Effects of sources and routes of administration of vitamins A, D and copper on postnatal status of these micronutrients in piglets.

Twenty-six nulliparous sows were fed conventional gestation and lactation diets supplemented (N = 13) or not (N = 13) with extra daily supplements of 25-hydroxy-cholecalciferol (25-OH-D3; 4 ĸIU), ß-carotene (24 ĸIU), and copper (Cu)-proteinate (45 mg) from day 90 of gestation to 21 d of lactation (L21). In each litter, 10 piglets were divided into 5 pairs received, at 2 (L2) and 8 d (L8) of age, one of the five combinations of micronutrient sources and routes of administration (N = 260 piglets total). These neonatal treatments (N = 26 pairs or 52 piglets each) consisted of oral vitamin D3, retinol acetate and CuSO4 (T1); oral 25-OH-D3, ß-carotene, and Cu proteinate (T2); exposure to ultraviolet light (UVB), oral retinol palmitate and Cu gluconate (T3); intramuscular vitamin D3 and retinyl propionate and oral Cu acetate (T4); oral saline (CTRL). Oral or intramuscular provisions corresponded to 12 mg of Cu and 70 and 12 ĸIU of vitamins A and D, respectively. Blood samples were collected from all piglets at L2, L8, and L21 for determination of serum Cu, retinol, and 25-OH-D3. Body weight was measured at birth, L2, L8, and L21. Piglets were weaned at L21, and liver and blood samples were collected 2 d later to evaluate oxidative enzymes in blood and liver and hepatic ATP concentrations and expression of genes associated with antioxidant status. Sow treatments had marginal or no impacts on Cu, retinol, 25-OH-D3, or antioxidant status in piglet blood serum and liver. However, when supplements were given to piglets, hepatic Cu was 38% greater in for all treated piglets compared to CTRL (P < 0.01), hepatic retinol was 3 times higher in T1 than in CTRL (P < 0.01) and intermediate for other treatments whereas serum 25-OH-D3 was markedly increased with T2 and T3 at L8 and L21, respectively, compared to CTRL (Piglet treatment × Age interaction, P < 0.01). Concerning antioxidant activities, glutathione peroxidase, and superoxide dismutase were increased (P < 0.03) in plasma of T2 piglets whereas the highest values (P < 0.03) for indicators of oxidative damage to proteins were observed in T4 piglets. The study revealed that oral Cu proteinate from T2, oral retinol acetate from T1, oral 25-hydroxy-cholecalciferol from T2, and UVB light exposure from T3 were the most efficient ways of increasing the postnatal status of these micronutrients in suckling piglets and this may have some impacts on their peri-weaning antioxidant status.

FGF21 underlies a hormetic response to metabolic stress in methylmalonic acidemia.

Methylmalonic acidemia (MMA), an organic acidemia characterized by metabolic instability and multiorgan complications, is most frequently caused by mutations in methylmalonyl-CoA mutase (MUT). To define the metabolic adaptations in MMA in acute and chronic settings, we studied a mouse model generated by transgenic expression of Mut in the muscle. Mut-/-;TgINS-MCK-Mut mice accurately replicate the hepatorenal mitochondriopathy and growth failure seen in severely affected patients and were used to characterize the response to fasting. The hepatic transcriptome in MMA mice was characterized by the chronic activation of stress-related pathways and an aberrant fasting response when compared with controls. A key metabolic regulator, Fgf21, emerged as a significantly dysregulated transcript in mice and was subsequently studied in a large patient cohort. The concentration of plasma FGF21 in MMA patients correlated with disease subtype, growth indices, and markers of mitochondrial dysfunction but was not affected by renal disease. Restoration of liver Mut activity, by transgenesis and liver-directed gene therapy in mice or liver transplantation in patients, drastically reduced plasma FGF21 and was associated with improved outcomes. Our studies identify mitocellular hormesis as a hepatic adaptation to metabolic stress in MMA and define FGF21 as a highly predictive disease biomarker.

