Major urinary protein 5, a scent communication protein, is regulated by dietary restriction and subsequent re-feeding in mice.

Major urinary proteins (Mups) are important for rodent scent communication and sexual behaviour. Recent evidence suggests that Mup1 may be regulated by fasting and re-feeding (RF). However, other Mup isoforms are poorly investigated, and data on the impact of long-term dietary restriction (DR) and ad libitum RF on Mup expression are missing. We investigated the effects of long-term 25 per cent DR and subsequent RF on Mup expression in male C57BL6 mice. DR significantly decreased Mup gene expression, hepatic and urinary protein levels compared with ad libitum (AL) fed control mice, with the greatest downregulation found for Mup5 expression. The decline in Mup expression was inverted by six months of RF. Because of inhibitory glucocorticoid response elements in the genomic sequence of the Mup5 gene, the observed inverse correlation of nuclear glucocorticoid receptor levels with Mup expression in response to DR and subsequent RF is a possible regulatory mechanism. Additionally, gene-expression-inhibiting histone deacetylation (H3K9) occurred in the region of the Mup5 gene in response to DR. We assume that Mup may act as a molecular switch linking nutritional status to sexual behaviour of mice, and thereby regulating male fertility and reproduction in response to food supply.

Effect of quercetin on traits of the metabolic syndrome, endothelial function and inflammation in men with different APOE isoforms.

BACKGROUND AND AIMS: The polyphenol quercetin may prevent cardiovascular diseases due to its vasorelaxant and anti-oxidative properties. We investigated the effects of quercetin on risk factors of atherosclerosis, biomarkers of inflammation and oxidative stress, depending on the apolipoprotein E (APOE) genotype. METHODS AND RESULTS: In a double-blind crossover study 49 healthy male subjects with APOE genotype 3/3 (n = 19), 3/4 (n = 22) and 4/4 (n = 8) consumed 150 mg/d quercetin or placebo for 8 weeks each, intermitted by a three-week washout phase. After each intervention, endothelial function, anthropometry, metabolic and inflammatory parameters were measured in the fasting and postprandial state following a standardized lipid-rich meal. Endothelial function was not changed. In all subjects combined, quercetin significantly decreased waist circumference (P = 0.004) and postprandial systolic blood pressure (P = 0.044). Postprandial triacylglycerol concentrations were significantly decreased and HDL-cholesterol concentrations increased after quercetin as compared to placebo consumption (P = 0.025). Quercetin also moderately increased levels of TNFα (P = 0.024). There was a significant gene-diet interaction for waist circumference and for body mass index (BMI). CONCLUSIONS: Quercetin supplementation improved some risk factors of cardiovascular disease, yet exerted slightly pro-inflammatory effects. Genotype-dependent effects were seen only on waist circumference and BMI.

In-vitro antidiabetic activity of a Bistorta officinalis Delarbre root extract can not be confirmed in the in-vivo models hen's egg test and Drosophila melanogaster.

The potential of plant bioactives for the prevention and therapy of diabetes is increasingly being recognized. In the present study we investigated the antidiabetic properties of an aqueous Bistorta officinalis Delarbre extract (BODE) by employing both in-vitro assays and in-vivo models. Multiple targets in glucose homeostasis which are involved in the regulation of the blood glucose level were affected by BODE in-vitro. The extract exhibited inhibitory activities towards the intestinal carbohydrate-hydrolysing enzymes α-amylase and α-glucosidase with IC50 values of 81.5 µg/mL and 8.4 µg/mL, respectively. Furthermore, moderate reduction of the dipeptidyl peptidase-4 (DPP4) enzyme activity was evident when tested in the presence of 1.0 mg/mL BODE. A significant inhibition of the intestinal glucose transporter sodium-dependent glucose transporter 1 (SGLT1) in response to 1.0 mg/mL BODE was shown for Caco-2 cells mounted in Ussing chambers. High performance liquid chromatography-mass spectrometry analyses of the BODE revealed several plant bioactives including gallotannins, catechins and chlorogenic acid. Although our in-vitro data were promising, BODE-supplementation in the model organism Drosophila melanogaster lacked to confirm the antidiabetic effect of the extract in-vivo. Moreover, BODE failed to reduce blood glucose levels in chicken embryos (in-ovo). Hence, BODE is probably not a suitable candidate for developing a pharmaceutical against diabetes mellitus.

