Cyanidin-3-O-(2″-xylosyl)-glucoside, an anthocyanin from Siberian ginseng (Acanthopanax senticosus) fruits, inhibits UVB-induced COX-2 expression and AP-1 transactivation

Cyanidin-3-O-(2″-xylosyl)-glucoside (C-3-O-(2″-xylosyl)-G) was analyzed as an active constituent from the fruit of Siberian ginseng (Acanthopanax senticosus) using a HPLC diode array detection-electrospray ionization/mass spectrometry analysis system and the effect of C-3-O-(2″-xylosyl)-G on UVB-induced inflammatory signaling in JB6 P+ cells was investigated. C-3-O-(2″-xylosyl)-G inhibited UVB-induced cyclooxygenase (COX)-2 expression and promoter binding activity in JB6 P+ cells and JB6 P+ cells stably transfected with the COX-2 luciferase reporter plasmid. It inhibited both the UVB-induced activator protein-1 (AP-1) and nuclear factor (NF)-κB transactivation in JB6 P+ cells stably transfected with the AP-1 and NF-κB luciferase reporter plasmids. Additionally, C-3-O-(2″-xylosyl)-G significantly suppressed UVB-induced upregulated phosphorylation of c-Jun N terminal kinase, MEK/extracellular signaling kinase, and mitogen activated protein kinase kinase (MAPKK) 3/6 in JB6 P+ cells. These results indicate that C-3-O-(2″-xylosyl)-G may be a promising chemopreventive material that acts by suppressing COX-2 expression and AP-1 and NF-κB transactivation and JNK, MAPKK3/6, and MEK/ERK1/2 phosphorylation.     

In vivo investigation on the potential of galangin,kaempferol and myricetin for protection of d-galactose-induced cognitive impairment

The potential of three natural flavonols (galangin, kaempferol and myricetin) to protect against d-galactose-induced cognitive impairment in mice was investigated. After 8 weeks treatment, the mice were assessed by behavioural tests. The levels of oxidative stress, the amount of Na⁺,K⁺-ATPase and extracellular signal-regulated kinases (ERK)-cyclic AMP response element binding protein (CREB) signaling pathway in hippocampus were also analysed. It was found that all the three dietary flavonols could ameliorate the oxidative stress, enhance the activity of Na⁺,K⁺-ATPase and regulate the expression of ERK-CREB pathway in mice. However, only kaempferol and myricetin could significantly improve the learning and memory capability when compared with d-galactose model. Our results suggest that the presence of hydroxyl groups in the B ring of flavonols may have contribution to the neuroprotective activity.     

The Hsp90/Cdc37p chaperone system is a determinant of molybdate resistance in Saccharomyces cerevisiae

Saccharomyces cerevisiae lacks enzymes that contain the molybdopterin co‐factor and therefore any requirement for molybdenum as a trace mineral supplement. Instead, high molybdate levels are inhibitory to its growth. Low cellular levels of heat shock protein 90 (Hsp90), an essential chaperone, were found to enhance this sensitivity to molybdate. Certain Hsp90 point mutations and co‐chaperone protein defects that partially compromise the function of the Hsp90/Cdc37p chaperone system also rendered S. cerevisiae hypersensitive to high molybdate levels. Sensitivity was especially apparent with mutations close to the Hsp90 nucleotide binding site, with the loss of the non‐essential co‐chaperone Sti1p (the equivalent of mammalian Hop), and with the abolition of residue Ser14 phosphorylation on the essential co‐chaperone Cdc37p. While it remains to be proved that these effects reflect direct inhibition of the Hsp90 of the cell by the MoO₄²⁺ oxyanion in vivo; this possibility is suggested by molybdate sensitivity arising with a mutation in the Hsp90 nucleotide binding site that does not generate stress sensitivity or an impaired stress response. Molybdate sensitivity may therefore be a useful phenotype to score when studying mutations in this chaperone system. Copyright © 2009 John Wiley & Sons, Ltd.     

