Disruption of the uncoupling protein-2 gene in mice reveals a role in immunity and reactive oxygen species production.

The gene Ucp2 is a member of a family of genes found in animals and plants, encoding a protein homologous to the brown fat uncoupling protein Ucp1 (refs 1-3). As Ucp2 is widely expressed in mammalian tissues, uncouples respiration and resides within a region of genetic linkage to obesity, a role in energy dissipation has been proposed. We demonstrate here, however, that mice lacking Ucp2 following targeted gene disruption are not obese and have a normal response to cold exposure or high-fat diet. Expression of Ucp2 is robust in spleen, lung and isolated macrophages, suggesting a role for Ucp2 in immunity or inflammatory responsiveness. We investigated the response to infection with Toxoplasma gondii in Ucp2-/- mice, and found that they are completely resistant to infection, in contrast with the lethality observed in wild-type littermates. Parasitic cysts and inflammation sites in brain were significantly reduced in Ucp2-/- mice (63% decrease, P<0.04). Macrophages from Ucp2-/- mice generated more reactive oxygen species than wild-type mice (80% increase, P<0.001) in response to T. gondii, and had a fivefold greater toxoplasmacidal activity in vitro compared with wild-type mice (P<0.001 ), which was absent in the presence of a quencher of reactive oxygen species (ROS). Our results indicate a role for Ucp2 in the limitation of ROS and macrophage-mediated immunity.

Catechins: natural free-radical scavengers against ochratoxin A-induced cell damage in a pig kidney cell line (LLC-PK1).

Besides aflatoxin B1, recent findings suggested that oxidative stress plays an important role in the toxicity of an other mycotoxin: ochratoxin A (OTA). The protective effect of two catechins (epigallocatechin gallate, EGCG, and epicatechin gallate, ECG) against OTA-induced cytotoxicity was investigated in a pig kidney cell line (LLC-PK1). The ability of the catechins to reduce ROS production and DNA fragmentation induced by OTA was also investigated. Our experiments proved the significant cytoprotective effects of the molecules in vitro from OTA-induced cell damage. In particular a 24h pre-treatment with EGCG or ECG restored cell viability with respect to OTA alone. Pre-treatment with EGCG at low concentration for 8 days protected cells from OTA-induced cell death. Moreover both catechins reduced OTA-induced ROS production. A reduction of OTA-induced DNA fragmentation was found for LLC-PK1 cells pre-treated with EGCG and ECG. The free-radical scavenging capacity of both catechins was tested with the Briggs-Rauscher oscillating method (pH approximately 2) and the TEAC assay (pH 7.4). The results show a good scavenging power according with inhibition of ROS production. Catechins could be useful to develop alimentary strategies for both humans and animals to prevent OTA-induced cytotoxicity.

Protective role of uncoupling protein 2 in atherosclerosis.

BACKGROUND: Uncoupling protein 2 (UCP2) regulates the production of reactive oxygen species in macrophages. However, its role in atherosclerosis is unknown. METHODS AND RESULTS: Irradiated low-density lipoprotein receptor deficient mice (LDLR-/-) were transplanted with bone marrow from either UCP2 deficient mice (Ucp2-/-) or wild type mice (Ucp2+/+). Mice were fed an atherogenic diet for 7 weeks. Engraftment of bone marrow cells was confirmed by the presence of UCP2 protein expression in spleen cell mitochondria of Ucp2+/+ transplanted mice and its absence in Ucp2-/- transplanted mice. Leukocyte counts and plasma cholesterol levels were comparable in both groups. We found a marked increase in atherosclerotic lesion size in the thoracic aorta of Ucp2-/- transplanted mice compared with control Ucp2+/+ transplanted mice (8.3+/-0.9% versus 4.3+/-0.4%, respectively; P<0.005), as well as in the aortic sinus (150 066+/-12 388 microm2 versus 105 689+/-9 727 microm2, respectively; P<0.05). This was associated with increased nitrotyrosine staining, which suggests enhanced oxidative stress. Analysis of plaque composition revealed a significant increase in macrophage accumulation (P<0.05) and apoptosis (P<0.05), along with a decrease in collagen content (P<0.05), suggesting a potentially more vulnerable phenotype. CONCLUSION: These results suggest a protective role for UCP2 against atherosclerosis.