Cyanidin does not affect sulforaphane-mediated Nrf2 induction in cultured human keratinocytes.

There is increasing interest in the gene-regulatory activities of isothiocyanates and flavonoids in human skin. Nrf2 agonists, such as isothiocyanate sulforaphane (SFN), have been shown to promote chemopreventive effects in skin both in vitro and in vivo. Recent data indicate that different secondary plant compounds may either antagonise or enhance SFN-induced Nrf2 activation. We therefore studied the interactions of a flavonoid, cyanidin and the potent Nrf2 inductor SFN in cultured human keratinocytes (HaCaT cells). We observed that cyanidin does not induce the activation of Nrf2 and its target genes, γ-glutamylcysteine synthetase (γGCS), NAD(P)H:quinone oxidoreductase 1 and haem oxygenase-1 in HaCaT cells. Furthermore, SFN-mediated Nrf2 activation and its target gene expression were not further enhanced by the co-application of SFN with cyanidin.

A postbiotic from Aspergillus oryzae attenuates the impact of heat stress in ectothermic and endothermic organisms.

Heat stress is detrimental to food-producing animals and animal productivity remains suboptimal despite the use of heat abatement strategies during summer. Global warming and the increase of frequency and intensity of heatwaves are likely to continue and, thus, exacerbate the problem of heat stress. Heat stress leads to the impairment of physiological and cellular functions of ectothermic and endothermic animals. Therefore, it is critical to conceive ways of protecting animals against the pathological effects of heat stress. In experiments with endothermic animals highly sensitive to heat (Bos taurus), we have previously reported that heat-induced systemic inflammation can be ameliorated in part by nutritional interventions. The experiments conducted in this report described molecular and physiological adaptations to heat stress using Drosophila melanogaster and dairy cow models. In this report, we expand previous work by first demonstrating that the addition of a postbiotic from Aspergillus oryzae (AO) into the culture medium of ectothermic animals (Drosophila melanogaster) improved survival to heat stress from 30 to 58%. This response was associated with downregulation of genes involved in the modulation of oxidative stress and immunity, most notably metallothionein B, C, and D. In line with these results, we subsequently showed that the supplementation with the AO postbiotic to lactating dairy cows experiencing heat stress decreased plasma concentrations of serum amyloid A and lipopolysaccharide-binding protein, and the expression of interleukin-6 in white blood cells. These alterations were paralleled by increased synthesis of energy-corrected milk and milk components, suggesting enhanced nutrient partitioning to lactogenesis and increased metabolic efficiency. In summary, this work provides evidence that a postbiotic from AO enhances thermal tolerance likely through a mechanism that entails reduced inflammation.

Cellular uptake, stability, visualization by 'Naturstoff reagent A', and multidrug resistance protein 1 gene-regulatory activity of cyanidin in human keratinocytes.

There is increasing interest in the role of anthocyanidins as potential skin protective phytochemicals. However, little is known if and to what extent anthocyanidins are taken up by the human skin. In the present study cellular uptake (as determined by HPLC), stability, and gene-regulatory activity of cyanidin were determined in human HaCaT keratinocytes in culture. Using the fluorescent dye Naturstoff reagent A cyanidin was visualized in order to determine its cellular accumulation via flow cytometry and fluorescence microscopy. Cyanidin was rapidly taken up by HaCaT cells at relatively low concentrations. Following incubation, cellular cyanidin levels decreased time-dependently most likely due to degradation into protocatechuic acid and phloroglucinol aldehyde. Confocal laser scanning microscopy data demonstrated that cyanidin was mainly present in the cytoplasm. Cellular uptake of cyanidin was accompanied by an inhibition of multidrug resistance protein 1 (involved in cellular efflux of flavonoids) mRNA-levels indicating its gene-regulatory activity. Naturstoff reagent A seems to be a promising fluorescent dye to visualize cyanidin in keratinocytes.

Dietary flavonoids do not affect vitamin E status in growing rats.