Docosahexaenoic acid downregulates phenobarbital-induced cytochrome P450 2B1 gene expression in rat primary hepatocytes via the c-Jun NH2-terminal kinase mitogen-activated protein kinase pathway

Mitogen-activated protein kinase (MAPK) pathways play central roles in the transduction of extracellular stimuli into cells and the regulation of expression of numerous genes. Docosahexaenoic acid (DHA) was shown to be involved in the regulation of expression of drug metabolizing enzymes (DMEs) in rat primary hepatocytes in response to xenobiotics. Cytochrome P450 2B1 (CYP 2B1) is a DME that is dramatically induced by phenobarbital-type inducers. The constitutive androstane receptor (CAR) plays a critical role in regulating the expression of DMEs, and the phosphorylation/dephosphorylation of CAR is an important event in CYP 2B1 expression. In the present study, we determined the effect of DHA on MAPK transactivation and its role in CYP 2B1 expression induced by phenobarbital. c-Jun NH2-terminal kinase (JNK) JNK1/2 and ERK1/2 were activated by phenobarbital in a dose-dependent manner. DHA (100 muM) inhibited JNK1/2 and ERK2 activation induced by phenobarbital in a time-dependent manner. Both SP600125 (a JNK inhibitor) and SB203580 (a p38 MAPK inhibitor) inhibited CYP 2B1 protein and mRNA expression induced by phenobarbital. SB203580 significantly increased the intracellular 3'-5'-cyclic adenosine monophosphate (cAMP) concentration compared with a control group (p < 0.05). Our results suggest that inhibition of JNK activation by DHA is at least part of the mechanisms of DHA's downregulation of CYP 2B1 expression induced by phenobarbital.     

Determination of hyperoside and 2″-acetylhyperoside from Ligularia fischeri by high performance liquid chromatography

Ligularia fischeri and its main flavonoids, hyperoside and 2″-acetylhyperoside, posses antioxidant properties. This study was carried out to investigate the contents of hyperoside and 2″-acetylhyperoside in L. fischeri by using high performance liquid chromatography (HPLC). An HPLC–photodiode array (PDA) detection method was established for the simultaneous determination of hyperoside and 2″-acetylhyperoside in L. fischeri. Two flavonoids were successfully separated in less than 20 min using an YMC RP 18 column. The mobile phase was composed of water (A) and acetonitrile (B) with isocratic elution system (23% B) at a flow rate of 1 mL/min. Their calibration curves showed good linear regression (r > 0.9992) within the test ranges. The method was validated for specificity, accuracy, precision, and limits of detection. The determined two compounds were well separated with a linear range of 18–180 μg/mL. The contents of hyperoside and 2″-acetylhyperoside were 0.387 ± 0.002 and 0.526 ± 0.006 mg/g in L. fischeri, respectively.     

Antidiabetic effect of Ficus racemosa Linn. stem bark in high-fat diet and low-dose streptozotocin-induced type 2 diabetic rats: A mechanistic study

The present study was designed to investigate the effects of the ethanol extract of Ficus racemosa (FRE) on biochemical parameters in type 2-like diabetes, induced by a combination of standardised high-fat diet and low-dose streptozotocin (25 mg kg⁻¹, i.p.) in rats. To elucidate the mode of action of FRE, its effects on a battery of targets involved in glucose homeostasis was evaluated. FRE (200 and 400 mg kg⁻¹, p.o.), in a dose-dependent manner, altered the biochemical parameters and significantly improved glucose tolerance and HDL-c levels. In different bioassays, FRE showed inhibition of PTP-1B (IC₅₀ 12.1 μg/mL) and DPP-IV (42.5%). FRE exhibited 82.6% binding to PPAR-γ. Furthermore FRE exhibited stimulation of glucose uptake by skeletal muscles (hemi-diaphragm). Bergenin was quantified in bioactive-FRE by high-performance liquid chromatography (0.15% w/w). This is the first report demonstrating the effectiveness of F. racemosa stem bark in type 2 diabetes and targets involved in it.     

