Dietary conjugated linoleic acid downregulates the AlCl3-induced hyperactivation of compensatory and maladaptive signalling in the mouse brain cortex.

Oxidative stress, hyperactivation of compensatory mechanisms (unfolded protein response, UPR; nuclear factor erythroid 2-related factor 2, Nrf2) and the stimulation of maladaptive response (inflammation/apoptosis) are interconnected pathogenic processes occurring during Alzheimer's disease (AD) progression. The neuroprotective ability of dietary Conjugated linoleic acid (CLAmix) in a mouse model of AlCl3-induced AD was recently described but, the effects of AlCl3 or CLAmix intake on these pathogenic processes are still unknown. The effects of dietary AlCl3 or CLAmix - alone and in combination - were examined in the brain cortex of twenty-eight BalbC mice divided into 4 groups (n = 7 each). The neurotoxic effects of AlCl3 were investigated in animals treated for 5 weeks with 100 mg/kg/day (AL). CLAmix supplementation (600 mg/kg bw/day) for 7 weeks (CLA) was aimed at evaluating its modulatory effects on the Nrf2 pathway while its co-treatment with AlCl3 during the last 5 weeks of CLAmix intake (CLA + AL) was used to investigate its neuroprotective ability. Untreated mice were used as controls. In the CLA group, the NADPH oxidase (NOX) activation in the brain cortex was accompanied by the modulation of the Nrf2 pathway. By contrast, in the AL mice, the significant upregulation of oxidative stress markers, compensatory pathways (UPR/Nrf2), proinflammatory cytokines (IL-6, TNFα) and the proapoptotic protein Bax levels were found as compared with control. Notably, in CLA + AL mice, the marked decrease of oxidative stress, UPR/Nrf2 markers and proinflammatory cytokines levels were associated with the significant increase of the antiapoptotic protein Bcl2. The involvement of NOX in the adaptive response elicited by CLAmix along with its protective effects against the onset of several pathogenic processes triggered by AlCl3, broadens the knowledge of the mechanism underlying the pleiotropic activity of Nrf2 activators and sheds new light on their potential therapeutic use against neurodegenerative disorders.

Conjugated linoleic acid downregulates Alzheimer's hallmarks in aluminum mouse model through an Nrf2-mediated adaptive response and increases brain glucose transporter levels.

Mitochondrial dysfunction, oxidative stress, inflammation and glucose dysmetabolism are pathological signs of Alzheimer's disease (AD). Dietary aluminum (Al) overload is often used to induce AD in rodents and trigger the onset of oxidative-stress hallmarks resembling those of the human disease. The Nuclear factor erythroid 2-related factor 2 (Nrf2), owing to its key role in redox homeostasis, mitochondrial function and inflammation, is a promising drug target for neurological disorders, but only a few data are available on its modulatory effects on glucose transporter expression levels. While it has been found that the protective effect of Conjugated linoleic acid (CLA) occurs through the activation of an Nrf2-mediated adaptive response, its beneficial effect on the considered pathological signs in the Al-induced model has not been established yet. Thirty-five male BalbC mice were divided into 5 groups: two Al-intoxicated groups were treated for 5 weeks with low or high Al doses (8 or 100 mg/kg/day in drinking water, respectively; L or H). Two groups of animals, orally supplemented with CLA (600 mg/kg bw/day) for 7 weeks (2 preliminary weeks plus the 5-week treatment with Al; CLA + L, CLA + H) were used to investigate its protective effect, while untreated mice were used as control (Cntr). We provide evidence that mitochondrial dysfunction, Nrf2 alteration, inflammation and Acetylcholinesterase (AChE) hyperactivation can occur even from L exposure. Interestingly, animal pre-treatment with an allometric CLA dose led to significant downregulation of the toxic effects elicited by L or H, likely through the activation of an adaptive response. In conclusion, CLA ability to increase the level of glucose transporters - along with its antioxidant and anti-inflammatory effect - expands the therapeutic targets of these molecules and comes out as an intriguing suitable candidate for the treatment of multifactorial disease.

Application of response surface methodology (RSM) for the optimization of supercritical CO2 extraction of oil from patè olive cake: Yield, content of bioactive molecules and biological effects in vivo.

