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Background: The miR-106a-363 cluster, encoding six miRNAs (miR- 106a, miR-18b, miR-20b, miR-19b-2, miR-92-2 and miR-363), has been shown to be overexpressed in various tumours. In oral carcinoma cells, however only miR- 106a was detectable from this cluster. We have investigated how effects of transfection of oral carcinoma cells with a non-expressed member of this cluster affect mRNA transcriptomes and cellular selected functions., Methods: Investigate effects of miR-20b and miR-363 mimics on cellular respiration, glycolysis and mobility. Effects on mRNA transcriptomes were monitored using microarrays., Results: The studies show that in oral carcinoma cells transfected with miR-20b -, or miR-363-3p or miR-363-5p mimic different mRNAs were differentially expressed. Nevertheless, bioinformatics analysis suggested significant associations of differentially expressed genes to inhibition of cellular proliferation, cell cycle and cellular migration. These results were also experimentally confirmed., Conclusions: Transfection of miRNA mimics for unexpressed members of the miR-106a-363 cluster (miR20b, miR-363-3p and miR-363-5p) exhibit an anti-proliferative effect on oral carcinoma cells, although likely mediated by different regulatory mechanisms.

The main objective of this study was to map global gene expression in order to provide information about the populations of mRNA species participating in murine tooth development at 24 h intervals, starting at the 11th embryonic day (E11.5) up to the 7th post-natal day (P7). The levels of RNA species expressed during murine tooth development were mesured using a total of 58 deoxyoligonucleotide microarrays. Microarray data was validated using real-time RT-PCR. Differentially expressed genes (p < 0.05) were subjected to bioinformatic analysis to identify cellular activities significantly associated with these genes. Using ANOVA the microarray data yielded 4362 genes as being differentially expressed from the 11th embryonic day (E11.5) up to 7 days post-natal (P7), 1921 of these being genes without known functions. The remaining 2441 genes were subjected to further statistical analysis using a supervised procedure. Bioinformatic analysis results for each time-point studied suggests that the main molecular functions associated with genes expressed at the early pre-natal stages (E12.5-E18.5) were cell cycle progression, cell morphology, lipid metabolism, cellular growth, proliferation, senescence and apoptosis, whereas most genes expressed at post-natal and secretory stages (P0-P7) were significantly associated with regulation of cell migration, biosynthesis, differentiation, oxidative stress, polarization and cell death. Differentially expressed genes (DE) not described earlier during murine tooth development; Inositol 1, 4, 5-triphosphate receptor 3 (Itpr3), metallothionein 1(Mt1), cyclin-dependent kinase 4 (Cdk4), cathepsin D (Ctsd), keratin complex 2, basic, gene 6a (Krt2-6a), cofilin 1, non-muscle (Cfl1), cyclin 2 (Ccnd2), were verified by real-time RT-PCR.

Transfection of human oral squamous carcinoma cells (clone E10) with mimics for unexpressed miR-20b or miR-363-5p, encoded by the miR-106a-363 cluster (miR-20b, miR-106a, miR-363-3p, or miR-363-5p), caused 40-50% decrease in proliferation. Transfection with mimics for miR-18a or miR-92a, encoded by the miR-17-92 cluster (all members being expressed in E10 cells), had no effect on proliferation. In contrast, mimic for the sibling miRNA-19a yielded about 20% inhibition of proliferation. To investigate miRNA involvement profiling of miRNA transcriptomes were carried out using deoxyoligonucleotide microarrays. In transfectants for miR-19a, or miR-20b or miR-363-5p most differentially expressed miRNAs exhibited decreased expression, including some miRNAs encoded in paralogous miR-17-92-or miR-106b-25 cluster. Only in cells transfected with miR-19a mimic significantly increased expression of miR-20b observed-about 50-fold as judged by qRT-PCR. Further studies using qRT-PCR showed that transfection of E10 cells with mimic for miRNAs encoded by miR-17-92 - or miR-106a-363 - or the miR-106b-25 cluster confirmed selective effect on expression on sibling miRNAs. We conclude that high levels of miRNAs encoded by the miR-106a-363 cluster may contribute to inhibition of proliferation by decreasing expression of several sibling miRNAs encoded by miR-17-92 or by the miR-106b-25 cluster. The inhibition of proliferation observed in miR-19a-mimic transfectants is likely caused by the miR-19a-dependent increase in the levels of miR-20b and miR-106a. Bioinformatic analysis of differentially expressed miRNAs from miR-106a, miR-20b and miR-363-5p transfectants, but not miR-92a transfectants, yielded significant associations to "Cellular Growth and Proliferation" and "Cell Cycle." Western blotting results showed that levels of affected proteins to differ between transfectants, suggesting that different anti-proliferative mechanisms may operate in these transfectants.

Expression of clusterin (Clu) in the murine first molar tooth germ was markedly increased at postnatal developmental stages. The time-course of expression of this gene paralleled those of other genes encoding proteins involved during the secretory phase of odontogenesis, as described previously. Immunohistochemical studies of clusterin in murine molar tooth germs suggested this protein to be located in outer enamel epithelium, regressing enamel organ, secretory ameloblasts, and the dental epithelium connecting the tooth to the oral epithelium at an early eruptive stage. Immunolabelling of transforming growth factor beta-1 (TGF-β1) revealed it to be located close to clusterin. The levels of expression of Clu and Tgfb1 were markedly decreased following in-vivo transfection with anti-miR-214. In contrast, the expression of several genes associated with regulation of growth and development were increased by this treatment. We suggest that clusterin has functions during secretory odontogenesis and the early eruptive phase. Bioinformatic analysis after treatment with anti-miR-214 suggested that, whilst cellular activities associated with tooth mineralization and eruption were inhibited, activities associated with an alternative developmental activity (i.e. biosynthesis of contractile proteins) appeared to be stimulated. These changes probably occur through regulation mediated by a common cluster of transcription factors and support suggestions that microRNAs (miRNAs) are highly significant as regulators of differentiation during odontogenesis., (© 2013 Eur J Oral Sci.)

