Effects of quorum-sensing inhibition on experimental periodontitis induced by mixed infection in mice.

This study aimed to verify, in in vivo settings, whether quorum-sensing inhibition molecules could attenuate alveolar bone loss induced by Porphyromonas gingivalis/Fusobacterium nucleatum co-infection and reduce the bacterial colonization of periodontal tissues. In BALB/c mice, periodontitis was induced through oral inoculation with P. gingivalis and F. nucleatum six times during a 42-d period. Quorum sensing inhibitors (a furanone compound and D-ribose) were administered simultaneously with bacterial infection. Linear and volumetric modifications of interproximal alveolar bone levels were compared between groups using micro-computed tomography. Total bacteria, and P. gingivalis and F. nucleatum DNA in periodontal tissues, were quantified using real-time PCR. Radiographic linear measurements demonstrated a significant reduction of alveolar bone loss, of approximately 40%, in mice treated with quorum sensing inhibitors when compared with the co-infection group. This was confirmed by a significant increase of residual bone volume in the test group. While total bacterial genes in the treatment group significantly decreased by 93% in periodontal tissue samples when quorum sensing inhibitors were administered, no significant differences of P. gingivalis DNA were found. Quorum sensing inhibitors reduced periodontal breakdown and bacterial infection in periodontal tissues after co-infection with P. gingivalis and F. nucleatum.

Cinnamomum osmophloeum Kanehira ethanol extracts prevents human liver-derived HepG2 cell death from oxidation stress by induction of ghrelin gene expression.

Diabetes patients associated with liver disease carry a significant risk of morbidity and mortality. Cinnamon has been reported to reduce fructose-induced oxidative stress in the rat liver. However, the mechanism by which cinnamon protects the liver in a high-saccharide environment remains to be investigated. HepG2 cells were cultured with 30 mM D-ribose to mimic the high-oxidative-stress environment, typical of a liver in a diabetic patient. Three different chemical types of C. osmophloeum ethanol extracts (CEEs) were added in HepG2 culture media and the administration of all three CEEs protected HepG2 cells from D-ribose damage and increased cell survival by approximately 20 percent. Exclusively, the transcript variant 1 of the ghrelin gene, but not variant 3, was 2-3 times induced by the addition of these CEEs. Moreover, the mRNAs of ghrelin processing enzyme, furin, and mboat4 were detected in HepG2 cells. The ghrelin hormones in the culture media were increased 4-9 times by the addition of CEEs. The protective effects of ghrelin on HepG2 cells in D-ribose environment were further confirmed by recombinant ghrelin transfection. We conclude that the CEEs induce ghrelin gene expression and protect HepG2 cells from D-ribose-induced oxidative damage through ghrelin signalling.

Catechol-O-methyltransferase in complex with substituted 3'-deoxyribose bisubstrate inhibitors.

The biological activity of catechol neurotransmitters such as dopamine in the synapse is modulated by transporters and enzymes. Catechol-O-methyltransferase (COMT; EC 2.1.1.6) inactivates neurotransmitters by catalyzing the transfer of a methyl group from S-adenosylmethionine to catechols in the presence of Mg²âº. This pathway also inactivates L-DOPA, the standard therapeutic for Parkinson's disease. Depletion of catechol neurotransmitters in the prefrontal cortex has been linked to schizophrenia. The inhibition of COMT therefore promises improvements in the treatment of these diseases. The concept of bisubstrate inhibitors for COMT has been described previously. Here, ribose-modified bisubstrate inhibitors were studied. Three high-resolution crystal structures of COMT in complex with novel ribose-modified bisubstrate inhibitors confirmed the predicted binding mode but displayed subtle alterations at the ribose-binding site. The high affinity of the inhibitors can be convincingly rationalized from the structures, which document the possibility of removing and/or replacing the ribose 3'-hydroxyl group and provide a framework for further inhibitor design.

Carnosine inhibits modifications and decreased molecular chaperone activity of lens alpha-crystallin induced by ribose and fructose 6-phosphate.

