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Crocetin is a main bioactive component with a carotenoid skeleton in Gardenia jasminoides, a typical traditional Chinese medicine with a long history in Southeast Asia. Crocetin is being commonly consumed as spices, dyes, and food colorants. Recent pharmacological studies had implied that crocetin may possess potent anti-inflammatory properties; however, the underlying molecular mechanism is not fully elucidated. In the present study, the regulatory effect of crocetin on redox balance was systematically investigated in lipopolysaccharide- (LPS-) stimulated RAW264.7 cells. The results showed that crocetin dose-dependently inhibited LPS-induced nitric oxide production and inducible nitric oxide synthase (iNOS) expression in RAW264.7 cells. Molecular data revealed that crocetin exerted its anti-inflammatory property by inhibiting the MEK1/JNK/NF-κB/iNOS pathway and activating the Nrf2/HO-1 pathway. The shRNA-knockdown (KD) of MEK1 and ERK1 confirmed that the activation of MEK1 and inhibition of JNK mediated the anti-inflammatory effect of crocetin. Moreover, the pull-down assay and computational molecule docking showed that crocetin could directly bind to MEK1 and JNK1/2. It is noticed that both KD and knockout (KO) of HO-1 gene blocked this action. More detailed data have shown that HO-1-KO blocked the inhibition of p-IκB-α by crocetin. These data indicated that crocetin exerted its anti-inflammatory property via modulating the crosstalk between the MEK1/JNK/NF-κB/iNOS pathway and the Nrf2/HO-1 pathway, highlighting HO-1 as a major player. Therefore, the present study reveals that crocetin can act as a potential candidate for redox-balancing modulation in charge of its anti-inflammatory and chemopreventive effect, which strengthens its potency in the subsequent clinic application in the near future.

The study compared the effects of selected proteins replacing fish meal in low-protein diets on piglets' growth performance, intestinal digestive physiology, and nitrogen digestion and metabolism. Five reduced CP, amino acid (AA)-supplemented diets containing 4% of either S50, HP300, concentrated degossypolized cottonseed protein (CDCP), P50, or fish meal were assigned to six pens with 11 pigs for a 28-day study period. Compared with fish meal, dietary proteins did not affect growth performance, apparent total tract digestibility (ATTD) of nutrients, serum hormone levels and biochemical parameters, apparent ileal digestibility (AID) of CP and most AA, colonic short-chain fatty acid (SCFA) contents, duodenal and ileal morphology, digestive enzyme activity, and pH in small intestine of piglets. However, HP300, CDCP, and P50 decreased (p < 0.05) fecal N excretion per weight gain. AID of Ile in S50 and HP300 and Glu in P50 were improved (p < 0.05), and AID of Gly in other proteins was reduced (p < 0.05). S50 and P50 lowered (p < 0.05) the contents of colonic isobutyric and isovaleric. S50 and HP300 reduced (p < 0.05) jejunal villus height. CDCP increased (p < 0.05) the pepsin activity in stomach. S50, HP300, and CDCP decreased (p < 0.05) pH in the proximal colon. Overall, the selected proteins could completely replace fish meal in low-protein diet without impairing piglets' growth via maintaining intestinal digestive physiology, and nitrogen digestion and metabolism.

Recent evidences suggest that gut microbiota plays an important role in regulating physiological and metabolic activities of pregnant sows, and β-carotene has a potentially positive effect on reproduction, but the impact of β-carotene on gut microbiota in pregnant sows remains unknown. This study aimed to explore the effect and mechanisms of β-carotene on the reproductive performance of sows from the aspect of gut microbiota. A total of 48 hybrid pregnant sows (Landrace × Yorkshire) with similar parity were randomly allocated into three groups (n = 16) and fed with a basal diet or a diet containing 30 or 90 mg/kg of β-carotene from day 90 of gestation until parturition. Dietary supplementation of 30 or 90 mg/kg β-carotene increased the number of live birth to 11.82 ± 1.54 and 12.29 ± 2.09, respectively, while the control group was 11.00 ± 1.41 (P = 0.201). Moreover, β-carotene increased significantly the serum nitric oxide (NO) level and glutathione peroxidase (GSH-Px) activity (P < 0.05). Characterization of fecal microbiota revealed that 90 mg/kg β-carotene increased the diversity of the gut flora (P < 0.05). In particular, β-carotene decreased the relative abundance of Firmicutes including Lachnospiraceae AC2044 group, Lachnospiraceae NK4B4 group and Ruminococcaceae UCG-008, but enriched Proteobacteria including Bilophila and Sutterella, and Actinobacteria including Corynebacterium and Corynebacterium 1 which are related to NO synthesis. These data demonstrated that dietary supplementation of β-carotene may increase antioxidant enzyme activity and NO, an important vasodilator to promote the neonatal blood circulation, through regulating gut microbiota in sows.

Non-alcoholic fatty liver disease (NAFLD) is a disease that is prevalent worldwide, and its prevention by dietary administration has recently been considered as an important strategy. In this study, we administered mice with vine tea polyphenol (VTP) extracted from Ampelopsis grossedentata, a Chinese herb, to investigate the preventive effect on western diet (WD)-induced NAFLD. Male C57BL/6N mice were fed either a normal diet (ND) or WD with or without VTP for 12 weeks. The results revealed that VTP supplementation decreased the serum levels of cholesterol and triglycerides, and reduced the accumulation of hepatic lipid droplets caused by WD. Molecular data revealed that VTP enhanced fatty acid oxidation by reactivating the WD-suppressed phosphorylation of AMP-activated protein kinaseα (AMPKα) and the expressions of peroxisome proliferator-activated receptor alpha (PPARα), carnitine palmitoyl transferase IA (CPT1A) and cytochrome P450, family 4, subfamily a1 (CYP4A1). VTP inhibited hepatic lipogenesis by reducing the WD-enhanced level of mature sterol regulatory element-binding protein 1 (SREBP1) and fatty acid synthase (FAS). Moreover, VTP activated nuclear factor (erythroid-derived 2)-like 2 (Nrf2)-mediated expressions of hemeoxygenase-1 (HO-1) and quinone oxidoreductase (NQO1), and reduced hepatic TBARS levels to prevent hepatic oxidative stress. On the other hand, VTP also increased intestinal zonula occludens-1 (ZO-1) expression and the relative abundance of gut Akkermansia, and reduced the ratio of Firmicutes/Bacteroidetes. Thus, VTP might prevent WD-induced NAFLD by balancing fatty acid oxidation and lipogenesis, hepatic oxidative stress, and gut microbiome, at least. These results suggest that vine tea, containing a high content of the bioactive compound dihydromyricetin, is a potential food resource for preventing NAFLD.

