Effect of melatonin on DNA damage of bovine cumulus cells during in vitro maturation (IVM) and on in vitro embryo development

The effect of melatonin during in vitro maturation (IVM) on DNA damage of cumulus cells (CCs) from bovine cumulus-oocyte complexes (COCs) and embryo development was evaluated. COCs from abattoir ovaries were cultured in maturation medium (MM) with 0.5 mu g/ml FSH and 5.0 mu g/ml LH (FSH-LH); 10(-9) M melatonin (MEL) or FSH-LH + MEL (FSH-LH-MEL). After 24 h of in vitro maturation, the CCs surrounding the oocyte were subjected to DNA analysis by Comet assay. After in vitro fertilization and in vitro embryo culture, the embryo development rates were evaluated on day 2 post insemination (cleavage) and days 7-8 (blastocyst). The percentage of CCs with no DNA damage was significantly superior in MEL group (37.6 +/- 2.4) than in FSH-LH-MEL (28.0 +/- 2.4) and FSH-LH (17.8 +/- 2.41) groups. Cleavage and blastocysts rates were similar among groups. Melatonin during IVM protects the CCs from DNA damage but this effect did not influence embryo development in vitro. (C) 2010 Elsevier Ltd. All rights reserved.

Diurnal and nocturnal herbivore induction on maize elicit different innate response of the fall armyworm parasitoid, Campoletis flavicincta

Herbivore-attacked plants produce specific volatile substances that represent important cues for host finding by natural enemies. The fall armyworm Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) is a voracious herbivore and usually feed on maize in all periods of the day. Given that plant needs light to synthesize de novo herbivore-induced volatiles, volatile blend may be changed depending on time of the day the plant is induced, what could interfere in natural enemy foraging. In this sense, the current study aimed to investigate differential attractiveness of maize elicited by fall armyworm regurgitant under light and dark conditions to its specialist larval parasitoid Campoletis flavicincta (Ashmead) (Hymenoptera: Ichneumonidae). All bioassays were conducted in Y-tube olfactometer to assess parasitoid response to odors from undamaged maize, mechanical damage, and regurgitant-treated plants at 0-1, 5-6, and 24-25 h after induction. The results showed that na < ve wasps were attracted to volatiles emitted by nocturnal regurgitant-treated maize at 5-6 h, but not to odors from diurnal regurgitant-treated plants. The differential attractiveness is likely due to blend composition as nocturnal regurgitant-treated plants emit aromatic compounds and the homoterpene (3E)-4,8-dimethyl-1,3,7-nonatriene in larger amounts than diurnal-treated plants.

Correlation between mandibular gland secretion and cuticular hydrocarbons in the stingless bee Melipona quadrifasciata

We investigated whether Melipona quadrifasciata worker mandibular gland secretions contribute directly to their cuticular hydrocarbon profile. The mandibular gland secretion composition and cuticular surface compounds of newly emerged worker bees, nurse bees, and foragers were determined by gas chromatography and mass spectrometry and compared. Both the mandibular gland secretions and the cuticular surface compounds of all worker stages were found to be composed almost exclusively of hydrocarbons. Although the relative proportion of hydrocarbons from the cuticular surface and gland secretion was statistically different, there was a high similarity in the qualitative composition between these structures in all groups of bees.

In vivo hydroquinone exposure causes tracheal hyperresponsiveness due to TNF secretion by epithelial cells

