Behavioral effects of acute stimulation of kappa-opioid receptors during lactation

The behavioral effects of the K-opioid receptor agonist U69593 were examined in lactating rats. On day 5 of lactation, animals were treated with 0.1 mg/kg of U69593 to determine whether it influences general activity and maternal latencies toward pups. Because little attention has been given to the possibility that pre-mating treatment with morphine may modulate the response to K-opioid receptor stimulation, another group of animals was submitted to the same acute challenge after abrupt withdrawal from repeated treatment with morphine sulfate during the pre-mating period (5 mg/kg on alternate days for a total of five doses). Acute F;opioid stimulation reduced total locomotion, rearing frequency, and time spent self-grooming and increased immobility duration. These K agonist effects were not observed in animals pretreated with morphine. Similarly, latencies to retrieve pups were longer only in animals pretreated with saline and challenged acutely with U69593. None of these effects were observed in morphine sulfate-pretreated animals. The present results suggest that pre-mating repeated exposure to morphine produces a tolerance-like effect on behavioral responses to low-dose K-opioid receptor stimulation in active reproductive females. (c) 2008 Elsevier Inc. All rights reserved.

Bovine oocyte vitrification: Effect of ethylene glycol concentrations and meiotic stages

Success in oocyte cryopreservation is limited and several factors as cryoprotectant type or concentration and stage of oocyte meiotic maturation are involved. The aim of the present study was to evaluate the effect of maturation stage and ethylene glycol (EG) concentration on survival of bovine oocytes after vitrification. In experiment 1, kinetics of oocyte in vitro maturation (IVM) was evaluated. Germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase I (MI), and metaphase II (MII) oocytes were found predominantly at 0, 0-10, 10-14, and 18-24 h of INK respectively. In experiment 2, in vitro embryo development after in vitro fertilization (IVF) of oocytes exposed to equilibrium (ES) and vitrification solution VS-1 (EG 30%), or VS-2 (EG 40%) at 0, 12 or 18 It of IVM was evaluated. Only blastocyst rate from oocytes vitrified in SV-2 after 18 h of IVM was different from control oocytes. Hatched blastocyst rates from oocytes vitrified in VS-1 after 12 and 18 h, and SV-2 after 18 h of IVM were different from unvitrified oocytes. In experiment 3, embryo development was examined after IVF of oocytes vitrified using VS-I or VS-2 at 0, 12 or 18 h of IVM. Rates of blastocyst development after vitrification of oocytes in VS-1 at each time interval were similar. However, after vitrification in VS-2, blastocyst rates were less at 18 h than 0 h. Both cleavage rates and blastocyst rates were significantly less in all vitrification groups when compared to control group and only control oocytes hatched. In conclusion, both EG concentration and stage of meiotic maturation affect the developmental potential of oocytes after vitrification. (C) 2007 Elsevier B.V. All rights reserved.

The Role of Toll-Like Receptor 2 in the Recognition of Aggregatibacter actinomycetemcomitans

Background: Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans) is a Gram-negative bacterium present in the oral cavity and is usually associated with localized aggressive periodontitis. Isolated antigens from A. actinomycetemcomitans can activate innate immune cells through Toll-like receptors (TLRs), which are molecules that recognize structural components conserved among microorganisms. In this study, we evaluate the role of TLR2 in the recognition of A. actinomycetemcomitans. Methods: Macrophages and neutrophils from knockout mice with targeted disruption of TLR2 (TLR2(-/-) mice) and wild-type mice were collected and used for the subsequent assays. The production of cytokines and chemokines was evaluated by enzyme-linked immunosorbent assay (ELISA), and the presence of apoptotic cells was determined by flow cytometry. In addition, the mechanisms that modulate the outcome of A. actinomycetemcomitans-induced periodontal disease in TLR2(-/-) mice were examined. Results: The results show that TLR2-deficient mice developed more severe periodontitis after A. actinomycetemcomitans infection, characterized by significantly higher bone loss and inflammatory cell migration to periodontal tissues. The inflammatory cell influx into the peritoneal cavities of TLR2(-/-) mice was three-fold lower than that observed for the littermate controls. A significantly diminished production of the cytokines tumor necrosis factor-alpha and interleukin-1 beta as well as the chemokine CC-ligand-5 in the peritoneal cavities of TLR2(-/-) mice was observed. In addition, a high frequency of apoptotic cells in the inflammatory exudates from TLR2(-/-) mice was observed. Phagocytosis and nitric oxide production was diminished in cells from TLR2(-/-) mice, facilitating the dissemination of the pathogen to the spleen. Conclusion: The results of this study highlight the involvement of TLR2 in recognizing A. actinomycetemcomitans and its essential role in controlling A. actinomycetemcomitans infection. J Periodontot 2009,80:2070-2019.

