Characterization of Collagen from Sakhalin Taimen Skin as Useful Biomass.

RESEARCH BACKGROUND: Animal collagen has been widely utilized in foods, cosmetics and biomedical fields. The non-edible portion, such as fish skin and bones, are obtained during cooking. Most of them are currently discarded as wastes, although the nutritional value of the skin and bones is high. The non-edible portion needs to be reused in order to reduce environmental impact, as it is one of the sources of environmental pollution. EXPERIMENTAL APPROACH: Collagen was prepared by cold acetone treatment from Sakhalin taimen skin as a waste produced during cooking. Next, the colour, SDS-polyacrylamide gel electrophoresis, ultraviolet absorption, subunit composition, amino acid composition, denaturation temperature and attenuated total reflectance-Fourier transform infrared spectroscopy analyses were conducted to explore the properties of the collagen. Lastly, we attempted to improve the functional properties of the collagen for future applications using chemical modification technique (succinylation). RESULTS AND CONCLUSIONS: Cold acetone treatment easily removed the fats and pigments from the skin. The odourless and pure white collagen was obtained with high yield. The α3 chain did not exist in the collagen. Sakhalin taimen skin collagen had rich α-helix and low ß-sheet structures. Succinylation caused the secondary structural changes of the collagen molecule. Moreover, it made it possible not only to increase the viscosity of the collagen solution but also to improve the solubility of the collagen under the physiological conditions around pH=6. NOVELTY AND SCIENTIFIC CONTRIBUTION: This finding is the first report on the absence of the α3 chain from salmonid fish skin collagens. The succinylated collagen from Sakhalin taimen skins as useful biomass has potential to utilize in foods, cosmetics and related industries.

Phenylethylchromones with In Vitro Antitumor Promoting Activity from Aquilaria filaria.

A new chromone, 2-(2-hydroxy-2-phenylethyl)chromone (1), was isolated together with ten known phenylethyl chromones from MeOH extracts of agarwood (Aquilaria filaria). The selected compounds were evaluated in an antiproliferative assay against five human tumor cell lines, including a multidrug-resistant cell line. They were also tested for antitumor promoting activity, as mediated by 12-O-tetradecanoylphorbol-13-acetate-induced activation of the Epstein-Barr virus early antigen in Raji cells. Among all compounds, 4',7-dimethyoxy-6-hydroxychromone (2) displayed broad spectrum antiproliferative activity against all tumor cell lines tested with IC50 values of 25-38 µM, while 8 was selectively inhibitory against multidrug-resistant cells. All tested compounds suppressed tumor promotion at noncytotoxic concentrations. 4',6-Dihydroxyphenylethylchromone (7) exhibited the most potent effect with an IC50 value of 319 mol ratio relative to 12-O-tetradecanoylphorbol-13-acetate. This study is the first to report the antitumor promoting activity of 2-(2-phenylethyl)chromone derivatives, as well as the selective antiproliferative activity of 8 against a multidrug-resistant tumor cell line.

Total Synthesis and in Vitro Anti-Tumor-Promoting Activities of Racemic Acetophenone Monomers from Acronychia trifoliolata.

Six acetophenone derivatives, acronyculatins I (1), J (2), K (3), L (4), N (5), and O (6), were recently isolated from Acronychia trifoliolata, and the structure of the known acronyculatin B (7) was revised. Because of the limited quantities of isolated products as well as their structure similarity, racemic acronyculatins I-L, N, O, and B (1-7) were synthesized to confirm their structures and to obtain sufficient material for biological evaluation. Trihydroxyacetophenone was converted to the target compounds by various sequences of hydroxy group protection, allylation or prenylation, and epoxidation followed by cyclization. C-Prenylations were carried out by direct addition of a prenyl group or through 1,3- or 3,3-sigmatropic rearrangement. The synthesized racemic compounds were evaluated in an anti-tumor-promoting assay using the Epstein-Barr virus early antigen (EBV-EA) activation induced by 12-O-tetradecanoylphorbol-13-acetate in Raji cells. All tested compounds significantly inhibited EBV-EA activation. Especially, racemic acronyculatin I (1) displayed the most potent inhibitory effects, with an IC50 value of 7.3 µM.