Tannin 1-alpha-O-galloylpunicalagin induces the calcium-dependent activation of endothelial nitric-oxide synthase via the phosphatidylinositol 3-kinase/Akt pathway in endothelial cells

Many polyphenols have been found to increase endothelial nitric oxide (NO) production. In our present study, we investigated the effects of 1-alpha-O-galloylpunicalagin upon endothelial nitric oxide synthase (eNOS) activity in endothelial cells (ECs). Both 1-alpha-O-galloylpunicalagin and punicalagin induced NO production in a dose-dependent manner in ECs. Despite having similar chemical structures, punicalagin induced lower levels of NO production than 1-alpha-O-galloylpunicalagin. After 1-alpha-O-galloylpunicalagin addition, a rise in the intracellular Ca(2+) concentration preceded NO production. The Ca(2+) ionophore A23187 stimulated eNOS phosphorylation and augmented NO production. Pretreatment with Ca(2+) chelators inhibited 1-alpha-O-galloylpunicalagin-induced eNOS phosphorylation and NO production. Treatment with 1-alpha-O-galloylpunicalagin did not alter the eNOS protein levels but, unlike punicalagin, induced a sustained activation of eNOS Ser(1179) phosphorylation. 1-alpha-O-galloylpunicalagin was also found to activate ERK1/2, JNK and Akt in ECs. Moreover, simultaneous treatment of these cells with specific phosphatidylinositol-3-kinase inhibitors significantly inhibited the observed increases in eNOS activity and phosphorylation levels. In contrast, the inhibition of (ERK)1/2, JNK and p38 had no influence on eNOS Ser(1179) phosphorylation. Our present results thus indicate that the 1-alpha-O-galloylpunicalagin-induced calcium-dependent activation of eNOS is primarily mediated via a phosphatidylinositol 3-kinase/Akt-dependent increase in eNOS activity, and occurs independently of the eNOS protein content.     

Effect of phosphorus on the fruit yield and food value of two landraces of Trichosanthes cucumerina L.- Cucurbitaceae

Studies were conducted in the early season of 2002 and 2003 at the Teaching and Research Farm, Obafemi Awolowo University, Ile-Ife, Nigeria to evaluate the effect of phosphorus (P) on fruit yield and chemical composition of two landraces of Trichosanthes cucumerina L. For the purpose of the study, two landraces of T. cucumerina named Landrace I and Landrace II were used. The five levels of phosphorus evaluated were 0, 30, 60, 90 and 120 kg P₂O₅ ha⁻¹ using single super phosphate fertilizer (8% P). Statistical analysis showed that 90 kg P₂O₅ ha⁻¹ gave statistically significant higher fruit yield (16.4 tons ha⁻¹) compared to other P levels. The fruit yield of the two Landraces did not differ significantly. Except for crude protein content, the 90 kg P₂O₅ ha⁻¹ produced significantly higher ether extract (1.22 g 100 g⁻¹), crude fibre (1.93 g 100 g⁻¹), moisture content (90.5 g 100 g⁻¹), ash (0.90 g 100 g⁻¹), total sugars (0.81 g 100 g⁻¹) and ascorbic acid (28.7 mg 100 g⁻¹) than other P levels. The essential amino acids compositions were also significantly higher at 90 g 100 g⁻¹ compared to other lower P levels. Landrace I had significantly higher ether extract (0.90 g 100 g⁻¹) content than Landrace II (0.62 g 100 g⁻¹) while Landrace II in turn had significantly higher total sugar (0.76 g 100 g⁻¹) compared to Landrace I (0.61 g 100 g⁻¹). The essential amino acids composition is high and the oxalate composition is low. The high ascorbic acid and amino acid content together with a low oxalate composition suggested a strong basis for encouraging the cultivation of this indigenous fruit vegetable to augment nutrient requirement, improve diet and consequently alleviate poverty, preserve the biodiversity and increase the gene bank of neglected wild species of high quality nutrient sources.     