Phosphorylation of target proteins by protein kinases leads to the activation of cellular signaling pathways, which induce a great number of biological processes including cell growth, differentiation, development, and death. Although the activities of protein kinases are stringently regulated in homeostasis system, protein kinases can be deregulated under pathological conditions, leading to perturbation of protein kinase-mediated cell signaling pathways and resulting in various disorders including cancer, diabetes, and inflammation. The data from the Human Genome Project has revealed that 518 protein kinases are encoded in the human genome, thereby creating an enormous repertoire of potential targets for chemoprevention. Polyphenols, as integral constituents of the diet, have been proposed to exert beneficial effects in a multitude of disease states. Recent studies have suggested that the cellular effects of polyphenols are, at least partly, mediated by their interactions with protein kinases central to intracellular signaling cascades. In the present study, we investigated the interaction of polyphenols-protein kinases by pull down assay, affinity assay and computer simulation. The results revealed that some polyphenolic compounds suppressed the phosphorylation of various protein kinases with different affinity and selectivity. Finally, we discussed the impact of these polyphenols-protein kinases interactions on cellular signaling and chemoprevention.

6-(Methylsulfinyl)hexyl isothiocyanate (6-MSITC), a major bioactive compound in Wasabi [Wasabia japonica (Miq.) Matsum.], has revealed the inhibitory effect on colon carcinogenesis in rat cancer model although the underlying mechanism is unclear. In this study, we used two types of human colorectal cancer cells (HCT116 p53+/+ and HCT116 p53-/- ) to investigate the anticancer activity and molecular mechanisms of 6-MSITC. Interestingly, 6-MSITC inhibited the cell proliferation in both types of cells with similar IC50 value although a light increase in the phosphorylation and accumulation of P53 protein was observed in HCT116 p53+/+ cells at 24 h after treatment. In addition, 6-MSITC increased the ratio of proapoptotic cells in both types of cells with the same fashion in a p53-independent manner. The data from mitochondrial analysis revealed that 6-MSITC enhanced the ratio of proapoptotic B-cell lymphoma-2-associated X protein/antiapoptotic myeloid cell leukemia 1, and sequentially caused mitochondrial membrane potential (ΔΨm ) loss, cytochrome c release, and caspase-3 activation in both types of cells. Taken together, Wasabi 6-MSITC induced apoptosis of human colorectal cancer cells in p53-independent mitochondrial dysfunction pathway. These findings suggest that 6-MSITC might be a potential agent for colon cancer chemoprevention although with p53 mutation. © 2018 BioFactors, 2018.

Magnolol rich in Magnolia officinalis is a bioactive polyphenolic compound. The aim of this study was to examine the effects of magnolol additive (MA) on growth performance, expression levels of antioxidant-related genes, and intestinal mucosal morphology of Linwu ducks aged from 49 to 70 days, comparing with that of an antibiotic additive (colistin sulfate [CS]). A total of 275, 49-day-old ducks were assigned to 5 groups with 5 cages of 11 ducks each and fed diets supplemented with 0, 100, 200 and 300 mg of MA/kg and 300 mg of CS/kg for 3 weeks, respectively. The results showed that the average daily body weight gain (ADG) was increased significantly in MA-fed groups (200 and 300 mg/kg), compared with the basal diet (BD) group (P

8-prenyl quercetin (PQ) is a typical prenylflavonoid distributed in plant foods. It shows higher potential bioactivity than its parent quercetin (Q) although the mechanisms are not fully understood. This study aims to clarify the anti-inflammatory effects and molecular mechanisms of PQ in cell and animal models, compared to Q.RAW264.7 cells were treated with PQ or Q to investigate the influence on the production of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and protein kinases by Western blotting. Nitric oxide (NO) and prostaglandin E2 (PGE2 ) were measured by the Griess method and ELISA, respectively. Cytokines were assayed by the multiplex technology. Mouse paw edema was induced by LPS. The results revealed that PQ had stronger inhibition on the production of iNOS, COX-2, NO, PGE2 , and 12 kinds of cytokines, than Q. PQ also showed in vivo anti-inflammatory effect by attenuating mouse paw edema. Molecular data revealed that PQ had no competitive binding to Toll-like receptor 4 with LPS, but directly targeted SEK1-JNK1/2 (where SEK is stress-activated protein kinase and JNK1/2 is Jun-N-terminal kinase 1/2) and MEK1-ERK1/2 (where ERK is extracellular signal regulated kinase).PQ as a potential inhibitor revealed anti-inflammatory effect in both cell and animal models at least by targeting SEK1-JNK1/2 and MEK1-ERK1/2.

Blue honeysuckle (Lonicera caerulea L) is a (poly) phenol-rich edible berry. Based on the properties of phenolic compounds, we investigated the anti-inflammatory and antioxidant effects of blue honeysuckle extract (BHE) in adjuvant-induced arthritis (AIA) rat model and macrophage-like (RAW264.7) cell model. Oral administration of BHE in the range of 75–300 µg/g BW attenuated AIA symptom as reducing paw edema in rats. The serum levels of pro-inflammatory factors including tumor necrosis factor alpha (TNF-α), interleukin (IL)-1β, IL-6, and nitric oxide (NO) were significantly reduced in BHE-fed rat. The production of inflammatory enzymes, inducible nitric oxide synthases (iNOS) and cyclooxygenase-2 (COX-2), in the spleen was also significantly suppressed. Moreover, serum transaminases including glutamic oxaloacetic transaminase (GOT), glutamate-pyruvate transaminase (GPT) and gamma-glutamyl transferase (GGT) were inhibited, and the antioxidant enzymes including superoxide dismutase (SOD) and glutathione peroxidase (GPx) were recovered. Finally, the major bioactive components in BHE were identified as (−)-epicatechin (EC) and cyanidin 3-glucoside (C3G), which were proven to suppress the expression of COX-2 and iNOS, accompanied by enhancing the level of Nrf2 and HO-1 in RAW264.7 cells. Taken together, our data demonstrated that BHE rich with EC and C3G attenuated rat AIA symptom with crosstalk of anti-inflammatory and antioxidant effects.

Fresh loquat leaves have been used as folk health herb in Asian countries for long time, although the evidence supporting their functions is still minimal. This study aimed to clarify the chemopreventive effect of loquat tea extract (LTE) by investigating the inhibition on proliferation, and underlying mechanisms in human promyelocytic leukemia cells (HL-60). LTE inhibited proliferation of HL-60 in a dose-dependent manner. Molecular data showed that the isolated fraction of LTE induced apoptosis of HL-60 as characterized by DNA fragmentation; activation of caspase-3, -8, and -9; and inactivation of poly(ADP)ribose polymerase. Moreover, LTE fraction increased the ratio of pro-apoptotic Bcl-2-associated X protein (Bax)/anti-apoptotic myeloid cell leukemia 1 (Mcl-1) that caused mitochondrial membrane potential loss and cytochrome c released to cytosol. Thus, our data indicate that LTE might induce apoptosis in HL-60 cells through a mitochondrial dysfunction pathway. These findings enhance our understanding for chemopreventive function of loquat tea.