Hydroquinone (HQ) is the main oxidative substance in cigarette smoke and a toxic product of benzene biotransformation. Although the respiratory tract is an inlet pathway of HQ exposure, its effect on airway muscle responsiveness has not been assessed. We thus investigated the effects of low dose in vivo HQ-exposure on tracheal responsiveness to a muscarinic receptor agonist. Male Swiss mice were exposed to aerosolised 5% ethanol/saline solution (HQ vehicle; control) or 0.04 ppm HQ (1 h/day for 5 days) and tracheal rings were collected 1 h after the last exposure. HQ exposure caused tracheal hyper-responsiveness to methacholine (MCh), which was abolished by mechanical removal of the epithelium. This hyperresponsiveness was not dependent on neutrophil infiltration, but on tumour necrosis factor (TNF) secretion by epithelial cells. This conclusion was based on the following data: (1) trachea from HQ-exposed mice presented a higher amount of TNF, which was abrogated following removal of the epithelium; (2) the trachea hyperresponsiveness and TNF levels were attenuated by in vivo chlorpromazine (CPZ) treatment, an inhibitor of TNF synthesis. The involvement of HQ-induced TNF secretion in trachea mast cell degranulation was also demonstrated by the partial reversion of tracheal hyperresponsiveness in sodium cromoglicate-treated animals, and the in vivo HQ-exposure-induced degranulation of trachea connective tissue and mucosal mast cells, which was reversed by CPZ treatment. Our data show that in vivo HQ exposure indirectly exacerbates the parasympathetic-induced contraction of airway smooth muscle cells, mediated by TNF secreted by tracheal epithelial cells, clearly showing the link between environmental HQ exposure and the reactivity of airways. (C) 2012 Elsevier Ireland Ltd. All rights reserved.

Effects of neonatal castration and androgenization on sexual dimorphism in bone, leptin and corticosterone secretion

This study investigated the role of neonatal sex steroids in rats on sexual dimorphism in bone, as well as on leptin and corticosterone concentrations throughout the lifespan. Castration of males and androgenization of females were used as models to investigate the role of sex steroids shortly after birth. Newborn Wistar rats were divided into four groups, two male groups and two female groups. Male pups were cryoanesthetized and submitted to castration or sham-operation procedures within 24 h after birth. Female pups received a subcutaneous dose of testosterone propionate (100 mu g) or vehicle. Rats were euthanized at 20, 40, or 120 postnatal days. Body weight was also measured at 20, 40, and 120 days of age, and blood samples and femurs were collected. The length and thickness of the femurs were measured and the areal bone mineral density (areal BMD) was determined by dual-energy X-ray absorptiometry (DEXA). Biomechanical three-point bending testing was used to evaluate bone breaking strength, energy to fracture, and extrinsic stiffness. Blood samples were submitted to a biochemical assay to estimate calcium, phosphorus, alkaline phosphatase, leptin, and corticosterone levels. Weight gain, areal BMD and bone biomechanical properties increased rapidly with respect to age in all groups. In control animals, skeletal sexual dimorphism, leptin concentration, and dimorphic corticosterone concentration patterns were evident after puberty. However, androgen treatment induced changes in growth, areal BMD, and bone mass properties in neonatal animals. In addition, neonatally-castrated males had bone development and mechanical properties similar to those of control females. These results suggest that the exposure to neonatal androgens may represent at least one covariate that mediates dimorphic variation in leptin and corticosterone secretions. The study indicates that manipulation of the androgen environment during the critical period of sexual differentiation of the brain causes long-lasting changes in bone development, as well as serum leptin and corticosterone concentrations. In addition, this study provides useful models for the investigation of bone disorders induced by hypothalamic hypogonadism. (C) 2011 Elsevier Inc. All rights reserved.

Ubiquitin proteasome system and the atypical kinase PfPK7 are involved in melatonin signaling in Plasmodium falciparum

We previously reported that melatonin modulates the Plasmodium falciparum erythrocytic cycle by increasing schizont stage population as well as diminishing ring stage population. In addition, the importance of calcium and cAMP in melatonin signaling pathway in P. falciparum was also demonstrated. Nevertheless, the molecular effectors of the indoleamine signaling pathway remain elusive. We now demonstrate by real-time PCR that melatonin treatment up-regulates genes related to ubiquitin/proteasome system (UPS) components and that luzindole, a melatonin receptor antagonist, inhibits UPS transcription modulation. We also show that protein kinase PfPK7, a P. falciparum orphan kinase, plays a crucial role in the melatonin transduction pathway, since following melatonin treatment of P. falciparum parasites where pfpk7 gene is disrupted (pfpk7- parasites) (i) the ratio of asexual stages remain unchanged, (ii) the increase in cytoplasmatic calcium in response to melatonin was strongly diminished and (iii) up-regulation of UPS genes did not occur. The wild-type melatonin-induced alterations in cell cycle features, calcium rise and UPS gene transcription were restored by re-introduction of a functional copy of the pfpk7 gene in the pfpk7- parasites.