Identification and characterization of a novel fermented substance produced by edible Aspergillus oryzae AO-1 that inhibits DPP-IV activity

We identified a novel fermented substance (FKA) produced by the edible Aspergillus oryzae strain AO-1 that inhibited dipeptidyl peptidase IV (DPP-IV) (IC₅₀ = 3.41 mg·mL^− 1). HPLC analysis of FKA showed specific one metabolite (WYK-1), which inhibited DPP-IV (IC₅₀ = 6.98 μM). WYK-1 was identified as a tetrahydroxyisoquinoline derivative. Interestingly, we examined 60 strains of A. oryzae, AO-1 was the only A. oryzae strain that produces WYK-1. This is the first report that an edible A. oryzae strain produces a DPP-IV inhibitor such as WYK-1. This study also suggests that FKA has applications in the development of novel antihyperglycemic therapeutics or functional foods.     

Characterisation of monoclonal antibody against aflatoxin B1 produced in hybridoma 2C12 and its single-chain variable fragment expressed in recombinant Escherichia coli

An anti-aflatoxin B₁ monoclonal antibody (anti-AFB₁ mAb) from the hybridoma 2C12 was established and its inhibition concentration fifty (IC₅₀) for AFB₁ and relative cross-reactivities (CRs) to other mycotoxins were estimated to be 8 ng/mL and less than 4% compared with AFB₁ by a competitive direct enzyme-linked immunosorbent assay. For production of anti-AFB₁ single-chain variable fragment (anti-AFB₁ scFv) in recombinant Escherichia coli, its scFv-coding genes were cloned from the hybridoma 2C12. The anti-AFB₁ scFv formed inclusion bodies in the cytoplasm of E. coli required in vitro refolding process and hence recovered to retain binding activity successfully. Surface plasmon resonance analysis resulted that anti-AFB₁ scFv possessed 1.16 × 10⁻⁷ M of equilibrium dissociation constant (K_D), which was about 17 times higher than the parental anti-AFB₁ mAb of 6.95 × 10⁻⁹ M.     

Changes in phosphorylation of myofibrillar proteins during postmortem development of porcine muscle

A gel-based phosphoproteomic study was performed to investigate the postmortem (PM) changes in protein phosphorylation of the myofibrillar proteins in three groups of pigs with different pH decline rates, from PM 1 to 24 h. The global phosphorylation level in the group with a fast pH decline rate was higher than that in the slow and intermediate groups at early PM time, but became the lowest at 24 h. The protein phosphorylation level of seven individual protein bands was only significantly (p < 0.05) affected by PM time, and two protein bands were subjected to a synergy effect between PM time and pH decline rate. A total of 35 non-redundant highly abundant proteins were identified from 19 protein bands; most of the identified proteins were sarcomeric function-related proteins. Myosin-binding protein C, troponin T, tropomyosin and myosin regulatory light chain 2 were identified in the highly phosphorylated protein bands with the highest scores. The results indicate that the phosphorylation pattern of myofibrillar proteins in PM muscle is mainly changed with PM time, but only to a minor extent influenced by the rate of pH decline, suggesting that the phosphorylation of myofibrillar proteins may be related to the meat rigor mortis and quality development.     

Production of enniatins A,A1, B,B1, B4, J1 by Fusarium tricinctum in solid corn culture: Structural analysis and effects on mitochondrial respiration

Enniatins (ENs) are secondary fungal metabolites with hexadepsipeptidic chemical structure and they possess a number of potent biological activities that can contaminate several kind of food and foodstuffs increasing the exposure risk for consumers. ENs are produced by several Fusariun strains including Fusarium subglutinans, Fusarium proliferatum and Fusarium tricinctum. Production of a mixture of ENs was performed by culturing F. tricinctum ITEM 9496 on white corn as substrate. The solid culture components were dried and subsequently extracted with water/methanol (50/50 v/v, 0.5% NaCl), homogenised, filtered, extracted by ethyl acetate and analysed by liquid chromatography with diode array detection (LC-DAD). The crude extract was first separated by low pressure liquid chromatography (LPLC) and then further purified by liquid chromatography (LC), resulting in six compounds with a purity higher than 95% as calculated by ¹H NMR, and with a yield of 30–300 mg per compound. The chemical structures of the ENs were determined by liquid chromatography coupled to mass spectrometry (LC–MS) and nuclear magnetic resonance (NMR). The biological activity of the resulting ENs was determined using a mitochondrial respiration test. We discovered that all the ENs studied induced an increase in the mitochondrial respiration resulting in uncoupling of the oxidative phosphorylation. This effect was most likely due to flux of K⁺ ions into the mitochondrial matrix. The order of potency of the ENs derivatives was: A₁ > B₁ > B > A > B₄ > J₁. These results suggest a correlation between the chemical structures and bioactivity and confirm the severe risks for human associated with consumption of enniatins.     

