Anti-tumor-promoting activities of agaro-oligosaccharides on two-stage mouse skin carcinogenesis.

We have previously reported that agaro-oligosaccharides (AGOs) suppressed the elevated levels of nitric oxide (NO), prostaglandin E2(PGE2), and pro-inflammatory cytokines in activated monocytes/macrophages, via heme oxygenase-1 induction. In this report, we initially demonstrated that AGOs intake inhibited NO production in activated peritoneal macrophages. Then, we tested for the ability of AGOs to prevent tumor promotion on the two-stage mouse skin carcinogenesis model. As a result, AGOs feeding led to delayed tumor appearance and decreased tumor number. It is known that PGE2 is one of key players in carcinogenesis. Thus, we confirmed that PGE2 production was suppressed by AGOs intake in TPA-induced ear edema model. We also demonstrated that cyclooxygenase-2 and microsomal PGE synthase-1, rate-limiting enzymes in PGE2 production, were down-regulated by AGOs in human monocytes. Consequently, AGOs are expected to prevent tumor promotion by inhibiting PGE2 elevation in chronic inflammation site.

Angelica keiskei extract improves insulin resistance and hypertriglyceridemia in rats fed a high-fructose drink.

Angelica keiskei is a traditional herb peculiar to Japan and abundantly contains vitamins, dietary fiber and such polyphenols as chalcone. We investigated in the present study the effect of A. keiskei on insulin resistance and hypertriglyceridemia in fructose-drinking rats as a model for the metabolic syndrome. Male Wistar rats were given a 15% fructose solution as drinking water for 11 weeks. Fructose significantly increased the levels of serum insulin and triglyceride (TG) compared with the control level. Treatment with an ethanol extract of A. keiskei (AE) significantly reduced the levels of blood glucose (-16.5%), serum insulin (-47.3%), HOMA-R (-56.4%) and TG (-24.2%). A hepatic gene analysis showed that fructose reduced the expression of the genes related to fatty acid ß-oxidation and high-density lipoprotein (HDL) production. Treatment with AE enhanced the expression of the acyl-CoA oxidase 1 (ACO1), medium-chain acyl-CoA dehydrogenase (MCAD), ATP-binding membrane cassette transporter A1 (ABCA1) and apolipoprotein A1 (Apo-A1) genes. These results suggest that AE improved the insulin resistance and hypertriglyceridemia of the fructose-drinking rats.

Six new chalcones from Angelica keiskei inducing adiponectin production in 3T3-L1 adipocytes.

Angelica keiskei (Ashitaba in Japanese), a traditional herb in Japan, contains abundant prenylated chalcones. It has been reported that the chalcones from A. keiskei showed such bioactivities as anti-bacterial, anti-cancer and anti-diabetic effects. Xanthoangelol, 4-hydroxyderricin and six new chalcones were isolated in this study from an ethanol extract of A. keiskei by octadecyl silyl (ODS) and silica gel chromatography, and identified by 1D- and 2D-nuclear magnetic resonance (NMR) and high-resolution mass spectrometric analyses. The chalcones from A. keiskei markedly increased the expression of the adiponectin gene and the production of adiponectin in 3T3-L1 adipocytes. These results suggest that the chalcones from A. keiskei might be useful for preventing the metabolic syndrome.

Reduced genome of the thioautotrophic intracellular symbiont in a deep-sea clam, Calyptogena okutanii.

Although dense animal communities at hydrothermal vents and cold seeps rely on symbioses with chemoautotrophic bacteria [1, 2], knowledge of the mechanisms underlying these chemosynthetic symbioses is still fragmentary because of the difficulty in culturing the symbionts and the hosts in the laboratory. Deep-sea Calyptogena clams harbor thioautotrophic bacterial symbionts in their gill epithelial cells [1, 2]. They have vestigial digestive tracts and nutritionally depend on their symbionts [3], which are vertically transmitted via eggs [4]. To clarify the symbionts' metabolic roles in the symbiosis and adaptations to intracellular conditions, we present the complete genome sequence of the symbiont of Calyptogena okutanii. The genome is a circular chromosome of 1,022,154 bp with 31.6% guanine + cytosine (G + C) content, and is the smallest reported genome in autotrophic bacteria. It encodes 939 protein-coding genes, including those for thioautotrophy and for the syntheses of almost all amino acids and various cofactors. However, transporters for these substances to the host cell are apparently absent. Genes that are unnecessary for an intracellular lifestyle, as well as some essential genes (e.g., ftsZ for cytokinesis), appear to have been lost from the symbiont genome. Reductive evolution of the genome might be ongoing in the vertically transmitted Calyptogena symbionts.