Tissue-specific effects of leptin administration on the abundance of mitochondrial proteins during neonatal development.

Many tissues undergo a rapid transition after birth, accompanied by dramatic changes in mitochondrial protein function. In particular, uncoupling protein (UCP) abundance increases at birth in the lung and adipose tissue, to then gradually decline, an adaptation that is important in enabling normal tissue function. Leptin potentially mediates some of these changes and is known to promote the loss of UCP1 from brown fat but its effects on UCP2 and related mitochondrial proteins (i.e. voltage-dependent anion channel (VDAC) and cytochrome c) in other tissues are unknown. We therefore determined the effects of once-daily jugular venous administration of ovine recombinant leptin on mitochondrial protein abundance as determined by immunoblotting in tissues that do (i.e. the brain and pancreas) and do not (i.e. liver and skeletal muscle) express UCP2. Eight pairs of 1-day-old lambs received either 100 mug leptin or vehicle daily for 6 days, before tissue sampling on day 7. Administration of leptin diminished UCP2 abundance in the pancreas, but not the brain. Leptin administration had no affect on the abundance of VDAC or cytochrome c in any tissue examined. In leptin-administered animals, but not controls, UCP2 abundance in the pancreas was positively correlated with VDAC and cytochrome c content, and UCP2 abundance in the brain with colonic temperature. In conclusion, leptin administration to neonatal lambs causes a tissue-specific loss of UCP2 from the pancreas. These effects may be important in the regulation of neonatal tissue development and potentially for optimising metabolic control mechanisms in later life.

Anti-inflammatory, anti-nociceptive and antioxidant activities of Balanites aegyptiaca (L.) Delile.

The anti-inflammatory and anti-nociceptive activities of methanol (ME), butanol (BE) extracts and of two new saponins isolated from Balanites aegyptiaca bark were evaluated. The study was carried out in vivo and in vitro. The samples, extracts and pure substances, were intra-gastrically administered to animals. Two different animal models, the carrageenin-induced edema, in the rat, and acetic acid-induced writhing test in mice, were adopted. Moreover, the antioxidant power of extracts, fractions and individual constituents from Balanites aegyptiaca has been evaluated in vitro, using a method based on the Briggs-Rauscher (BR) oscillating reaction. Results obtained demonstrate that both ME or BE have a significant effect at the highest dose on the number of abdominal writhes induced by acetic acid, with a 38 and 54% inhibition respectively, but no significant difference was observed for extracts at the lowest dose and for the pure compounds compared with control animals. The same extracts exhibit a significant reduction on the rat paw edema. The inhibition produced by ME is about the same (28+/-3% lowest dose, 32+/-3% highest dose) after administration. A more evident effect is obtained by BE (41+/-3% and 68+/-6% respectively) and single saponins B1 and B2 (62+/-5% and 59+/-6% respectively) after oral administration. The antioxidant activity obtained seems to be in good accordance with the pharmacological results. The histological sections of rat paw confirm the antiflogistic activity of the plant extracts.

Rm values of phenols. Their relationship with log P values and activity.

The experimental Rm values for a series of phenols were obtained by a reversed-phase TLC system. The extrapolation from a range of linear relationship between experimental Rm values and acetone concentration provided a set of extrapolated Rm values. This were used for studying the relationship between structure and activity in vitro and in vivo. The possibility to obtain by means of the extrapolation technique the Rm values in a standard system for serveral series of chemotherapeutic agents is pointed out.

Rm values of steroids as an expression of their lipophilic character in structure-activity studies.

The chromatographic Rm values of three series of steroids were determined by means of a reversed-phase system. The Rm values at 45% acetone in the mobile phase were shown to be correlated with the partition coefficients in an ether-water system. However, an almost equally good correlation was found when using extrapolated Rm values. The extrapolation technique could provide a standard system. The relationship between biological data and Rm values pointed out the important role of the lipophilic character in regulating the activity of steroids. In particular, the dependence of protein binding absorption and biotransformation on lipophilic character might strongly influence the availability of steroids at the site of action.

