Lipidomic and Proteomic Alterations Induced by Even and Odd Medium-Chain Fatty Acids on Fibroblasts of Long-Chain Fatty Acid Oxidation Disorders.

Medium-chain fatty acids (mc-FAs) are currently applied in the treatment of long-chain fatty acid oxidation disorders (lc-FAOD) characterized by impaired ß-oxidation. Here, we performed lipidomic and proteomic analysis in fibroblasts from patients with very long-chain acyl-CoA dehydrogenase (VLCADD) and long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHADD) deficiencies after incubation with heptanoate (C7) and octanoate (C8). Defects of ß-oxidation induced striking proteomic alterations, whereas the effect of treatment with mc-FAs was minor. However, mc-FAs induced a remodeling of complex lipids. Especially C7 appeared to act protectively by restoring sphingolipid biosynthesis flux and improving the observed dysregulation of protein homeostasis in LCHADD under control conditions.

Prostaglandin E receptor EP4 stimulates lymphangiogenesis to promote mucosal healing during DSS-induced colitis.

In the intestine, the formation of new lymphatic vessels from pre-existing lymphatic vasculature (lymphangiogenesis) is related to the progression of inflammatory bowel disease (IBD). However, it remains unclear whether lymphangiogenesis contributes to mucosal repair after acute colitis. Prostaglandin Ereceptor EP4 suppresses the development of experimental colitis. In this study, we investigated whether EP4 exerts this effect by contributing to lymphangiogenesis, in turn promoting mucosal tissue repair, following acute colitis. We elicited experimental colitis in male C57/BL6 mice by administering dextran sulphate sodium (DSS) via the drinking water for 5 days, followed by normal water for 9 additional days. From Day 5 through Day 13, the experimental mice received a daily dose of EP4-selective agonist, EP4-selective antagonist, or vehicle. On Day 14, mice treated with vehicle had recovered 95 % of body weight and exhibited moderate increases in disease activity and histological score relative to untreated controls. Compared with vehicle, post-treatment with EP4 antagonist increased signs of colitis, colonic tissue destruction, and CD11b+ cell infiltration, associated with elevated lymphatic vessel density (LVD) and reduced percentage of lymphatic vessel area (LVA%). By contrast, post-treatment with EP4 agonist improved disease activity, suppressed CD11b+ infiltration, and decreased levels of inflammatory cytokines; these changes were associated with upregulation of lymphatic growth factors and lymphangiogenesis, as evidenced by increases in LVA% and lymphatic drainage function. Inhibition of vascular endothelial growth factor receptor 3 (VEGFR3) caused a delay in mucosal repair, accompanied by impaired lymphangiogenesis. These results suggest that EP4 stimulation aids in mucosal repair from DSS-induced acute colitis by promoting lymphangiogenesis.

Differential Mechanism of ATP Production Occurs in Response to Succinylacetone in Colon Cancer Cells.

Our aim was to verify the potential ability of succinylacetone (SA) to inhibit mitochondrial function, thereby suppressing cancer cell proliferation. SA treatment caused apoptosis in HCT116 and HT29 cells, but not in SW480 cells, with mitochondria playing a key role. We checked for dysfunctional mitochondria after SA treatment. Mitochondria of HT29 cells were swollen, indicating damage, whereas in HCT116 cells, several mitochondria had a diminished size. Damaged mitochondria decreased ATP production and induced reactive oxygen species (ROS) in the cells. To understand SA-induced reduction in ATP production, we investigated the electron transfer chains (ETC) and pyruvate dehydrogenase kinase (PDK) activity, which prevents the transfer of acetyl-CoA to the TCA (tricarboxylic acid) cycle by inhibiting PDH (pyruvate dehydrogenase) activity. In each cell line, the inhibitory mechanism of ATP by SA was different. The activity of complex III consisting of the mitochondrial ETCs in HT29 cells was decreased. In contrast, PDH activity in HCT116 cells was reduced. Nicotinamide nucleotide transhydrogenase (NNT)-removing reactive oxygen species (ROS) was upregulated in HT29 cells, but not in HCT116 cells, indicating that in HT29 cells, a defense mechanism was activated against ROS. Collectively, our study showed a differential mechanism occurs in response to SA in colon cancer cells.