PURPOSE: Alpha-crystallin, a major structural protein in the lens, prevents heat- and oxidative stress-induced aggregation of proteins and inactivation of enzymes by acting as a molecular chaperone. Modification of alpha-crystallin by some posttranslational modifications results in conformational changes and decreases in chaperone activity, which may contribute to cataractogenesis in vivo. Carnosine (beta-alanyl-L-histidine), an endogenous histidine dipeptide, prevents protein modifications including glycation and oxidation. The purpose of this study was to further explore whether carnosine can protect alpha-crystallin against glycation by a sugar and a sugar phosphate, and in particular to find whether it can protect against its decreased chaperone activity. Additionally, we investigated whether carnosine could directly react with a sugar and a sugar phosphate. METHODS: Bovine lens alphaL-crystallin was separated by size-exclusion chromatography on a Sephacryl S-300 HR column. alphaL-crystallin was incubated with different concentrations of fructose 6-phosphate (F6P) and ribose with or without carnosine for different times. The chaperone activity of alphaL-crystallin was monitored using the prevention of thermal aggregation of betaL-crystallin. The modified alphaL-crystallin was examined by SDS-PAGE and fluorescence measurements. The absorbance spectra of solutions of carnosine and sugars were investigated. RESULTS: Carnosine inhibited the crosslinking of alphaL-crystallin induced by F6P and ribose in a dose- and time-dependent manner. It protected alphaL-crystallin against its decreased chaperone activity induced by 100 mM F6P during four days incubation, but not against ribose-induced change. Control alphaL-crystallin gave 96% protection against aggregation of betaL-crystallin after four days incubation, but only 85% protection was achieved in the presence of F6P, rising to 96% (p=0.0004) in the presence of carnosine. After more extensive modification by sugar and a sugar phosphate, there was no significant protective effect of carnosine on alphaL-crystallin cross-linking or chaperone activity. The tryptophan fluorescence of modified alphaL-crystallin was remarkably decreased in the presence of F6P and ribose. However, the decrease was less when 50 mM carnosine was present during eight days incubation with F6P. Carnosine did not maintain the fluorescence when ribose was used. The nontryptophan fluorescence was increased with a shift to longer wavelengths in a time-dependent manner. Carnosine readily reacted with F6P and ribose thereby inhibiting glycation-mediated protein modification as revealed electrophoretically. The increased absorbance was time-dependent, suggesting adducts may be formed between F6P, ribose, and carnosine. CONCLUSIONS: This is the first report showing that carnosine can protect the chaperone activity of alpha-crystallin. This chaperone may protect against cataractous changes. In addition to demonstrating the effects of carnosine on prevention crosslinking, our studies also bring out important evidence that carnosine reacts with F6P and ribose, which suggests carnosine's potential as a possible nontoxic modulator of diabetic complications.

Glycation-dependent, reactive oxygen species-mediated suppression of the insulin gene promoter activity in HIT cells.

Prolonged poor glycemic control in non-insulin-dependent diabetes mellitus patients often leads to a decline in insulin secretion from pancreatic beta cells, accompanied by a decrease in the insulin content of the cells. As a step toward elucidating the pathophysiological background of the so-called glucose toxicity to pancreatic beta cells, we induced glycation in HIT-T15 cells using a sugar with strong deoxidizing activity, D-ribose, and examined the effects on insulin gene transcription. The results of reporter gene analyses revealed that the insulin gene promoter is more sensitive to glycation than the control beta-actin gene promoter; approximately 50 and 80% of the insulin gene promoter activity was lost when the cells were kept for 3 d in the presence of 40 and 60 mM D-ribose, respectively. In agreement with this, decrease in the insulin mRNA and insulin content was observed in the glycation-induced cells. Also, gel mobility shift analyses using specific antiserum revealed decrease in the DNA-binding activity of an insulin gene transcription factor, PDX-1/IPF1/STF-1. These effects of D-ribose seemed almost irreversible but could be prevented by addition of 1 mM aminoguanidine or 10 mM N-acetylcysteine, thus suggesting that glycation and reactive oxygen species, generated through the glycation reaction, serve as mediators of the phenomena. These observations suggest that protein glycation in pancreatic beta cells, which occurs in vivo under chronic hyperglycemia, suppresses insulin gene transcription and thus can explain part of the beta cell glucose toxicity.

Glucocorticoid agonists as well as antagonists are effective inducers of mouse mammary tumor virus RNA in mouse mammary tumor cells treated with inhibitors of ADP-ribosylation.

Glucocorticoids increase expression of specific genes by a mechanism involving binding to and "activation" of a specific receptor protein. Other steroids, such as RU 486, bind to the glucocorticoid receptor but the resultant steroid-receptor complex is unable to activate glucocorticoid sensitive genes. In the present study we have observed that steroid regulation of the glucocorticoid-regulated mouse mammary tumor virus (MMTV) genome in cultured mouse mammary tumor cells is altered by treatment of the cells with inhibitors of (ADP-ribose)n synthetase. The ability of glucocorticoid agonists to increase MMTV is about 2-fold increased by the inhibitor treatment. Interestingly, RU 486 and other steroids that are normally inactive in control cells are very good inducers of MMTV in the treated cells. This alteration in MMTV expression is associated with a 37% increase in nuclear binding of the glucocorticoid, triamcinolone acetonide, and also RU 486 in the inhibitor-treated cells. Steroids that do not bind to the glucocorticoid receptor are not inducers in control or in treated cells. The results point to a role for ADP-ribosylation of proteins as a negative regulator of MMTV expression and suggest a mechanism for activation of steroid-sensitive genomes.