Delta-like 1 homolog (Dlk1) and insulin-like growth factor 2 (Igf2) are two of six well-studied mouse imprinted gene clusters that are paternally expressed. Their expression is also linked to their maternally expressed non-coding RNAs, encoded by Gene trap locus 2 (Gtl2) and Imprinted maternally expressed transcript (H19), co-located as imprinted gene clusters. Using deoxyoligonucleotide microarrays and real-time RT-PCR analysis we showed Dlk1 and Gtl2 to exhibit a time-course of expression during tooth development that was similar to that of Igf2 and H19. Western blot analysis of proteins encoded by Dlk1 and Igf2 suggested that the levels of these proteins reflected those of the corresponding mRNAs. Immunohistochemical studies of DLK1 in murine molars detected the protein in both epithelial and mesenchymal regions, in developing cusp mesenchyme, and in newly synthesized enamel and dentin tubules. IGF2 protein was detected primarily at prenatal stages, suggesting that it may be active before birth. Analysis of methylation of cytosine-phosphate-guanine (CpG) islands in both Dlk1 and Igf2 suggested the presence of an increasing fraction of hypermethylated bases with increasing time of development. The increased levels of hypermethylation coincided both with the diminished levels of expression of Dlk1 and Igf2 and with decreased levels of DLK1 and IGF2 proteins in the tooth germ, suggesting that their expression is regulated via methylation of CpG islands present in these genes., (© 2012 Eur J Oral Sci.)

The aim of this study was to describe the expression of genes, including ameloblastin (Ambn), amelogenin X chromosome (Amelx), and enamelin (Enam) during early (pre-secretory) tooth development. The expression of these genes has predominantly been studied at post-secretory stages. Deoxyoligonucleotide microarrays were used to study gene expression during development of the murine first molar tooth germ at 24 h intervals, starting at the 11th embryonic day (E11.5), and up to the 7th day after birth (P7). The profile search function of Spotfire software was used to select genes with similar expression profile as the enamel genes (Ambn, Amelx, and Enam). Microarray results where validated using real-time reverse transcription-polymerase chain reaction (real-time RT-PCR), and translated proteins identified by Western-blotting. In situ localization of the Ambn, Amelx, and Enam mRNAs were monitored from E12.5 to E17.5 using deoxyoligonucleotide probes. Bioinformatics analysis was used to associate biological functions with differentially expressed (DE; p ≤ 0.05) genes. Microarray results showed a total of 4362 genes including Ambn, Amelx, and Enam to be significant DE throughout the time-course. The expression of the three enamel genes was low at pre-natal stages (E11.5-P0) increasing after birth (P1-P7). Profile search lead to isolation of 87 genes with significantly similar expression to the three enamel proteins. These mRNAs were expressed in dental epithelium and epithelium derived cells. Although expression of Ambn, Amelx, and Enam were lower during early tooth development compared to secretory stages enamel proteins were detectable by Western-blotting. Bioinformatic analysis associated the 87 genes with multiple biological functions. Around 35 genes were associated with 15 transcription factors.

The use of deoxyoligonucleotide microarrays facilitates rapid expression profiling of gene expression using samples of about 1 μg of total RNA. Here are described practical aspects of the procedures involved, including essential reagents. Analysis of results is discussed from a practical, experimental, point of view together with software required to carry out the required statistical analysis to isolate populations of differentially expressed genes.

The six microRNAs (miRNA) encoded by the miR-17-92 cluster, also named oncomir-1, have been associated with carcinogenesis and typically exhibit-increased expression in tumors. Despite the well-established role for the miR-17-92 cluster in an oncogenic network, the physiological function of these miRNAs in normal tissues remains unresolved. In order to investigate whether there are similar patterns of miR-17-92 expression during embryogenesis and carcinogenesis, we have preformed a systematic study of the expression in cultured carcinoma cells, cultured primary human keratinocytes (KC), and during development of some murine tissues. Both levels of expression of the primary transcript (pri-miRNA) and levels of expression of the individual members of the cluster were monitored. Irrespectively of tissue examined we found that the level of expression decreased markedly during development. With cultured primary human KCs their levels of expression of some of these microRNAs decreased as the number of cell passages increased. Their levels of expression in cultured carcinoma cells, in contrasts, increased, or remained unchanged, with increasing number of cell passages. The results suggest these microRNAs are involved in the regulation of foetal development and that they may promote proliferation and inhibit differentiation during embryogenesis and carcinogenesis. Additionally, the six microRNAs exhibit variable tissue expression, suggesting selective processing of these microRNAs., (Copyright © 2010 Wiley-Liss, Inc.)

MicroRNAs (miRNAs) are an abundant class of noncoding RNAs that are believed to be important in many biological processes through regulation of gene expression. Little is known of their function in tooth morphogenesis and differentiation. MicroRNA-214 (miR-214), encoded by the polycistronic Dnm30os gene, is highly expressed during development of molar tooth germ and was selected as a target for silencing with anti-miR-214. Mandibular injection of 1-100 pmol of anti-miR-214 close to the developing first molar in newborn mice resulted in significant decrease in expression of miR-214, miR-466h, and miR-574-5p in the tooth germ. Furthermore, levels of miR-199a-3p, miR-199a-5p, miR-690, miR-720, and miR-1224 were significantly increased. Additionally, the expression of 863 genes was significantly increased and the expression of 305 genes was significantly decreased. Among the genes with increased expression was Twist-1 and Ezh2, suggested to regulate expression of miR-214. Microarray results were validated using real-time RT-PCR and Western blotting. Among genes with decreased expression were Amelx, Calb1, Enam, and Prnp; these changes also being reflected in levels of corresponding encoded proteins in the tooth germ. In the anti-miR-214-treated molars the enamel exhibited evidence of hypomineralization with remnants of organic material and reduced surface roughness after acid etching, possibly due to the transiently decreased expression of Amelx and Enam. In contrast, several genes encoding contractile proteins exhibited significantly increased expression. mRNAs involved in amelogenesis (Ambn, Amelx, Enam) were not found among targets of miRNAs that were differentially expressed following treatment with anti-miR-214. It is therefore suggested that effects of miR-214 on amelogenesis are indirect, perhaps mediated by the observed miR-214-dependent changes in levels of expression of numerous transcription factors.