Loquat (Eriobotrya japonica) leaves have been used as traditional medicine for chemoprevention and treatment of chronic inflammatory diseases although the evidence supporting their functions is still poor. This study aims to clarify the anti-inflammatory effects and molecular mechanisms of loquat tea extract (LTE), in both cell and animal models. LTE, especially C fraction, inhibited the production of pro-inflammatory factors including inducible nitric oxide synthase (iNOS), nitric oxide (NO), IL-6, RANTES and TNF-α. Cellular signaling data revealed that the downregulation of TGF-β-activated kinase (TAK1)-mediated both mitogen-activated protein kinase (MAPK) and NF-κB pathways were involved in the inhibition of pro-inflammatory factors by C fraction. Mouse paws edema model further confirmed the in vivo anti-inflammatory effects of LTE.

Oolong tea theasinensins are a group of tea polyphenols different from green tea catechins and black tea theaflavins, and they are considered as bioactive compounds in Oolong tea. In the present study, based on the properties of theasinensin and information about inflammatory processes, we investigated the anti-inflammatory activity and molecular mechanisms of theasinensin A (TSA) in both cell and animal models. In the cell model, TSA reduced the levels of pro-inflammatory mediators including inducible nitric oxide synthase (iNOS), nitric oxide (NO), interleukin-12 (IL-12) (p70), tumor necrosis factor alpha (TNF-α), and monocyte chemotactic protein-1 (MCP-1) induced by lipopolysaccharide (LPS). Cellular signaling analysis revealed that TSA downregulated MAPK/ERK kinase (MEK)-extracellular signal-regulated kinase (ERK) signaling. Pull-down assay and affinity data revealed that TSA might directly bind to MEK-ERK for the inhibitory action. In the animal model, TSA suppressed the production of IL-12 (p70), TNF-α, and MCP-1 and attenuated mouse paw edema induced by LPS.

Scope Garlic is used for both culinary and medicinal purposes by many cultures. The garlic organosulfur compounds (GOSCs) are thought to be bioactive components. This study aims to clarify the antiinflammatory effects and molecular mechanisms of GOSCs in both cell and animal models. Methods and results RAW264.7 cells were treated with six kinds of GOSCs to screen their influence on cyclooxygenase-2 and inducible nitric oxide synthase expression by Western blotting. Prostaglandin E2 and nitrite were measured by ELISA and Griess reaction, respectively. Cytokines in culture medium were assayed by the multiplex technology. Proteins were detected by Western blotting. Mouse paw edema was induced by LPS. The results revealed that diallyl trisulfide (DATS) was a strongest inhibitor for cyclooxygenase and inducible nitric oxide synthase among GOSCs, and reduced the levels of LPS-induced IL-6, IL-10, IL-12(p70), KC, MCP-1, and TNF-α. Cellular signaling analysis revealed that DATS downregulated AKT1/TGF-β-activated kinase-mediated mitogen-activated protein kinase (MAPK) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathways. Furthermore, DATS activated Nrf2-mediated expression of HO-1 and NQO1 and reduced LPS-induced intracellular reactive oxygen species, which may contribute to suppress inflammatory mediator production. Finally, in vivo data demonstrated that DATS attenuated LPS-induced mouse paw edema. Conclusion DATS as a potential inhibitor revealed antiinflammatory effect in both cell and animal models by downregulating AKT1/TGF-β-activated kinase-mediated NFκB and MAPK signaling pathways.

Purified polyphenolic-rich extracts from four native Australian fruits, Illawarra Plum ( Podocarpus elatus Endl., Podocarpaceae ), Kakadu Plum ( Terminalia ferdinandiana Exell, Combretaceae ), Muntries ( Kunzea pomifera F. Muell., Myrtaceae ) and Native Currant ( Acrotriche depressa R.Br., Epacridaceae ), were screened for their ability to modulate anti-inflammatory activity in LPS-activated murine macrophages (RAW 264.7). The Kakadu Plum extract inhibited the expression of inducible nitric oxide synthase (iNOS), and importantly was the only fruit in this study that displayed differential inhibition of the expression of cyclooxygenase (COX)-2 but not COX-1. Illawarra Plum (COX-2 and iNOS) and Native Currant (iNOS only) also inhibited inflammatory enzymes, while Muntries exhibited none of these activities under the same conditions. All evaluated extracts inhibited the production of prostaglandin E 2 and nitric oxide, suggesting the involvement of alternative pathways in their regulation for the Muntries extract. Further molecular investigations, showed that Kakadu Plum inhibited the NF-кB pathway, but not the p44/42 mitogen activated protein kinase (MAPK) pathway. Collectively, these results demonstrate potential anti-inflammatory activities of native Australian fruits, in particular Kakadu Plum, in LPS-activated murine macrophages, thus confirming the potential biological activities of these fruits.

Protein kinases play crucial roles in the regulation of multiple cell signaling pathways and cellular functions. Deregulation of protein kinase function has been implicated in carcinogenesis. The inhibition of protein kinases has emerged as an important target for cancer chemoprevention and therapy. Accumulated data revealed that flavonoids exert chemopreventive effects through acting at protein kinase signaling pathways, more than as conventional hydrogen-donating antioxidants. Recent studies show that flavonoids can bind directly to some protein kinases, including Akt/protein kinase B (Akt/PKB), Fyn, Janus kinase 1 (JAK1), mitogen-activated protein kinase kinase 1 (MEK1), phosphoinositide 3-kinase (PI3K), mitogen-activated protein (MAP) kinase kinase 4 (MKK4), Raf1, and zeta chain-associated 70-kDa protein (ZAP-70) kinase, and then alter their phosphorylation state to regulate multiple cell signaling pathways in carcinogenesis processes. In this review, we report recent results on the interactions of flavonoids and protein kinases, especially their direct binding and molecular modeling. The data suggest that flavonoids act as protein kinase inhibitors for cancer chemoprevention that were thought previously as conventional hydrogen-donating antioxidant. Moreover, the molecular modeling data show some hints for creating natural compound-based protein kinase inhibitors for cancer chemoprevention and therapy.