Early life course determinants of young adults' gambling behaviour. An Australian Longitudinal Study

Problem gambling represents a public concern as both a social and health issue. Available evidence shows problem gambling is associated with a range of psychological disorders, criminality, and disruption to families. While gambling itself may represent a pleasurable pursuit for the majority, for a proportion, gambling-related activities may assume many of the characteristics of an addiction and have the capacity to undermine individuals� mental and physical health, social relationships, financial independence, as well as the financial and psychological wellbeing of their families and/or friends. The objectives of this study are based on the need to increase our understanding of gambling behaviour, its antecedents, as well its influence on the health and wellbeing of gamblers and their families. One of the most important and unresolved issues in gambling research is whether the mental health and social/family correlates of gambling precede or follow gambling behaviour. This report focuses on this issue.

Matrix metalloproteinases and their inhibitors in oral lichen planus

Background: Oral lichen planus (OLP) is characterized by a subepithelial lymphocytic infiltrate, basement membrane (BM) disruption, intra-epithelial T-cell migration and apoptosis of basal keratinocytes. BM damage and T-cell migration in OLP may be mediated by matrix metalloproteinases (MMPs). Methods: We examined the distribution, activation and cellular sources of MMPs and their inhibitors (TIMPs) in OLP using immunohistochemistry, ELISA, RT-PCR and zymography. Results: MMP-2 and -3 were present in the epithelium while MMP-9 was associated with the inflammatory infiltrate. MMP-9 and TIMP-1 secretion by OLP lesional T cells was greater than OLP patient (p

Nitric oxide synthase inhibitors improve prepulse inhibition responses of Wistar rats

Introduction: Cognitive and attentional deficits in schizophrenia include impairment of the sensorimotor filter as measured by prepulse inhibition (PPI). In this way, the study of animals that naturally present low PPI responses could be a useful approach for screening new antipsychotic drugs. Several pieces of evidence suggest that dopamine and nitric oxide (NO) can modulate PPI but their role in those animals is unknown. Objectives: The aim of this study was to investigate the role of dopamine and NO in Wistar rats with naturally low PPI response. Methods: Male Wistar rats with low PPI responses received an i.p. injection of the antipsychotics haloperidol (0.1, 0.3 or 1 mg/kg) or clozapine (0.5, 1.5 or 5 mg/kg), the anxiolytic diazepam (1 or 3 mg/kg) or the NO synthase (NOS) inhibitors, N(G)- nitro-L-arginine (L-NOARG; 40 mg/kg, acutely or sub-chronically) or 7-Nitroindazole (7-NI; 3, 10 or 30 mg/kg). All animals were submitted to the PPI test 1 h after injection. Striatal and cortical dopamine, DOPAC, and noradrenaline levels of rats with low PPI responses were compared to rats with normal PPI responses. Results: We found increased levels of catecholamines on the striatum and prefrontal cortex of Wistar rats with low PPI. In these animals, both antipsychotics, typical and atypical, and NOS inhibitors significantly increased PPI. Conclusion: Taken together, our findings suggest that the low PPI phenotype may be driven by an over-active catecholamine system. Additionally, our results corroborate the hypothesis of dopamine and NO interaction on PPI modulation and suggest that Wistar rats with low PPI may represent an interesting non-pharmacological model to evaluate new potential antipsychotics. (C) 2010 Elsevier B.V. All rights reserved.