This study aimed at investigating potential effects of the flavonoids genistein, quercetin and catechin and the role of co-ingested dietary fat on vitamin E concentrations in rats. In experiment 1, genistein, quercetin and catechin were fed to rats, incorporated into semisynthetic diets at concentrations of 2 g/kg, either as individual compounds or in combination to investigate their individual and possible synergistic actions towards alpha-tocopherol in plasma and selected tissues. For experiments 2 and 3, quercetin was selected as a representative model flavonoid to study the effects of the quantity (5% vs. 10%) and type of dietary fat (coconut fat plus corn oil vs. rapeseed oil; experiment 2) and the role of cholesterol (experiment 3) on potential flavonoid-vitamin E interactions. The concentrations of alpha-tocopherol and gamma-tocopherol in the plasma, liver, lung and cortex of flavonoid-fed rats were not significantly different from the concentrations measured in control rats in all three experiments. However, increasing the amount of coconut fat plus corn oil from 5 to 10% resulted in lower alpha-tocopherol concentrations in plasma and tissue. The alpha-tocopherol concentrations in the rats fed rapeseed oil were significantly higher than in rats fed coconut fat plus corn oil. The addition of 0.2% cholesterol to the diet did not influence the tocopherol concentrations in plasma and tissue in both quercetin-supplemented and control rats. Additionally, the mRNA levels of alpha-TTP, CYP3A4, CYP4F and Mdr2, which are integral proteins involved in vitamin E homeostasis were measured. Only genistein reduced the Mdr2 mRNA level, but none of the other transcripts. All other flavonoids were without effect. In conclusion, co-ingested dietary fat appears to influence vitamin E concentrations in rats, but does not seem to be an important determinant of flavonoid-vitamin E interactions.

Effects of dietary gamma-cyclodextrin on voluntary activity and muscle strength in mice.

Gamma-cyclodextrin (γCD) is a cyclic oligosaccharide consisting of eight α-(1,4)-linked glucopyranose subunits, which is often used in the food and pharmaceutical industries. However, little is known regarding the metabolic activity of "empty" γCD per se. Therefore, in the present study young C57BL/6 male mice received a control diet (CON) or an experimental diet that was supplemented with 12.88% γCD exchanged against corn starch. After 6 weeks of treatment, the voluntary wheel running activity was monitored and the muscle strength of mice was measured by employing Kondziela's inverted screen test and forelimb grip strength assay. The γCD-treated mice covered a significantly larger distance per night (CON 8.6 km, γCD 12.4 km) and were significantly longer active (CON 340 min, γCD 437 min). Moreover, γCD-treated mice significantly performed better at the inverted screen test indicated by an enhanced Kondziela score (CON 3.10, γCD 4.63). These data suggest that dietary γCD leads to an increased endurance. We also found a slightly anti-glycemic effect of γCD during oral glucose tolerance test. However, our mice from the γCD group exhibited no difference in terms of GLUT2 protein level in ileum tissue nor increased muscle glycogen storage. Furthermore, γCD exhibited no DPP-4 inhibitory activity in vitro. By analysing candidate muscle genes and proteins related to endurance and muscle performance we did not observe any differences in terms of Sirt1, Pgc1α, Cpt1b, Mef2c, Myh1 and Myh2 gene expression levels as well as total oxidative phosphorylation (OXPHOS), mtTFA and GLUT4 protein expression levels in skeletal muscle in response to γCD. We could not fully establish the exact underlying molecular mechanisms of the fitness improvement by dietary γCD which warrants further investigations.

Ochratoxin A impairs Nrf2-dependent gene expression in porcine kidney tubulus cells.