Supplements of vitamins B9 and B12 affect hepatic and mammary gland gene expression profiles in lactating dairy cows.

BACKGROUND: A combined supplement of vitamins B9 and B12 was reported to increase milk and milk component yields of dairy cows without effect on feed intake. The present study was undertaken to verify whether this supplementation positively modifies the pathways involved in milk and milk component synthesis. Thus, by studying the transcriptome activity in these tissues, the effect of supplements of both vitamins on the metabolism of both liver and mammary gland, was investigated. For this study, 24 multiparous Holstein dairy cows were assigned to 6 blocks of 4 animals each according to previous 305-day milk production. Within each block, cows were randomly assigned to weekly intramuscular injections of 5 mL of either saline 0.9 % NaCl, 320 mg of vitamin B9, 10 mg of vitamin B12 or a combination of both vitamins (B9 + B12). The experimental period began 3 weeks before the expected calving date and lasted 9 weeks of lactation. Liver and mammary biopsies were performed on lactating dairy cows 64 ± 3 days after calving. Samples from both tissues were analyzed by microarray and qPCR to identify genes differentially expressed in hepatic and mammary tissues. RESULTS: Microarray analysis identified 47 genes in hepatic tissue and 16 genes in the mammary gland whose expression was modified by the vitamin supplements. Gene ontology (GO) categorizes genes in non-overlapping domains of molecular biology. Panther is one of the online GO resources used for gene function classification. It classifies the 63 genes according to Molecular Function, Biological Process and Protein Class. Most of the biological processes modulated by the vitamin supplements were associated to developmental process, protein metabolic process, transport and response to inflammation. In the liver, most of the genes modulated by the vitamin treatments involved protein metabolic process while developmental process appeared to be more affected by the treatments in mammary gland. Out of 25 genes analysed by qPCR, 7 were validated. CONCLUSION: The results indicate that several metabolic processes were modulated by the supplementation of vitamins in early-lactating dairy cows. In addition, the results suggest that the vitamin supplements promoted liver regeneration and reduced catabolism of lipids in early lactation.

Optimization and validation of a reversed-phase high performance liquid chromatography method for the measurement of bovine liver methylmalonyl-coenzyme a mutase activity.

BACKGROUND: Methylmalonyl-CoA mutase (MCM) is an adenosylcobalamin-dependent enzyme that catalyses the interconversion of (2R)-methylmalonyl-CoA to succinyl-CoA. In humans, a deficit in activity of MCM, due to an impairment of intracellular formation of adenosylcobalamin and methylcobalamin results in a wide spectrum of clinical manifestations ranging from moderate to fatal. Consequently, MCM is the subject of abundant literature. However, there is a lack of consensus on the reliable method to monitor its activity. This metabolic pathway is highly solicited in ruminants because it is essential for the utilization of propionate formed during ruminal fermentation. In lactating dairy cows, propionate is the major substrate for glucose formation. In present study, a reversed-phase high performance liquid chromatography (RP-HPLC) was optimized and validated to evaluate MCM activity in bovine liver. The major aim of the study was to describe the conditions to optimize reproducibility of the method and to determine stability of the enzyme and its product during storage and processing of samples. RESULTS: Specificity of the method was good, as there was no interfering peak from liver extract at the retention times corresponding to methylmalonyl-CoA or succinyl-CoA. Repeatability of the method was improved as compared to previous RP-HPLC published data. Using 66 µg of protein, intra-assay coefficient of variation (CV) of specific activities, ranged from 0.90 to 8.05% and the CV inter-day was 7.40%. Storage and processing conditions (frozen homogenate of fresh tissue vs. fresh homogenate of tissue snapped in liquid nitrogen) did not alter the enzyme activity. The analyte was also stable in liver crude extract for three frozen/thawed cycles when stored at -20°C and thawed to room temperature. CONCLUSIONS: The improved method provides a way for studying the effects of stages of lactation, diet composition, and physiology in cattle on MCM activity over long periods of time, such as a complete lactation period. Interestingly, this sensitive and accurate method could benefit the study of the cobalamin status in experimental studies and clinical cases.