Chemical Chaperones Curcumin and 4-Phenylbutyric Acid Improve Secretion of Mutant Factor H R127H by Fibroblasts from a Factor H-Deficient Patient

Factor H (FH) is one of the most important regulatory proteins of the alternative pathway of the complement system. Patients with FH deficiency have a higher risk for development of infections and kidney diseases because of the uncontrolled activation and subsequent depletion of the central regulatory component C3 of the complement system. In this study, we investigated the consequences of the Arg(127)His mutation in FH (FHR127H) previously described in an FH-deficient patient, on the secretion of this protein by skin fibroblasts in vitro. We observed that, although the patient cells stimulated with IFN-gamma were able to synthesize FHR127H, the mutant protein was largely retained within the endoplasmic reticulum (ER), whereas normal human fibroblasts stimulated with IFN-gamma secrete FH without retention in the ER. Moreover, the retention of FHR127H provoked enlargement of ER cisterns after treatment with IFN-gamma. A similar ER retention was observed in Cos-7 cells expressing the mutant FHR127H protein. Despite this deficiency in secretion, we show that the FHR127H mutant is capable of functioning as a cofactor in the Factor I-mediated cleavage of C3. We then evaluated whether a treatment could increase the secretion of FH, and observed that the patient's fibroblasts treated with the chemical chaperones 4-phenylbutiric acid or curcumin increased the secretion rate of FH. We propose that these chemical chaperones could be used as alternative therapeutic agents to increase FH plasma levels in FH-deficient patients caused by secretion delay of this regulatory protein. The Journal of Immunology, 2012, 189: 3242-3248.

Do Higher Temperatures Increase Growth in the Nocturnal Gecko Homonota darwini (Gekkota: Phyllodactylidae)? A Skeletochronological Assessment Analyzed at Temporal and Geographic Scales

Recent studies predict that several lineages of tropical animals are at particular risk given current estimates of global climate change. Yet, much uncertainty exists on the effects of climate shifts in ectothermic species from cool temperate regions such as Patagonia. In this study, we focus on the impact of environmental temperature on growth, age at sexual maturity, and life-span of the Patagonian gecko Homonota darwini. Skeletochronological methods were used to assess the bone growth rates Of individuals from three populations at different geographic and temporal scales: two populations from Chubut (warm site; 1941 and 2010) and one population from Rio Negro (cold site; 1997-1998). Populations displayed similar bone arrangement and the growth patterns fit a von Bertalanffy curve. Three populations attained reproductive size at a minimum age of 3 yr, but at the cold site two specimens were shown to mature in 4 yr. We found no differences in juvenile growth rates in body size or bone zone width between juveniles of 1 to 3 yr of age from the 1941 warm site and the 2010 warm site. However, these traits appeared to be higher at these two warm sites than at the cold site, which is consistent with the climatic differences among the three localities. Our results suggest that higher temperatures positively affect growth, denoting that global warming might benefit H. darwini, especially the southern populations.

ANTIFUNGAL ACTIVITY OF LECTINS AGAINST YEAST OF VAGINAL SECRETION

Lectins are carbohydrate-binding proteins of non-imune origin. This group of proteins is distributed widely in nature and they have been found in viruses, microorganisms, plants and animals. Lectins of plants have been isolated and characterized according to their chemical, physical-chemical, structural and biological properties. Among their biological activities, we can stress its fungicidal action. It has been previously described the effect of the lectins Dviol, DRL, ConBr and LSL obtained from the seeds of leguminous plants on the growth of yeasts isolated from vaginal secretions. In the present work the experiments were carried out in microtiter plates and the results interpreted by both methods: visual observations and a microplate reader at 530nm. The lectin concentrations varied from 0.5 to 256 mu g/mL, and the inoculum was established between 65-70% of trammitance. All yeast samples isolated from vaginal secretion were evaluated taxonomically, where were observed macroscopic and microscopic characteristics to each species. The LSL lectin did not demonstrate any antifungal activity to any isolate studied. The other lectins DRL, ConBr and DvioL, showed antifungal potential against yeast isolated from vaginal secretion. These findings offering offer a promising field of investigation to develop new therapeutic strategies against vaginal yeast infections, collaborating to improve women's health.