The mycotoxin, ochratoxin A (OTA), which is produced by Aspergillus and Penicillium subspecies, is frequently present in feedstuffs. Ochratoxin A exhibits a wide range of toxic activities including nephrotoxicity. However, the underlying molecular mechanisms of OTA-induced cellular nephrotoxicity have yet not been fully elucidated. Nrf2 is a basic leucine zipper transcriptional activator essential for the coordinated transcriptional induction of antioxidant and xenobiotic metabolizing enzymes in the kidney. Therefore, in the present study, the effects of OTA on the nuclear translocation and transactivation of the transcription factor Nrf2 as well as mRNA levels of Nrf2 target genes including glutathione-S-transferase and gamma-glutamylcysteinyl synthetase have been studied in cultured porcine kidney tubulus cells (LLC-PK1). Nrf2 was induced by sulforaphane, a well-known activator of this transcription factor. Ochratoxin A significantly decreased gamma-glutamylcysteinyl synthetase and glutathione-S-transferase mRNA levels in LLC-PK1 cells. Decreased mRNA levels of gamma-glutamylcysteinyl synthetase and glutathione-S-transferase were accompanied by a lowered nuclear translocation and transactivation of Nrf2. Furthermore, OTA also lowered Nrf2 mRNA levels. Current data indicate that OTA nephrotoxicity may be, at least partly, mediated by an Nrf2-dependent signal transduction pathway.

Effects of resveratrol and genistein on growth, nutrient utilization and fatty acid composition of rainbow trout.

The replacement of the finite and costly resource fish oil is an important task for aquaculture nutrition. A promising approach could be the use of plant bioactives that may have the potential to influence the metabolism and the synthesis of n-3 long chain polyunsaturated fatty acids, especially EPA (20:5n-3) and DHA (22:6n-3). In this study, the two phytochemicals resveratrol (RV) and genistein (G) were investigated for their effects on fish growth, nutrient utilization and body nutrient composition alongside their effects on whole body fatty acid (FA) composition. In a feeding trial lasting 8 weeks, rainbow trout (initial BW: 81.4±0.5 g) were held in a recirculating aquaculture system and fed six experimental diets with varying fish oil levels as plain variants or supplemented with 0.3% of dry matter (DM) of either RV or G. The six diets were as follows: diet F4 had 4% DM fish oil, diet F0 had 0% DM fish oil, diets F4+RV, F4+G, F0+RV and F0+G were equal to the diets F4 and F0, respectively, and supplemented with the phytochemicals RV and G. The feeding of the F0+RV diet resulted in reduced feed intake, growth rate and slightly reduced whole body lipid levels. At the same time, the amount of polyunsaturated FA and the n-3/n-6 ratio were significantly increased in whole body homogenates of rainbow trout fed diet F0+RV in comparison to the F0 control. The feeding of the F0+G diet led to reduced feed intake, slightly increased protein utilization but did not significantly affect the whole body FA composition. Overall, feeding the fish oil-free diet supplemented with the phytochemicals resulted in more pronounced effects on fish performance and FA composition than the single factors per se (dietary fish oil level or phytochemical). Present data indicate that G might not be of profitable use for trout nutrition. In terms of FA composition, RV could be a potentially useful complement for fish oil. However, the impairment of growth and performance parameters as observed in the present study discourages its use in trout diets.

Dietary resveratrol impairs body weight gain due to reduction of feed intake without affecting fatty acid composition in Atlantic salmon.

Recent studies suggest that the use of vegetable oils at expense of fish oil in aquaculture feeds might have potential negative effects on fish redox homeostasis and adiposity. Resveratrol (RESV) is a lipid-soluble phytoalexin present in fruits and vegetables with proven in vivo antioxidant function in animals. The present study aims to assess the potential use of RESV in Atlantic salmon feeds. To this end, post-smolt salmons with an initial BW of 148±3 g were fed four experimental diets for 15 weeks. A diet low in fish oil served as a control and was supplemented with 0, 0.5, 1.5 and 2.5 g/kg of RESV, respectively. The effect of the experimental diets on animal performance, tissue fatty acid composition, and the expression of genes encoding proteins involved in antioxidant signalling, lipid peroxidation, and metabolism were studied. Resveratrol significantly reduced feed intake and final BW of the salmon. Feeding RESV did not affect the sum of saturated and monounsaturated fatty acids or total lipids in the fillet. While the content of total polyunsaturated fatty acids was not affected, the percentages of some fatty acids in the liver and fillet were changed by RESV. Furthermore, in liver, the relative expression of glutathione peroxidase 4b, nuclear factor-like 2, and arachidonate 5-lipoxygenase remained unchanged across treatment groups. In conclusion, the negative impact of dietary RESV on FI and hence reduction of the BW discourages its inclusion in low fish oil diets for Atlantic salmon.

