Chewing unflavored gum does not reduce cortisol levels during a cognitive task but increases the response of the sympathetic nervous system.

OBJECTIVES: Stress might be caused by various lifestyle factors and physical challenges and can result in severe diseases. The body responds to stressful events by release of hormones, like cortisol, as well as reaction of the sympathetic nervous system. One strategy to counteract stress is chewing gum. The present study aimed at investigating the influence of mastication on biomarkers of stress during performance of a stress test. METHODS: A two-armed cross-over study with 40 young male volunteers was performed. Hormone plasma concentrations were determined after an initial resting phase (2:30p.m.), immediately before (3:00p.m.) and two times after (3:20, 3:50p.m.) performance of a multitasking test using magnetic beads and ELISA methods. In addition, visual analog scales were used to rate subjective mood and the breathing and heart rates were monitored throughout the entire study period using a sensor chest belt. RESULTS: Performance of the stress test led to an increase in plasma cortisol concentrations from 223±23.3 to 325±38.4ng/ml (p=0.023) and caused changes in subjective mood ratings as well as breathing rates. Although chewing gum base had no impact on the plasma hormone concentrations, it induced a stronger elevation of average heart rates compared to not chewing (p=0.016). DISCUSSION: The effect of chewing gum on a mild stress load was less pronounced than in previous studies. Besides the detection of cortisol in saliva, not in plasma, in previous studies, flavored gum was used. Aroma active compounds might have substantially contributed to the beneficial effects of gum on biomarker of stress shown before.

Identification of Magnolia officinalis L. bark extract as the most potent anti-inflammatory of four plant extracts.

This study was designed to compare the anti-inflammatory potential of a Magnolia officinalis L. bark extract solely or in combination with extracts prepared from either Polygonum aviculare L., Sambucus nigra L., or Isodon japonicus L. in bacterial lipopolysaccharide (LPS) stimulated human gingival fibroblasts (HGF-1) and human U-937 monocytes, as cell models of periodontal disease. HGF-1 and U-937 cells were incubated with LPS from either Porphyromonas gingivalis or Escherichia coli together with the four plant extracts alone or in combination. Secretion of anti-inflammatory cytokines from HGF-1 and U-937 cells was measured by means of a multiplexed bead assay system. Magnolia officinalis L. bark extract, at concentrations of 1 µg/mL and 10 µg/mL, reduced interleukin 6 (IL-6) and interleukin-8 (IL-8) secretion from HGF-1 cells to 72.5 ± 28.6% and reduced matrix metalloproteinase 2 (MMP-2) and matrix metalloproteinase 9 (MMP-9) secretion from U-937 cells to 8.87 ± 7.97% compared to LPS-treated cells (100%). The other three extracts also reduced secretion of these inflammatory markers but were not as effective. Combination of 9 µg/mL Magnolia officinalis L. extract with 1 µg/mL of each of the other extracts maintained the anti-inflammatory effect of Magnolia officinalis L. extract. Combination of 5 µg/mL Magnolia officinalis L. extract with 5 µg/mL Isodon japonicus L. extract also maintained the anti-inflammatory potential of the Magnolia officinalis L. extract, whereas increasing concentrations of any of the other plant extracts in the combination experiments reduced the Magnolia officinalis L. extract efficacy in U-937 cells.

Identification of 1,8-cineole, borneol, camphor, and thujone as anti-inflammatory compounds in a Salvia officinalis L. infusion using human gingival fibroblasts.

Drinking or gargling Salvia officinalis L. infusion (sage infusion) is thought to soothe a sore throat, tonsillitis, and inflamed, red gums, although structure-based scientific evidence for the key anti-inflammatory compounds in sage infusion is scarce. Human gingival fibroblasts (HGF-1) were treated with sage infusion (SI) or SI fractions containing either its volatile components and water (aqueous distillate, AD) or its dry matter (DM) for six hours. SI, AD, and DM reduced a mean phorbol-12-myristate-13-acetate/ionomycin (PMA/I)-stimulated release of the pro-inflammatory interleukins IL-6 and IL-8 by more than 50% (p < 0.05). Cellular uptake experiments and subsequent GC-MS analysis using stable-isotope-labeled internal standards revealed the presence of 1,8-cineole, borneol, camphor, and α-/ß-thujone in SI-treated cells; LC-MS analysis demonstrated the presence of rosmarinic acid. A significant, more than 50% mean inhibition of PMA/I-induced IL-6 and IL-8 release was demonstrated for the volatile compounds 1,8-cineole, borneol, camphor, and thujone, but not for the nonvolatile rosmarinic acid when applied in concentrations representative of sage infusion. Therefore, the volatile compounds were found to be more effective than rosmarinic acid. 1,8-Cineole, borneol, camphor, and α-/ß-thujone chiefly contribute to the anti-inflammatory activity of sage infusion in human gingival fibroblasts.