NF-κB Drives the Synthesis of Melatonin in RAW 264.7 Macrophages by Inducing the Transcription of the Arylalkylamine-N-Acetyltransferase (AA-NAT) Gene

We demonstrate that during inflammatory responses the nuclear factor kappa B (NF-kappa B) induces the synthesis of melatonin by macrophages and that macrophage-synthesized melatonin modulates the function of these professional phagocytes in an autocrine manner. Expression of a DsRed2 fluorescent reporter driven by regions of the aa-nat promoter, that encodes the key enzyme involved in melatonin synthesis (arylalkylamine-N-acetyltransferase), containing one or two upstream kappa B binding sites in RAW 264.7 macrophage cell lines was repressed when NF-kappa B activity was inhibited by blocking its nuclear translocation or its DNA binding activity or by silencing the transcription of the RelA or c-Rel NF-kappa B subunits. Therefore, transcription of aa-nat driven by NF-kappa B dimers containing RelA or c-Rel subunits mediates pathogen-associated molecular patterns (PAMPs) or pro-inflammatory cytokine-induced melatonin synthesis in macrophages. Furthermore, melatonin acts in an autocrine manner to potentiate macrophage phagocytic activity, whereas luzindole, a competitive antagonist of melatonin receptors, decreases macrophage phagocytic activity. The opposing functions of NF-kappa B in the modulation of AA-NAT expression in pinealocytes and macrophages may represent the key mechanism for the switch in the source of melatonin from the pineal gland to immune-competent cells during the development of an inflammatory response.

Suppression of Inflammatory Cytokine Secretion by an NF-kappa B Inhibitor DHMEQ in Nasal Polyps Fibroblasts

Background: NF-kappa B is an essential transcription factor strongly associated to inflammatory response in chronic rhinosinusitis with nasal polyps (CRSwNP). DHMEQ is a NF-kappa B inhibitor that has been previously described with a greatpotential indecreasing inflammation in diseases other than CRSwNP. The aim of study isto evaluate the ability of DHMEQ to reducethe inflammatory recruiters on CRSwNP and to compare its anti-inflammatory profile as a single-agent or in association with fluticasone propionate (FP). Methods: nasal polyp fibroblasts were cultured in TNF-alpha enriched media. Cells were submitted to three different concentrations (1, 10 and 100nM) of either FP, DHMEQ or both. Inflammatory response was accessed by VCAM-1, ICAM-1 and RANTES expression (by RTQ-PCR) and protein levels by ELISA. Nuclear translocation of NF-kappa B was also evaluated. Results: both FP and DHMEQ inhibited inflammatory recruiters' production and NF-kappa B nuclear translocation. Interestingly, the anti-inflammatory effect from the association steroids plus DHMEQ was more intense than of each drug in separate. Conclusion: DHMEQ seems efficient in modulating the inflammatory process in CRSwNP. The synergic anti-inflammatory effect of DHMEQ and steroids may be a promising strategy to be explored, particularly in the setting of steroid-resistant NP. Copyright (c) 2012 S. Karger AG, Basel

Glia-Pinealocyte Network: The Paracrine Modulation of Melatonin Synthesis by Tumor Necrosis Factor (TNF)

The pineal gland, a circumventricular organ, plays an integrative role in defense responses. The injury-induced suppression of the pineal gland hormone, melatonin, which is triggered by darkness, allows the mounting of innate immune responses. We have previously shown that cultured pineal glands, which express toll-like receptor 4 (TLR4) and tumor necrosis factor receptor 1 (TNFR1), produce TNF when challenged with lipopolysaccharide (LPS). Here our aim was to evaluate which cells present in the pineal gland, astrocytes, microglia or pinealocytes produced TNF, in order to understand the interaction between pineal activity, melatonin production and immune function. Cultured pineal glands or pinealocytes were stimulated with LPS. TNF content was measured using an enzyme-linked immunosorbent assay. TLR4 and TNFR1 expression were analyzed by confocal microscopy. Microglial morphology was analyzed by immunohistochemistry. In the present study, we show that although the main cell types of the pineal gland (pinealocytes, astrocytes and microglia) express TLR4, the production of TNF induced by LPS is mediated by microglia. This effect is due to activation of the nuclear factor kappa B (NF-kB) pathway. In addition, we observed that LPS activates microglia and modulates the expression of TNFR1 in pinealocytes. As TNF has been shown to amplify and prolong inflammatory responses, its production by pineal microglia suggests a glia-pinealocyte network that regulates melatonin output. The current study demonstrates the molecular and cellular basis for understanding how melatonin synthesis is regulated during an innate immune response, thus our results reinforce the role of the pineal gland as sensor of immune status.