In order to gain insight into possible cellular functions of the prion protein (PrP) during normal development, the expression of Prnp (encoding the PrP) and the distribution of the PrP were studied in murine tooth germs. Expression of Prnp in the mouse first molar tooth germ was highly dynamic, increasing several-fold during the secretory phase of odontogenesis, exhibiting a time-course of expression similar to that of genes coding for other extracellular proteins [e.g. enamel matrix proteins (Amelx, Ambn, Enam), Aplp1, Clstn1, and Clu]. Western blot analysis suggested that the amounts of PrP and amyloid beta (A4) precursor-like protein 1 (APLP1) in the tooth germ followed time-courses similar to those of the corresponding mRNAs. Immunohistochemical studies of the distribution of PrP in murine molar and incisor tooth germs at embryonic day (E)18.5 suggested that this protein was located in the cervical loop, outer enamel epithelium, pre-ameloblasts, and dental papilla. Different degrees of immunolabelling of pre-ameloblasts on the mesial and distal aspects of a lower molar cusp may be related to different enamel configurations on the two aspects. It is concluded that the dynamic patterns of expression of Prnp, and of distribution of PrP, suggest that PrP may have functions during secretory odontogenesis, perhaps in relation to amelogenesis., (© 2010 Eur J Oral Sci.)

At the mouse incisor tip the initially differentiated ameloblasts produce a thin, prism-free enamel, while further apically, in the immediate adjacent segment, the enamel thickness increases and the four-layered enamel of mouse incisor is formed. Comparative gene-expression profiling was carried out on RNA isolated from these two segments of incisor tooth germs at embryonic day (E)17.5 and at postnatal days (P)0, 1, 2, and 10 using microarrays to measure messenger RNA (mRNA) and microRNA (miRNA) species present in the segments. Validation of expression data was achieved using real-time reverse transcription-polymerase chain reaction (RT-PCR) and western blotting. Bioinformatic data suggested enhanced cellular apoptosis in the incisal tip segment, which, together with diminished expression of the Amelx and Enam genes, may contribute to the production of the thin enamel seen in this tooth segment. For genes exhibiting higher levels of expression in the adjacent segment where complex enamel is being formed, bioinformatic analysis suggested significant associations with cellular functions involving the actin cytoskeleton, cellular development, morphology, and movement. This is suggested to reflect that ameloblasts with Tomes' process are being organized in transverse rows, facilitating the transverse movement that results in prism decussation in the inner enamel of the adjacent segment. Bioinformatic analysis of miRNA expression data lends support to these suggestions.

Objective: In search for possible novel genes that may be involved in tooth development, we analysed the genome-wide transcriptome of developing mandibular tooth germs of mouse during embryonic and early life and selected family-with-sequence-similarity-46, member A (Fam46a) gene for further expression analysis., Methods: We applied microarray, quantitative real time polymerase chain reaction and in situ hybridisation methods for the expression study of the mouse Fam46a gene., Results: We found the family-with-sequence-similarity-46, member A (Fam46a) gene to be highly expressed and further verify its temporo-spatial expression in the mouse tooth., Conclusion: We have shown that Fam46a is expressed in ameloblasts' nuclei of tooth germs and hypothesise that it might act together with morphogenetic factors important for the formation of enamel in mouse tooth.

Trefoil factor family 3 (TFF3) is secreted in saliva. The peptide improves the mechanical and chemical resistance of mucins, and it may act as a motility signal for oral keratinocytes during wound healing. This study aimed to identify novel functions of TFF3 in oral keratinocytes. To achieve this, we used phosphoprotein and messenger RNA (mRNA) arrays to compare TFF3-treated and untreated oral keratinocytes. Analysis of the phosphoprotein array indicated that TFF3 signals through the mitogen-activated protein kinases (MAPKs) c-Jun N-terminal kinase (JNK), p38, and extracellular signal-regulated kinase (ERK1/2), and through the phosphoinositide 3-kinase (PI3K)/protein kinase B (PKB) pathway. Microarray analysis of mRNA showed that TFF3 stimulation induced changes in the expression of genes functionally related to cell death/survival, cell growth and proliferation, and cell movement. The reverse transcription-polymerase chain reaction (RT-PCR) results indicated that the transcription of some immediate-early genes (IEGs) was downregulated, whereas the IEGs FBJ osteosarkoma oncogene (FOS) and C-MYC binding protein (MYCBP2) were transiently upregulated by TFF3 stimulation. Together, the results of the arrays indicate that TFF3 is a modifying factor in pathways regulating cell survival, cell growth and proliferation, and cell migration of oral keratinocytes. Trefoil factor family 3 may therefore promote oral wound healing and it should be considered for the treatment of oral ulcerating diseases, or of other diseases.

Pentadecafluorooctanoic acid is an established peroxisome proliferator. Little is known about effects of treatment with 1H,1H,2H,2H-heptadecafluorodecan-1-ol, which is metabolized to pentadecafluorooctanoic acid. We compared effects of various dosages (3, 10, or 25 mg/kg body wt) of each of these compounds on hepatic gene expression in rats with microarrays. Microarray data were validated by real-time RT-PCR. Expression data were also correlated with hepatic activities of selected enzymes and with hepatic levels of pentadecafluorooctanoic acid and 1H,1H,2H,2H-heptadecafluorodecan-1-ol. Pentadecafluorooctanoic acid caused the more powerful change in gene expression, in terms of both number of genes affected and extent of change in expression. Across the dosages used pentadecafluorooctanoic acid and 1H,1H,2H,2H-heptadecafluorodecan-1-ol caused significant (P < or = 0.05) changes in expression for 441 and 105 genes, respectively. With 1H,1H,2H,2H-heptadecafluorodecan-1-ol approximately 38% of the 105 genes exhibited decreased expression with a dose of 25 mg/kg body wt, these genes also appearing less responsive to treatment at the lower dosages. Bioinformatic analysis suggested that these genes are associated with regulatory functions. With pentadecafluorooctanoic acid, increasing dosage up to 10 mg/kg body wt brought about progressive increase in expression of affected genes. Pathways analysis suggested similar effects of the two compounds on lipid and amino acid metabolism. Marked differences were also found, particularly with respect to effects on genes related to oxidative phosphorylation, oxidative metabolism, free radical scavenging, xenobiotic metabolism, and complement and coagulation cascades.