Isolation and characterization of a lectin with antifungal activity from Egyptian Pisum sativum seeds

A plant lectin isolated in its pure state from the Egyptian seeds of Pisum sativum (PSL) produced two bands in SDS–PAGE (5.53 and 19.3 kDa; i.e. α and β chain) but one peak by gel filtration chromatography on Sephadex G-100, corresponding to 50 kDa, i.e., a dimeric structure of two monomers, each consisting of one α and one β subunit. PSL is a glycoprotein bound with glucose (2 mol/mol of protein) and stabilized by 2 atoms of each of Ca²⁺ and Mn²⁺ per molecule of protein. It highly agglutinated human, rabbit and rat erythrocytes but weakly agglutinated chicken erythrocytes, while no agglutination occurred with sheep erythrocytes. Hemagglutination was markedly affected by acidic pH, but was heat stable below 60 °C for 30 min. Among the various tested sugars, PSL agglutination was most inhibited by mannose. PSL is rich in hydroxyl amino acids while totally lacking sulfur amino acids. PSL inhibited the growth of Aspergillus flavus, Trichoderma viride and Fusarium oxysporum.     

Polymorphic AP-1 binding site in bovine CSN1S1 shows quantitative differences in protein binding associated with milk protein expression

Polymorphisms in 5′-flanking regions of milk protein encoding genes can influence the binding activity of the affected response elements and thus have an impact on the expression of the gene products. However, precise quantitative data concerning the binding properties of such variable response elements have so far not been described. In this study we present the results of a quantitative fluorescent electromobility shift assay comparing the allelic variants of a polymorphic activator protein-1 binding site in the promoter region of the bovine α_s1-casein encoding gene (CSN1S1), which is affected by an A→G exchange at −175 bp (CSN1S1^−175bp). A supershift assay using a commercial c-jun antibody was carried out to verify the specificity of protein binding. The gel shift analysis revealed specific and significantly reduced protein binding of oligonucleotides containing the G variant of the CSN1S1^−175bp binding site. Further investigations comprised genotyping of the variable CSN1S1^−175bp activator protein-1 element by an NmuCl restriction fragment length polymorphism in 62 cows of the breed Simmental and 80 cows of the breed German Holstein. Single milk proteins from at least 4 milk samples per cow were quantified by alkaline urea polyacrylamide gel electrophoresis. Homozygotes for CSN1S1^−175bp*G were not observed, and the allele frequencies were 0.19 in Simmental and 0.05 in German Holstein. Carriers of CSN1S1^−175bp*G showed higher content (%) as well as quantity (g/d) of α_s1-casein than CSN1S1^−175bp*A homozygotes, independent of breed. We assume that the positive association of the CSN1S1^−175bp*G variant with CSN1S1 expression is likely to be caused by a reduced affinity of the affected response element to a c-jun-containing CSN1S1 dimer with repressor properties.     