Promotion of vesicular zinc efflux by ZIP13 and its implications for spondylocheiro dysplastic Ehlers-Danlos syndrome.

Zinc is essential but potentially toxic, so intracellular zinc levels are tightly controlled. A key strategy used by many organisms to buffer cytosolic zinc is to store it within vesicles and organelles.It is yet unknown whether vesicular or organellar sites perform this function in mammals. Human ZIP13, a member of the Zrt/Irt-like protein (ZIP) metal transporter family, might provide an answer to this question. Mutations in the ZIP13 gene, SLC39A13, previously were found to cause the spondylocheiro dysplastic form of Ehlers­Danlos syndrome (SCD-EDS), a heritable connective tissue disorder.Those previous studies suggested that ZIP13 transports excess zinc out of the early secretory pathway and that zinc overload in the endoplasmic reticulum (ER) occurs in SCD-EDS patients. In contrast,this study indicates that ZIP13's role is to release labile zinc from vesicular stores for use in the ER and other compartments. We propose that SCD-EDS is the result of vesicular zinc trapping and ER zinc deficiency rather than overload.

High dose of dietary resveratrol enhances insulin sensitivity in healthy rats but does not lead to metabolite concentrations effective for SIRT1 expression.

SCOPE: trans-Resveratrol has been shown to improve insulin sensitivity and to enhance cellular glucose uptake. Evidence from recent studies indicates that these effects depend on SIRT1-pathways. METHODS AND RESULTS: Since ingestion of resveratrol leads to the presence of resveratrol and resveratrol metabolites in the body, we aimed at investigating (i) whether a daily dose of 300 mg resveratrol/kg body weight in healthy male Wistar rats for a period of 8 wk affects the selected parameters of glucose and lipid metabolism and (ii) whether the resulting plasma concentrations of resveratrol metabolites were effective in modulating SIRT1 expression. The dietary dose was based on the results from preceding toxicity studies. The results from the feeding experiment revealed plasma concentrations of resveratrol and its metabolites below 1 µmol/L and showed that fasting glucose and insulin levels were decreased by 35 and 41%, respectively, in the resveratrol group compared with controls. Insulin sensitivity was enhanced by 70%, whereas liver SIRT1 protein expression was not affected. Treatment of HepG2 cells with 10 µM resveratrol (1.49-fold) or its diglucuronides (1.21-fold) increased SIRT1 expression. CONCLUSION: These results suggest that the improved insulin sensitivity after dietary administration of 300 mg resveratrol/kg body weight does not involve increased protein expression of SIRT1.

Interactions between copper-binding sites determine the redox status and conformation of the regulatory N-terminal domain of ATP7B.

Copper-transporting ATPase ATP7B is essential for human copper homeostasis and normal liver function. ATP7B has six N-terminal metal-binding domains (MBDs) that sense cytosolic copper levels and regulate ATP7B. The mechanism of copper sensing and signal integration from multiple MBDs is poorly understood. We show that MBDs communicate and that this communication determines the oxidation state and conformation of the entire N-terminal domain of ATP7B (N-ATP7B). Mutations of copper-coordinating Cys to Ala in any MBD (2, 3, 4, or 6) change the N-ATP7B conformation and have distinct functional consequences. Mutating MBD2 or MBD3 causes Cys oxidation in other MBDs and loss of copper binding. In contrast, mutation of MBD4 and MBD6 does not alter the redox status and function of other sites. Our results suggest that MBD2 and MBD3 work together to regulate access to other metal-binding sites, whereas MBD4 and MBD6 receive copper independently, downstream of MBD2 and MBD3. Unlike Ala substitutions, the Cys-to-Ser mutation in MBD2 preserves the conformation and reduced state of N-ATP7B, suggesting that hydrogen bonds contribute to interdomain communications. Tight coupling between MBDs suggests a mechanism by which small changes in individual sites (induced by copper binding or mutation) result in stabilization of distinct conformations of the entire N-ATP7B and altered exposure of sites for interactions with regulatory proteins.

The N-terminal metal-binding site 2 of the Wilson's Disease Protein plays a key role in the transfer of copper from Atox1.