Birth weight, current body mass index, and insulin sensitivity and secretion in young adults in two Latin American populations

Background and aims: Although studies have shown association of birth weight (BW) and adult body mass index (BMI) with insulin sensitivity in adults, there is limited evidence that BW is associated with insulin secretion. We assessed the associations between BW and current BMI with insulin sensitivity and secretion in young Latin American adults. Methods and results: Two birth cohorts, one from Ribeirao Preto, Brazil, based on 1984 participants aged 23-25 years, and another from Limache, Chile, based on 965 participants aged 22-28 years were studied. Weight and height at birth, and current fasting plasma glucose and insulin levels were measured. Insulin sensitivity (HOMA%S) and secretion (HOMA%beta) were estimated using the Homeostatic Model Assessment (HOMA2). Multiple linear regression analyses were carried out to test the associations between BW and adult BMI z-scores on log HOMA%S and log HOMA%beta. BW z-score was associated with HOMA%S in the two populations and HOMA%beta in Ribeirao Preto when adult BMI z-score was included in the model. BW z-score was associated with decreasing insulin secretion even without adjusting for adult BMI, but only in Ribeirao Preto. BMI z-score was associated with low HOMA%S and high HOMA%beta. No interactions between BW and BMI z-scores on insulin sensitivity were shown. Conclusions: This study supports the finding that BW may affect insulin sensitivity and secretion in young adults. The effect size of BW on insulin status is small in comparison to current BMI. (C) 2010 Elsevier B.V. All rights reserved.

Physical exercise and pancreatic islets Acute and chronic actions on insulin secretion

Diabetes mellitus (DM) is a great public health problem, which attacks part of the world population, being characterized by an imbalance in body glucose homeostasis. Physical exercise is pointed as a protective agent and is also recommended to people with DM. As pancreatic islets present an important role in glucose homeostasis, we aim to study the role of physical exercise (chronic adaptations and acute responses) in pancreatic islets functionality in Wistar male rats. First, animals were divided into two groups: sedentary (S) and aerobic trained (T). At the end of 8 weeks, half of them (S and T) were submitted to an acute exercise session (exercise until exhaustion), being subdivided as acute sedentary (AS) and acute trained (AT). After the experimental period, periepididymal, retroperitoneal and subcutaneous fat pads, blood, soleus muscle and pancreatic islets were collected and prepared for further analysis. From the pancreatic islets, total insulin content, insulin secretion stimulated by glucose, leucine, arginine and carbachol were analyzed. Our results pointed that body adiposity and glucose homeostasis improved with chronic physical exercise. In addition, total insulin content was reduced in group AT, insulin secretion stimulated by glucose was reduced in trained groups (T and AT) and insulin secretion stimulated by carbachol was increased in group AT. There were no significant differences in insulin secretion stimulated by arginine and leucine. We identified a possible modulating action on insulin secretion, probably related to the association of chronic adaptation with an acute response on cholinergic activity in pancreatic islets.

Protein secretion in Lactococcus lactis : an efficient way to increase the overall heterologous protein production

Anderson MIYOSHI, Daniela FREITAS, Luciana RIBEIRO, Jane E. GABRIEL, Sophie LECLERCQ, Maricê N. OLIVEIRA, and Valeria D. GUIMARÃES were recipients of a CAPES fellowship (project CAPESCOFECUB #319II). Luis BERMUDEZ and Sébastien NOUAILLE were recipients of a fellowship from the French Ministry of Education and Research. INRA and Région IledeFrance also financed L. BERMUDEZ and V. GUIMARAES. Cathy CHARLIER is recipient of a fellowship from INRA and Région Bretagne.