Heptanoate is neuroprotective in vitro but triheptanoin post-treatment did not protect against middle cerebral artery occlusion in rats.

Triheptanoin, the medium-chain triglyceride of heptanoate, has been shown to be anticonvulsant and neuroprotective in several neurological disorders. In the gastrointestinal tract, triheptanoin is cleaved to heptanoate, which is then taken up by the blood and most tissues, including liver, heart and brain. Here we evaluated the neuroprotective effects of heptanoate and its effects on mitochondrial oxygen consumption in vitro. We also investigated the neuroprotective effects of triheptanoin compared to long-chain triglycerides when administered after stroke onset in rats. Heptanoate pre-treatment protected cultured neurons against cell death induced by oxygen glucose deprivation and N-methyl-D-aspartate. Incubation of cultured astrocytes with heptanoate for 2 h increased mitochondrial proton leak and also enhanced basal respiration and ATP turnover, suggesting that heptanoate protects against oxidative stress and is used as fuel. However, continuous 72 h infusion of triheptanoin initiated 1 h after middle cerebral artery occlusion in rats did not alter stroke volume at 3 days or neurological deficit at 1 and 3 days relative to long-chain triglyceride control treatment.

Potential role of heme metabolism in the inducible expression of heme oxygenase-1.

BACKGROUND: The degradation of heme significantly contributes to cytoprotective effects against oxidative stress and inflammation. The enzyme heme oxygenase-1 (HO-1), involved in the degradation of heme, forms carbon monoxide (CO), ferrous iron, and bilirubin in conjunction with biliverdin reductase, and is induced by various stimuli including oxidative stress and heavy metals. We examined the involvement of heme metabolism in the induction of HO-1 by the inducers sulforaphane and sodium arsenite. METHODS: We examined the expression of HO-1 in sulforaphane-, sodium arsenite- and CORM3-treated HEK293T cells, by measuring the transcriptional activity and levels of mRNA and protein. RESULTS: The blockade of heme biosynthesis by succinylacetone and N-methyl protoporphyrin, which are inhibitors of heme biosynthesis, markedly decreased the induction of HO-1. The knockdown of the first enzyme in the biosynthesis of heme, 5-aminolevulinic acid synthase, also decreased the induction of HO-1. The cessation of HO-1 induction occurred at the transcriptional and translational levels, and was mediated by the activation of the heme-binding transcriptional repressor Bach1 and translational factor HRI. CO appeared to improve the expression of HO-1 at the transcriptional and translational levels. CONCLUSIONS: We demonstrated the importance of heme metabolism in the stress-inducible expression of HO-1, and also that heme and its degradation products are protective factors for self-defense responses. GENERAL SIGNIFICANCE: The key role of heme metabolism in the stress-inducible expression of HO-1 may promote further studies on heme and its degradation products as protective factors of cellular stresses and iron homeostasis in specialized cells, organs, and whole animal systems.

Differential effects of octanoate and heptanoate on myocardial metabolism during extracorporeal membrane oxygenation in an infant swine model.

Nutritional energy support during extracorporeal membrane oxygenation (ECMO) should promote successful myocardial adaptation and eventual weaning from the ECMO circuit. Fatty acids (FAs) are a major myocardial energy source, and medium-chain FAs (MCFAs) are easily taken up by cell and mitochondria without membrane transporters. Odd-numbered MCFAs supply carbons to the citric acid cycle (CAC) via anaplerotic propionyl-CoA as well as acetyl-CoA, the predominant ß-oxidation product for even-numbered MCFA. Theoretically, this anaplerotic pathway enhances carbon entry into the CAC, and provides superior energy state and preservation of protein synthesis. We tested this hypothesis in an immature swine model undergoing ECMO. Fifteen male Yorkshire pigs (26-45 days old) with 8-h ECMO received either normal saline, heptanoate (odd-numbered MCFA), or octanoate (even-numbered MCFA) at 2.3 µmol·kg body wt(-1)·min(-1) as MCFAs systemically during ECMO (n = 5/group). The 13-carbon ((13)C)-labeled substrates ([2-(13)C]lactate, [5,6,7-(13)C3]heptanoate, and [U-(13)C6]leucine) were systemically infused as metabolic markers for the final 60 min before left ventricular tissue extraction. Extracted tissues were analyzed for the (13)C-labeled and absolute concentrations of metabolites by nuclear magnetic resonance and gas chromatography-mass spectrometry. Octanoate produced markedly higher myocardial citrate concentration, and led to a higher [ATP]-to-[ADP] ratio compared with other groups. Unexpectedly, octanoate and heptanoate increased the flux of propionyl-CoA relative to acetyl-CoA into the CAC compared with control. MCFAs promoted increases in leucine oxidation, but were not associated with a difference in protein synthesis rate. In conclusion, octanoate provides energetic advantages to the heart over heptanoate.