The two-phase technology for olive oil extraction generates large amounts of patè olive cake (POC), a by-product that is rich in bioactive health-promoting compounds. Here, response surface methodology (RSM) was used to maximize supercritical-CO2 oil extraction from POC, while minimizing operative temperature, pressure and time. Under the optimal parameters (40.2 °C, 43.8 MPa and time 30 min), the oil yield was 14.5 g·100 g-1 dw (~65% of the total oil content of the freeze-dried POC matrix), as predicted by RSM. Compared with freeze-dried POC, the oil contained more phytosterols (13-fold), tocopherols (6-fold) and squalene (8-fold) and was a good source of pentacyclic triterpenes. When the biological effects of POC oil intake (20-40 µL·die-1) were evaluated in the livers of BALB/c mice, no significant influence on redox homeostasis was observed. Notably, a decline in liver triglycerides alongside increased activities of NAD(P)H:Quinone Oxidoreductase 1, Carnitine Palmitoyl-CoA Transferase and mitochondrial respiratory complexes suggested a potential beneficial effect on liver fatty acid oxidation.

Relative effectiveness of insects versus hummingbirds as pollinators of Rubiaceae plants across elevation in Dominica, Caribbean.

Most angiosperms rely on animal pollination for reproduction, but the dependence on specific pollinator groups varies greatly between species and localities. Notably, such dependence may be influenced by both floral traits and environmental conditions. Despite its importance, their joint contribution has rarely been studied at the assemblage level. At two elevations on the Caribbean island of Dominica, we measured the floral traits and the relative contributions of insects versus hummingbirds as pollinators of plants in the Rubiaceae family. Pollinator importance was measured as visitation rate (VR) and single visit pollen deposition (SVD), which were combined to assess overall pollinator effectiveness (PE). In the wet and cool Dominican highland, we found that hummingbirds were relatively more frequent and effective pollinators than insects, whereas insects and hummingbirds were equally frequent and effective pollinators at the warmer and less rainy midelevation. Furthermore, floral traits correlated independently of environment with the relative importance of pollinators, hummingbirds being more important in plant species having flowers with long and wide corollas producing higher volumes of dilute nectar. Our findings show that both environmental conditions and floral traits influence whether insects or hummingbirds are the most important pollinators of plants in the Rubiaceae family, highlighting the complexity of plant-pollinator systems.

Pollinator-independent orchid attracts biotic pollinators due the production of lipoidal substances.

Flowering plants often depend on the attraction of biotic pollinators for sexual reproduction. Consequently, the emergence and maintenance of selected floral attributes related to pollinator attraction and rewarding are driven by pollinator pressure. In this paper we explore the effect of pollinators, rainfall, temperature and air humidity on the reproduction of a Brazilian terrestrial orchid, Cranichis candida based on data of phenology, flower resources, olfactory and visual attraction cues, pollinators and breeding system. The flowers of C. candida are strongly protandrous and pollinated by workers of the social native bee Tetragonisca angustula. The bees collect labellar lipoidal substances (wax scales), which are transported to the nest. The lipoidal substance is composed of sterols, hydrocarbons and terpenes. The last presumably protects the bees and their nests against pathogens and other arthropods. C. candida sets fruits through biotic self- and cross-pollination, and spontaneously due the action of raindrops on flowers. Our results indicate that in C. candida, although rain-mediated spontaneous self-pollination happens, fructification mediated by biotic pollinations also occurs, which may result in fruit set by cross-pollination. A mixed pollination system must result in higher genetic variability when compared to species whose fruits are produced entirely by self-pollination. On the other hand, autogamy is a form of reproductive assurance, and has commonly evolved where pollination services are rare or absent.

Protective effect of Rumenic acid rich cow's milk against colitis is associated with the activation of Nrf2 pathway in a murine model.