Much is known about the bioactive properties of green tea flavan-3-ol. However, very little work has been done to determine the properties of proanthocyanidins, another kind of polyphenols in green tea. In this study, we have investigated the anti-inflammatory effect of tea prodelphinidin B-4 3'-O-gallate (PDG) by demonstrating the inhibitory effects on cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) in lipopolysaccharide (LPS)-activated murine macrophage RAW264 cells. PDG caused a dose-dependent inhibition of COX-2 and iNOS at both mRNA and protein levels with the attendant decrease of prostaglandin E2 (PGE2) and nitric oxide (NO) production. Molecular data revealed that PDG downregulated NF-kappaB signaling pathway. Electrophoretic mobility shift assay (EMSA) showed that PDG reduced the binding complex of NF-kappaB-DNA in the promoter of COX-2 and iNOS. Immunochemical analysis revealed that PDG suppressed LPS-induced phosphorylation and degradation of IkappaBalpha, and subsequent nuclear translocation of p65. Consequently, PDG suppressed phosphorylation of IkappaB kinase alpha/beta (IKKalpha/beta) and TGF-beta-activated kinase (TAK1). Taken together, our data indicated that PDG is involved in the inhibition of COX-2 and iNOS via the downregulation of TAK1-NF-kappaB pathway, revealing partial molecular basis for the anti-inflammatory properties of tea PDG.

The inhibitory effects of green tea proanthocyanidins on cyclooxygenase-2 (COX-2) expression and prostaglandin E(2) (PGE(2)) release were investigated in lipopolysaccharide (LPS)-activated murine macrophage RAW264 cells. Prodelphinidin B2 3,3' di-O-gallate (PDGG) caused a dose-dependent inhibition of COX-2 at both mRNA and protein levels with the attendant release of PGE(2). Molecular evidence revealed that PDGG inhibited the degradation of Ikappa-B, nuclear translocation of p65 and CCAAT/enhancer-binding protein (C/EBP)delta, and phosphorylation of c-Jun, but not CRE-binding protein (CREB), which regulate COX-2 expression. Moreover, PDGG suppressed the activations of mitogen-activated protein kinase (MAPK) including c-Jun NH(2)-terminal kinase (JNK), extracellular signal-regulated kinase (ERK) and p38 kinase. The results demonstrated that PDGG suppressed COX-2 expression via blocking MAPK-mediated activation of nuclear factor-kappaB (NF-kappaB), activator protein-1 (AP-1) and C/EBPdelta. Furthermore, studies on structure-activity relationship using five kinds of proanthocyanidins revealed that the galloyl moiety of proanthocyanidins appeared important to their inhibitory actions. Thus, our findings provide the first molecular basis that green tea proanthocyanidins with the galloyl moiety might have anti-inflammatory properties through blocking MAPK-mediated COX-2 expression.

The effects of anthocyanidins, the aglycon nucleuses of anthocyanins widely occurring in reddish fruits and vegetables, on the expression of cyclooxygenase-2 (COX-2) were investigated in lipopolysaccharide (LPS)-activated murine macrophage RAW264 cells. Of five anthocyanidins, delphinidin and cyanidin inhibited LPS-induced COX-2 expression, but pelargonidin, peonidin and malvidin did not. The structure-activity relationship suggest that the ortho-dihydroxyphenyl structure of anthocyanidins on the B-ring appears to be related with the inhibitory actions. Delphinidin, the most potent inhibitor, caused a dose-dependent inhibition of COX-2 expression at both mRNA and protein levels. Western blotting analysis indicated that delphinidin inhibited the degradation of IkappaB-alpha, nuclear translocation of p65 and CCAAT/enhancer-binding protein (C/EBP)delta and phosphorylation of c-Jun, but not CRE-binding protein (CREB). Moreover, delphinidin suppressed the activations of mitogen-activated protein kinase (MAPK) including c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK) and p38 kinase. MAPK inhibitors (U0126 for MEK1/2, SB203580 for p38 kinase and SP600125 for JNK) specifically blocked LPS-induced COX-2 expression. Thus, our results demonstrated that LPS-induced COX-2 expression by activating MAPK pathways and delphinidin suppressed COX-2 by blocking MAPK-mediated pathways with the attendant activation of nuclear factor-kappaB (NF-kappaB), activator protein-1 (AP-1) and C/EBPdelta. These findings provide the first molecular basis that anthocyanidins with ortho-dihydroxyphenyl structure may have anti-inflammatory properties through the inhibition of MAPK-mediated COX-2 expression.

Phenolic composition and radical scavenging activity in the shochu distillery by-products of sweetpotato (Ipomoea batatas L.) treated with koji (Aspergillus awamori mut.) and cellulase (Cellulosin T2) were investigated to develop new uses. Koji and Cellulosin T2 treatment of shochu distillery by-products from sweetpotatoes, rice, and barley increased phenolic content. Caffeic acid was identified as a dominant phenolic component in the shochu distillery by-products of the sweetpotato. Adding koji and/or Cellulosin T2 to the shochu distillery by-product indicated that koji was involved in caffeic acid production. Caffeic acid was not detected in raw or steamed roots of "Koganesengan", the material of sweetpotato for shochu production, suggesting that it is produced during shochu fermentation. The phenolic content and radical scavenging activity the shochu distillery by-product treated with koji and Cellulosin T2 were superior to those of commercial vinegar. These results suggest that koji treatment of sweetpotato-derived shochu distillery by-products has potential for food materials with physiological functions. Further koji treatment of sweetpotato shochu-distillery by-products may be applicable to mass production of caffeic acid.

Rhein is an anthraquinone compound enriched in the rhizome of rhubarb, a traditional Chinese medicine herb showing anti-tumor promotion function. In this study, we first reported that rhein could induce apoptosis in human promyelocytic leukemia cells (HL-60), characterized by caspase activation, poly(ADP)ribose polymerase (PARP) cleavage, and DNA fragmentation. The efficacious induction of apoptosis was observed at 100 μM for 6 h. Mechanistic analysis demonstrated that rhein induced the loss of mitochondrial membrane potential (ΔΨ m ), cytochrome c release from mitochondrion to cytosol, and cleavage of Bid protein. Rhein also induced generation of reactive oxygen species (ROS) and the phosphorylation of c-Jun N-terminal kinase (JNK) and p38 kinase. However, these actions seem not to be associated with the apoptosis induction because antioxidants including N -acetyl cysteine (NAC), Tiron, and catalase did not block rhein-induced apoptosis, although they could block the generation of ROS and the phosphorylation of JNK and p38 kinase. Our data demonstrate that rhein induces apoptosis in HL-60 cells via a ROS-independent mitochondrial death pathway.