Heme-mediated inhibition of Bach1 regulates the liver specificity and transience of the Nrf2-dependent induction of zebrafish heme oxygenase 1.

The induction of the gene encoding heme oxygenase 1 (Hmox1, HO-1) by Nrf2 is unique compared with other Nrf2 targets. We previously showed that the Nrf2a-mediated induction of zebrafish hmox1a was liver specific and transient. We screened transcription factors that could repress the induction of hmox1a but not other Nrf2a targets and concluded that Bach1b was a prime candidate. In bach1b-knocked-down larvae, the induction of hmox1a was observed ectopically in nonliver tissues and persisted longer than normal fish, suggesting that Bach1 is the only regulator for both the liver-specific and transient induction of hmox1a. Co-knockdown of bach1b with its co-ortholog bach1a enhanced these effects. To determine why Bach1 could not repress the hmox1a induction in the liver, we analyzed the effects of a heme biosynthesis inhibitor, succinylacetone, and a heme precursor, hemin. Succinylacetone decreased the Nrf2a-mediated hmox1a induction, whereas pre-treatment with hemin caused ectopic induction of hmox1a in nonliver tissues, implying that the high heme levels in the liver may release the repressive activity of Bach1. Our results suggested that Bach1 regulates the liver specificity and transience of the Nrf2a-dependent induction of hmox1a and that heme mediates this regulation through Bach1 inhibition based on its level in each tissue.

Influence of aromatase inhibition on the bone-protective effects of testosterone.

The influence of the aromatase enzyme in androgen-induced bone maintenance after skeletal maturity remains somewhat unclear. Our purpose was to determine whether aromatase activity is essential to androgen-induced bone maintenance. Ten-month-old male Fisher 344 rats (n = 73) were randomly assigned to receive Sham surgery, orchiectomy (ORX), ORX + anastrozole (AN; aromatase inhibitor), ORX + testosterone-enanthate (TE, 7.0 mg/wk), ORX + TE + AN, ORX + trenbolone-enanthate (TREN; nonaromatizable, nonestrogenic testosterone analogue; 1.0 mg/wk), or ORX + TREN + AN. ORX animals exhibited histomorphometric indices of high-turnover osteopenia and reduced cancellous bone volume compared with Shams. Both TE and TREN administration suppressed cancellous bone turnover similarly and fully prevented ORX-induced cancellous bone loss. TE- and TREN-treated animals also exhibited greater femoral neck shear strength than ORX animals. AN co-administration slightly inhibited the suppression of bone resorption in TE-treated animals but did not alter TE-induced suppression of bone formation or the osteogenic effects of this androgen. In TREN-treated animals, AN co-administration produced no discernible effects on cancellous bone turnover or bone volume. ORX animals also exhibited reduced levator ani/bulbocavernosus (LABC) muscle mass and elevated visceral adiposity. In contrast, TE and TREN produced potent myotrophic effects in the LABC muscle and maintained fat mass at the level of Shams. AN co-administration did not alter androgen-induced effects on muscle or fat. In conclusion, androgens are able to induce direct effects on musculoskeletal and adipose tissue, independent of aromatase activity.

Disruption of fumarylacetoacetate hydrolase causes spontaneous cell death under short-day conditions in Arabidopsis.