Dietary supplementation with pure cis9, trans11 isomer of Conjugated Linoleic Acid -known as Rumenic Acid (RA)- improves cytoprotective defenses downstream through the activation of nuclear factor-E2-related factor-2(Nrf2). This capability, when Rumenic Acid is consumed in the form of foods, is still unknown. The ability of standard (St) or cow milk naturally-enriched in RA (En) to activate Nrf2 pathway and its impact on dextran sodium sulfate (DSS)-induced colitis was comparatively evaluated. Activity of Nrf2 pathway was investigated in colonic tissue of BALB/c mice, receiving 4-week supplement with skimmed milk (SK), St or St reinforced with pure RA (RSt) providing increasing RA dose (0, 124 or 404mg RA/kg-1 b.w, respectively). Next, the anti-oxidant/ anti-inflammatory effect produced by St or En treatment (383mg RA/kg-1 b.w.) was explored. Finally, macroscopic and histomorphologic features of colitis were evaluated in animals challenged with 5% (w/v) DSS, at the end of St or En treatment. Significant activation of Nrf2 pathway is associated with RSt and En intake (P<0.05), but not with SK or En treatment. En pre-treatment offers better protection, in comparison with St, against pro-oxidant, pro-inflammatory signs (P<0.01) and macroscopic signs triggered by DSS. It can be concluded that Nrf2 activation by higher RA amount contained in En is, at least in part, responsible for the improved protection associated with En intake against DSS-induced colitis.

In vitro study of AFB1 and AFM1 effects on human lymphoblastoid Jurkat T-cell model.

Aflatoxin B(1) (AFB(1)) is a mycotoxin produced by Aspergillus spp. that can occur as a natural contaminant in foods and feeds of vegetable origin. Post-ingestion, AFB(1) can be metabolized in the liver of mammals into hydroxylated aflatoxin M(1) (AFM(1)) that is excreted with milk. Although several studies have been carried out to evaluate effects of AFB(1) on the immune system, studies regarding AFM(1) are moreover lacking. The aim of the current study was to investigate effects of AFB(1) and AFM(1) on immune function using a lymphoblastoid Jurkat T-cell line as an experimental model. Both AFB(1) and AFM(1) produced significant decreases in Jurkat cell proliferation, whereas only minor effects were noted on interleukin (IL)-2 and interferon (IFN)-γ cytokines mRNA expression in stimulated cells that had been pre-incubated with AFB(1) and AFM(1). Particularly, AFB(1), but not AFM(1), at the highest concentration (50 µM) induced a marked increase in IL-8 mRNA expression. The results of the current study suggested the existence of a concentration threshold for AFB(1) and AFM(1) needed to exert biological activity on cell viability and innate immunity.

Small peptide inhibitors of acetyl-peptide hydrolase having an uncommon mechanism of inhibition and a stable bent conformation.

Acyl peptide hydrolase (APEH) catalyzes the removal of acetyl-amino acids from the N-terminus of peptides and cytoplasmic proteins. Due to the role played in several diseases, and to the growing interest around N-terminal acetylation, studies on APEH structure, function, and inhibition are attracting an ever increasing attention. We have therefore screened a random tetrapeptide library, N-capped with selected groups, and identified a trifluoroacetylated tetrapeptide (CF(3)-lmph) which inhibits the enzyme with a K(i) of 24.0 ± 0.8 µM. The inhibitor is selective for APEH, shows an uncommon uncompetitive mechanism of inhibition, and in solution adopts a stable bent conformation. CF(3)-lmph efficiently crosses cell membranes, blocking the cytoplasmic activity of APEH; however, it triggers a mild pro-apoptotic effect as compared to other competitive and noncompetitive inhibitors. The unusual inhibition mechanism and the stable structure make the new compound a novel tool to investigate enzyme functions and a useful scaffold to develop more potent inhibitors.

MEK inhibition enhances ABT-737-induced leukemia cell apoptosis via prevention of ERK-activated MCL-1 induction and modulation of MCL-1/BIM complex.