Using microarrays, miRNA expression profiles have been established at selected times during development (E15.5, P0 and P5) of the murine first molar mandibular tooth germ and the right submandibular salivary gland (E15.5, P0, P5 and P25). Microarray data was validated using real-time PCR, also facilitating RT-PCR profiling of nine selected miRNAs. In general, good agreement between microarray data and real-time PCR data was found. Further, miRNA expression profiles of foetal and adult liver were also investigated, and found to agree with published data. In tooth germ and salivary gland up to 88 different miRNAs were detected. In all tissues examined miRNA expression was highly dynamic; miRNA profiles changing extensively with time of development. Additionally, the expression of some miRNAs was tissue-specific. Bioinformatic analysis of clusters of miRNAs was attempted using the miRGate software, the results suggesting miRNAs to be involved in the regulation of essential developmental processes, e.g., epithelical cell proliferation, mesodermal cell fate determination and salivary gland morphogenesis.

In a matter of a few days the murine tooth germ develops into a complex, mineralized, structure. Murine 30K microarrays were used to examine gene expression in the mandibular first molar tooth germs isolated at 15.5dpc and at 2DPN. Microarray results were validated using real-time RT-PCR. The results suggested that only 25 genes (3 without known functions) exhibited significantly higher expression at 15.5dpc compared to 2DPN. In contrast, almost 1400 genes exhibited significantly (P<0.015) higher expression at 2DPN compared to 15.5dpc, about half of which were genes with unknown functions. More than 50 of the 783 known genes exhibited higher than 10-fold increase in expression at 2DPN, amongst these were genes coding for enamel matrix proteins which were expressed several 100-fold higher at 2DPN. GO and KEGG analysis showed highly significant associations between families of the 783 known genes and cellular functions relating to energy metabolism, protein metabolism, regulation of cell division, cell growth and apoptosis. The use of bioinformatics analysis therefore yielded a functional profile in agreement with known differences in tissue morphology and cellular composition between these two stages. Such data is therefore useful in directing attention towards genes, or cellular activities, which likely are worthy of further studies as regards their involvement in odontogenesis.

Objective: The responsiveness of the hypothalamic-pituitary-adrenal (HPA) axis has been found to play a significant role for susceptibility and resistance to periodontal disease. In the present study we have investigated the effects of two different treatment strategies, which have been found to down-regulate the HPA axis, on ligature-induced periodontitis., Methods: In experiment 1, newborn rats were treated with the synthetic glucocorticoid hormone dexamethasone-21-phosphate, which permanently down-regulates HPA axis responsiveness. In experiment 2, adult rats were treated with the novel antidepressant drug tianeptine, which opposes the action of stress. Periodontitis was inflicted upon all rats. Just before decapitation the animals received gram-negative bacterial lipopolysaccharide (LPS) to induce a robust immune and HPA axis response., Results: Compared to the saline-treated control rats, dexamethasone-treated rats had significantly less periodontal bone loss (p < 0.01), reduced expression of glucocorticoid receptors in the hippocampus (p < 0.001), lower corticosterone (p=0.01) and higher plasma levels of the cytokine tumor necrosis factor (TNF)-alpha (p < 0.05) after LPS challenge. Also the tianeptine-treated rats showed significantly reduced periodontal bone loss (p=0.01), enhanced plasma levels of TNF-alpha (p < 0.05), and transforming growth factor-1beta (p < 0.01), whereas no significant difference was found in corticosterone levels., Conclusion: An individual's responsiveness to danger signals, whether they are of immunological, chemical, or psychological origin, may be an important factor for explaining variability in susceptibility to periodontal disease. The results may provide new insight into the mechanisms of periodontal disease development, and open new vistas for disease prevention.

Donepezil is a selective inhibitor of acetylcholinesterase (AChE) clinically used for treating Alzheimer's disease. Cholinergic effects after short-term exposure of donepezil (up to 12 h) have been extensively studied in rats, but few have addressed the potential long-term effects. After 14 days administration (1x3 mg/kg, decapitation 4 h after the last injection) the cerebral acetylcholine level was increased by 35% and the AChE activity was decreased by 66% and 32% in brain and blood, respectively. No change was detected in choline acetyltransferase activity, or the levels of vesicular acetylcholine transporter, choline transporter, or muscarinic receptors. Expression of various cholinergic genes was unaffected. Preliminary results of AChE activity in human blood showed 60-97% and 43-89% of pre-exposed level after one and three days of donepezil administration (5 mg daily), respectively. In conclusion, donepezil exposure in rats at doses that do not inhibit brain AChE continuously during the day, will not lead to tolerance development.

Background: In man, elevated levels of plasma plipoprotein (a) (Lp(a)) is a cardiovascular risk factor, and oxidized phospholipids are believed to play a role as modulators of inflammatory processes such as atherosclerosis. Polyamines are potent antioxidants and anti-inflammatory agents. It was therefore of interest to examine polyamines and their metabolism in LPA transgenic mice. Concentration of the polyamines putrescine, spermidine and spermine as well as the activity of peroxisomal polyamine oxidase and two other peroxisomal enzymes, acyl-CoA oxidase and catalase were measured. The mice were fed either a standard diet or a diet high in fat and cholesterol (HFHC). Some of the mice in each feeding group were in addition given aminoguanidine (AG), a specific inhibitor of diamine oxidase, which catalyses degradation of putrescine, and also inhibits non-enzymatic glycosylation of protein which is implicated in the aetiology of atherosclerosis in diabetic patients. Non-transgenic mice were used as controls., Results: Intestinal peroxisomal polyamine oxidase activity was significantly higher in LPA transgenic mice than in the non-transgenic mice, while intestinal peroxisomal catalase activity was significantly lower. Hepatic beta-oxidation increased in Lp(a) transgenic mice fed the HFHC diet, but not in those on standard diet. Hepatic spermidine concentration was increased in all mice fed the HFHC diet compared to those fed a standard diet, while spermine concentration was decreased. With exception of the group fed only standard diet, transgenic mice showed a lower degree of hepatic steatosis than non-transgenic mice. AG had no significant effect on hepatic steatosis., Conclusion: The present results indicate a connection between peroxisomal enzyme activity and the presence of the human LPA gene in the murine genome. The effect may be a result of changes in oxidative processes in lipid metabolism rather than resulting from a direct effect of the LPA construct on the peroximal gene expression.