Oligosaccharides from agar inhibit pro-inflammatory mediator release by inducing heme oxygenase 1.

We investigated whether agaro-oligosaccharides have any immunological effects on RAW264.7 mouse macrophages and human monocytes in vitro. We demonstrate that agaro-oligosaccharides suppressed the elevated levels of nitric oxide, prostaglandin E(2), and such pro-inflammatory cytokines as tumor necrosis factor-alpha, interleukin-1beta and interleukin-6 in lipopolysaccharide-stimulated monocytes and macrophages. We also demonstrate that those effects of agaro-oligosaccharides on activated monocytes and macrophages may have been caused by heme oxygenase-1 induction. It is therefore proposed that agaro-oligosaccharides might be a good candidate for a functional food to prevent inflammatory diseases.

Transient gene expression mediated by integrase-defective retroviral vectors.

Nonintegrating retroviral vectors were produced from a Moloney murine leukemia virus (MoMLV)-based retroviral vector system by introducing a point mutation into the integrase (IN) gene of the packaging plasmid. The efficacy of IN-defective retroviral vectors was measured through the transient expression of ZsGreen or luciferase in human cell lines. The IN-defective retroviral vectors could transduce target cells efficiently, but their gene expression was transient and lower than that seen with the integrating vectors. IN-defective retroviral vector gene expression decreased to background levels in fewer than 10 days. Southern blot analysis of transduced K562 cells confirmed the loss of a detectable vector sequence by 15 days. The residual integration activity of the IN-defective vector was 1000- to 10,000-fold lower than that of the integrating vector. These results demonstrate that the IN-defective retroviral vectors can provide a useful tool for efficient transient gene expression targeting of primary hematopoietic stem cells and lymphoid cells.

The expression profile of microRNAs in mouse embryos.

MicroRNAs (miRNAs), which are non-coding RNAs 18-25 nt in length, regulate a variety of biological processes, including vertebrate development. To identify new species of miRNA and to simultaneously obtain a comprehensive quantitative profile of small RNA expression in mouse embryos, we used the massively parallel signature sequencing technology that potentially identifies virtually all of the small RNAs in a sample. This approach allowed us to detect a total of 390 miRNAs, including 195 known miRNAs covering approximately 80% of previously registered mouse miRNAs as well as 195 new miRNAs, which are so far unknown in mouse. Some of these miRNAs showed temporal expression profiles during prenatal development (E9.5, E10.5 and E11.5). Several miRNAs were positioned in polycistron clusters, including one particular large transcription unit consisting of 16 known and 23 new miRNAs. Our results indicate existence of a significant number of new miRNAs expressed at specific stages of mammalian embryonic development and which were not detected by earlier methods.

Investigation of the molecular mechanism of ICAN, a novel gene amplification method.

Isothermal and Chimeric primer-initiated Amplification of Nucleic acids (ICAN) allows the amplification of target DNA under isothermal conditions at around 55 degrees C using only a pair of 5'-DNA-RNA-3' chimeric primers, thermostable RNaseH and a DNA polymerase with strand-displacing activity (H. Mukai et al. J. Biochemistry, in the preceding paper in this issue). Here we elucidated the mechanism of ICAN by analysing the nicking site of RNaseH, behaviour of chimeric primers and extension products. We found that the ICAN reaction was composed of two unique mechanisms, multi-priming and template-switching, that were responsible for the highly efficient amplifying capability of ICAN. The simultaneous occurrence of two types of reactions, one based on multi-priming and the other based on template-switching, is likely to drive the DNA amplification in ICAN.

Highly efficient isothermal DNA amplification system using three elements of 5'-DNA-RNA-3' chimeric primers, RNaseH and strand-displacing DNA polymerase.

We developed an efficient method of isothermally amplifying DNA termed ICAN, Isothermal and Chimeric primer-initiated Amplification of Nucleic acids. This method allows the amplification of target DNA under isothermal conditions at around 55 degrees C using only a pair of 5'-DNA-RNA-3' chimeric primers, a thermostable RNaseH and a DNA polymerase with strong strand-displacing activity. ICAN is capable of amplifying DNA at least several times greater than the amount produced with PCR by increasing primer concentration. This method would be applicable for on-site DNA detection including gene diagnosis, and would also be suitable for 'real time' detection when combined with a cycling probe.