Recently, strategies for acute myeloid leukemia (AML) therapy have been developed that target anti-apoptotic BCL2 family members using BH3-mimetic drugs such as ABT-737. Though effective against BCL2 and BCL-X(L), ABT-737 poorly inhibits MCL-1. Here we report that, unexpectedly, ABT-737 induces activation of the extracellular receptor activated kinase and induction of MCL-1 in AML cells. MEK inhibitors such as PD0325901 and CI-1040 have been used successfully to suppress MCL-1. We report that PD0325901 blocked ABT-737-induced MCL-1 expression, and when combined with ABT-737 resulted in potent synergistic killing of AML-derived cell lines, primary AML blast and CD34+38-123+ progenitor/stem cells. Finally, we tested the combination of ABT-737 and CI-1040 in a murine xenograft model using MOLM-13 human leukemia cells.Whereas control mice and CI-1040-treated mice exhibited progressive leukemia growth, ABT-737, and to a significantly greater extent, ABT-737+CI-1040 exerted major anti-leukemia activity. Collectively, results demonstrated unexpected anti-apoptotic interaction between the BCL2 family-targeted BH3-mimetic ABT-737 and mitogen-activated protein kinase signaling in AML cells: the BH3 mimetic is not only restrained in its activity by MCL-1, but also induces its expression. However, concomitant inhibition by BH3 mimetics and MEK inhibitors could abrogate this effect and may be developed into a novel and effective therapeutic strategy for patients with AML.

[Comparative safety study of two commercialised vaccines against canine babesiosis induced by Babesia canis]. / Etude comparative de l'innocuité de deux vaccins commerciaux contre la babésiose canine provoquée par Babesia canis.

The safety of two vaccines available on the French market against canine babesiosis - Nobivac Piro® (NP) and Pirodog® (P) - have been evaluated. Their local, general and biochemical impacts have been compared in a controlled experimental study. Three groups were used: a control group (T) and two groups vaccinated twice at 21 days interval. All dogs presented moderate local reaction. However, either clinical and biological parameters showed that the NP group presented a significantly more intense reaction at the injection site compared to the P group. No statistical difference has been revealed between the groups P and T evolutions.

Trichothecenes NIV and DON modulate the maturation of murine dendritic cells.

Nivalenol (NIV) and Deoxynivalenol (DON), mycotoxins of the trichothecene family are considered very common food contaminants. In this work, we investigated whether the immunotoxic effects ascribed to these trichothecenes may be mediated by perturbations in the activity of dendritic cells (DCs). Murine bone marrow-derived DCs were used to evaluate the effects of NIV and DON on the LPS-induced maturation process. We found that the expression of the class II MHC and of the accessory CD11c molecules, but not of the costimulatory CD86 marker, was down-regulated by NIV and DON exposure in LPS-treated DCs, as well as nitric oxide (NO) production. Interestingly, NIV, but not DON, induced DC necrosis. Moreover, the analysis of the cytokine pattern showed that IL-12 and IL-10 expressions induced by LPS exposure were suppressed by both trichothecenes in a dose-dependent fashion. On the other hand, the secretion of the proinflammatory cytokine TNF-alpha was increased as a direct consequence of DON and NIV exposure. Taken together, our data indicated that the immunotoxicity of NIV and DON was related to the capacity of both trichothecenes to interfere with phenotypic and functional features of maturing DCs.

Interactive effects of fumonisin B1 and alpha-zearalenol on proliferation and cytokine expression in Jurkat T cells.

Mycotoxins are secondary metabolites of fungi that grow on various food and feed. These compounds elicit a wide spectrum of toxicological effects, including the capacity to alter normal immune function. Feed commodities are usually contaminated with more than one mycotoxin; however, extensive information on the interaction between concomitantly occurring mycotoxins and the consequence for their toxicity is lacking. In the present study, we examined the effects in vitro of fumonisin B1 (FB1) and alpha-zearalenol (alpha-ZEA), alone or in combination, on the immune function in the human lymphoblastoid Jurkat T cell line. Treatment of cells with increasing concentrations of FB1 resulted in a dose-dependent induction of proliferation. In contrast, alpha-ZEA showed a marked inhibitory effect on cell proliferation, even at very low doses, essentially mediated by apoptosis. In stimulated cells pre-incubated with FB1, the levels of IL-2 and IFN gamma mRNAs were similar to control whereas a reduction of cytokine transcripts was reported following alpha-ZEA treatment. Interestingly, co-administration of mycotoxins resulted in further inhibition of both proliferation and IFN gamma mRNA expression when compared with alpha-ZEA alone. In conclusion, FB1 and alpha-ZEA showed different immunomodulation abilities when individually administered. Combination of mycotoxins resulted instead in interactive effects.

Bondable reinforcement ribbon: clinical applications.