Synthesis and biological activities of the amide derivative of aplog-1, a simplified analog of aplysiatoxin with anti-proliferative and cytotoxic activities.

Aplog-1 is a simplified analog of the tumor-promoting aplysiatoxin with anti-proliferative and cytotoxic activities against several cancer cell lines. Our recent findings have suggested that protein kinase Cδ (PKCδ) could be one of the target proteins of aplog-1. In this study, we synthesized amide-aplog-1 (3), in which the C-1 ester group was replaced with an amide group, to improve chemical stability in vivo. Unfortunately, 3 exhibited seventy-fold weaker binding affinity to the C1B domain of PKCδ than that of aplog-1, and negligible anti-proliferative and cytotoxic activities even at 10(-4) M. A conformational analysis and density functional theory calculations indicated that the stable conformation of 3 differed from that of aplog-1. Since 27-methyl and 27-methoxy derivatives (1, 2) without the ability to bind to PKC isozymes exhibited marked anti-proliferative and cytotoxic activities at 10(-4) M, 3 may be an inactive control to identify the target proteins of aplogs.

Characterization of collagen from emu (Dromaius novaehollandiae) skins.

Collagen from emu skins as a by-product was prepared. The skins were hardly solubilized in acetic acid, however were successfully solubilized on digestion with 10 % pepsin (w/w) for 4 days. The yield of pepsin-solubilized collagen (PSC) was about 27.3 %, on a raw weight basis. By SDS-PAGE and CM-Toyopearl 650 M column chromatography, the presence of a fourth subunit that was previously designated α4 was confirmed. The denaturation temperature of the PSC was 31.5 °C, about 6-7 °C lower than that from the porcine skins. ATR-FTIR analysis indicated that the helical arrangements of the PSC from emu skins existed and its structures of PSC were changed slightly due to the loss of N- and C-terminus domains in similar to that from the porcine skins. That is, the PSC from emu skins did not possess telopeptide chains as major portion of antigenic sites in collagen. The present study indicates that a large quantity of emu skins as by-products have potential as a good alternative source of high-quality collagen for industrial purposes in the foods, cosmetics, and pharmaceutical and biomedical fields.

A novel mouse model for Down syndrome that harbor a single copy of human artificial chromosome (HAC) carrying a limited number of genes from human chromosome 21.

Down syndrome (DS), also known as Trisomy 21, is the most common chromosome aneuploidy in live-born children and displays a complicated symptom. To date, several kinds of mouse models have been generated to understand the molecular pathology of DS, yet the gene dosage effects and gene(s)-phenotype(s) correlation are not well understood. In this study, we established a novel method to generate a partial trisomy mice using the mouse ES cells that harbor a single copy of human artificial chromosome (HAC), into which a small human DNA segment containing human chromosome 21 genes cloned in a bacterial artificial chromosome (BAC) was recombined. The produced mice were found to maintain the HAC carrying human genes as a mini-chromosome, hence termed as a Trans-Mini-Chromosomal (TMC) mouse, and HAC was transmitted for more than twenty generations independent from endogenous mouse chromosomes. The three human transgenes including cystathionine ß-synthase, U2 auxiliary factor and crystalline alpha A were expressed in several mouse tissues with various expression levels relative to mouse endogenous genes. The novel system is applicable to any of human and/or mouse BAC clones. Thus, the TMC mouse carrying a HAC with a limited number of genes would provide a novel tool for studying gene dosage effects involved in the DS molecular pathogenesis and the gene(s)-phenotype(s) correlation.

A-ring modified betulinic acid derivatives as potent cancer preventive agents.

Ten new 3,4-seco betulinic acid (BA) derivatives were designed and synthesized. Among them, compounds 7-15 exhibited enhanced chemopreventive ability in an in vitro short-term 12-O-tetradecanoylphorbol-13-acetate (TPA) induced Epstein-Barr virus early antigen (EBV-EA) activation assay in Raji cells. Specifically, analogs with a free C-28 carboxylic acid, including 7, 8, 11, and 13, inhibited EBV-EA activation significantly. The most potent compound 8 displayed 100% inhibition at 1×10(3) mol ratio/TPA and 73.4%, 35.9%, and 8.4% inhibition at 5×10(2), 1×10(2), and 1×10 mol ratio/TPA, respectively, comparable with curcumin at high concentration and better than curcumin at low concentration. The potent chemopreventive activity of novel seco A-ring BAs (8 and 11) was further confirmed in an in vivo mouse skin carcinogenesis assay.