The influence of physicochemical parameters on the biliary excretion of a series of nitroimidazoles.

The relationship between physicochemical parameters and biliary excretion of nitroimidazoles was investigated. The unmetabolized form of each drug was detected in the bile by means of a UV procedure. A highly significant reversed parabolic relationship was shown between the Rm values and the biliary excretion of the test compounds. In other words, the compounds closer to the optimal Rm value are excreted less than those characterized by higher or lower Rm values. Since the Rm values seem to account for both the lipophilic and polar character of nitroimidazoles, the reversed parabola could be due to plasma protein binding and/or some protein binding within the hepatocyte. In fact, both the lipophilic and polar character seem to play an important role in protein binding of chemicals.

Relationship between lipophilic character and urinary excretion of nitroimidazoles and nitrothiazoles in rats.

Many processes are involved in the renal excretion of drugs, but very little is known about their quantitative structure-activity relationship. The relationship between urinary excretion and lipophilic character of a series of nitroimidazoles and nitrothiazoles was studied. The unmetabolized forms of the drugs were detected in the urine by means of UV and HPLC procedures. The urinary excretion of unmetabolized forms is parabolically related with the log P, as an expression of lipophilic character of molecules.

Rm values and structure-activity relationship of benzodiazepines.

Quantitative structure-activity relationships (QSAR) have been formulated for the activities of a series of benzodiazepines in rats. The lipophilic character of molecules was expressed by means of the chromatographic Rm values which were very well correlated with experimental or calculated log P values. The ideal lipophilic character for activity of benzodiazepines in the exploratory behavior test is not far from that of compounds acting in the central nervous system as unspecific depressant agents. The results of both the conflict and exploratory behavior studies might support the hypothesis of different sites of action for the antianxiety and sedative effects of benzodiazepines.

Influence of genotype on the differential ontogeny of uncoupling protein 2 and 3 in subcutaneous adipose tissue and muscle in neonatal pigs.

The present study aimed to determine whether porcine genotype and/or postnatal age influenced mRNA abundance or protein expression of uncoupling protein (UCP)2 or 3 in subcutaneous adipose tissue (AT) and skeletal muscle (SM) and the extent to which these differences are associated with breed-specific discordance in endocrine and metabolic profiles. Piglets from commercial and Meishan litters were ranked according to birth weight. Tissue samples were obtained from the three median piglets from each litter on either day 0, 4, 7, 14 or 21 of neonatal life. UCP2 protein abundance in AT was similar between genotypes on the first day of life, but it was elevated at all subsequent postnatal ages (P<0.05) in AT of Meishan piglets. In contrast, UCP2 mRNA abundance was lower in Meishans up to 14 days of age. UCP2 mRNA expression was not correlated with protein abundance in either breed at any age. UCP3 mRNA in AT was similar between breeds up to day 7; thereafter, expression was higher (general linear model, P<0.05) in Meishan piglets. Conversely, UCP3 mRNA expression in SM was higher in commercial piglets after day 7. Colonic temperature remained lower in Meishan than commercial piglets throughout the study; this was most obvious in the immediate post-partum period when Meishan piglets had lower (P<0.05) plasma triiodothyronine. In conclusion, we have demonstrated that porcine genotype influences the expression and abundance of UCP2 and 3, an influence which may, in part, be due to the distinctive endocrine profiles associated with each genotype.

Comparison between chinese medical herb Pueraria lobata crude extract and its main isoflavone puerarin antioxidant properties and effects on rat liver CYP-catalysed drug metabolism.

