sulfolipids in the sulfoquinovosylacylglycerol class from a fern and an alga are potent inhibitors of DNA polymerase alpha and beta and potent anti-neoplastic agents. In developing a procedure for chemical synthesis of sulfolipids, we synthesized many derivatives and stereoisomers of sulfoquinovosylmonoacylglycerol (SQMG) / sulfoquinovosyldiacylglycerol (SQDG). Some of these molecules were stronger inhibitors than the SQMG / SQDG originally reported as natural compounds. In this study, we examined the structure-inhibitory function relationship of synthetic SQMG / SQDG and its relationship to cytotoxic activity. The inhibitory effect is probably mainly dependent on the fatty acid effect, which we reported previously, although each of the SQMG / SQDG was a much stronger inhibitor than the fatty acid alone that was present in the SQMG / SQDG. The inhibitory effect could be influenced by the chain size of fatty acids in the SQMG / SQDG. The sulfate moiety in the quinovose was also important for the inhibition. Lineweaver-Burk plots of SQMG / SQDG indicated that DNA polymerase alpha was non-competitively inhibited, but the SQMG / SQDG were effective as antagonists of both template-primer DNA-binding and nucleotide substrate-binding of DNA polymerase beta. The SQMG had an cytotoxic effect, but the SQDG tested did not. The SQDG might not be able to penetrate into cells. Based on these results, we discuss the molecular action of SQMG / SQDG and propose drug design strategies for developing new anti-neoplastic agents.

Differentiation of rat pheochromocytoma cells by fomitellic acids, specific DNA polymerase inhibitors.

Fomitellic acid (FA) A and B are specific inhibitors of DNA polymerase alpha and beta. They showed cytotoxicity against rat pheochromocytoma cells (PC-12 cells) in a concentration-dependent manner. However, after PC-12 cells were cultivated with low concentrations of FAs, the cells extended neurites in greater degree similar to the cells cultivated with nerve growth factor. Another DNA polymerase alpha inhibitor, aphidicolin, also induced neurite outgrowth. Furthermore, PC-12 cells were strongly immunostained with anti-alpha-tubulin or anti-tau antibody after the treatment with FAs. These results suggest that weak inhibition of DNA polymerase activity induces the neurite outgrowth in PC-12 cells.

Novel triterpenoids inhibit both DNA polymerase and DNA topoisomerase.

As described previously, we found that new triterpenoid compounds, designated fomitellic acids A and B, which selectively inhibit the activities of mammalian DNA polymerases alpha and beta [Mizushina, Tanaka, Kitamura, Tamai, Ikeda, Takemura, Sugawara, Arai, Matsukage, Yoshida and Sakaguchi (1998) Biochem. J. 330, 1325-1332; Tanaka, Kitamura, Mizushina, Sugawara and Sakaguchi (1998) J. Nat. Prod. 61, 193-197] and that a known triterpenoid, ursolic acid, is an inhibitor of human DNA topoisomerases I and II (A. Iida, Y. Mizushina and K. Sakaguchi, unpublished work). Here we report that all of these triterpenoids are potent inhibitors of calf DNA polymerase alpha, rat DNA polymerase beta and human DNA topoisomerases I and II, and show moderate inhibitory effects on plant DNA polymerase II and human immunodeficiency virus reverse transcriptase. However, these compounds did not influence the activities of prokaryotic DNA polymerases such as Escherichia coli DNA polymerase I or other DNA metabolic enzymes such as human telomerase, T7 RNA polymerase and bovine deoxyribonuclease I. These triterpenoids were not only mammalian DNA polymerase inhibitors but also inhibitors of DNA topoisomerases I and II even though the enzymic characteristics of DNA polymerases and DNA topoisomerases, including their modes of action, amino acid sequences and three-dimensional structures, differed markedly. These triterpenoids did not bind to DNA, suggesting that they act directly on these enzymes. Because the three-dimensional structures of fomitellic acids were shown by computer simulation to be very similar to that of ursolic acid, the DNA-binding sites of both enzymes, which compete for the inhibitors, might be very similar. Fomitellic acid A and ursolic acid prevented the growth of NUGC cancer cells, with LD(50) values of 38 and 30 microM respectively.

Dehydroaltenusin, a mammalian DNA polymerase alpha inhibitor.

Dehydroaltenusin was found to be an inhibitor of mammalian DNA polymerase alpha (pol alpha) in vitro. Surprisingly, among the polymerases and DNA metabolic enzymes tested, dehydroaltenusin inhibited only mammalian pol alpha. Dehydroaltenusin did not influence the activities of the other replicative DNA polymerases, such as delta and epsilon; it also showed no effect even on the pol alpha activity from another vertebrate (fish) or plant species. The inhibitory effect of dehydroaltenusin on mammalian pol alpha was dose-dependent, and 50% inhibition was observed at a concentration of 0.5 microm. Dehydroaltenusin-induced inhibition of mammalian pol alpha activity was competitive with the template-primer and non-competitive with the dNTP substrate. BIAcore analysis demonstrated that dehydroaltenusin bound to the core domain of the largest subunit, p180, of mouse pol alpha, which has catalytic activity, but did not bind to the smallest subunit or the DNA primase p46 of mouse pol alpha. These results suggest that the dehydroaltenusin molecule competes with the template-primer molecule on its binding site of the catalytic domain of mammalian pol alpha, binds to the site, and simultaneously disturbs dNTP substrate incorporation into the template-primer.