Bondable reinforcement ribbon can be used in dentistry to splint periodontally involved teeth, to provide postorthodontic splinting, to reinforce provisional prostheses, and to fabricate direct adhesive prostheses. Four cases illustrate esthetically and functionally successful use of reinforced ribbon.

CD4-mediated anchoring of the seminal antigen gp17 onto the spermatozoon surface.

A soluble 1, kDa glycoprotein, namely gp17, was previously isolated from human semen and used to obtain mouse monoclonal or chicken polyclonal antibodies. This protein was shown to bind CD4+ T-cells and to soluble recombinant CD4 in vitro. Here, we report that the anti-gp17 monoclonal antibodies are captured by ejaculated spermatozoa and that gp17-like antigens are released by cell acid extraction. Immunoblotting experiments with monoclonal antibodies indicated that SDS-lysates from spermatozoa contain proteins with the same electrophoretic and antigenic properties of CD4 and gp17. Anti-CD4 mouse monoclonal antibodies were used to coprecipitate from NP40-lysate proteins reacting with chicken anti-gp17 antibodies. Analytical chromatography demonstrated that a number of gp17-like forms are present in the seminal plasma, put that only the 1 kDa species can be detected in the spermatozoa lysate. This protein was localised by immunofluorescence on the post-acrosomal region of the spermatozoon. The same surface domain was also reactive with anti-CD4 antibodies. After treatment to induce in vitro capacitation, gp17 was detected all over the spermatozoon head. Conversely, only a minor part of the treated spermatozoa exhibited CD4 immunostaining, which remained localised on the post-acrosomal region. The possible function of CD4 and gp17 on male germ cells is discussed.

Purification of a 240 kDa protein from serum and follicular fluid of water buffalo and its identification as haptoglobin.

The fluids from healthy growing follicles of water buffalo were previously found free of the polypeptides H (M(r) 36,000) and L (M(r) 21,000) which were instead detected in fluids from atretic follicles and blood. Here we report evidence that these two polypeptides, as selected from serum by specific anti-L antibodies, are the subunits of an oligomeric protein. The protein was purified from serum or follicular fluid, and its molecular weight (240 kDa), isoelectric point (6.5), and amino acid composition were determined. The NH2-terminal sequences of the subunits L and H were analyzed: 100% and 90% homology with alpha and beta chains of bovine haptoglobin, respectively, was found. Thus, haptoglobin can be used as a novel molecular marker to assess the physiological state of the blood-follicle barrier or discriminate between atretic and healthy follicles.

Association of cumulus-oocyte complexes with the intrafollicular levels of a blood protein in Bubalus bubalis.

The protein pattern of the follicular fluid (FF) and the ultrastructure of the inner cumulus-oocyte complex (COC) has been analysed in single antral follicles (n = 146) of buffalo B. bubalis ovaries. The protein population of FF was fractionated by SDS-PAGE; the resulting pattern was Coomassie stained and processed for densitometry. Comparative analysis of sera and autologous FFs showed a marked difference in the level (measured as the percentage of total proteins) of one 21 kDa polypeptide band, called 'L'. Concentration of L, which was mainly higher in the serum (2.05 +/- 1.5%) than in the surrounding FF (0.98 +/- 0.94%), fluctuated widely in fluids from the same ovary. On gel filtration of FF and SDS-PAGE of the fractions collected, the L polypeptide was found and eluted together with a 36 kDa polypeptide, called 'H', with an exclusion volume lower than that of albumin. The levels of both polypeptides in the eluted fractions were measured by gel densitometry, and the same ratio H/L was found (2:1). These data suggest that H and L are subunits of a complex high-molecular-weight protein. The presence of L levels in male sera comparable to those detected in females indicates that this putative protein does not originate in the ovary but is transported from the blood. Moreover, a correlation between the increase in the percentage of Lf (calculated as %L in FF/%L in serum) and atresia was observed. COCs (n = 86) obtained during the collection of the single FF samples were processed for transmission electron microscopy. The ultrastructure of each COC was compared with the SDS-PAGE data of the associated FF. Healthy COCs were found to be related to very low levels of Lf (between 0 and 14% of those measured in serum). COCs with an early atretic ultrastructure undetectable at the dissection microscope, were associated with FFs having Lf levels between 24% and 60%; advanced atresia was associated with Lf values up to 70%. Finally, the acrosome reaction of buffalo precipicitated spermatozoa in vitro was monitored by adding one volume of FF with high (FF+; Lf = 80%) or undetectable (FF-) values of Lf to the sperm suspension.(ABSTRACT TRUNCATED AT 400 WORDS)

Localisation and capacitation-dependent loss of buffalo sperm-coating antigens shared with rat sperm.