LINE1 family member is negative regulator of HLA-G expression.

Class Ia molecules of human leucocyte antigen (HLA-A, -B and -C) are widely expressed and play a central role in the immune system by presenting peptides derived from the lumen of the endoplasmic reticulum. In contrast, class Ib molecules such as HLA-G serve novel functions. The distribution of HLA-G is mostly limited to foetal trophoblastic tissues and some tumour tissues. The mechanism required for the tissue-specific regulation of the HLA-G gene has not been well understood. Here, we investigated the genomic regulation of HLA-G by manipulating one copy of a genomic DNA fragment on a human artificial chromosome. We identified a potential negative regulator of gene expression in a sequence upstream of HLA-G that overlapped with the long interspersed element (LINE1); silencing of HLA-G involved a DNA secondary structure generated in LINE1. The presence of a LINE1 gene silencer may explain the limited expression of HLA-G compared with other class I genes.

Functional properties of autolysate and enzymatic hydrolysates from yam tsukuneimo (Dioscorea opposita Thunb.) tuber mucilage tororo: antioxidative activity and antihypertensive activity.

Yam tsukuneimo tuber mucilage tororo hydrolysates were prepared by autolysis and three different peptic enzymes. Except for pepsin hydrolysate, tororo was perfectly digested. Each hydrolysate for 100 mg/ml significantly prolonged the induction period of auto-oxidation of linoleic acid, which was similar to 5 mM ascorbic acid. These hydrolysates also possessed high scavenging activities such as superoxide anion radicals, hydroxyl radicals, and DPPH radicals. Moreover, high antihypertensive activities were detected in these hydrolysates except for autolysate, which were similar to various fermented foods such as miso, natto, sake, cheese, and so on. Present findings suggest that yam tsukuneimo tuber mucilage tororo may be useful for preventing diseases associated with reactive oxygen species and blood pressure in the body system and it can fully absorb the useful components from it to digest using the gastrointestinal enzymes.

Effects of the methoxy group in the side chain of debromoaplysiatoxin on its tumor-promoting and anti-proliferative activities.

Debromoaplysiatoxin (DAT) is a tumor promoter isolated from sea hare and exhibits anti-proliferative activity against several cancer cell lines. To clarify key residues that are responsible for its tumor-promoting activity, we focused on the chiral methoxy group in the side chain, whose role had not yet been discussed or examined before. Demethoxy-DAT (8) was derived from DAT and we evaluated its tumor-promoting activity, anti-proliferative activity, and ability to bind to protein kinase C (PKC) isozymes. Compound 8 showed somewhat weaker tumor-promoting activity than that of DAT both in vitro and in vivo, but showed higher anti-proliferative activity against several cancer cell lines. Although the affinity to novel PKC isozymes of 8 was comparable to that of DAT, the affinity to conventional PKC isozymes decreased slightly. These results suggest that the methoxy group of DAT is one of the key residues critical for tumor-promoting activity but not for anti-proliferative activity. Since the methoxy group has little influence on the molecular hydrophobicity, this is the first report showing that structural factors other than hydrophobicity in the side chain of DAT affected its biological activities.

Structure-activity studies on the side chain of a simplified analog of aplysiatoxin (aplog-1) with anti-proliferative activity.

We have recently developed a simplified analog of aplysiatoxin (aplog-1) as an activator of protein kinase C (PKC) with anti-proliferative activity like bryostain 1. To identify sites in aplog-1 that could be readily modified to optimize therapeutic performance and to develop a molecular probe for examining the analog's mode of action, substituent effects on the phenol ring were systematically examined. Whereas hydrophilic acetamido derivatives were less active than aplog-1 in inhibiting cancer cell growth and binding to PKCδ, introduction of hydrophobic bromine and iodine atoms enhanced both biological activities. The anti-proliferative activity was found to correlate closely with molecular hydrophobicity, and maximal activity was observed at a logP value of 4.0-4.5. On the other hand, an induction test with Epstein-Barr virus early antigen demonstrated that these derivatives have less tumor-promoting activity in vitro than aplog-1 regardless of the hydrophobicity of their substituents. These results would facilitate rapid preparation of molecular probes to examine the mechanism of the unique biological activities of aplog-1.