Effect of ochratoxin A on redox-regulated transcription factors, antioxidant enzymes and glutathione-S-transferase in cultured kidney tubulus cells.

Ochratoxin A (OTA), a mycotoxin mostly produced by Aspergillus ochraceus and Penicillium verrucosum, is a worldwide contaminant of food and feedstuff. OTA is nephrotoxic and a renal carcinogen in rodents. The underlying molecular and cellular mechanisms by which OTA exhibits its toxicity have yet not been fully clarified. In the present study the effects of ochratoxin A on the activity of redox-regulated transcription factors, antioxidant enzymes, as well as glutathione-S-transferase (GST) have been studied in cultured kidney tubulus cells (LLC-PK1). Confluent LLC-PK1 cells were incubated with increasing concentrations of OTA for 24h. OTA decreased SOD activity and enhanced intracellular levels of reactive oxygen species (ROS) as measured by flow cytometry. Furthermore OTA resulted in a down-regulation of GST mRNA and activity levels. Lower GST levels were accompanied by a decreased transactivation of activator protein-1 (AP-1) and NF-E2-related factor-2 (Nrf2), which mediate GST gene transcription. Present data indicate that enhanced ROS production and an impairment of GST activity, possibly due to an AP-1 and Nrf2 dependent signal transduction pathway, may be centrally involved in OTA induced nephrotoxicity.

Dietary green tea polyphenols do not affect vitamin E status, antioxidant capacity and meat quality of growing pigs.

Supplementation of pigs with vitamin E, the most important lipid-soluble antioxidant, has been shown to improve meat quality and animal health. Previous studies in cultured cells and laboratory animals indicate synergistic effects between polyphenols and vitamin E. The present feeding trial was undertaken to investigate the effects of dietary green tea polyphenols (GTP) on vitamin E status, antioxidative capacity and parameters of meat quality in growing pigs. Eighteen castrated, crossbred, male pigs received a flavonoid-poor diet based on corn starch, caseinate and rapeseed oil with a total vitamin E content of 17 IU/kg diet over a period of 5 weeks. This basal diet was supplemented with green tea extract to provide daily doses of 0 (control), 10 and 100 mg GTP/kg body weight. Dietary supplementation of growing pigs with GTP did not affect serum, liver, lung and muscle vitamin E (alpha- and gamma-tocopherol) concentrations, plasma antioxidant capacity (ferric reducing ability of plasma, trolox equivalent antioxidant capacity) or parameters of meat quality including meat temperature, pH, conductivity, colour and drip loss. In conclusion, supplementation of pig diets with green tea catechins is not associated with improved antioxidant status and meat quality under practice-oriented conditions.

Effects of a six-week intraduodenal supplementation with quercetin on liver lipid metabolism and oxidative stress in peripartal dairy cows.

The purpose of this study was to evaluate possible effects of quercetin (Q) on liver lipid metabolism and antioxidative status in periparturient dairy cows. The periparturient period is associated with enormous metabolic changes for dairy cows. Energy needs for incipient lactation are too high to be balanced by feed intake, leading to negative energy balance and body fat mobilization. It has been estimated that this leads to the development of fatty liver in about 50% of cows, which are at high risk for disease. Furthermore, the antioxidative status of these cows may be impaired. Quercetin is a plant flavonoid having hepatoprotective and antioxidative potential and the ability to reduce liver lipid accumulation in monogastric animals. Little information is available in regard to these effects in ruminants. To prevent microbial Q degradation in the rumen, Q was administered via a duodenal fistula to improve systemic availability. Five cows of the Q-treated group received, daily, 100 mg of quercetin dehydrate/kg BW in a 0.9% sodium chloride solution from d -20 until d 20 relative to calving, whereas 5 control (CTR) cows received only a sodium chloride solution. Blood samples were taken weekly and liver biopsies were performed in wk -4, -2, and 3 relative to calving. Cows treated with Q showed a tendency ( = 0.082) for lower liver fat content compared with CTR cows. Liver glycogen, glutathione concentrations, and relative mRNA abundance of genes related to hepatic lipid metabolism and antioxidative status as well as parameters of antioxidative status in plasma were not affected ( > 0.1) by Q supplementation. In conclusion, liver fat content in dairy cows tended to be reduced by Q supplementation, but potential underlying mechanisms remain unclear because analyzed parameters related to hepatic lipid metabolism and antioxidative defense were not altered by Q supplementation.