Fumarylacetoacetate hydrolase (FAH) hydrolyzes fumarylacetoacetate to fumarate and acetoacetate, the final step in the tyrosine (Tyr) degradation pathway that is essential to animals. Deficiency of FAH in animals results in an inborn lethal disorder. However, the role for the Tyr degradation pathway in plants remains to be elucidated. In this study, we isolated an Arabidopsis (Arabidopsis thaliana) short-day sensitive cell death1 (sscd1) mutant that displays a spontaneous cell death phenotype under short-day conditions. The SSCD1 gene was cloned via a map-based cloning approach and found to encode an Arabidopsis putative FAH. The spontaneous cell death phenotype of the sscd1 mutant was completely eliminated by further knockout of the gene encoding the putative homogentisate dioxygenase, which catalyzes homogentisate into maleylacetoacetate (the antepenultimate step) in the Tyr degradation pathway. Furthermore, treatment of Arabidopsis wild-type seedlings with succinylacetone, an abnormal metabolite caused by loss of FAH in the Tyr degradation pathway, mimicked the sscd1 cell death phenotype. These results demonstrate that disruption of FAH leads to cell death in Arabidopsis and suggest that the Tyr degradation pathway is essential for plant survival under short-day conditions.

Heme status affects human hepatic messenger RNA and microRNA expression.

AIM: To assess effects of heme on messenger RNA (mRNA) and microRNA (miRNA) profiles of liver cells derived from humans. METHODS: We exposed human hepatoma cell line Huh-7 cells to excess iron protoporphyrin (heme) (10 µmol/L) or induced heme deficiency by addition of 4, 6-dioxoheptanoic acid (500 µmol/L), a potent inhibitor of aminolevulinic acid dehydratase, for 6 h or 24 h. We harvested total RNA from the cells and performed both mRNA and miRNA array analyses, with use of Affymetrix chips, reagents, and instruments (human genome U133 plus 2.0 and miRNA 2.0 arrays). We assessed changes and their significance and interrelationships with Target Scan, Pathway Studios, and Ingenuity software. RESULTS: Changes in mRNA levels were most numerous and striking at 6 h after heme treatment but were similar and still numerous at 24 h. After 6 h of heme exposure, the increase in heme oxygenase 1 gene expression was 60-fold by mRNA and 88-fold by quantitative reverse transcription-polymerase chain reaction. We found striking changes, especially up-regulation by heme of nuclear erythroid-2 related factor-mediated oxidative stress responses, protein ubiquitination, glucocorticoid signaling, P53 signaling, and changes in RNAs that regulate intermediary metabolism. Fewer mRNAs were down-regulated by heme, and the fold decreases were less exuberant than were the increases. Notable decreases after 24 h of heme exposure were patatin-like phospholipase domain-containing protein 3 (-6.5-fold), neuronal PAS domain protein 2 (-1.93-fold), and protoporphyrinogen oxidase (-1.7-fold). CONCLUSION: Heme excess exhibits several toxic effects on liver and kidney, which deserve study in humans and in animal models of the human porphyrias or other disorders.

Heptanoate as a neural fuel: energetic and neurotransmitter precursors in normal and glucose transporter I-deficient (G1D) brain.

It has been postulated that triheptanoin can ameliorate seizures by supplying the tricarboxylic acid cycle with both acetyl-CoA for energy production and propionyl-CoA to replenish cycle intermediates. These potential effects may also be important in other disorders associated with impaired glucose metabolism because glucose supplies, in addition to acetyl-CoA, pyruvate, which fulfills biosynthetic demands via carboxylation. In patients with glucose transporter type I deficiency (G1D), ketogenic diet fat (a source only of acetyl-CoA) reduces seizures, but other symptoms persist, providing the motivation for studying heptanoate metabolism. In this work, metabolism of infused [5,6,7-(13)C(3)]heptanoate was examined in the normal mouse brain and in G1D by (13)C-nuclear magnetic resonance spectroscopy, gas chromatography-mass spectrometry (GC-MS), and liquid chromatography-mass spectrometry (LC-MS). In both groups, plasma glucose was enriched in (13)C, confirming gluconeogenesis from heptanoate. Acetyl-CoA and glutamine levels became significantly higher in the brain of G1D mice relative to normal mice. In addition, brain glutamine concentration and (13)C enrichment were also greater when compared with glutamate in both animal groups, suggesting that heptanoate and/or C5 ketones are primarily metabolized by glia. These results enlighten the mechanism of heptanoate metabolism in the normal and glucose-deficient brain and encourage further studies to elucidate its potential antiepileptic effects in disorders of energy metabolism.