Cancer chemopreventive effect of bergenin from Peltophorum pterocarpum wood.

The aqueous extract of Peltophorum pterocarpum (Fabaceae) wood exhibited potent inhibitory effects against EpsteinBarr virus early antigen (EBV-EA) activation induced with 12-O-tetradecanoylphorbol-13-acetate (TPA) in Raji cells and against melanogenesis in α-melanocyte-stimulating hormone (α-MSH)-stimulated B16 melanoma cells, as well as potent 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical-scavenging activity. Two phenolic acid derivatives, bergenin (1) and gallic acid (2), were isolated from the ethyl acetate (AcOEt)-soluble fraction obtained from the extract. Compound 1 exhibited potent inhibitory effect against EBV-EA activation and against skin tumor promotion in an in vivo two-stage mouse skin carcinogenesis test based on 7,12-dimethylbenz[a]anthracene (DMBA) as initiator, and with TPA as promoter. Both compounds 1 and 2 exhibited melanogenesis-inhibitory activities in α-MSH-stimulated B16 melanoma cells, and, in addition, compound 2 showed strong DPPH radical-scavenging activity.

Biological activities of phenolic compounds and triterpenoids from the galls of Terminalia chebula.

Nine phenolic compounds, including two phenolic carboxylic acids, 1 and 2, seven hydrolyzable tannins, 3-9, eight triterpenoids, including four oleanane-type triterpene acids, 10-13, and four of their glucosides, 14-17, isolated from a MeOH extract of the gall of Terminalia chebula Retz. (myrobalan tree; Combretaceae), were evaluated for their inhibitory activities against melanogenesis in B16 melanoma cells induced by α-melanocyte-stimulating hormone (α-MSH), against the Epstein-Barr virus early antigen (EBV-EA) activation induced by 12-O-tetradecanoylphorbol 13-acetate (TPA) in Raji cells, and against TPA-induced inflammation in mice. Their 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging activities and cytotoxic activities against four human cancer cell lines were also evaluated. Compounds 6-9 and 12 exhibited potent inhibitory activities against melanogenesis (39.3-66.3% melanin content) with low toxicity to the cells (74.5-105.9% cell viability) at a concentration of 10 µM. Western-blot analysis revealed that isoterchebulin (8) reduced the protein levels of MITF (=microphtalmia-associated transcription factor), tyrosinase, and TRP-1 (=tyrosine-related protein 1), mostly in a concentration-dependent manner. Eight triterpenoids, 10-17, showed potent inhibitory effects on EBV-EA induction with the IC50 values in the range of 269-363 mol ratio/32 pmol TPA, while these compounds exhibited no DPPH scavenging activities (IC50 >100 µM). On the other hand, the nine phenolic compounds, 1-9, exhibited potent radical-scavenging activities (IC50 1.4-10.9 µM) with weak inhibitory effects on EBV-EA induction (IC50 460-518 mol ratio/32 pmol TPA). The tannin 6 and seven triterpenoids, 10-16, have been shown to inhibit TPA-induced inflammation (1 µg/ear) in mice with the ID50 values in the range of 0.06-0.33 µmol/ear. Arjungenin (10) exhibited inhibitory effect on skin-tumor promotion in an in vivo two-stage mouse-skin carcinogenesis test based on 7,12-dimethylbenz[a]anthracene (DMBA) as initiator and with TPA as promoter. Compounds 1, 2, 4, 5, 7-9, 12, and 13, against HL60 cell line, compounds 1 and 4, against AZ521 cell line, and compounds 1, 11, and 12, against SK-BR-3 cell line, showed moderate cytotoxic activities (IC50 13.9-73.2 µM).

Structure elucidation and characterization of microbial 2-tridecyl sophorosides (biosurfactants).