Mice with genetic deletion of the heparin-binding growth factor midkine exhibit early preclinical features of Parkinson`s disease

There is considerable evidence showing that the neurodegenerative processes that lead to sporadic Parkinson`s disease (PD) begin many years before the appearance of the characteristic motor symptoms and that impairments in olfactory, cognitive and motor functions are associated with time-dependent disruption of dopaminergic neurotransmission in different brain areas. Midkine is a 13-kDa retinoic acid-induced heparin-binding growth factor involved in many biological processes in the central nervous system such as cell migration, neurogenesis and tissue repair. The abnormal midkine expression may be associated with neurochemical dysfunction in the dopaminergic system and cognitive impairments in rodents. Here, we employed adult midkine knockout mice (Mdk(-/-)) to further investigate the relevance of midkine in dopaminergic neurotransmission and in olfactory, cognitive and motor functions. Mdk(/-) mice displayed pronounced impairments in their olfactory discrimination ability and short-term social recognition memory with no gross motor alterations. Moreover, the genetic deletion of midkine decreased the expression of the enzyme tyrosine hydroxylase in the substantia nigra reducing partially the levels of dopamine and its metabolites in the olfactory bulb and striatum of mice. These findings indicate that the genetic deletion of midkine causes a partial loss of dopaminergic neurons and depletion of dopamine, resulting in olfactory and memory deficits with no major motor impairments. Therefore, Mdk(-/-) mice may represent a promising animal model for the study of the early stages of PD and for testing new therapeutic strategies to restore sensorial and cognitive processes in PD.

Neonatal handling reduces the number of cells in the medial preoptic area of female rats

Early-life events may induce alterations in neuronal function in adulthood. A crucial aspect in studying long-lasting effects induced by environmental interventions imposed to the animal several weeks before is finding a stable change that could be causally related to the phenotype observed in adulthood. In order to explain an adult trait, it seems necessary to look back to early life and establish a temporal line between events. The neonatal handling procedure is an experimental tool to analyze the long-lasting impact of early-life events. Aside from the neuroendocrine response to stress, neonatal handling also alters the functionality of the hypothalamus-pituitary-gonad (HPG) axis. Reductions in ovulation and surge of the luteinizing hormone (LH) on the proestrous day were shown in female rats. Considering the importance of the medial preoptic area (MPA) for the control of ovulation, the present study aimed to verify the effects of neonatal handling on the numerical density and cell size in the MPA in 11-day-old and 90-day-old female rats. Cellular proliferation was also assessed using BrdU (5-bromo-2`-deoxyuridine) in 11-day-old pups. Results showed that neonatal handling induces a stable reduction in the number of cells and in the size of the cell soma, which were lower in handled females than in nonhandled ones at both ages. Cellular proliferation in the MPA was also reduced 24 h after the last manipulation. The repeated mother-infant disruption imposed by the handling procedure ""lesioned"" the MPA. The dysfunction in the ovulation mechanisms induced by the handling procedure could be related to that neuronal loss. The study also illustrates the impact of an environmental intervention on the development of the brain. (C) 2008 Elsevier B.V. All rights reserved