Ge-gen (Radix Puerariae; RP) is used in traditional oriental medicine for various medicinal purposes. The drug is the root of a wild leguminous creeper, Pueraria lobata (Willd) Ohwi. It possesses a high content of flavonoid derivatives, the most abundant of which is puerarin (PU). Here, using the enhanced chemiluminescence technique based on horseradish peroxidase and a luminol-oxidant-enhancer reagent, we evaluated in vitro the antioxidant activity of PU and RP crude extract. Both biological samples inhibited the steady-state chemiluminescent reaction in a dose-dependent fashion. However, different inhibition mechanism were postulated, since only RP behaved like conventional antioxidants. This activity was supposed to be due the presence of compounds other than PU in the crude extract. Using each of the specific substrates to different cytochrome P450 (CYP) isoforms or the regio- and stereo-selective hydroxylation of testosterone as polyfunctional probe we found that when intragastrically administered in male Wistar rats, PU (100 or 200 mg/kg b.w.) and RP (700 or 1,400 mg/kg b.w.) significantly altered hepatic CYP-linked monooxygenases. While both CYP content and NADPH-(CYP)-c-reductase activity were significantly increased in all situations, a complex pattern of CYP modulation was observed, including both induction (PU: CYP2A1, 1A1/2, 3A1, 2C11; RP: CYP1A2, 3A1, 2B1) and inactivation (PU and RP: CYP3A, 2E1, 2B1), the latter being due to either parental agents or metabolites, as demonstrated by in vitro studies. Overall, these findings indicate that RP contains compounds with potent antioxidant activity and that both PU and RP impairs CYP-catalysed drug metabolism.

Mechanism for the prevention of cholestasis involving cytochrome P4503A overexpression.

BACKGROUND: To clarify the preventive effect of taurohyodeoxycholic acid on liver cholestasis induced by toxic bile acids in rats, we evaluated whether modulation of cytochrome P4503A-linked oxidases is involved in the hepatic bile acid retention and secretion mechanism. We investigated whether the safe or the toxic taurochenodeoxycholic acid, administered singly or together, affects cytochrome P450-catalyzed drug metabolism or biliary parameters. We also considered whether the inhibition of the P-glycoprotein export pump by vinblastine might be related to cytochrome P4503A overexpression. METHODS: Hydroxylation of testosterone and N-demethylation of aminopyrine were studied in subcellular rat liver preparations after intravenous infusion of hepatoprotective and toxic bile acids administered singly or together. Bile flow, calcium secretion, biliary enzymes activity, and secretion rates of the endogenous and administrated bile acids were determined. CYP3A-dependent monooxygenases were also measured in the same coinfusion model in the presence of vinblastine. RESULTS: Although wide modulation of the activities of different P450 subfamily of isoenzymes was seen, P4503A-associated monooxygenases showed similar patterns in the various situations, i.e., induction by taurohyodeoxycholic acid, reduction by taurochenodeoxycholic acid, and protection (intermediate induction) in the coinfusion experiments. This correlates well with biliary parameters demonstrating the hepatoprotective ability of taurohyodeoxycholic acid. Coadministration of bile acids and vinblastine significantly modifies CYP3A-linked activities. CONCLUSIONS: Bile acid structure seems to be linked with hepatotoxicity/hepatoprotection and P4503A modulation. Taurohyodeoxycholic acid could be therapeutic in cholestatic liver disease by inducing P4503A; we can hypothesize that an associated P-glycoprotein expression might facilitate biliary excretion of toxic taurochenodeoxycholic acid accumulated in the liver during cholestasis.

Anti-inflammatory and cicatrizing activity of Echinacea pallida Nutt. root extract.

Among the different species belonging to the Echinacea family, largely used in traditional medicine, Echinacea pallida, Echinacea purpurea and Echinacea angustifolia were investigated. These different species, due to their difficult identification, were commonly confused in the past and probably used indifferently for the same therapeutic purposes. In fact, the three species have in common, some pharmacological activities, based on the presence of active compounds that act additively and synergistically. Nevertheless, the composition of each species has slight variation in the amount of each active component. In particular, echinacoside, a caffeoyl derivative, is present in E. pallida and only in traces in E. angustifolia. It seems to have protective effects on skin connective tissue and to enhance wound healing. The anti-inflammatory and wound healing activities of echinacoside, compared with the ones of the total root extract of E. pallida and E. angustifolia, were examined in rats, after topical application. The tissues of the treated animals were evaluated after 24, 48 and 72 h treatment and excised for histological observation at the end of the experiment. Results confirm the good anti-inflammatory and wound healing properties of E. pallida and of its constituent echinacoside, with respect to E. purpurea and control. This activity probably resides in the antihyaluronidase activity of echinacoside.

Efficacy of different Cynara scolymus preparations on liver complaints.