Objective: Previous experiments in rats suggest that hypothalamic-pituitary-adrenal (HPA) axis over-responsiveness, which leads to increased secretion of immunoregulatory glucocorticoid hormones, increases periodontal disease susceptibility, whereas HPA axis under-responsiveness is associated with increased resistance to the disease. The present study was designed to investigate whether MK-801 (dizocilipine malate), an antagonist of the glutamate receptor N-methyl-D-aspartate (NMDA) in the brain, which has been found to play an important role in the regulation of the HPA axis, would influence the outcome of experimental ligature-induced periodontal disease in a rat model., Methods: Experimental periodontal disease was induced in periodontal disease susceptible and HPA axis high-responding Fischer 344 rats 2 days before chronic treatment with MK-801(1 mg/kg intraperitoneally). The periodontal breakdown was assessed after the ligatures had been in place for 23 days. Following intraperitoneal Gram-negative bacterial lipopolysaccharide stimulation (Escherichia coli, 250 microg/kg), concentrations of glucocorticoid receptors (GRs) in the hippocampus, and levels of the cytokine tumour necrosis factor alpha (TNF-alpha), as well as the HPA axis-derived hormone corticosterone, were measured in serum., Results: Compared to vehicle-treated controls, MK-801-treated rats had significantly increased periodontal tissue destruction (p < 0.01). MK-801-treated rats also showed significantly increased expression of GRs in the hippocampus (p < 0.05), elevated levels of corticosterone (p < 0.001) and reduced levels of TNF-alpha (p < 0.01) in serum 2 h after lipopolysaccharide stimulation., Conclusion: These findings may implicate glutamate receptor-dependent mechanisms in periodontal disease, and support the concept of a bidirectional immune-brain-immune regulatory network with importance for periodontal health and disease.

Liver mitochondria isolated from rats treated in vivo with trimethyltin chloride show stimulation of respiration using glutamate/malate as substrate, and a transient inhibition on rates of respiration using palmitoyl-L-carnitine as substrate. This phenomenon was observed with both ADP- and FCCP-stimulated respiration. In contrast, rates of respiration by liver mitochondria isolated from rats treated in vivo with trimethyltin chloride, following prior treatment with clofibrate, were inhibited when glutamate/malate was respiratory substrates. With palmitoyl-L-carnitine no effect of trimethyltin chloride was observed. In vitro treatment of rat liver mitochondria, or of rat liver homogenates, led to the expected, powerful inhibition of respiration. The synthesis of ATP by liver mitochondria isolated from rats treated in vivo with trimethyltin chloride was not inhibited compared to mitochondria isolated from control rats. Similarly, ATP synthesis by mitochondria isolated from rats treated with clofibrate, before treatment with trimethyltin chloride, was not inhibited. We, therefore, conclude that the powerful inhibitory effects of trimethyltin found in vitro, is not expressed in vivo during the first 36 hr following administration. In vivo treatment of rats with trimethyltin chloride caused a marked increase in hepatic levels of taurine and glycine, while levels of glutathione and glutamine were diminished. This is consistent with an enhanced oxidative stress in the liver. Our findings lead to the conclusion that increased oxidative stress, rather than inhibition of the mitochondrial ATPase, is a likely major cause of the in vivo toxic effects due to trimethyltin chloride.

Some metabolic effects of dietary marine oils, or of dietary eicosapentaenoic or docosahexaenoic acid are reviewed. It is pointed out that docosahexaenoic acid appears more effective as regards induction of peroxisomal beta-oxidation. Similarly, docosahexaenoic appears more powerful in terms of suppression of hepatic delta9-desaturase activity and mRNA-levels. The potential inhibitory effect of polyunsaturated fatty acids, particularly docosahexaenoic acid, on mitochondrial beta-oxidation is discussed. Experiments with rats suggesting that the hypolipidaemic response of eicosapentaenoic acid is more marked when the fatty acid was given to fed rats, as compared to fasted rats, are discussed.

(1) Effects of dietary treatment of male albino rats with eicosapentaenoic acid (EPA) or docosahexaenoic acid on hepatic mitochondrial lipid metabolism have been investigated. (2) Mitochondria isolated from rats given these treatments were shown to have increased ability to respire on acyl-CoA esters in the presence of malonate. This effect was expressed with most of the long-chain acyl-CoA esters used as substrates. When malonate in the incubations was replaced with malate, mitochondria from treated animals were found to exhibit diminished rates of respiration on polyunsaturated acyl-CoA esters, in particular linolenoyl-, eicosapentaenoyl- and docosahexaenoyl-CoA. This phenomenon could not be attributed to changes in activity of carnitine palmitoyltransferase I or in peroxisomal beta-oxidation. (3) Uncoupled respiration on glutamate, malate or succinate was also affected by treatment with EPA. With liver mitochondria isolated from rats that had been treated with a omega-3 fatty acid in the fasted state, the respiratory rates were lower than those observed with mitochondria isolated from control rats. Respiratory rates with mitochondria isolated from rats given the omega-3 fatty acid in the fed state was not significantly different from control rates. (4) In rats treated with EPA in the fed state, the amount of EPA incorporated into mitochondrial lipids was markedly more increased as compared to rats given omega-3 fatty acid in the fasted state. Incorporation of dietary EPA into tissue lipids was investigated, also following mildronate treatment of rats (an inhibitor of carnitine biosynthesis). (5) A hypolipidaemic effect of dietary EPA was only observed when the fatty acid was given to fed rats. Rats treated with EPA in the fasted state, in contrast, exhibited hypoglycaemia, the hypolipidaemic effects now being absent. (6) These results suggest that hypolipidaemia is most pronounced when the metabolic state favours incorporation of dietary EPA into body lipids rather than its beta-oxidation, as mediated by the fed/fasted transition or by treatment with mildronate.