The c-myb proto-oncogene product (c-Myb) is a transcriptional activator. Vertebrate c-Myb is a key regulator of the G(1)/S transition in cell cycle, while Drosophila Myb (dMyb) is important for the G(2)/M transition. Here we report that dMyb induces expression of cyclin B, a critical regulator of the G(2)/M transition, in Drosophila eye imaginal disc. In the wild-type eye disc, dmyb mRNA was expressed in the stripes both anterior and posterior to the morphogenetic furrow. Ectopic expression of C-terminal-truncated dMyb in the eye disc caused ectopic expression of cyclin B and the rough eye phenotype. This rough eye phenotype correlated with prolonged M phase, caused by overexpression of cyclin B. Cyclin B expression was lost in dmyb-deficient clones. In Schneider cells, the activity of the cyclin B promoter was dramatically reduced by loss of dMyb using the RNA interference method. Mutations of the multiple AACNG sequences in the cyclin B promoter also abolished the promoter activity. These results indicate that dMyb regulates the G(2)/M transition by inducing cyclin B expression via binding to its promoter.

6-(Methylsulfinyl)hexyl isothiocyanate (6-MSITC) is a major bioactive compound in wasabi (Wasabia japonica), which is a typical Japanese pungent spice. Recently,in vivoandin vitrostudies demonstrated that 6-MSITC has several biological properties, including anti-inflammatory, antimicrobial, antiplatelet, and anticancer effects. We previously reported that 6-MSITC strongly suppresses cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and cytokines, which are important factors that mediate inflammatory processes. Moreover, molecular analysis demonstrated that 6-MSITC blocks the expressions of these factors by suppressing multiple signal transduction pathways to attenuate the activation of transcriptional factors. Structure-activity relationships of 6-MSITC and its analogues containing an isothiocyanate group revealed that methylsulfinyl group and the length of alkyl chain of 6-MSITC might be related to high inhibitory potency. In this paper, we review the anti-inflammatory properties of 6-MSITC and discuss potential molecular mechanisms focusing on inflammatory responses by macrophages.

Theasinensin A is one of the oolong tea theasinensins, which differ from green tea catechins and black tea theaflavins. In a previous study, we found that theasinesin A had a potential effect on antiinflammation since theasinensin A suppressed LPS-induced COX2 and PGE(2) production. To clarify the molecular mechanisms, we investigated the gene expression profiling in macrophage-like cells treated with theasinensin A through a genome-wide DNA microarray in the present study. Among 22,050 oligonucleotides, the expression levels of 406 genes were increased by ≥3-fold in LPS-activated RAW264 cells, 259 gene signals of which were attenuated by theasinensin A treatment (≥2-fold). Expression levels of 717 genes were decreased by ≥3-fold in LPS-activated cells, of which 471 gene signals were restored by theasinensin A treatment (≥2-fold). These genes were further categorized as "defense, inflammatory response, cytokines activities, and receptor activities," and some of them were confirmed by real-time polymerase chain reaction. Furthermore, pathways analysis revealed that theasinensin A regulated the relevant expression networks of chemokines, interleukins, and interferons to exert its antiinflammatory effects.

Chymotrypsinogen is a principal precursor of pancreatic proteolytic enzymes. We previously isolated a cDNA clone for human prechymotrypsinogen from a human pancreatic cDNA library. In the present study, we used this cDNA sequences to isolate genomic DNA clones. Three overlapping cosmid clones spanning approximately 65-kb genomic sequences were isolated from a human cosmid library. The genomic DNA clones were characterized by restriction enzyme mapping and by hybridizing them to subfragments of the cDNA. The sequence tagged sites for human chymotrypsinogen gene were created by designing two oligonucleotides. Furthermore, the isolated genomic clones were confirmed to be localized on chromosome 16q23 by fluorescence in situ hybridization and G-banding analysis.

Oolong tea theasinensins are a group of tea polyphenols different from green tea catechins and black tea theaflavins. The present study reports the inhibitory effects of oolong tea theasinensins on the expression of cyclooxygenase-2 (COX-2) and underlying molecular mechanisms in lipopolysaccharide (LPS)-activated murine macrophage RAW264 cells. The structure-activity data revealed that the galloyl moiety of theasinensins played an important role in the inhibitory actions. Theasinensin A, a more potent inhibitor, caused a dose-dependent inhibition of mRNA, protein, and promoter activity of COX-2. An electrophoretic mobility shift assay (EMSA) revealed that theasinensin A reduced the complex of NF-κB- and AP-1-DNA in the promoter of COX-2. Signaling analysis demonstrated that theasinensin A attenuated IκB-α degradation, nuclear p65 accumulation, and c-Jun phosphorylation. Furthermore, theasinensin A suppressed the phosphorylation of MAPKs, IκB kinase α/β (IKKα/β), and TGF-β activated kinase (TAK1). These data demonstrated that the down-regulation of TAK1-mediated MAPKs and NF-κB signaling pathways might be involved in the inhibition of COX-2 expression by theasinensin A. These findings provide the first molecular basis for the anti-inflammatory properties of oolong tea theasinensins.

6-(Methylsulfinyl)hexyl isothiocyanate (6-MSITC) is a bioactive ingredient of wasabi [Wasabia japonica (Miq.) Matsumura], which is a popular pungent spice of Japan. To evaluate the anti-inflammatory function and underlying genes targeted by 6-MSITC, gene expression profiling through DNA microarray was performed in mouse macrophages. Among 22,050 oligonucleotides, the expression levels of 406 genes were increased by ≥3-fold in lipopolysaccharide (LPS)-activated RAW264 cells, 238 gene signals of which were attenuated by 6-MSITC (≥2-fold). Expression levels of 717 genes were decreased by ≥3-fold in LPS-activated cells, of which 336 gene signals were restored by 6-MSITC (≥2-fold). Utilizing group analysis, 206 genes affected by 6-MSITC with a ≥2-fold change were classified into 35 categories relating to biological processes (81), molecular functions (108) and signaling pathways (17). The genes were further categorized as 'defense, inflammatory response, cytokine activities and receptor activities' and some were confirmed by real-time polymerase chain reaction. Ingenuity pathway analysis further revealed that wasabi 6-MSITC regulated the relevant networks of chemokines, interleukins and interferons to exert its anti-inflammatory function.