Fumarylacetoacetate inhibits the initial step of the base excision repair pathway: implication for the pathogenesis of tyrosinemia type I.

Hereditary tyrosinemia type I (HT1) is an autosomal recessive disease caused by a deficiency in human fumarylacetoacetate (FAA) hydrolase (FAH), which is the last enzyme in the catabolic pathway of tyrosine. Several reports suggest that intracellular accumulation of intermediates of tyrosine catabolism, such as FAA and succinylacetone (SA) is important for the pathogenesis in liver and kidney of HT1 patients. In this work, we examined the effect of FAA and SA on DNA glycosylases initiating base excision repair (BER), which is the most important pathway for removing mutagenic DNA base lesions. In vitro assays monitoring DNA glycosylase activities demonstrated that FAA but not SA inhibited base removal. In particular, the Neil1 and Neil2 DNA glycosylases were strongly inhibited, whereas inhibition of Nth1 and Ogg1 were less efficient. These DNA glycosylases initiate excision of a broad range of mutagenic oxidative base lesions. Further, FAA showed a modest inhibitory effect on the activity of the alkylbase DNA glycosylase Aag and no significant inhibition of the uracil DNA glycosylase Ung2. These data indicate that FAA inhibition of DNA glycosylases removing oxidative base lesions in HT1 patients may increase mutagenesis, suggesting an important mechanism for development of hepatocarcinoma and somatic mosaicism.

Heme and heme biosynthesis intermediates induce heme oxygenase-1 and cytochrome P450 2A5, enzymes with putative sequential roles in heme and bilirubin metabolism: different requirement for transcription factor nuclear factor erythroid- derived 2-like 2.

Cytochrome P450 2A5 (CYP2A5) oxidizes bilirubin to biliverdin and represents a putative candidate for maintaining bilirubin at safe but adequate antioxidant levels. Curiously, CYP2A5 is induced by both excessive heme and chemicals that inhibit heme synthesis. We hypothesized that heme homeostasis is a key modifier of Cyp2a5 expression via transcription factor nuclear factor erythroid-derived 2-like 2 (Nrf2) and characterized the coordination of CYP2A5 and heme oxygenase-1 (HMOX1) responses using wild-type and Nrf2(-/-) primary mouse hepatocytes. HMOX1 was rapidly elevated by exogenous hemin, thereby limiting the transactivation of Cyp2a5 until high heme (> 5µM) exposure. Nrf2 was mandatory for CYP2A5 but not for HMOX1 induction by heme. CYP2A5 was intensively and HMOX1 moderately elevated in heme synthesis blockades by succinylacetone and N-methyl protoporphyrin IX, and Nrf2 partially mediated the induction of CYP2A5. Immunoelectron microscopy revealed that CYP2A5 is targeted Nrf2 dependently both to the endoplasmic reticulum (ER) and mitochondria. However, excessive heme increased CYP2A5 predominantly in the ER. Phenobarbital, dibutyryl-cAMP, and peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) overexpression stimulate heme biosynthesis and induce CYP2A5. Acute but not chronic CYP2A5 induction by phenobarbital required Nrf2, whereas CYP2A5 induction by dibutyryl-cAMP and PGC-1α was potentiated by Nrf2 knockout. Collectively, heme homeostasis is established as a crucial regulator of hepatic Cyp2a5 expression mediated via Nrf2 activation, whereas Nrf2 is redundant for Hmox1 induction by heme. Similar subcellular targeting and coordination of CYP2A5 and HMOX1 responses suggest favorable conditions for enhanced CYP2A5-mediated bilirubin maintenance in altered heme homeostasis that predisposes to oxidative stress.

The intrinsic prostaglandin E2-EP4 system of the renal tubular epithelium limits the development of tubulointerstitial fibrosis in mice.