Salicylic acid enhances biocontrol efficacy of Rhodotorula glutinis against postharvest Rhizopus rot of strawberries and the possible mechanisms involved

The potential of using Rhodotorula glutinis alone or in combination with salicylic acid (SA) for the control of postharvest Rhizopus rot of strawberries, and their effects on enzyme activities of fruits were investigated. The combination of R. glutinis (1 × 10⁸ CFU ml⁻¹) with SA (100 μg ml⁻¹) resulted in a significant reduction in the disease incidence and lesion diameter of Rhizopus rot on the strawberry fruits at 20 °C and 4 °C, and more so than with SA or yeast alone. SA at the concentration of 100–1000 μg ml⁻¹ significantly inhibited spore germination of Rhizopus stolonifer. About 100 μg ml⁻¹ of SA did not inhibit the growth of the antagonistic yeast, and could significantly increase the population growth of R. glutinis in strawberry wounds at 20 °C. SA, combined with R. glutinis, increased the activity of strawberry host defence enzymes (POD) and cell wall lytic enzymes (β-1,3-glucanase).     

Effects of Lactobacillus kefiranofaciens M1 isolated from kefir grains on enterohemorrhagic Escherichia coli infection using mouse and intestinal cell models

A potential probiotic strain, Lactobacillus kefiranofaciens M1, was previously isolated from kefir grains, which are used to manufacture the traditional fermented drink kefir. The aim of this study was to investigate the effects of Lb. kefiranofaciens M1 on enterohemorrhagic Escherichia coli (EHEC) infection, using mice and intestinal cell models. BALB/c mice were daily administrated with either phosphate buffered saline or Lb. kefiranofaciens M1 at 2 × 10⁸ cfu/mouse per day intragastrically for 7 d. Intragastric challenges with EHEC (2 × 10⁹ cfu/mouse) were conducted on d 0, 4, and 7 after treatment. Administration of Lb. kefiranofaciens M1 was able to prevent EHEC infection-induced symptoms, intestinal damage, renal damage, bacterial translocation, and Shiga toxin penetration. Furthermore, the mucosal EHEC-specific IgA responses were increased after Lb. kefiranofaciens M1 administration in the EHEC-infected mouse system. Additionally, in vitro, Lb. kefiranofaciens M1 was shown to have a protective effect on Caco-2 intestinal epithelial cells and Caco-2 intestinal epithelial cell monolayers; the bacteria limited EHEC-induced cell death and reduced the loss of epithelial integrity. These findings support the potential of Lb. kefiranofaciens M1 treatment as an approach to preventing EHEC infection and its effects.     

The polymorphisms of stearoyl-CoA desaturase (SCD1) and sterol regulatory element binding protein-1 (SREBP-1) genes and their association with the fatty acid profile of muscle and subcutaneous fat in Fleckvieh bulls

The previously reported genetic polymorphisms of the stearoyl-CoA desaturase (SCD1) and sterol regulatory element binding protein-1 (SREBP-1) genes were investigated in Fleckvieh bulls using the PCR-RFLP and AS-PCR methods, respectively. The genomic DNA was obtained from a total of 370 bulls. The frequencies of alleles A and V of the single nucleotide polymorphism in exon 5 of the SCD1 gene (SNP 878C>T) were 0.555 and 0.445, respectively. In the 84-bp Ins/Del polymorphism in intron 5 of the SREBP-1 gene, the frequency of the L allele (insertion) was markedly higher (0.920) than that of the S allele (deletion; 0.080). Fatty acid profile was determined in a total of 367 samples of muscle fat (MSF) and 150 samples of subcutaneous fat (SCF). The AA genotype of SCD1 polymorphism showed a lower content of C18:0 (P < 0.01) and higher contents of C14:1 cis-9 (P < 0.001) and C18:1 cis-9 (P < 0.05) in MSF compared to the VV genotype. As a result, the bulls with genotypes AA or AV had lower SFA (P < 0.01), higher MUFA (P < 0.05) and higher MUFA/SFA (P < 0.01) than VV animals. The results obtained for SCF were similar. The SREBP-1 polymorphism was associated with a higher content of C14:1 cis-9 (P < 0.01) in the LS compared to LL genotype in SCF. The results of this study demonstrated the existence of the polymorphisms in the SCD1 and SREBP-1 genes in the population of Fleckvieh cattle and their associations with the concentrations of several MSF and SCF fatty acids.