The Wilson's disease protein (WNDP) is a copper-transporting ATPase regulating distribution of copper in the liver. Mutations in WNDP lead to a severe metabolic disorder, Wilson's disease. The function of WNDP depends on Atox1, a cytosolic metallochaperone that delivers copper to WNDP. We demonstrate that the metal-binding site 2 (MBS2) in the N-terminal domain of WNDP (N-WNDP) plays an important role in this process. The transfer of one copper from Atox1 to N-WNDP results in selective protection of the metal-coordinating cysteines in MBS2 against labeling with a cysteine-directed probe. Such selectivity is not observed when free copper is added to N-WNDP. Similarly, site-directed mutagenesis of MBS2 eliminates stimulation of the catalytic activity of WNDP by the copper-Atox1 complex but not by free copper. The Atox1 preference toward MBS2 is likely due to specific protein-protein interactions and is not due to unique surface exposure of the metal-coordinating residues or higher copper binding affinity of MBS2 compared with other sites. Competition experiments using a copper chelator revealed that MBS2 retained copper much better than Atox1, and this may facilitate the metal transfer process. X-ray absorption spectroscopy of the isolated recombinant MBS2 demonstrated that this sub-domain coordinates copper with a linear biscysteinate geometry, very similar to that of Atox1. Therefore, non-coordinating residues in the vicinity of the metal-binding sites are responsible for the difference in the copper binding properties of MBS2 and Atox1. The intramolecular changes that accompany transfer of a single copper to N-WNDP are discussed.

Functional properties of the human copper-transporting ATPase ATP7B (the Wilson's disease protein) and regulation by metallochaperone Atox1.

Wilson's disease protein (WNDP) is a copper-transporting P(1)-type ATPase which plays a key role in normal distribution of copper in a number of tissues, particularly in the liver and the brain. Copper has numerous effects on WNDP, altering its structure, activity, and intracellular localization. To better understand the function of this copper-transporting ATPase and its regulation by copper, we have recently developed the functional expression systems for WNDP and for Atox1, a cytosolic protein that serves as an intracellular donor of copper for WNDP. Here we summarize the results of our experiments on characterization of the enzymatic properties of WNDP and the effects of Atox1 on the WNDP activity.

Metallochaperone Atox1 transfers copper to the NH2-terminal domain of the Wilson's disease protein and regulates its catalytic activity.

Copper is essential for the growth and development of mammalian cells. The key role in the intracellular distribution of copper belongs to the recently discovered family of metallochaperones and to copper-transporting P-type ATPases. The mutations in the ATPase ATP7B, the Wilson's disease protein (WNDP), lead to intracellular accumulation of copper and severe hepatic and neurological abnormalities. Several of these mutations were shown to disrupt the protein-protein interactions between WNDP and the metallochaperone Atox1, suggesting that these interactions are important for normal copper homeostasis. To understand the functional consequences of the Atox1-WNDP interaction at the molecular level, we produced recombinant Atox1 and characterized its effects on WNDP. We demonstrate that Atox1 transfers copper to the purified amino-terminal domain of WNDP (N-WNDP) in a dose-dependent and saturable manner. A maximum of six copper atoms can be transferred to N-WNDP by the chaperone. Furthermore, the incubation of copper Atox1 with the full-length WNDP leads to the stimulation of the WNDP catalytic activity, providing strong evidence for the direct effect of Atox1 on the function of this transporter. Our data also suggest that Atox1 can regulate the copper occupancy of WNDP. The incubation with apo-Atox1 results in the removal of copper from the metalated N-WNDP and apparent down-regulation of WNDP activity. Interestingly, at least one copper atom remains tightly bound to N-WNDP even in the presence of excess apo-Atox1. We suggest that this incomplete reversibility reflects the functional non-equivalency of the metal-binding sites in WNDP and speculate about the intracellular consequences of the reversible Atox1-mediated copper transfer.

Human copper-transporting ATPase ATP7B (the Wilson's disease protein): biochemical properties and regulation.

Wilson's disease protein (WNDP) is a product of a gene ATP7B that is mutated in patients with Wilson's disease, a severe genetic disorder with hepatic and neurological manifestations caused by accumulation of copper in the liver and brain. In a cell, WNDP transports copper across various cell membranes using energy of ATP-hydrolysis. Copper regulates WNDP at several levels, modulating its catalytic activity, posttranslational modification, and intracellular localization. This review summarizes recent studies on enzymatic function and copper-dependent regulation of WNDP. Specifically, we describe the molecular architecture and major biochemical properties of WNDP, discuss advantages of the recently developed functional expression of WNDP in insect cells, and summarize the results of the ligand-binding studies and molecular modeling experiments for the ATP-binding domain of WNDP. In addition, we speculate on how copper binding may regulate the activity and intracellular distribution of WNDP, and what role the human copper chaperone Atox1 may play in these processes.