Inflammatory responses in the kidney lead to tubulointerstitial fibrosis, a common feature of chronic kidney diseases. Here we examined the role of prostaglandin E(2) (PGE(2)) in the development of tubulointerstitial fibrosis. In the kidneys of wild-type mice, unilateral ureteral obstruction leads to progressive tubulointerstitial fibrosis with macrophage infiltration and myofibroblast proliferation. This was accompanied by an upregulation of COX-2 and PGE(2) receptor subtype EP(4) mRNAs. In the kidneys of EP(4) gene knockout mice, however, obstruction-induced histological alterations were significantly augmented. In contrast, an EP(4)-specific agonist significantly attenuated these alterations in the kidneys of wild-type mice. The mRNAs for macrophage chemokines and profibrotic growth factors were upregulated in the kidneys of wild-type mice after ureteral obstruction. This was significantly augmented in the kidneys of EP(4)-knockout mice and suppressed by the EP(4) agonist but only in the kidneys of wild-type mice. Notably, COX-2 and MCP-1 proteins, as well as EP(4) mRNA, were localized in renal tubular epithelial cells after ureteral obstruction. In cultured renal fibroblasts, another EP(4)-specific agonist significantly inhibited PDGF-induced proliferation and profibrotic connective tissue growth factor production. Hence, an endogenous PGE(2)-EP(4) system in the tubular epithelium limits the development of tubulointerstitial fibrosis by suppressing inflammatory responses.

Prostaglandin E(2) receptors as potential bone anabolic targets - selective EP4 receptor agonists.

Prostaglandin E(2) is known to be a potent metabolite in bone biology. Its effects are mediated via four receptor subtypes with different properties, effects and mechanisms of action. The EP2 and EP4 receptors have been extensively investigated as bone anabolic therapy targets in the literature. The aim of this review was to analyse the available evidence supporting the use of selective agonists for those receptors for anabolic bone application purposes. Although several studies report on the presence of the EP2 receptor in several cell types, efforts to directly confirm the presence of this receptor in human bone cells have not been successful. The EP4 receptor however has been identified in human bone cells and its significant role in bone biology has been demonstrated with the use of selective agonists, antagonists and transgenic small animals. The use of selective EP4 agonists reversed established osteoporotic changes, enhanced the boneimplant interface strength and was shown to have a synergistic effect when used with other bone cell targeting pharmacological agents such as BMP-2 and bisphosphonates. Further elucidation of the side-effect profile of prostanoid and non-prostanoid agonists is required for these agents to proceed towards clinical applications.

Functional significance of the ATP-binding cassette transporter B6 in hepatocellular carcinoma.

ABCB6 is a mitochondrial transporter that regulates porphyrin biosynthesis. ABCB6 expression is upregulated in hepatocellular carcinoma (HCC) but the significance of this upregulation to HCC is not known. In the present study, we investigated: 1) ABCB6 expression in 18 resected human hepatocellular carcinoma (HCC) tissues and 3 human hepatoma cell lines; 2) pattern of ABCB6 expression during liver disease progression; and 3) functional significance of ABCB6 expression to HCC using the hepatoma cell line Huh7. ABCB6 expression was determined by real-time quantitative reverse transcription-polymerase chain reaction and western blotting. ABCB6 expression was upregulated in all the HCC specimens and the three-hepatoma cell lines. Increased ABCB6 expression correlated with liver disease progression with the pattern of expression being HCC > cirrhosis > steatosis. Small hairpin RNA (shRNA)-mediated knockdown of ABCB6 in Huh7 cells lead to decreased cellular proliferation and colony formation. Attenuation of ABCB6 expression did not affect Huh7 apoptosis but lead to a delay in G2/M phase of the cell cycle. In contrast, ABCB6 overexpression resulted in increased growth and proliferation of Huh7 cells. Since ABCB6 expression is induced in multiple tumor types we explored the role of ABCB6 in other cancer cells. ShRNA mediated knockdown of ABCB6 in HEK293 and K562 cells reduced cellular proliferation leading to a delay in G2/M phase, while ABCB6 overexpression promoted cell growth and proliferation. Collectively, these findings, obtained by loss of function and gain of function analysis, suggest that ABCB6 plays a role in cell growth and proliferation by targeting the cell cycle.

Prostaglandin E(2) receptor EP(4)-selective agonist (ONO-4819) increases bone formation by modulating mesenchymal cell differentiation.