To produce novel types of sophorose lipids containing an odd number of carbon atoms in the lipophilic moiety, Candida bombicola ATCC 22214 was grown in 500-ml flask cultures with glucose as main carbon source, and additionally, 2-tridecanone as co-substrate. After solvent extraction, the crude product mixture was separated into pure fractions, and each fraction was analysed via NMR and mass spectroscopy. This effective strategy generated five new glycolipids, 2-tridecyl sophorosides, which differed in the number of glucose units, and acetyl and hydroxy groups, respectively. Based on these compounds, a proposal for the possible biosynthetic pathway was deduced. Two compounds of the mixture, mono- and diacetylated 2-tridecyl sophorosides, respectively, were able to lower the surface tension of water from 72 mN m(-1) to 32 mN m(-1) and the interfacial tension between water and n-hexadecane from 43 mN m(-1) down to 4 and 3 mN m(-1). Thus, both compounds possess a very good surfactant behaviour. Moreover, it was observed that the new products inhibit the growth of particular Gram-positive bacteria, and they indicate potential for antitumour-promoting activity.

IL-21-induced Bepsilon cell apoptosis mediated by natural killer T cells suppresses IgE responses.

Epidemiological studies have suggested that the recent increase in the incidence and severity of immunoglobulin (Ig)E-mediated allergic disorders is inversely correlated with Mycobacterium bovis bacillus Calmette Guerin (BCG) vaccination; however, the underlying mechanisms remain uncertain. Here, we demonstrate that natural killer T (NKT) cells in mice and humans play a crucial role in the BCG-induced suppression of IgE responses. BCG-activated murine Valpha14 NKT cells, but not conventional CD4 T cells, selectively express high levels of interleukin (IL)-21, which preferentially induces apoptosis in Bepsilon cells. Signaling from the IL-21 receptor increases the formation of a complex between Bcl-2 and the proapoptotic molecule Bcl-2-modifying factor, resulting in Bepsilon cell apoptosis. Similarly, BCG vaccination induces IL-21 expression by human peripheral blood mononuclear cells (PBMCs) in a partially NKT cell-dependent fashion. BCG-activated PBMCs significantly reduce IgE production by human B cells. These findings provide new insight into the therapeutic effect of BCG in allergic diseases.

Identification and biological activities of bryostatins from Japanese bryozoan.

Six bryostatins were isolated from Japanese bryozoan by evaluating their binding to the C1B domain of protein kinase Cδ (PKCδ). Structure-activity studies of bryostatins 4, 10, and 14 suggested that the ester group at C20 was not necessary for binding to and activating PKCδ. These bryostatins showed significant anti-tumor-promoting activity in induction tests with the Epstein-Barr virus early antigen.

Cell to cell transfer of the chromatin-packaged human beta-globin gene cluster.

Cell type-specific gene expression is regulated by chromatin structure and the transcription factors provided by the cells. In the present study, we introduced genes packaged into chromatin into target cells using a human artificial chromosome (HAC) and analyzed regulation of gene expression. The human beta-globin gene cluster was built into an HAC (globin-HAC) and introduced into mouse embryonic stem (ES) cells using microcell-mediated chromosome transfer (MMCT); the adult-type human beta-globin gene was expressed in bone marrow and spleen cells of the transgenic mice. In vitro differentiation of ES cells into mouse erythrocytes indicated that the natural sequential expression of epsilon, gamma and beta-globin genes was reproduced on the globin-HAC. Combination of MMCT and a novel chromosome transfection technique allowed transfer of globin-HAC from HT1080 cells into the human leukemia cell line K562, and from K562 cells back into HT1080 cells. Expression of the gamma-globin gene, repressed in HT1080 cells, was activated in K562 cells without any processes of differentiation into adult erythroid cells, and was completely repressed again in HT1080 cells when transferred back from K562 cells. Thus, transfer of target genes packaged into chromatin using a HAC was useful for functional analyses of gene regulation.

Anti-oxidative, anti-tumor-promoting, and anti-carcinogensis activities of nitroastaxanthin and nitrolutein, the reaction products of astaxanthin and lutein with peroxynitrite.