The acute antiparkinsonian and antidyskinetic effect of AFQ056, a novel metabotropic glutamate receptor type 5 antagonist, in L-Dopa-treated parkinsonian monkeys.

Overactivity of glutamatergic transmission has been implicated in Parkinson's disease (PD) and levodopa (L-Dopa)-induced dyskinesias. Striatal metabotropic glutamate receptors type 5 (mGluR5) are abundant and provide specific targets to modulate glutamatergic activity. This study investigated the acute effects of the novel mGluR5 antagonist AFQ056 on motor behavior in L-Dopa-treated monkeys with a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) lesion to model PD. Six Macaca fascicularis MPTP monkeys were treated repeatedly with L-Dopa; this treatment increased their locomotion and reduced their parkinsonian scores, but also induced dyskinesias. When AFQ056 (doses of 5, 25, 125 or 250mg/kg) was administered one hour prior to a high dose of L-Dopa, the antiparkinsonian activity of L-Dopa was maintained as measured with locomotion and antiparkinsonian scores, whereas dyskinesias were significantly reduced at 25, 125 and 250mg/kg AFQ056 for peak dyskinesia score and at 125 and 250mg/kg for the 1h peak period of dyskinesia score. Administration of AFQ056 one hour before L-Dopa led to peak or elevated plasma AFQ056 concentrations occurring close to L-Dopa peak-dose dyskinesias. We next investigated AFQ056 25mg/kg combined with a low dose of L-Dopa. The antiparkinsonian activity of L-Dopa was increased as measured with locomotion, while dyskinesias remained low at these doses. Our results show a beneficial motor effect of AFQ056 with L-Dopa in MPTP monkeys. This supports the therapeutic use of an mGluR5 antagonist to restore normal glutamatergic neurotransmission in PD and decrease dyskinesias.

Metabotropic glutamate receptor type 5 in levodopa-induced motor complications.

Metabotropic glutamate receptors type 5 (mGluR5) are implicated in regulation of synaptic plasticity and learning, and were the focus of our investigation in human Parkinson's disease (PD) patients with dyskinesias and wearing-off, and in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) monkeys with dyskinesias. Using the selective mGluR5 ligand [(3)H]ABP688 autoradiography, we measured mGluR5 in brain slices from 11 normal and 14 PD patients and from MPTP monkeys, in relation to motor complications (dyskinesias and wearing-off) associated with treatment with l-dopa. In 16 monkeys with a bilateral MPTP lesion and four controls, [(3)H]ABP688 specific binding was elevated in the striatum of dyskinetic l-dopa-treated MPTP monkeys but not in MPTP monkeys without dyskinesias compared to controls. PD patients with motor complications (either dyskinesias or wearing-off) had higher [(3)H]ABP688 specific binding compared to those without motor complications and controls in putamen, external and internal globus pallidus. Elevated glutamatergic transmission as measured with increased mGluR5 specific binding was associated with motor complications and its antagonism could be targeted for their treatment.

Effect of L-Dopa on metabotropic glutamate receptor 5 in the brain of parkinsonian monkeys.