Cynara scolymus leaves extracts have long been used in folk medicine for their choleretic and hepatoprotective activities, that are often related to the cynarin content. These therapeutic properties are also attributed to mono- and di-caffeoylquinic acids and since commercial C. scolymus preparations can differ for their activities, we studied four extracts to evaluate, if present, a relationship between the hepatobiliary properties of the different preparations and their content in phenolics. The antioxidant activity of the commercial preparations examined was also considered in an in vitro system. The results showed that the extract with the highest content in phenolic derivatives (GAE) exerted the major effect on bile flow and liver protection. Also the results of the antioxidant capacity (BR) of the different preparations are in good agreement with the results obtained in vivo. On the contrary, administering rats with doses of chlorogenic acid, equivalent to those present in this extract, we did not observe any choleretic or protective action. An histopathological analysis of liver sections confirmed the biochemical results. Perhaps caffeoyl derivatives have a role in the therapeutic properties of C. scolymus extracts, as reported in literature for "in vitro" studies, but when administered alone, they are not so effective in exerting this action.

ANÁLISIS PROXIMAL, PERFIL DE ÁCIDOS GRASOS DE LAS VíSCERAS DEL CUY (Cavia porcellus) Y SU USO POTENCIAL EN ALIMENTACIÓN ANIMAL / PROXIMATE ANALYSIS, FATTY ACID PROFILE OF GUINEA PIG (Cavia porcellus) VISCERA AND ITS POTENTIAL USE IN ANIMAL FEED

Con el fin de evaluar el valor nutritivo de materias primas no convencionales en la elaboración de concentrado animal, de bajo coste y que no compitan con la alimentación del hombre, en la presente investigación se analizó el contenido nutricional de las visceras abdominales de cuy (Cavia porcellus) y de su harina, usando análisis proximal y perfil de ácidos grasos mediante cromatografía de gases. Se compararon estos resultados con materias primas convencionales como la harina de pescado y con visceras de diversos animales. Los resultados obtenidos indican que la harina de visceras de cuy puede competir con harinas de diversas procedencias dadas sus cualidades nutricionales (58% proteína, 28% grasa, 4% cenizas); además, el alto contenido de grasa en las visceras frescas (55% base seca) está constituido principalmente ácidos grasos poliinsaturados tipo omega 3, 6 y 9, siendo relevante el contenido de ácido linolénico, ácidos grasos que en la dieta de los animales son escasos y de alto costo. Es posible concluir que los componentes nutricionales de las visceras de cuy la convierten en una materia prima promisoria en alimentación animal, estimulando asi el aprovechamiento de estos residuos.

In order to assess the nutritional value of unconventional raw materials in the production of animal feed, of low cost and that do not compete with human food, in the present work the nutritional content of abdominal guinea pig (Cavia porcellus) viscera by proximate analysis and fatty acid profile by gas chromatography were analyzed. These results were compared with conventional raw materials such as fish meal and different animal viscera. The results show that the guinea pig viscera flour can compete with animal feed of various sources given its nutritional qualities (58% protein, 28% fat, 4% ash); also the high fat content in fresh viscera (55% dry basis) are primarily composed by polyunsaturated fatty acids omega 3, 6 and 9, where was relevant the content of linolenic acid, fatty acids in the animal diet that are scarce and expensive. We conclude that given the nutritional components, the guinea pig viscera are a promising raw material for animal feed, stimulating the recycling of this waste.

Positive response to imatinib mesylate therapy for childhood chronic myeloid leukemia.

Chronic myeloid leukemia (CML) is rare in the pediatric population, accounting for 2-3% of childhood leukemia cases, with an annual incidence of one case per million children. The low toxicity profile of imatinib mesylate has led to its approval as a front-line therapy in children for whom interferon treatment has failed or who have relapsed after allogeneic transplantation. We describe the positive responses of 2 children (case 1 - from a 7-year-old male since May 2005; case 2 - from a 5-year-old female since June 2006) with Philadelphia-positive chromosome CML treated with imatinib (300 mg/day, orally) for up to 28 months, as evaluated by morphological, cytogenetic, and molecular approaches. Our patients are alive, are in the chronic phase, and are in continuous morphological complete remission.