Studies on a novel DNA polymerase inhibitor group, synthetic sulfoquinovosylacylglycerols: inhibitory action on cell proliferation.

Some chemically synthesized sulfoquinovosylmonoacylglycerols (SQMG)-sulfoquinovosyldiacylglycerols (SQDG) have been reported to selectively and strongly inhibit the activities of mammalian DNA polymerases alpha and beta in vitro. In this study, using human cancer cell lines, we investigated the effects of SQMG-SQDG on the DNA polymerase in the cells. In the presence of n-decane, the IC(50) values on cell growth were approximately 1-5 microM for SQMG and about 0.3-1 microM for SQDG. The values were almost the same as the in vitro enzyme inhibitory levels. The cell lines were arrested in early S-phase by SQMG-SQDG at the concentrations of 0.1-4.7 microM in a manner dependent on incubation time, suggesting that SQMG-SQDG blocked the primary step of DNA replication by inhibiting DNA polymerase, possibly alpha-type. We also demonstrated the localization of SQMG in the cell using the fluorescent SQMG analog, SQMGalpha-NBDD, which was synthesized in our laboratory. SQMGalpha-NBDD was localized in the nucleus and on the nuclear surface, but the binding site seemed not to be the DNA/chromatin, suggesting that the SQMG-SQDG might interact with molecules located close to the DNA/chromatin and on the nuclear surface. These results suggested a correlation between the in vitro biochemical action of the SQMG-SQDGs and their intracellular mode of action.

Epolactaene, a novel neuritogenic compound in human neuroblastoma cells, selectively inhibits the activities of mammalian DNA polymerases and human DNA topoisomerase II.

We chemically synthesized epolactaene, a neuritogenic compound in human neuroblastoma cells, and investigated its biochemical action in vitro. Epolactaene and its derivatives selectively inhibited the activities of mammalian DNA polymerase alpha and beta and human DNA topoisomerase II, with IC(50) values of 25, 94, and 10 microM, respectively. By comparison with its structural derivatives, the long alkyl side chain in epolactaene seemed to have an important role in this inhibitory effect. The compound did not influence the activities of plant or prokaryotic DNA polymerases or of other DNA metabolic enzymes such as telomerase, RNA polymerase, and deoxyribonuclease I. Epolactaene did not intercalate into DNA. These results suggested that the neuritogenic compound epolactaene influences both DNA polymerases and topoisomerase II despite the dissimilarity in both structure and properties of these two enzymes and that inhibition of these enzymes could be related to the neuritogenic effect in human neuroblastoma cells. The relationship between the neuritogenic mechanism and cell cycle regulation by epolactaene was also discussed.

(Z)-3-hexenyl and trans-linalool 3,7-oxide beta-primeverosides isolated as aroma precursors from leaves of a green tea cultivar.

6-O-beta-D-Xylopyranosyl-beta-D-glucopyranosides (beta-primeverosides) of (Z)-3-hexenol and trans-linalool 3,7-oxide were newly isolated from fresh leaves of a tea cultivar (Camellia sinensis var. sinensis cv. Yabukita). In addition, the already identified beta-primeverosides of benzyl alcohol, methyl salicylate, and trans-linalool 3,6-oxide from an oolong tea cultivar were isolated from the Yabukita cultivar. It was confirmed that all aglycones of the linalool oxide glycosides isolated here were of the optically active S-form by chiral GC after enzymatic hydrolysis with glycosidase.

Lucidenic acid O and lactone, new terpene inhibitors of eukaryotic DNA polymerases from a basidiomycete, Ganoderma lucidum.

Terpenoids, 1, 2 and 3, which selectively inhibit eukaryotic DNA polymerase activities, were isolated from the fruiting body of a basidiomycete, Ganoderma lucidum, and their structures were determined by spectroscopic analyses. New terpenes, lucidenic acid O (1) and lucidenic lactone (2), prevented not only the activities of calf DNA polymerase alpha and rat DNA polymerase beta, but also these of human immunodeficiency virus type 1 reverse transcriptase. Cerevisterol (3), which was reported to be a cytotoxic steroid, inhibited only the activity of DNA polymerase alpha. Although these compounds did not influence the activities of prokaryotic DNA polymerases and other DNA metabolic enzymes such as T7 RNA polymerase and deoxyribonuclease I.