Behavioral investigations of selective and potent metabotropic glutamate receptor type 5 (mGluR5) antagonists in animal models suggest involvement of mGluR5 in compensatory mechanisms of the basal ganglia circuitry in Parkinson's disease and levodopa (L-Dopa) induced motor complications. This study investigated mGluR5 changes in MPTP lesioned monkeys. The effect of a chronic 1 month treatment with L-Dopa on mGluR5-specific binding and mRNA levels was investigated in MPTP monkeys killed 4 or 24 h after their last L-Dopa administration. [(3)H]ABP688 specific binding in the putamen was elevated in L-Dopa-treated MPTP monkeys killed 24 h but not 4 h after their last L-Dopa dose compared with vehicle-treated MPTP monkeys. Caudate nucleus [(3)H]ABP688-specific binding was elevated in both groups of L-Dopa treated compared with vehicle-treated MPTP monkeys. In contrast, caudate nucleus and putamen mGluR5 mRNA levels were elevated only in L-Dopa-treated MPTP monkeys killed 4 h after their last L-Dopa administration. MPTP monkeys killed 4 h after their last L-Dopa treatment showed higher caudate nucleus and putamen L-Dopa concentrations compared with those killed after 24 h. Hence, mGluR5 in the putamen are sensitive to presence of L-Dopa leading to a rapid decrease of [(3)H]ABP688-specific binding possibly involving a direct mGluR5/dopamine receptors interaction.

Effect of non-dopaminergic drug treatment on Levodopa induced dyskinesias in MPTP monkeys: common implication of striatal neuropeptides.

Dopamine denervation in Parkinson's disease and repeated Levodopa (L-DOPA) administration that induces dyskinesias are associated with an enhancement of basal ganglia neuropeptide transmission. Various adjunct non-dopaminergic treatments to Levodopa were shown to reduce and/or prevent dyskinesias. The aim of this study was to seek if non-dopaminergic drug treatments to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) lesioned monkeys combined with L-DOPA to prevent dyskinesia were associated with changes of striatal neuropeptides. Chronic treatment with Ro 61-8048 a kynurenine hydroxylase inhibitor, docosahexaenoic acid (DHA) a polyunsaturated fatty acid (omega-3), naltrexone an opioidergic antagonist and CI-1041 an N-methyl-D-aspartate (NMDA) glutamate receptor antagonist with L-DOPA prevented dyskinesias to various extents except naltrexone whereas all MPTP monkeys treated with L-DOPA alone developed dyskinesias. Striatal preproenkephalin (PPE), preprodynorphin (PPD) and preprotachykinin A (PPT-A) mRNA levels were measured by in situ hybridization. An increase of PPE and PPD mRNA levels was observed in anterior caudate nucleus of L-DOPA treated MPTP monkeys compared to controls and to Saline-treated MPTP monkeys whereas PPT-A mRNA levels were unchanged. Striatal PPE and PPD mRNA levels remained elevated in L-DOPA plus naltrexone-treated MPTP monkeys, while co-treatment with DHA, CI-1041 or Ro 61-8048 prevented their increase to various extents. Maximal dyskinesias scores of MPTP monkeys correlated significantly with striatal PPE and PPD mRNA levels but not with PPT-A mRNA levels. These results show that drugs displaying a wide range of pharmacological activities can modulate L-DOPA induced dyskinesias and this activity is correlated with striatal PPD and PPE mRNA levels suggesting a convergent mechanism.

Nur77 mRNA levels and L-Dopa-induced dyskinesias in MPTP monkeys treated with docosahexaenoic acid.

We have previously shown that docosahexaenoic acid (DHA) significantly reduced L-Dopa-induced dyskinesia (LID) in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) monkeys (Samadi et al., Ann. Neurol. 59:282-288, 2006). In the present study, we measured for the first time mRNA levels of Nur77, an orphan nuclear receptor that participates to adaptive and/or aberrant dopamine-related behaviors, and retinoid X receptor gamma1 (RXRgamma1), a putative brain receptor for DHA and transcriptional partner of Nur77, in MPTP monkeys treated with L-Dopa and DHA. The RXRgamma1 mRNA is strongly expressed in monkey caudate nucleus and putamen, but no change in levels of RXRgamma1 was observed following MPTP and L-Dopa treatments. On the other hand, denervation reduced Nur77 mRNA levels, whereas chronic L-Dopa treatment strongly induced Nur77 transcripts. These modulations are taking place in substance P positive cells and are associated with both caudate-putamen matrix and striosome compartments. Interestingly, combination of L-Dopa with DHA further increases Nur77 mRNA levels in the anterior caudate-putamen, and mainly in striosomes. This is accompanied by a significant inverse correlation between Nur77 mRNA levels and dyskinetic scores. Taken together, our results show that Nur77 expression is modulated following dopamine denervation and chronic L-Dopa therapy in a non-human primate model of Parkinson's disease, and suggest that strong modulation of Nur77 expression might be linked to a reduced risk to develop LIDs.