Antidiabetic activities of chalcones isolated from a Japanese Herb, Angelica keiskei.

Diabetes mellitus is a chronic disease that is characterized by hyperglycemia caused by insufficient insulin action. We have explored the edible ingredients from folk medicines in Japan that contain substances complementing insulin action, such as the induction of adipocyte differentiation and the enhancement of glucose uptake. We eventually found that the ethanol extract from a Japanese herb "Ashitaba", Angelica keiskei, contained two major chalcones of 4-hydroxyderricin (4-HD) and xanthoangelol that showed strong insulin-like activities via a pathway independent of the peroxisome proliferator-activated receptor-gamma activation. The 4-HD especially showed the preventive effects on the progression of diabetes in genetically diabetic KK-Ay mice.

Transient expression of Ym1, a heparin-binding lectin, during developmental hematopoiesis and inflammation.

Ym1, a secretory protein transiently produced by activated peritoneal macrophages elicited by parasitic infections, has been identified as a novel heparin-binding lectin. X-ray crystallography study revealed that Ym1 has a beta/alpha barrel structure with a carbohydrate-binding cleft similar to that of triose-phosphate isomerases. To further delineate the physiological significance of Ym1, we examined its expression patterns during mouse embryonic development and inflammation states elicited by agents other than parasitic infections in the peritoneal cavity and brain. This is the first report revealing prominent expression of Ym1 in early myeloid precursor cells of hematopoietic tissues-initially in the yolk sac and subsequently in fetal liver, spleen, and bone marrow. In nonhematopoietic systems, Ym1 was not detected in most of the tissues examined, with the exception of lung. Although no expression was detected up to gestation day 16.5 (E16.5), an increasing level of Ym1 expression in lung was detected from E18.5 on and persisted through adulthood. While most resident macrophages in various tissues examined are Ym1-negative, transient expression of Ym1 may be induced in their activated counterparts during inflammation in response to different stimuli in vivo, ranging from various chemical agents to brain injuries. The temporal and spatial expression in myeloid precursors and its transient induction in activated macrophages support the notion that Ym1 may be involved in hematopoiesis and inflammation. In addition, its putative functional association with heparin/heparan sulfate is discussed.

A marine strain of flavobacteriaceae utilizes brown seaweed fucoidan.

Fucoidan, a mixture of sulfated fucose-containing polysaccharides, was prepared from Kjellmaniella crassifolia (class Phaeophyceae, order Laminariales, family Laminariaceae) with a yield of about 3.8% dry weight. To isolate enzymes that degrade fucoidan, we first screened marine bacteria for their ability to utilize fucoidan, and isolated one strain of Flavobacteriaceae from seawater that could do this. Phylogenetic analysis of the 16S ribosomal DNA sequence suggested that this strain appeared to belong to a new genus, and was tentatively named Fucobacter marina. The strain utilized L-fucose (17%), D-mannose (91%), D-galactose (46%), and D-glucuronic acid (66%) in the fucoidan from K. crassifolia. The strain partially utilized fucoidan from 2 other seaweeds that belong to the order Laminariales, Undaria pinnatifida (10%) and Lessonia nigrescens (48%).

Isolation and characterization of a fucoidan-degrading marine bacterial strain and its fucoidanase.

A marine bacterial strain that degraded fucoidan from Kjellmaniella crassifolia (class Phaeophyceae, order Laminariales, family Laminariaceae) was isolated in our laboratory. The strain was gram-negative, ubiquinone 8 was the predominant respiratory quinone, and the GC-content of its genomic DNA was 36%. The cells of the strain were rod-shaped (2.0 microm long x 1.0 microm wide), and each cell was motile by means of one polar flagellum. Phylogenetic analysis of its 16S ribosomal DNA sequence indicated that it was a member of the family Alteromonadaceae. It produced a type of extracellular fucoidanase, an endosulfated fucan-digesting enzyme. The enzyme was purified with 3500-fold purity at 12.0% yield. Optimum conditions for the enzyme reaction were approximately pH 6.5 to 8.0 and temperature 30 degrees to 35 degrees C. The enzyme was activated by calcium ions, and maximum activity was observed in the presence of greater than 30 mM calcium ion.

Isolation and characterization of a fucoidan-degrading marine bacterium.