Analysis of the TGF beta functional pathway in epithelial ovarian carcinoma

Epithelial ovarian carcinoma is often diagnosed at an advanced stage of disease and is the leading cause of death from gynaecological neoplasia. The genetic changes that occur during the development of this carcinoma are poorly understood. It has been proposed that IGFIIR, TGF beta1 and TGF beta RII act as a functional unit in the TGF beta growth inhibitory pathway, and that somatic loss-of-function mutations in any one of these genes could lead to disruption of the pathway and subsequent loss of cell cycle control. We have examined these 3 genes in 25 epithelial ovarian carcinomas using single-stranded conformational polymorphism analysis and DNA sequence analysis. A total of 3 somatic missense mutations were found in the TGF beta RII gene, but none in IGFRII or TGF beta1. An association was found between TGF beta RII mutations and histology, with 2 out of 3 clear cell carcinomas having TGF beta RII mutations. This data supports other evidence from mutational analysis of the PTEN and beta -catenin genes that there are distinct developmental pathways responsible for the progression of different epithelial ovarian cancer histologic subtypes. (C) 2001 Cancer Research Campaign.

The leisure participation of clients with a dual diagnosis

People with a dual diagnosis experience disruption in carrying out their daily occupations. This article describes a study in which an occupational therapist explored the leisure participation of clients with a dual diagnosis. In-depth, semi-structured interviews were conducted with four outpatients from an alcohol and drug rehabilitation programme. Inductive analysis of the informants’ interviews identified two main themes: leisure as part of the recovery process and the barriers to leisure participation. This study provides support for the need to understand the leisure occupations of the clients with whom occupational therapists work. Further research is required to examine the interventions that assist clients with a dual diagnosis to develop meaningful leisure activities.

Abolition of (-)-CGP 12177-evoked cardiostimulation in double beta(1)/beta(2)-adrenoceptor knockout mice. Obligatory role of beta(1)-adrenoceptors for putative beta 4-adrenoceptor pharmacology

Some beta (1)- and beta (2)-adrenoceptor-blocking agents, such as (-)-CGP 12177, cause cardiostimulant effects at concentrations considerably higher than those that antagonise the effects of catecholamines. The cardiostimulant effects of these non-conventional partial agonists are relatively resistant to blockade by (-)-propranolol and have been proposed to be mediated through putative beta (4)-adrenoceptors or through atypical states of either beta (1)- or beta (2)-adrenoceptors. We investigated the effects of (-)-CGP 12177 on sinoatrial rate and left atrial contractile force as well as the ventricular binding of (-)-[H-3]CGP 12177 in tissues from wild-type, beta (2)-adrenoceptor knockout and beta (1)/beta (2)-adrenoceptor double knockout mice. The cardiostimulant effects of (-)-CGP 12177 were present in wildtype and beta (2)-adrenoceptor knockout mice but were absent in beta (1)/beta (2)-adrenoceptor double knockout mice. Thus, the presence of beta (1)-adrenoceptors is obligatory for the cardiostimulant effects of (-)-CGP 12177. It appears therefore that an atypical state of the beta (1)-adrenoceptor contributes to the mediation of the cardiostimulant effects induced by non-conventional partial agonists. Ventricular beta (1)- and beta (2)-adrenoceptors, labelled in wild-type with a K(D)similar to0.5 nmol/l (similar to 16 fmol/mg protein), were absent in beta (1)/beta (2)-adrenoceptor double knockout mice. However, a high density binding site (similar to 154-391 fmol/mg protein) that did not saturate completely (K(D)similar to 80-200 nM) was labelled by (-)-[H-3]CGP 12177 in the three groups of mice, being distinct from beta (1)- and beta (2)-adrenoceptors, as well as from the site mediating the agonist effects of(-)-CGP 12177.

Ataxia telangiectasia mutated (ATM) kinase and ATM and Rad3 related kinase mediate phosphorylation of Brca1 at distinct and overlapping sites - In vivo assessment using phospho-specific antibodies