Prostaglandin E(2) (PGE(2)) positively regulates bone resorption and formation mainly mediated through the EP(4) receptor, a subtype of PGE(2) receptors. ONO-4819, an EP(4) receptor-selective agonist, has been shown to increase bone volume, density, and strength; however, the mechanism of these effects has yet to be fully elucidated. To explore this matter, ONO-4819 (10µg/kg) was injected into intact rats twice a day for 5weeks, and their bones were then analyzed by morphological techniques. The effects of ONO-4819 on the differentiation of bone cells were also examined in vitro. Bone morphometric analysis showed that osteoblast number, bone volume, mineral apposition rate, and bone formation rate were significantly increased by ONO-4819, whereas osteoclast number was not affected. Immunohistochemical examination demonstrated that ONO-4819 increased the number of Runx2- and Osterix-positive osteoblasts in rats. In vitro studies using the multipotent mesenchymal cell line C3H10T1/2 showed that ONO-4819 induced alkaline phosphatase (ALPase) activity and up-regulated the mRNA expression of ALPase and Osterix. In contrast, ONO-4819 reduced the mRNA expression of peroxisome proliferator-activated receptor γ (PPARγ) and inhibited adipocyte differentiation of C3H10T1/2 cells, which findings are consistent with the observation that the age-dependent increase in adipocyte number in the bone marrow was significantly suppressed in the ONO-4819-treated animals. ONO-4819 also dose-dependently increased osteoclast-like cell formation in vitro, but the required concentrations were much higher than those to induce osteoblast differentiation. These results collectively suggest that ONO-4819 increased bone formation by stimulating osteoblast differentiation and function, possibly through modulating mesenchymal cell differentiation in the bone.

Plastid-associated porphobilinogen synthase from Toxoplasma gondii: kinetic and structural properties validate therapeutic potential.

Apicomplexan parasites (including Plasmodium spp. and Toxoplasma gondii) employ a four-carbon pathway for de novo heme biosynthesis, but this pathway is distinct from the animal/fungal C4 pathway in that it is distributed between three compartments: the mitochondrion, cytosol, and apicoplast, a plastid acquired by secondary endosymbiosis of an alga. Parasite porphobilinogen synthase (PBGS) resides within the apicoplast, and phylogenetic analysis indicates a plant origin. The PBGS family exhibits a complex use of metal ions (Zn(2+) and Mg(2+)) and oligomeric states (dimers, hexamers, and octamers). Recombinant T. gondii PBGS (TgPBGS) was purified as a stable approximately 320-kDa octamer, and low levels of dimers but no hexamers were also observed. The enzyme displays a broad activity peak (pH 7-8.5), with a K(m) for aminolevulinic acid of approximately 150 microM and specific activity of approximately 24 micromol of porphobilinogen/mg of protein/h. Like the plant enzyme, TgPBGS responds to Mg(2+) but not Zn(2+) and shows two Mg(2+) affinities, interpreted as tight binding at both the active and allosteric sites. Unlike other Mg(2+)-binding PBGS, however, metal ions are not required for TgPBGS octamer stability. A mutant enzyme lacking the C-terminal 13 amino acids distinguishing parasite PBGS from plant and animal enzymes purified as a dimer, suggesting that the C terminus is required for octamer stability. Parasite heme biosynthesis is inhibited (and parasites are killed) by succinylacetone, an active site-directed suicide substrate. The distinct phylogenetic, enzymatic, and structural features of apicomplexan PBGS offer scope for developing selective inhibitors of the parasite enzyme based on its quaternary structure characteristics.

Assessing the DNA methylation status of single cells with the comet assay.

The comet assay (single cell gel electrophoresis) is a cost-effective, sensitive, and simple technique that is traditionally used for analyzing and quantifying DNA damage in individual cells. The aim of this study was to determine whether the comet assay could be modified to detect changes in the levels of DNA methylation in single cells. We used the difference in methylation sensitivity of the isoschizomeric restriction endonucleases HpaII and MspI to demonstrate the feasibility of the comet assay to measure the global DNA methylation level of individual cells. The results were verified with the well-established cytosine extension assay. We were able to show variations in DNA methylation after treatment of cultured cells with 5-azacytidine and succinylacetone, an accumulating metabolite in human tyrosinemia type I.