Antioxidant activity and biologic properties of a procyanidin-rich extract from pine (Pinus maritima) bark, pycnogenol.

There is growing interest in the biologic activities of plant extracts such as that obtained from the bark of the French maritime pine Pinus maritima, Pycnogenol. Pycnogenol (PYC) is a standardized extract composed of a mixture of flavonoids, mainly procyandins and phenolic acids. Studies indicate that PYC components are highly bioavailable. Uniquely PYC displays greater biologic effects as a mixture than its purified components do individually indicating that the components interact synergistically. PYC has been reported to have cardiovascular benefits, such as a vasorelaxant activity, angiotensin-converting enzyme (ACE) inhibiting activity, and the ability to enhance the microcirculation by increasing capillary permeability. Investigations of the cellular mechanisms of these therapeutic effects have demonstrated that PYC has strong free radical-scavenging activity against reactive oxygen and nitrogen species. The oligomeric components of PYC contribute significantly to the ESR free radical signal. PYC also participates in the cellular antioxidant network as indicated by its ability to regenerate the ascorbyl radical and to protect endogenous vitamin E and glutathione from oxidative stress. PYC modulates NO metabolism in activated macrophages by quenching the NO radical and inhibiting both iNOS mRNA expression and iNOS activity. The spectrum of different effects of NO in the circulation and the nervous system suggest the potential applications of PYC in immune and circulatory disorders as well as in neurodegenerative disease. PYC can bind to proteins, altering their structure and thereby modulating the activity of key enzymes and proteins involved in metabolic pathways. PYC effects redox-sensitive signal transduction pathways and alters gene expression. Aspects of PYC's activity are presented and discussed together with possible future implications and directions in the field of flavonoid research.

Activity of monomeric, dimeric, and trimeric flavonoids on NO production, TNF-alpha secretion, and NF-kappaB-dependent gene expression in RAW 264.7 macrophages.

Flavonoids are potent antioxidants and have been associated with lowering the risk of cardiovascular diseases. In this study, the effect of flavonoids (monomers, dimers and a trimer) as well as French maritime pine bark extract, Pycnogenol, on NO production, tumor necrosis factor-alpha (TNF-alpha) secretion and nuclear factor (NF)-kappaB activity was compared. Monomers and dimers repressed NO production, TNF-alpha secretion and NF-kappaB-dependent gene expression induced by interferon gamma, whereas the trimeric procyanidin C2 and Pycnogenol enhanced these parameters. In addition, in unstimulated RAW 264.7 macrophages, both procyanidin C2 and Pycnogenol increased TNF-alpha secretion in a concentration- and time-dependent manner. These results demonstrate that procyanidins act as modulators of the immune response in macrophages.

Ferulic acid excretion as a marker of consumption of a French maritime pine (Pinus maritima) bark extract.

French maritime pine (Pinus maritima) bark extract (PBE) is a polyphenol-rich food supplement patented under the name of Pycnogenol and known to have strong antioxidant activity and different beneficial effects on human health. Although its biological properties have begun to be extensively studied both in vitro, in laboratory animals and more recently in humans, little is known about its bioavailability. The present study investigated the urinary excretion of free and conjugated ferulic acid, present in quantitatively detectable amounts in PBE, after oral PBE administration to human subjects. Eleven healthy adult subjects (4 women and 7men) consumed either a single dose (200 mg PBE) or two doses of PBE (100 and 200 mg, respectively) within a 48-h interval. Two days before the oral administration of PBE and during the urine sample collection period volunteers adhered to a diet low in polyphenols. Aliquots of all urine production were collected over 24 h. Free and conjugated ferulic acid was assessed in urine by HPLC using diode array detection. A close association between the dietary intake of PBE and the urinary excretion of ferulic acid was detected. Moreover, the results indicate that a considerable proportion of ferulic acid is excreted as glucuronide or sulfate after PBE consumption, varying over the range 2 to 20% between individuals. The kinetics of excretion associated with the administration of 100 mg PBE was quite similar to that obtained after 200 mg PBE. A a biphasic trend was evident in a number of subjects. All subjects studied here displayed a significant, although variable level of excretion of ferulic acid after supplementation with PBE, Thus, the data provide evidence that at least a part of the phenolic components of PBE are absorbed, metabolized, and eliminated by humans.