1. We investigated the nature and roles of various xenobiotic acyl-CoA hydrolases in liver subcellular fractions from rat treated with sulphur-substituted (thia) fatty acids. To contribute to our understanding of factors influencing enzymes involved in the degradation of activated fatty acids, the effects on these activities of the oppositely acting thia fatty acid analogues, the peroxisome proliferating 3-thia fatty acids (tetradecylthioacetic acid and 3-dithiacarboxylic acid), which are blocked for beta-oxidation, and a non-peroxisome-proliferating 4-thia fatty acid (tetradecylthiopropionic acid), which undergoes one cycle of beta-oxidation, were studied. 2. The hepatic subcellular distributions of palmitoyl-CoA, tetradecylthioacetyl-CoA and tetradecylthiopropionyl-CoA hydrolase activities were similar to each other in the control and 3-thia fatty acid-treated rat. In control animals, most of these hydrolases were located in the microsomal fraction, but after treatment with the 3-thia fatty acids, the specific activities of the mitochondrial, peroxisomal, and cytosolic palmitoyl-CoA, tetradecylthioacetyl-CoA, and tetradecylthiopropionyl-CoA hydrolase activities were significantly increased. This increase in activity was seen mostly for the enzymes using tetradecylthiopropionyl-CoA and tetradecylthioacetyl-CoA as substrates. The increased mitochondrial activities for these two substrates were seen already after 1 day of treatment, whereas the peroxisomal activities increased after 3 days. No stimulation was seen after treatment with the 4-thia fatty acid analogue, tetradecylthiopropionic acid, but a decrease in peroxisomal hydrolase activities for all three substrates was observed. 3. The cellular distributions of clofibroyl-CoA, POCA-CoA, and sebacoyl-CoA hydrolase activities were different from those of the 'long-chain acyl-CoA' hydrolases mentioned above both in the normal and 3-thia fatty acid treated rat. This group of hydrolases was found in the mitochondrial, peroxisomal, and cytosolic fractions. 3-Thia fatty acid treatment increased the activities of clofibroyl-CoA and sebacoyl-CoA hydrolases in all three fractions. Clofibroyl-CoA and sebacoyl-CoA hydrolase activities were increased after 1 day of treatment. Only the cytosolic POCA-CoA hydrolase was stimulated after 3-thia fatty acid treatment after only 1 day of treatment, whereas treatment with the 4-thia fatty acid led to an increase of enzyme activity in the mitochondrial and peroxisomal fractions. 4. Based on the subcellular distributions and specific activities, we suggest that several enzymes exist which may act as regulators of intracellular acyl-CoA levels.

(1) During peroxisomal beta-oxidation of [U-14C]hexadecanoate, at concentrations higher than 100 microM, long-chain 3-oxoacyl-CoA-esters and 3-oxobutyryl-CoA accumulate. Only 3-oxobutyryl-CoA accumulates at a low concentration of [U-14C]hexadecanoate. Accumulation of long chain 3-oxoacyl-CoA esters is most extensive when the supply of CoA can be considered limiting for beta-oxidation. (2) Added acetyl-CoA was found to inhibit peroxisomal beta-oxidation. This inhibition was not significantly relieved by added L-carnitine and carnitine acetyltransferase (EC 2.3.17). (3) Added L-carnitine, at concentrations below 0.2 mM, was found to stimulate peroxisomal beta-oxidation of [U-14C]hexadecanoate by up to 20%, causing the conversion of acetyl-CoA into acetylcarnitine. Higher concentrations of L-carnitine were progressively inhibitory to beta-oxidation. This effect was specific for L-carnitine as both D-carnitine and aminocarnitine neither caused stimulation at low concentrations, nor inhibition at higher concentrations. Added L-carnitine caused accumulation of acylcarnitines of chain-lengths ranging from 4 to 16 carbon-atoms. The inhibition observed with higher concentrations of added L-carnitine is likely due to conversion of [U-14C]hexadecanoate into [U-14C]hexadecanoylcarnitine. (4) Low concentrations of added hexadecanoylcarnitine was shown to inhibit peroxisomal beta-oxidation by about 15%, while added acetylcarnitine did not inhibit at concentrations up to 100 microM. (5) These data are interpreted to indicate significant control being exerted on flux at the stage of thiolysis either directly by means of CoA availability, or indirectly by means of the rate of acetyl-CoA generation.

The activities of acyl-CoA hydrolase (EC 3.1.2.2.) and acyl-CoA:1-acyl- lysophospholipid acyltransferase (EC 2.3.1.23) have been studied in subcellular fractions of human platelets. The acyl-CoA:1-acyl-lysophospholipid acyltransferase activity was higher in the 'dense-tubular-system-enriched' fraction than in the 'light-mitochondrial' fraction, using endogenously acyl-CoAs formed from labelled fatty acids, ATP, CoA and various lysophospholipids. No activity was found in the 'particle-free' fraction. No difference in specificities was observed between the incorporation of various fatty acids into different lysoPLs in the subcellular fractions compared with the platelet lysates. Generally, arachidonic, linoleic and eicosapentaenoic acids were better substrates for the acyl-CoA:1-acyl-lysophospholipid acyltransferases than oleic, docosahexaenoic and palmitic acids. The opposite was observed with the acyl-CoA hydrolase activity, palmitoyl-CoA was the substrate giving the highest activity, and eicosapentaenoyl-CoA and arachidonoyl-CoA the lowest. About 85% of the hydrolase activity was detected in the 'particle-free' fraction, with each of the six acyl-CoA derivatives tested.

1. The mechanism of xylitol-dependent inhibition of glycolysis in Streptococcus sobrinus OMZ 176 was investigated in aerobically and anaerobically grown cells. 2. Glucose-stimulated glycolysis was followed polarographically, by radio-HPLC-analyses of glycolytic intermediates, by measurement of ATP generated, and spectrophotometric monitoring of extent of NAD(P)+/NADPH-status. 3. Xylitol added to suspensions of S. sobrinus inhibited O2 uptake by approximately 20%, and led to a corresponding decrease in rate of lactate formation in aerobic and anaerobic cells. 4. Xylitol also delayed the onset of the glucose-dependent rapid reduction of NAD(P)+ by approximately 1 min, although the total extent of reduction was not significantly affected compared to control cells. 5. The inhibitory effect of xylitol on glucose dependent ATP synthesis, however, was decreased by 70-80%. 6. Hence the dramatic decrease in glucose-dependent synthesis of ATP may be the direct cause of decreased bacterial growth in the presence of xylitol. 7. A mechanism explaining the observed phenomena is proposed.