Fucoidan, a mixture of sulfated fucose-containing polysaccharides, was prepared from the algal bodies of Cladosiphon okamuranus (class Phaeophyceae, order Chordariales, family Chordariaceae) with a yield of 2.0% of the wet weight of the alga. To obtain enzymes that digest the fucoidan, we screened bacteria in the guy contents of the sea cucumber Stichopus japonicus for their ability to decrease the fucoidan in their culture media, and successfully isolated one bacterial strain that could decrease it. The bacterial strain was gram-negative and possessed menaquinone 7 as the predominant respiratory quinone, and the GC content of its genomic DNA was 52%. The results of the phylogenetic analysis of its 16S ribosomal DNA sequence indicated that the bacterial strain was a member of the division "Verrucomicrobia." However, as the bacterial strain is phylogenetically and phenotypically distinct from verrucomicrobial species described previously, the strain was assumed to be a new member of the division "Verrucomicrobia." When the bacterial strain was cultivated in an algal fucoidan-containing medium, the strain decreased fucoidan from C. okamuranus (44%), Nemacystus decipiens (19%), Laminaria japonica (31%), Kjellmaniella crassifolia (23%), sporophyl of Undaria pinnatifida (22%), Fucus vesiculosus (42%), and Ascophyllum nodosum (61%).

Purification of sulfated fucoglucuronomannan lyase from bacterial strain of Fucobacter marina and study of appropriate conditions for its enzyme digestion.

A marine bacterial strain, Fucobacter marina, produced extracellular sulfated fucoglucuronomannan (SFGM) lyase when cultivated in the presence of crude SFGM obtained from fucoidan of Kjellmaniella crassifolia (brown algae) by cetyl pyridinium chloride fractionation. For the SFGM lyase assay, SFGM fraction separated from K. crassifolia fucoidan by anion exchange column chromatography was used as the substrate. The extracellular SFGM lyase was purified to homogeneity on an electrophoresis gel with 4240-fold purity at 13.8% yield. The enzyme proved to be a monomer, since gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis gave the same relative molecular mass of 67,000. The enzyme specifically digested SFGM but did not digest any other uronic-acid-containing polysaccharides tested. The optimum conditions for the enzyme reaction were around pH 7.5, 43 degrees C, and 0.4 M NaCl concentration. The enzyme was strongly inhibited by CuCl(2) and ZnCl(2), and also by some sulfhydryl reagents.

Structures of oligosaccharides derived from Cladosiphon okamuranus fucoidan by digestion with marine bacterial enzymes.

A fucoidan-utilizing marine bacterium, Fucophilus fucoidanolyticus, was cultivated in medium containing fucoidan from Cladosiphon okamuranus. The C. okamuranus fucoidan was digested into oligosaccharides with the intracellular enzymes of F. fucoidanolyticus, and their structures were determined by nuclear magnetic resonance analyses. Some of their structures are represented by one general structural formula, (-3 L-Fuc palpha1-3 L-Fuc p(4- O-sulfate)alpha1-3 L-Fuc p(4- O-sulfate)alpha1-3( D-Glc pUAalpha1-2) L-Fuc palpha1)(m)-3 L-Fuc palpha1-3 L-Fuc p(4- O-sulfate)alpha1-3 L-Fuc p(4- O-sulfate) alpha1-3 L-Fuc p ( m = 0, 1, 2, or 3). We concluded that all oligosaccharides obtained were derived from a sulfated-fucose-containing polysaccharide of C. okamuranus, which has a repeating unit of (-3 L-Fuc palpha1-3 L-Fuc p(4- O-sulfate)alpha1-3 L-Fuc p(4- O-sulfate)alpha1-3( D-Glc pUAalpha1-2) L-Fuc palpha1-).

Marine bacterial sulfated fucoglucuronomannan (SFGM) lyase digests brown algal SFGM into trisaccharides.