Astaxanthin captured peroxynitrite to form nitroastaxanthins. 15-Nitroastaxanthin was a major reaction product of astaxanthin with peroxynitrite. Here, the anti-oxidative, anti-tumor-promoting, and anti-carcinogensis activities of 15-nitroastaxanthin were investigated. In addition to astaxanthin, 15-nitroastaxanthin showed excellent singlet oxygen quenching activity. Furthermore, 15-nitroastaxanthin showed inhibitory effects of in vitro Epstein-Barr virus early antigen activation and two-stage carcinogensis on mouse skin papillomas. These activities were slightly higher than those of astaxanthin. Similar results were obtained for the 15-nitrolutein, a major reaction product of lutein with peroxynitrite.

Cytotoxic activities and anti-tumor-promoting effects of microbial transformation products of prenylated chalcones from Angelica keiskei.

Three prenylated chalcones, 4-hydroxyderricin (1), xanthoangelol (2), and xanthoangelol F (3), isolated from Angelica keiskei, were transformed by the fungus Aspergillus saitoi. These chalcones were converted to flavanones (i.e., 4, 8, and 12), and prenyl-chain-hydrated (i.e., 5, 7, 9-11, and 13) and ring-B-hydroxylated (i.e., 6) chalcones. The structures of three new metabolites, 7, 9, and 13, were established as 2″,3″-dihydro-4,3″-dihydroxyderricin, 6″,7″-dihydro-7″-hydroxyxanthoangelol, and 6″,7″-dihydro-7″-hydroxyxanthoangelol F, respectively. Upon evaluation of cytotoxic activities of compounds 1-13, the metabolite 7 exhibited potent cytotoxicity against HL60 cells, and this cell death was revealed to be mostly due to apoptosis. In addition, compounds 1-4, 7-10, 12, and 13 were examined for their inhibitory effects on the induction of Epstein-Barr virus early antigen (EBV-EA) by 12-O-tetradecanoylphorbol-13-acetate (TPA) in Raji cells. All compounds tested showed inhibitory effects against EBV-EA activation with potencies higher than that of ß-carotene. Furthermore, the metabolite 13 exhibited inhibitory effect on skin tumor promotion in an in vivo two-stage mouse skin carcinogenesis test based on 7,12-dimethylbenz[a]anthracene (DMBA) as initiator, and with TPA as promoter.

Cucurbitane triterpenoids from the leaves of Momordica charantia, and their cancer chemopreventive effects and cytotoxicities.

Seventeen cucurbitane-type triterpenoids, 1-17, including six new compounds, (23E)-3ß,25-dihydroxy-7ß-methoxycucurbita-5,23-dien-19-al (1), (23S*)-3ß-hydroxy-7ß,23-dimethoxycucurbita-5,24-dien-19-al (6), (23R*)-23-O-methylmomordicine IV (7), (25ξ)-26-hydroxymomordicoside L (8), 25-oxo-27-normomordicoside L (9), and 25-O-methylkaravilagenin D (12), were isolated from a MeOH extract of the leaves of Japanese Momordica charantia. The structures of new compounds were elucidated on the basis of extensive spectroscopic analyses and comparison with literature. Compounds 1-17 were examined for their inhibitory effects on Epstein-Barr virus early antigen (EBV-EA) activation induced with 12-O-tetradecanoylphorbol-13-acetate (TPA) in Raji cells, a known primary screening test for inhibitors of tumor promotion. Four compounds, 1, (23E)-3ß,7ß-dihydroxy-25-methoxycucurbita-5,23-dien-19-al (2), karavilagenin D (11), and 12, showed potent inhibitory effects on EBV-EA induction with IC(50) values in the range of 242-264 mol ratio/32 pmol TPA. In addition, compounds 1 and 11 exhibited inhibitory effects on skin-tumor promotion in an in vivo two-stage mouse skin carcinogenesis test based on 7,12-dimethylbenz[a]anthracene (DMBA) as initiator, and with TPA as a promoter. Furthermore, upon evaluation of the cytotoxic activities of compounds 1-17 against human cancer cell lines, compounds 2, 5-7, 9, and 14 showed potent activities against HL60 cell line, and compound 2 against SK-BR-3 cell line.