Normal human lung fibroblast diploid cells, WI-38, become senescent after a definite number of divisions. VA-13 is a line of immortalized cells established by transformation of WI-38 cells by SV40 virus. To determine whether SV40 large T (SV40-T) antigen is essential for this immortalization of WI-38 cells we introduced an antisense gene for T antigen into VA-13. Two morphologically different types of antisense transformant (VA-AS5-8 and VA-AS37-8) were obtained. In both antisense transformants the expression of T antigen was reduced by more than 70% as compared to that in the parent cells. The morphology of the antisense transformants indicated a partial conversion to the senescent phenotype of WI-38. The relative number of cells in the S phase of the antisense transformants was decreased as compared to that in cultures of VA-13 and about 50% of cells were at G1/0. The doubling time of the transformants was prolonged to close to the doubling time of WI-38. The level of expression of retinoblastoma protein (pRB) complexed with SV40-T antigen of the antisense transformants was significantly decreased although the level of total pRB was much higher than that in VA-13. The pRB was present exclusively in the underphosphorylated form. Thus, the decreased level of formation of the complex between SV40-T and pRB or the underphosphorylation of pRB may explain the suppression of growth of antisense transformants. Together, these results show that an antisense gene for SV40-T antigen can efficiently block the cell proliferation and the cell immortalization of VA-13 cells.

A chymotrypsinogen showing the phenotype AA of chymotrypsin was purified from quail pancreas, and its chromatographic behavior, molecular weight, and electrophoretic mobility were very similar to those of another chymotrypsinogen, showing the aa phenotype of chymotrypsin. However, after activation, this chymotrypsinogen from AA showed band 5 of chymotrypsin, while the other chymotrypsinogen from aa did not. Peptide mapping demonstrated that the molecular structures of the two chymotrypsinogens are different. It was recognized that chymotrypsin variants result from activated products of different molecular structures of chymotrypsinogens.

1. 1. A chymotrypsinogen from pancreas of Japanese quail (Coturnix coturnix japonica) was purified by acid extraction, salt fractionation and chromatographic separation on CM-cellulose and Sephadex G-100, and gave a single protein band on SDS-PAGE. 2. 2. Quail chymotrypsinogen had a mol. wt of 26, 100 calculated from amino acid composition data, an isoelectric point of 7.68, a Km of 3.1 mM and K0 of 40.7 sec−1 for tyrosine ester substrate. 3. 3. The activated chymotrypsinogen of quail had a maximum activity at pH 7.0–8.0 and at 45°C, and was stable at pH 4.0–6.0 below 55°C. 4. 4. Comparison of quail and bovine chymotrypsinogens indicates that the activities of the enzymes from quail and bovine are more constant than their physical characteristics.

6-(methylsulfinyl)hexyl isothiocyanate (6-MITC) is a bioactive compound extracted from a typical Japanese spice, wasabi (Wasabia japonica (Miq.) Matsumura). In the present study, we found that 6-MITC suppressed the expression of cyclooxygenase-2 (COX-2) induced by lipopolysaccharide (LPS), interferon-gamma (IFN-gamma), but did not suppress that induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), in murine macrophage RAW264. Molecular mechanisms were investigated by targeting the transcriptional factors including activator protein-1 (AP-1), CCAAT/enhancer-binding protein delta (C/EBPdelta), CRE-binding protein (CREB) and nuclear factor kappaB (NF-kappaB), which bind to the core element of COX-2 promoter. LPS induced activation of all of these factors and 6-MITC suppressed LPS-induced activation of AP-1, C/EBPdelta, CREB, but not NF-kappaB. IFN-gamma did not induce any activation of these factors, but 6-MITC suppressed IFN-gamma-induced COX-2 expression, suggesting that the upstream region of the core element is linked for this suppression. Finally, TPA stimulated the activation of CREB and AP-1, but 6-MITC did not block TPA-induced COX-2 expression. These results suggest that LPS, IFN-gamma and TPA regulate COX-2 expression through different mechanisms, and 6-MITC acts as a potent inhibitor of COX-2 expression induced by LPS or IFN-gamma.

Schizandrae chinensis, a traditional Chinese medicine herb, has been used to treat hepatitis B disease in Chinese hospital clinic. We have isolated two bioactive compounds, deoxyschizandrin and gamma-schizandrin, from S. chinensis. In the present, we reported that deoxyschizandrin and gamma-schizandrin could induce apoptosis in human promyelocytic leukemia cells (HL-60), as characterized by DNA fragmentation and poly (ADP) ribose polymerase (PARP) cleavage. Further molecular analysis showed that deoxyschizandrin and gamma-schizandrin caused the loss of mitochondrial membrane potential (DeltaPsim), cytochrome c release from mitochondrion to cytosol, truncation of Bid protein, and activation of caspase-3 and -9. However, they did not increase the intracellular level of reactive oxygen species (ROS). Antioxidants such as N-acetyl cysteine (NAC) and catalase did not block the apoptosis induced by deoxyschizandrin or gamma-schizandrin. These findings suggest that deoxyschizandrin and gamma-schizandrin-induced apoptosis in HL-60 cells involved ROS-independent mitochondrial dysfunction pathway.

6-(Methylsulfinyl)hexyl isothiocyanate (6-MITC) is a chemopreventive compound occurring in Wasabi (Wasabia japonica (Miq.) Matsumura), which is a very popular pungent spice in Japan. We investigated the effects of 6-MITC on the expression of cyclooxygenase-2 (COX-2) in lipopolysaccharide (LPS)-activated murine macrophage RAW264 cells. Treatment with 6-MITC suppressed LPS-mediated induction of COX-2 protein in a dose-dependent manner. Transfections with various COX-2 promoter reporter constructs revealed that the inhibitory effects of 6-MITC on COX-2 gene expression were directed by the core promoter elements including nuclear factor kappaB (NF-kappaB), CCAAT/enhancer-binding protein (C/EBP) and cyclic AMP-response element (CRE) sites. Western blotting analysis showed that 6-MITC inhibited LPS-induced activation of MAPK (ERK, p38 kinase and JNK) and transcriptional factors (CREB, c-Jun and C/EBPdelta) binding the core elements of COX-2 promoter, substantiating the involvement of these signal transduction pathways in the regulation of COX-2 expression by 6-MITC. Moreover, Western blotting experiments with MAPK-specific inhibitors (U0126 for MEK1/2, SB203580 for p38 kinase and SP600125 for JNK) demonstrated that 6-MITC suppressed LPS-induced COX-2 expression by blocking the activation of JNK-mediated AP-1 and ERK/p38 kinase-mediated CREB or C/EBPdelta. Finally, the structure-activity study revealed that the inhibitory potency of methylsulfinyl isothiocyanates (MITCs) depended on the methyl chain length. These findings demonstrate for the first time that 6-MITC is an effective agent to attenuate COX-2 production, and enhance our understanding of the anti-inflammation properties of 6-MITC.