Solar ultraviolet-induced erythema in human skin and nuclear factor-kappa-B-dependent gene expression in keratinocytes are modulated by a French maritime pine bark extract.

The procyanidin-rich French maritime pine bark extract Pycnogenol (PBE) has been investigated for its effect in protecting human skin against solar UV-simulated light-induced erythema. Twenty-one volunteers were given an oral supplementation of Pycnogenol: 1.10 mg/kg body weight (b. wt.)/d for the first 4 weeks and 1.66 mg/kg b. wt./d for the next 4 weeks. The minimal erythema dose (MED) was measured twice before supplementation (baseline MED), once after the first 4 weeks of supplementation, and a last time at the end of the study. The UVR dose necessary to achieve 1 MED was significantly increased during PBE supplementation. Since the activation of the pro-inflammatory and redox-regulated transcription factor NF-kappaB is thought to play a major role in UVR-induced erythema, the effect of PBE was also investigated in the human keratinocyte cell line HaCaT. PBE, added to the cell culture medium, inhibited UVR-induced NF-kappaB-dependent gene expression in a concentration-dependent manner. However, NF-kappaB-DNA-binding activity was not prevented, suggesting that PBE affects the transactivation capacity of NF-kappaB. These data indicate that oral supplementation of PBE reduces erythema in the skin. Inhibition of NF-kappaB-dependent gene expression by PBE possibly contributes to the observed increase in MED.

Heat shock and 5-azacytidine inhibit nitric oxide synthesis and tumor necrosis factor-alpha secretion in activated macrophages.

To elucidate the role of stress response during macrophage activation, the effects of heat shock and the amino acid analog, 5-azacytidine on nitric oxide (NO) production, tumor necrosis factor-alpha (TNF-alpha) secretion, and heat shock protein (HSP) synthesis have been studied in murine peritoneal macrophages (C57BL/6). Heat shock (1 hr at 43 degrees C) or 5-azacytidine markedly inhibited the release of NO into the medium from interferon-gamma (IFN-gamma) plus lipopolysaccharide (LPS)-stimulated macrophages. Although heat shock significantly decreased TNF-alpha secretion only at the initiation stage of macrophage stimulation, 5-azacytidine treatment resulted in a more prolonged reduction in the secretion of TNF-alpha. When heat-shocked cells were stimulated with IFN-gamma plus LPS under normal culture conditions at 37 degrees C, the heat shock-induced inhibition of NO release reversed progressively with increasing recovery time. Although the total amount of cellular HSP72 measured by Western blot increased time-dependently over 7 hr, newly synthesized HSP72 measured by [35S]methionine incorporation was evident only after 1 and 3 hr of recovery time after heat shock treatment. At these time points, the lowest nitrite accumulation and TNF-alpha secretion into the medium was evident. It is concluded that signaling pathways related to newly synthesized HSP such as HSP72 are implicated in the down regulation of NO synthesis and TNF-alpha secretion in macrophages.

Interaction between cultured endothelial cells and macrophages: in vitro model for studying flavonoids in redox-dependent gene expression.

This article focused on two methods to measure the activity of NF-kB. Both methods evalute "post-IkB phosphorylation" stages in the NF-kB activation cascade. In fact, EMSA performed with nuclear extracts provides an information only on NF-kB nuclear translocation and its ability to bind kB-DNA sequences. Likewise, the reporter gene assay is limited to assessing NF-kB-dependent gene expression no matter the mechanism that originally activated NF-kB. Nevertheless, the latter assay represents a more physiological and more reproducible way of measuring NF-kB activity in mammalian cells than the EMSA does. In order to obtain further insights into NF-kB signal transduction pathways, investigating IkB degradation and phosphorylation are recommended. The cloning and characterization of IkB kinases provided new testing possibilities based on measure of their activity.