In the course of the last decade peroxisomal beta-oxidation has emerged as a metabolic process indispensable to normal physiology. Peroxisomes beta-oxidize fatty acids, dicarboxylic acids, prostaglandins and various fatty acid analogues. Other compounds possessing an alkyl-group of six to eight carbon atoms (many substituted fatty acids) are initially omega-oxidized in endoplasmic reticulum. The resulting carboxyalkyl-groups are subsequently chain-shortened by beta-oxidation in peroxisomes. Peroxisomal beta-oxidation is therefore, in contrast to mitochondrial beta-oxidation, characterized by a very broad substrate-specificity. Acyl-CoA oxidases initiate the cycle of beta-oxidation of acyl-CoA esters. The next steps involve the bi(tri)functional enzyme, which possesses active sites for enoyl-CoA hydratase-, beta-hydroxyacyl-CoA dehydrogenase- and for delta 2, delta 5 enoyl-CoA isomerase activity. The beta-oxidation sequence is completed by a beta-ketoacyl-CoA thiolase. The peroxisomes also contain a 2,4-dienoyl-CoA reductase, which is required for beta-oxidation of unsaturated fatty acids. The peroxisomal beta-hydroxyacyl-CoA epimerase activity is due to the combined action of two enoyl-CoA hydratases. (For a recent review of the enzymology of beta-oxidation enzymes see Ref. 225.) The broad specificity of peroxisomal beta-oxidation is in part due to the presence of at least two acyl-CoA oxidases, one of which, the trihydroxy-5 beta-cholestanoyl-CoA (THCA-CoA) oxidase, is responsible for the initial dehydrogenation of the omega-oxidized cholesterol side-chain, initially hydroxylated in mitochondria. Shortening of this side-chain results in formation of bile acids and of propionyl-CoA. In relation to its mitochondrial counterpart, peroxisomal beta-oxidation in rat liver is characterized by a high extent of induction following exposure of rats to a variety of amphipathic compounds possessing a carboxylic-, or sulphonic acid group. In rats some high fat diets cause induction of peroxisomal fatty acid beta-oxidation and of trihydroxy-5 beta-cholestanoyl-CoA oxidase. Induction involves increased rates of synthesis of the appropriate mRNA molecules. Increased half-lives of mRNA- and enzyme molecules may also be involved. Recent findings of the involvement of a member of the steroid hormone receptor superfamily during induction, suggest that induction of peroxisomal beta-oxidation represents another regulatory phenomenon controlled by nuclear receptor proteins. This will likely be an area of intense future research. Chain-shortening of fatty acids, rather than their complete beta-oxidation, is the prominent feature of peroxisomal beta-oxidation.(ABSTRACT TRUNCATED AT 400 WORDS)

1. 14C-labelled fatty acyl-CoA esters resulting from beta-oxidation of [U-14C]hexadecanoate by peroxisomal fractions isolated from rats treated with clofibrate showed the presence of the full range of saturated intermediates down to acetyl-CoA. 2. The pattern of intermediates generated was fairly constant. At low concentrations of [U-14C]hexadecanoate (50 microM), decanoyl-CoA was present in lowest amounts. At higher concentrations of [U-14C]hexadecanoate (greater than 100 microM), all intermediates of chain length shorter than 12 carbon atoms (except acetyl-CoA) were present at similar low concentrations; the process of beta-oxidation now resembling chain-shortening of hexadecanoate by two cycles of beta-oxidation. 3. In the absence of an NAD(+)-regenerating system [pyruvate and lactate dehydrogenase (EC 1.1.1.28)] 2-enoyl- and 3-hydroxyacyl-CoA esters were generated, suggesting that re-oxidation of NADH is essential for optimal rates of peroxisomal beta-oxidation in vitro. 4. At high concentrations of [U-14C]hexadecanoate (greater than 100 microM), 3-oxohexadecanoyl-CoA was produced, suggesting that thiolase (acetyl-CoA acetyltransferase; EC 2.3.1.9) can become rate-limiting for peroxisomal beta-oxidation.

1. The effects of 3-, 4- and 5-thia-substituted fatty acids on mitochondrial and peroxisomal beta-oxidation have been investigated. When the sulphur atom is in the 4-position, the resulting thia-substituted fatty acid becomes a powerful inhibitor of beta-oxidation. 2. This inhibition cannot be explained in terms of simple competitive inhibition, a phenomenon which characterizes the inhibitory effects of 3- and 5-thia-substituted fatty acids. The inhibitory sites for 4-thia-substituted fatty acids are most likely to be the acyl-CoA dehydrogenase in mitochondria and the acyl-CoA oxidase in peroxisomes. 3. The inhibitory effect of 4-thia-substituted fatty acids is expressed both in vitro and in vivo. The effect in vitro is instantaneous, with up to 95% inhibition of palmitoylcarnitine oxidation. The effect in vivo, in contrast, is dose-dependent and increases with duration of treatment. 4. Pretreatment of rats with a 3-thia-substituted fatty acid rendered mitochondrial beta-oxidation less sensitive to inhibition by 4-thia-substituted fatty acids.

The stimulation of hepatic polyamine metabolism observed 5 hr following intraperitoneal injection of clofibrate to rats was completely abolished following prior treatment with alpha-difluoromethylornithine. No induction of peroxisomal beta-oxidation could be observed 5 hr after injection of clofibrate, although appreciable induction occurred 10 hr after injection. Prior treatment with difluoromethylornithine partially inhibited this induction. On chronic treatment with clofibrate together with difluoro-methylornithine, clofibrate-dependent induction of peroxisomal beta-oxidation, as well as of the hepatomegaly, was partially inhibited. In hypophysectomized rats, no stimulation of polyamine metabolism was found following acute administration of a single dose of clofibrate. In thyroidectomized and in adrenalectomized animals, this stimulation was apparent, although the levels of activity were only some 10% of control levels. In hypophysectomized, in thyroidectomized and in adrenalectomized rats, appreciable induction of peroxisomal beta-oxidation occurred on chronic treatment with clofibrate. However, no hepatomegaly was observed in these animals.