6-(Methylsulfinyl)hexyl isothiocyanate (6-MITC) is an active ingredient of Wasabi (Wasabia japonica (Miq.) Matsumura), which is a very popular pungent spice in Japan. To clarify the cellular signaling mechanism underlying the anti-inflammatory action of 6-MITC, we investigated the effects of 6-MITC on the expression of inducible nitric oxide synthase (iNOS) in lipopolysaccharide (LPS)-activated murine macrophage RAW264 cells. 6-MITC showed a dose-dependent inhibition of LPS-induced nitric oxide (NO), iNOS mRNA and protein. LPS caused the c-Jun phosphorylation (a major component of AP-1) and IkappaB-alpha degradation. 6-MITC suppressed LPS-induced c-Jun phosphorylation, but did not inhibit IkappaB-alpha degradation. Cellular signaling analysis using MAPK-(U0126 for MEK1/2, SB203580 for p38 kinase and SP600125 for JNK) and Jak2-specific (AG490) inhibitors demonstrated that LPS stimulated iNOS expression via activating Jak2-mediated JNK, but not ERK and p38, pathway. 6-MITC suppressed iNOS expression through the inhibition of Jak2-mediated JNK signaling cascade with the attendant to AP-1 activation. In addition, the structure-activity study revealed that the inhibitory potency of methylsulfinyl isothiocyanates (MITCs) depended on the methyl chain length. These findings provide the molecular basis for the first time that 6-MITC is an effective agent to attenuate iNOS production.

Baicalein is one component of the dried root of Scutellaria Baicalensis Georgi. (Huang Qin) which is widely used in the traditional Chinese herbal medicine. In this study, we report that baicalein was able to induce apoptosis in human promyelocytic leukemia cells (HL-60), as characterized by poly-(ADP-ribose) polymerase (PARP) cleavage and DNA fragmentation. The efficacious induction of apoptosis was observed at 100 microM for 6 h. Mechanistic analysis demonstrated that baicalein induced the cleavage of Bid protein, cytochrome c release from mitochondria into cytosol, and activation of caspase-3, -8 and -9. Moreover, baicalein caused elevation of intracellular hydrogen peroxide level. Catalase could effectively block baicalein-induced DNA fragmentation. These data indicate that baicalein may trigger an apoptotic death program through reactive oxygen species (ROS)-mediated mitochondrial dysfunction pathway. The findings enhance our understanding of anticancer function of baicalein in herbal medicine.

Echinocystic acid (EA), a natural triterpone enriched in various herbs, has been showed to have cytotoxic activity in some cancer cells, and is used for medicinal purpose in many Asian countries. In the present study, we found that EA could induce apoptosis in human HepG2 cells, as characterized by DNA fragmentation, activation of caspase-3, -8, and -9, and PARP cleavage. The efficacious induction of apoptosis was observed at 45 microM for 24 h. Molecular data showed that EA induced the truncation of Bid protein and reduction of Bcl-2 protein. EA also caused the loss of mitochondrial membrane potential (DeltaPsi(m)) and cytochrome c release from mitochondria to cytosol. Moreover, EA could activate c-Jun NH(2)-terminal kinase (JNK) and p38 kinase, and JNK-specific inhibitor SP600125 and p38 kinase-specific inhibitor SB200235 could block serial molecular events of EA-induced apoptosis such as Bid truncation, Bcl-2 reduction, cytochrome c release, caspase activation, and DNA fragmentation in HepG2 cells. These findings indicate that JNK- and p38 kinase-mediated mitochondrial pathways might be involved in EA-induced apoptosis and enhance our understanding of the anticancer function of EA in herbal medicine.

Gossypol is a component present in cottonseeds and has been demonstrated to be an effective contraceptive drug in preventing spermatogenesis in mammalian species. In the present, we reported that gossypol could induce apoptosis in human promyelocytic leukemia cells (HL-60), as characterized by DNA fragmentation, poly(ADP) ribose polymerase (PARP) cleavage. The efficacious induction of apoptosis was observed at 50 microM for 6 h. Further molecular analysis showed that gossypol induced the truncation of Bid protein, the loss of mitochondrial membrane potential (DeltaPsi m), cytochrome c release from mitochondria into cytosol, and activation of caspase-3, -8, and -9. However, gossypol did not increase the level of reactive oxygen species (ROS), and antioxidants including N-acetyl cysteine (NAC) and catalase could not block gossypol-induced apoptosis in the HL-60 cells. These data suggest that gossypol induces apoptosis in HL-60 cells through ROS-independent mitochondrial dysfunction pathway.

Anthocyanidins are the aglycon nucleuses of anthocyanins, which are reddish pigments widely spread in colored fruits and vegetables. To investigate their anti-cancer effect, induction of apoptosis was tested in human promyelocytic leukemia cells (HL-60), which is a valid model for testing antileukemic or general antitumoral compounds. Of six anthocyanidins representing the aglycons of most of anthocyanins, only those with an ortho-dihydroxyphenyl structure on the B-ring induce apoptosis, suggesting that the ortho-dihydroxyphenyl structure of anthocyanidins may contribute to the induction of apoptosis. Delphinidin, the most potent inducer, causes apoptosis in a time- and dose-dependent manner. The efficacious induction of apoptosis was observed at 100 micro M for 6 h. Concomitant with the apoptosis, delphinidin stimulates JNK pathway activation including JNK phosphorylation and c-jun gene expression, and activates caspase-3. Antioxidants including N-acetyl-L-cysteine (NAC) and catalase effectively block delphinidin-induced JNK phosphorylation, caspase-3 activation, and DNA fragmentation. Moreover, anthocyanidins directly cause HL-60 cells to generate intracellular hydrogen peroxide. Thus, anthocyanidins may trigger an apoptotic death program through an oxidative stress-involved JNK signaling pathway. The induction of apoptosis by anthocyanins may be the pivotal mechanism by which its chemopreventive action against cancer is based.

Rhein (4,5-Dihydroxyanthraquinone-2-carboxylic acid), a constituent enriched in the rhizome of rhubarb (R. palmatum L. or R. tanguticum Maxim), is a traditional Chinese herb used as a laxative and stomachic drug. In the present study, we investigated the anti-carcinogenesis of rhein by using mouse epidermal cell JB6 line, an in vitro model for elucidating the molecular mechanisms of cancer chemopreventive agents. Rhein is shown to inhibit 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced cell transformation and activator protein-1 (AP-1) activation in a dose-dependent manner. Signal cascade analysis revealed that rhein inhibits the phosphorylation and abundance of c-Jun protein, c-Jun NH2-terminal kinase (JNK) phosphorylation, but does not inhibit the phosphorylation of extracellular signal-regulated protein kinase (ERK) and p38 kinase. Thus, these results provide the first evidence suggesting that rhein inhibits AP-1 activity and cell transformation through the inhibition of a JNK-dependent, ERK- and p38-independent molecular mechanism.