The heterodimeric sperm-coating protein CFS was previously localised on the middle-piece region of rat spermatozoa by anti-CFS rabbit antibodies. CFS-immunorelated antigens were detected in the secretion of the water buffalo seminal vesicle by protein electrophoresis and Western blotting. Spermatozoa from buffalo epididymal cauda were incubated with the rat antigen and, upon immunostaining with anti-CFS antibodies and goat anti-rabbit fluorescein isothiocyanate (FITC)-conjugated IgGs, CFS was found attached on both the post-acrosomal region and the tail. Indirect immunofluorescence analysis permitted the localisation of CFS-related antigens on the same domains of buffalo ejaculated spermatozoa. These results suggest that the buffalo antigens not only share some epitopes with the homologous rat antigen but may also have some of its functional properties. Ejaculated spermatozoa were capacitated in vitro and then assayed for their content of CFS-like antigens. An inverse relationship was found between the levels of capacitation and the amounts of antigens detected, thus suggesting that the in vitro treatment was effective at removing CFS-related proteins from the cell surface. Titration of these proteins to monitor plasma membrane changes during sperm manipulation or to evaluate sperm quality is proposed.

Intervening with high risk youth: a program model.

This paper describes a program model being implemented for older, high-risk adolescents who are beginning to exhibit significant substance use problems. The program provides an initial assessment and refers clients to appropriate services. Only a small percentage are being referred to inpatient treatment; most clients enter outpatient intervention groups, which are conducted by the program. These groups have three purposes: to allow staff to monitor and evaluate clients so that those who are in imminent danger can be placed in protective treatment settings; to help clients see themselves as candidates for recovery and to introduce them to treatment resources by accompanying them to AA and NA meetings; and to change behavior, a goal that seems more immediately achievable for youth with stable community ties who have more to lose if they get into further trouble. Besides describing the program model, this paper examines some of the difficulties of getting referrals from a variety of community agencies.

Detection of a sperm-coating antigen in the semen of Bubalus bubalis.

1. Surface antigens of B. bubalis spermatozoa were solubilized by Triton X-100 and EDTA; the sperm extract was used to raise antibodies in rabbits. 2. Two major polypeptides, immunoprecipitated from the seminal plasma by the antibodies against the sperm extract, exhibited the same electrophoretic mobilities of two immunorelated sperm surface antigens. 3. The two polypeptides were isolated from the seminal plasma, by a multi-step chromatographic procedure, and found subunits of a single protein (MW 30,000), called SP 30. 4. The SP 30 protein bound in vitro to the postacrosomal region of homologous spermatozoa from cauda epididymis. 5. The localization of the sperm-coating antigen on the cell surface is compatible with a role in the fertilization process.

Opiate effect on the threat display in the crab Carcinus mediterraneus.

The well-known defense response of a crab (laterus merus display, LMD) was easily evoked in Carcinus mediterraneus by striking the cephalothoraxic protogastric region between the eyestalks. Following a program aimed at investigating the regulatory action of diverse neuromodulators on the LMD of this crab, a study on the role of opioids was started by testing the effect of morphine administration. Injection of morphine HC1 (MP) (40, 50, 60, 70, or 100 micrograms/g) produced a dose-dependent reduction of the LMD so elicited that dissipated with the postinjection time. Only MP doses higher than 50 micrograms/g were effective 30 min after drug administration. The MP-induced inhibition of LMD was blocked by a 4.8-micrograms naloxone HC1/g dose injected 10 min before MP. These results and those previously obtained as the action of GABA on the LMD of this crab are discussed in connection with results reporting a similar effect of these drugs on another agonistic item of behavior in the crab Chasmagnatus granulatus. The possibility of demonstrating habituation of the LMD to an iterated stimulation in C. mediterraneus and of using such a process to elucidate the acting paths of the drugs is discussed.