1. A luminometric assay for acyl-CoA oxidase activity is described. The assay uses the luminol/microperoxidase system to monitor continuously acyl-CoA-dependent generation of H2O2. The assay is rapid, convenient, and lends itself to automation with an LKB 1251 luminometer. The assay is extremely sensitive, requiring at the most 10 micrograms of liver-homogenate protein per assay. 2. The assay can also be used to measure other oxidases, e.g. glycollate oxidase (EC 1.1.3.15), D-aspartate oxidase (EC 1.4.3.1) and urate oxidase (EC 1.7.3.3), the only modification being substitution of substrates to appropriate concentration. 3. With rat liver homogenates, spectrophotometrically measured rates of palmitoyl-CoA-dependent NAD+ reduction and acyl-CoA oxidase activity [Hryb & Hogg (1979) Biochem. Biophys. Res. Commun. 87, 1200-1206] was generally found in good agreement with luminometrically measured acyl-CoA oxidase activity. 4. With liver homogenates from streptozotocin-diabetic rats, however, rates of palmitoyl-CoA-dependent NAD+ reduction were consistently lower than the corresponding acyl-CoA oxidase activity. This difference was most marked with respect to luminometrically assayed acyl-CoA oxidase activity.

1. beta-Oxidation of gamma-linolenoylcarnitine, arachidonoylcarnitine and docosahexaenoylcarnitine by isolated rat liver mitochondria is inhibited by uncoupling conditions. Partial re-activation is obtained with added ATP. With mitochondria from clofibrate-treated rats ATP-stimulated rates of beta-oxidation of docosahexaenoylcarnitine are higher than ADP-stimulated rates. This is not observed with the beta-oxidation of oleoylcarnitine. 2. beta-Oxidation of docosahexaenoylcarnitine, in the presence of rotenone, is inhibited by added oxaloacetate, analogous to previous findings with pent-4-enoylcarnitine [see Osmundsen (1978) FEBS Lett. 88, 219-222]. In the absence of rotenone added oxaloacetate stimulates the beta-oxidation of docosahexaenoylcarnitine, but has the opposite effect on the beta-oxidation of palmitoylcarnitine. 3. beta-Oxidation of polyunsaturated acylcarnitines by isolated rat liver mitochondria is selectively increased after treatment of the animals with a low dietary dose (0.2%, w/w) of clofibrate. Treatment with a higher dose of clofibrate (0.5%, w/w) resulted in a general stimulation of beta-oxidation. 4. The results presented suggest that long-chain fatty acids possessing a delta 4-double bond are not readily beta-oxidized unless the 2,4-enoyl-CoA reductase (EC 1.3.1.-) is operating.

1. Oxidation of radioactivity labeled acyl-CoA esters by apparently intact peroxisomes, isolated from rats treated with clofibrate, was stimulated by about 100% by added oxaloacetate. Added pyruvate gave much smaller stimulation. This stimulation is proposed to be due to re-oxidation of NADH generated during peroxisomal beta-oxidation of acyl-CoA esters. 2. Additions of these alpha-ketoacids, and of CoA, were found to influence extent of peroxisomal beta-oxidation of [U-14C]palmitoyl-CoA. Various concentrations of added CoA was markedly less inhibitory to oxidation of [14-14C]erucoyl-CoA and [10-14C]elaidoyl-CoA, as compared to [U-14C]palmitoyl-CoA. 3. Added NADP+, at concentrations higher than 0.5 mM, added NADPH and NADH were inhibitory to peroxisomal beta-oxidation of [U-14C]palmitoyl-CoA. Significant inhibition was found at concentrations which were too high for this to be of significance in vivo.

Liver peroxisomes were prepared by using a Percoll gradient in a vertical rotor. beta-Oxidation was measured in peroxisomes isolated from livers of rats fed on either high-(15% by wt.) or low- (5% by wt.) fat diets. The feeding of high-fat diets gave a 1.4-2.4-fold increase in total liver peroxisomal beta-oxidation, and a similar increase in specific activity. A 1.5-4.5-fold increase was seen in the specific activity of purified peroxisomal preparations. The reasons for these increases are discussed.

beta-Oxidation of polyunsaturated fatty acids was studied with isolated rat liver mitochondria in state 3 or uncoupled conditions. 1. Incubation of mitochondria with docosahexaenoyl-, linolenoyl- or gamma-linolenoylcarnitine resulted in an increase of the absorbance at 340 minus 385 nm. This increased absorbance was due to an accumulation of beta-oxidation intermediates of the polyunsaturated fatty acids, and not to the reduction of nicotinamide nucleotides. 2. Experiments carried out with soluble fractions of liver mitochondria incubated with docosahexaenoyl-CoA and gamma-linolenoyl-CoA indicated that this ultraviolet light-absorption was at least partly caused by acyl-CoA esters having a 2,4(,7)-di(tri)enoyl-CoA structure. 3. The addition of glutamate to mitochondria oxidizing gamma-linolenoylcarnitine decreased the absorbance at 340 minus 385 nm, and simultaneously stimulated respiration. With liver mitochondria isolated from fasted rats, 6 mM glutamate increased the rate of acetoacetate production from gamma-linolenoylcarnitine by 130 and 210% under state 3 and uncoupled conditions, respectively. Glutamate did not have any significant effect on the degradation of oleoylcarnitine. The proposed explanation for these findings is that the glutamate dehydrogenase reaction can function as a source of NADPH for 2,4-dienoyl-CoA reductase. 4. The degradation of gamma-linolenoylcarnitine to ketone bodies was augmented in mitochondria isolated from rats treated with clofibrate or partially hydrogenated marine oil. 5. We conclude that 2,4-dienoyl-CoA reductase is an important auxiliary enzyme in the beta-oxidation of polyunsaturated fatty acids. Induction of this enzyme by clofibrate or by certain high-fat diets increases mitochondrial capacity for the degradation of polyunsaturated fatty acids.