Induction of phase II enzymes such as NADPH:quinone oxidoreductase (QR) can reduce carcinogen-induced mutagenesis and tumor formation. In our search for novel dietary anticarcinogens, fisetin, a flavonol widely distributed in fruits and vegetables was found to induce QR activity in murine hepatoma 1c1c7 cells. The cells were treated with various concentrations of fisetin, and then were assessed for cell growth, QR activity, QR mRNA expression and transcription activation of the QR gene. The results showed that fisetin induced QR activity in time- and dose-dependent manner in the concentration range of 0.1 to 10 microM, and the activity induction was associated with QR mRNA expression as detected by reverse transcription-PCR. Furthermore, transfection studies using a human QR antioxidant/electrophile-response element (ARE/EpRE) reporter construct demonstrated that fisetin activated the ARE/EpRE. These results show that fisetin increases QR activity by transcriptional activation of the ARE/EpRE, suggesting a novel mechanism by which dietary fisetin may be implicated in cancer chemoprevention.

Changes in the expression of cell adhesion molecule and albumin genes were investigated in primary cultures of rat hepatocytes with and without poly- N-p -vinylbenzyl-D-lactonamide (PVLA) coating of the dishes. In PVLA-coated cultures, hepatocytes aggregated into spheroids and expressed liver cadherin and albumin mRNAs at higher levels. In uncoated cultures, hepatocytes revealed low levels of cadherin and albumin mRNAs, but higher levels of integrin α-1 mRNA. The changes in mRNA levels of liver cadherin and integrin α-1 coordinated well with those in spheroid and monolayer formation of hepatocytes, respectively. These results suggest that, in the PVLA-coated culture, hepatocytes expressed cadherin at higher levels to promote cell–cell adhesion and further maintain the differentiated function, such as albumin secretion, for prolonged times.

Methylsulfinyl isothiocyanates (MITC) are a class of isothiocyanates occurring in a variety of cruciferous vegetables showing anticarcinogenic activity. To develop analogues of methylsulfinyl isothiocyanate with less toxicity and better biological activity, four types of methyl chain length (designated as 2-, 4-, 6- and 8-MITC) were synthesized. The murine hepatoma cells (Hepa 1c1c7) were treated with various concentrations of MITC, and then assessed for cell growth, enzyme activity and mRNA expression of the detoxifying enzyme NADPH:quinone oxidoreductase (QR). All of four MITC augmented the induction of QR activity and the expression of QR mRNA in a dose-dependent manner. In the non-toxic concentration range, an increase in the methyl chain length resulted in a higher QR induction in both activity and mRNA expression. However, increasing cytotoxicity was also observed by an increase in the methyl chain length. Our results suggested that 4- and 6-MITC as QR inducers appeared to be less toxic and even more potent.

It was found that the inhibition of the lysosomal acid lipase activity by rat apolipoprotein A-I (apo A-I) was increased with the degradation of apo A-I by the lysosomal proteases. We demonstrated that apo A-I could effectively inhibit the acid lipase activity even in the presence of the lysosomal proteases using the hepatic lysosomal fraction.

CBP (CREB-binding protein) is a transcriptional coactivator of CREB (cAMP response element-binding) protein, which is directly phosphorylated by PKA (cAMP-dependent protein kinase A). CBP interacts with the activated phosphorylated form of CREB but not with the nonphosphorylated form. We report here that CBP is also a coactivator of the c-myb proto-oncogene product (c-Myb), which is a sequence-specific transcriptional activator. CBP directly binds to the region containing the transcriptional activation domain of c-Myb in a phosphorylation-independent manner in vitro. The domain of CBP that touches c-Myb is also required for binding to CREB. A c-Myb/CBP complex in vivo was demonstrated by a yeast two-hybrid assay. CBP stimulates the c-Myb-dependent transcriptional activation. Conversely, the expression of antisense RNA of CBP represses c-Myb-induced transcriptional activation. In addition, adenovirus EIA, which binds to CBP, inhibits c-Myb-induced transcriptional activation. Our data thus identify CBP as a coactivator of c-Myb. These results suggest that CBP functions as a coactivator for more transcriptional activators than were thought previously.

A human chromosome 21-specific cosmid library from the Lawrence Livermore National Laboratory has been analyzed by two complementary methods, finger printing and hybridization; 40% coverage of the entire chromosome 21 has been achieved. To prepare a contig pool, approximately 9300 cosmid clones randomly selected from the library were fingerprinted and automatically assembled into 467 overlapping sets by the fluorescence-tagged restriction fragment method. The average size of the overlapping sets was 9.5 cosmids with minimal tiling paths consisting of 5.4 cosmids with a 10-kb extension each. However, as many as 10% of overlaps within members were estimated to be false. For regional localization, we hybridized gridded arrays of cosmids with inter-Alu-PCR probes obtained from YAC clones and somatic cell hybrids and assigned 592 cosmids to 26 subregions of 21q. Of these, 371 clones were incorporated into 139 contigs, anchoring the total 1864 cosmids to the subregion. The remaining 221 clones were mapped as orphans. To correlate the cytogenetic, YAC, and cosmid maps on 21q, the translocation breakpoints of the chromosomes contained in the somatic cell hybrids were mapped with respect to the STS content of the YACs. From the gene cluster regions, 176 ribosomal and 25 alphoid clones were isolated bymore » hybridization. Together, these sets of anchored contigs and cosmids will provide a valuable resource for construction of a high-resolution map and for isolation of genes of interest from chromosome 21. 34 refs., 4 figs., 1 tab.« less

Chymotrypsinogen is widely present in various animal pancreases. To study evolutionary relationship of chymotrypsinogen gene in species, we used a cDNA probe of human prechymotrypsinogen to investigate the species distribution of chymotrypsinogen gene, and designed oligodeoxynucleotide primers to investigate the genomic organization in the three domains of active sites. The genomic analyses showed that chymotrypsinogen gene is evolutionary conserved in species. On the basis of the deduced amino acid residues, a three-dimensional model for human chymotrypsinogen was further built by computer graphics. The model showed high similarity to the X-ray crystal structure of bovine chymotrypsinogen A, thus, demonstrated that the three-dimensional structure is more conserved in evolution than protein sequences.

A genetic variation was found in pancreatic proteinase of Japanese quail. In eight bands of proteinase isozymes, the variation of band 5 (presence or absence) was detected among quails. Band 5 was identified as a chymotrypsin. The presence and absence of band 5 are controlled by a pair of allelic genes (Prt-5A and Prt-5a) on an autosomal locus, and gene Prt-5A, causing expression of band 5, is dominant to gene Prt-5a, a null allele for band 5. Zymograms of proteinases and their zymogens were also compared; no variation of chymotrypsinogen; corresponding to chymotrypsin Prt 5 were detected. It is suggested that the Prt-5 variant of chymotrypsin may be formed during activation of chymotrypsinogen.