Three kinds of trisaccharides were prepared by digesting fucoidan from the brown alga Kjellmaniella crassifolia, with the extracellular enzymes of the marine bacterium Fucobacter marina. Their structures were determined as delta 4,5GlcpUA1-2(L-Fucp(3-O-sulfate)alpha 1-3)D-Manp, delta 4,5GlcpUA1-2(L-Fucp(3- O-sulfate)alpha 1-3)D-Manp(6-O-sulfate), and delta 4,5GlcpUA1-2(L-Fucp(2,4-O-disulfate)alpha 1-3)D-Manp(6-O-sulfate), which indicated the existence of a novel polysaccharide in the fucoidan and a novel glycosidase in the extracellular enzymes. In order to determine the complete structure of the polysaccharide and the reaction mechanism of the glycosidase, the fucoidan was partially hydrolyzed to obtain glucuronomannan, which is the putative backbone of the polysaccharide, and its sugar sequence was determined as (-4-D-GlcpUAbeta 1-2D-Manpalpha 1-)n, which disclosed that the main structure of the polysaccharide is (-4-D-GlcpUAbeta 1-2(L-Fucp(3-O-sulfate)alpha 1-3)D-Manpalpha 1-)n. Consequently, the glycosidase was deduced to be an endo-alpha-D-mannosidase that eliminatively cleaves the alpha-D-mannosyl linkage between D-Manp and D-GlcpUA residues in the polysaccharide and produces the above trisaccharides. The novel polysaccharide and glycosidase were tentatively named as sulfated fucoglucuronomannan (SFGM) and SFGM lyase, respectively.

Transfer of high-mannose-type oligosaccharides to disaccharides by endo-beta-N-acetylglucosaminidase from Arthrobacter protophormiae.

Endo-beta-N-acetylglucosaminidase from Arthrobacter protophormiae (Endo-A) has transglycosylation activity, and high-mannose-type oligosaccharides are transferred to suitable glycosides as acceptor substrates. The acceptor specificity of Endo-A-catalyzed transglycosylation toward various disaccharides was investigated. To identify an effective acceptor for the transglycosylation by Endo-A, the reaction was carried out using various disaccharides. Endo-A transferred high-mannose-type oligosaccharides more efficiently to beta-linked disaccharides (cellobiose, gentiobiose, sophorose, and laminaribiose) than to alpha-linked disaccharides (isomaltose, maltose, nigerose, kojibiose, and trehalose) as acceptor substrates. The transglycosylation products, (Man)6GlcNAc-Glc-beta-Glc, were more rapidly hydrolyzed than (Man)6GlcNAc-Glc-alpha-Glc. These results indicate that Endo-A recognizes the anomeric configuration of the acceptor substrates, and beta-linked glycosides are suitable for the synthesis of transglycosylation products.

[Development of the detection system for Mycobacterium tuberculosis DNA by using the isothermal DNA amplification method ICAN].

The isothermal and chimeric primer-initiated amplification of nucleic acids(ICAN) is a new isothermal DNA amplification method composed of exo- Bca DNA polymerase, RNaseH and DNA-RNA chimeric primers. We developed the detection system, combined the ICAN with luminescence detection by a probe hybridization, for Mycobacterium tuberculosis DNA targeting the IS6110 insertion element. We examined performance tests of the system. This system was able to detect one copy of Mycobacterium tuberculosis DNA for only 3.5 hours, and performance of the system was equivalent or better to the Roche PCR system. We also examined a detection system by using magnetic beads, which system could shorten detection time for 2.5 hours. It was shown that the ICAN system was an efficient and sensitive detection system for Mycobacterium tuberculosis DNA from mass samples.

Sustained inhibition of HIV-1 replication by conditional expression of the E. coli-derived endoribonuclease MazF in CD4+ T cells.

Gene therapy using a Tat-dependent expression system of MazF, an ACA nucleotide sequence-specific endoribonuclease derived from Escherichia coli, in a retroviral vector appears to be an alternative approach to the treatment of human immunodeficiency virus type 1 (HIV-1) infection. MazF can cleave HIV-1 RNA, since it has more than 240 ACA sequences. Significant inhibition of viral replication, irrespective of HIV-1 strains, was observed in CD4(+) T cells that had been transduced with the MazF-expressing retroviral vector (MazF-T cells). The growth and viability of MazF-T cells were not affected by HIV-1 infection. Interestingly, the infectivity of HIV-1 produced from MazF-T cells was found to be lower than that from control CD4(+) T cells. A long-term culture experiment with HIV-1-infected cells revealed that viral replication was always lower in MazF-T cells than in CD4(+) T cells transduced with or without a control vector for more than 200 days. MazF was expressed and mainly localized in the cytoplasm of the infected cells. Unlike in CD4(+) T cells, the expression level of Tat gradually decreased rather than increased in MazF-T cells after HIV-1 infection. As a consequence, the expression level of MazF appeared to be well regulated and sustained during HIV-1 infection in MazF-T cells. Furthermore, the levels of cellular mRNA were not affected by HIV-1 infection. Thus, the Tat-dependent MazF expression system has great potential for inhibition of HIV-1 replication in vivo without apparent toxicity and may be able to avoid the emergence of resistant strains.