Implication of NMDA receptors in the antidyskinetic activity of cabergoline, CI-1041, and Ro 61-8048 in MPTP monkeys with levodopa-induced dyskinesias.

This study assessed striatal N-methyl-D-aspartate (NMDA) glutamate receptors of 1-methyl 4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) monkeys with levodopa (L-DOPA)-induced dyskinesias (LID). In a first experiment, four MPTP monkeys receiving L-DOPA/Benserazide alone developed dyskinesias. Four MPTP monkeys received L-DOPA/Benserazide plus CI-1041 an NMDA antagonist selective for NR1/NR2B and four were treated with L-DOPA/Benserazide plus a small dose of cabergoline; one monkey of each group developed mild dyskinesias at the end of treatment. In a second experiment, a kynurenine 3-hydroxylase inhibitor Ro 61-8048, combined with L-DOPA/Benserazide, reduced dyskinesias in MPTP monkeys. Drug-treated MPTP monkeys were compared to intact monkeys and saline-treated MPTP monkeys. Glutamate receptors were investigated by autoradiography using [(3)H]CGP-39653 (NR1/NR2A antagonist) and [(3)H]Ro25-6981 (NR1/NR2B antagonist). In general, striatal [(3)H]CGP-39653 specific binding was unaltered in all experimental groups. MPTP lesion decreased striatal [(3)H]Ro25-6981 specific binding; these levels were enhanced in the L-DOPA-alone-treated MPTP monkeys and decreased in antidyskinetic drugs treated monkeys. Maximal dyskinesias scores of the MPTP monkeys correlated significantly with [(3)H]Ro25-6981 specific binding in the rostral and caudal striatum. Hence, MPTP lesion, L-DOPA treatment and prevention of LID with CI-1041 and cabergoline, or reduction with Ro 61-8048 were associated with modulation of NR2B/NMDA glutamate receptors.

[Efficiency of ethanol of Thonningia sanguinea against Cryptococcus neoformans]. / Efficacité de l'extrait éthanolique de Thonningia sanguinea sur Cryptococcus neoformans.

Herbal treatment of infectious diseases is a common practice in traditional society. One of the herbs used by African populations against skin and systemic infectious diseases is Thonningia sanguinea (THOS). In the Ivory Coast, Togo, and Ghana, this herb is used to prepare traditional remedies for diarrhoeas, asthma and mycoses. The latter account for a substantial number of the opportunistic infections associated with HIV/AIDS. Some species of eumycetes, such as Cryptococcus neoformans, infect mainly immunocompromised patients but can sometimes infect immunocompetent hosts and they are often fatal. In this study we evaluated the antimicrobial activity of aqueous extracts of T. sanguinea against C. neoformans (and several other microorganisms). We first assessed the fungal growth in vitro on agar medium. We then incubated the cultures for 48 h with various concentrations of THOS to determine the percentage of survival according to the concentration of THOS and the concentration required for 50% inhibition (IC50). The minimum fungicidal concentration (MFC) of the total aqueous extract was 6.25 mg/mL. (THOS was thus most effective against C. neoformans). Accordingly we sought to improve the extract by preparing it with ethanol. This solution yielded an IC50=0.06 mg/mL and an MFC=0.098 mg/mL against C. neoformans.