Recent studies have provided evidence that breast cancer susceptibility gene products (Brca1 and Brca2) suppress cancer, at least in part, by participating in DNA damage signaling and DNA repair. Brca1 is hyperphosphorylated in response to DNA damage and co-localizes with Rad51, a protein involved in homologous-recombination, and Nbs1.Mre11.Rad50, a complex required for both homologous-recombination and nonhomologous end joining repair of damaged DNA. Here, we report that there is a qualitative difference in the phosphorylation states of Brca1 between ionizing radiation (IR) and UV radiation. Brca1 is phosphorylated at Ser-1423 and Ser-1524 after IR and W; however, Ser-1387 is specifically phosphorylated after IR, and Ser-1457 is predominantly phosphorylated after W. These results suggest that different types of DNA-damaging agents might signal to Brca1 in different ways. We also provide evidence that the rapid phosphorylation of Brca1 at Ser-1423 and Ser-1524 after IR (but not after W) is largely ataxia telangiectasia mutated (ATM) kinase-dependent. The overexpression of catalytically inactive ATM and Rad3 related (ATR) kinase inhibited the UV-induced phosphorylation of Brca1 at these sites, indicating that ATR controls Brca1 phosphorylation in vivo after the exposure of cells to UV light. Moreover, ATR associates with Brca1; ATR and Brca1 foci co-localize both in cells synchronized in S phase and after exposure of cells to DNA-damaging agents. ATR can itself phosphorylate the region of Brca1 phosphorylated by ATM (Ser-Gln cluster in the C terminus of Brca1, amino acids 1241-1530), However, there are additional uncharacterized ATR phosphorylation site(s) between residues 521 and 757 of Brca1, Taken together, our results support a model in which ATM and ATR act in parallel but somewhat overlapping pathways of DNA damage signaling but respond primarily to different types of DNA lesion.

Vascular smooth muscle cell phenotypic modulation in culture is associated with reorganisation of contractile and cytoskeletal proteins

Smooth muscle cells (SMC) exhibit a functional plasticity, modulating from the mature phenotype in which the primary function is contraction, to a less differentiated state with increased capacities for motility, protein synthesis, and proliferation. The present study determined, using Western analysis, double-label immunofluorescence and confocal microscopy, whether changes in phenotypic expression of rabbit aortic SMC in culture could be correlated with alterations in expression and distribution of structural proteins. Contractile state SMC (days 1 and 3 of primary culture) showed distinct sorting of proteins into subcellular domains, consistent with the theory that the SMC structural machinery is compartmentalised within the cell. Proteins specialised for contraction (alpha -SM actin, SM-MHC, and calponin) were highly expressed in these cells and concentrated in the upper central region of the cell. Vimentin was confined to the body of the cell, providing support for the contractile apparatus but not co-localising with it. In line with its role in cell attachment and motility, beta -NM actin was localised to the cell periphery and basal cortex. The dense body protein alpha -actinin was concentrated at the cell periphery, possibly stabilising both contractile and motile apparatus. Vinculin-containing focal adhesions were well developed, indicating the cells' strong adhesion to substrate. In synthetic state SMC (passages 2-3 of culture), there was decreased expression of contractile and adhesion (vinculin) proteins with a concomitant increase in cytoskeletal proteins (beta -non-muscle [NM] actin and vimentin). These quantitative changes in structural proteins were associated with dramatic chan-es in their distribution. The distinct compartmentalisation of structural proteins observed in contractile state SMC was no longer obvious, with proteins more evenly distributed throughout die cytoplasm to accommodate altered cell function. Thus, SMC phenotypic modulation involves not only quantitative changes in contractile and cytoskeletal proteins, but also reorganisation of these proteins. Since the cytoskeleton acts as a spatial regulator of intracellular signalling, reorganisation of the cytoskeleton may lead to realignment of signalling molecules, which, in turn, may mediate the changes in function associated with SMC phenotypic modulation. (C) 2001 Wiley-Liss, Inc.

Initiation of cell locomotility is a morphogenetic checkpoint in thyroid epithelial cells regulated by ERK and PI3-kinase signals

Epithelial locomotility is a fundamental determinant of tissue patterning that is subject to strict physiological regulation. The current, study sought to identify cellular signals that initiate cell migration in cultured thyroid epithelial cells. Porcine thyroid cells cultured as 3-dimensional follicles convert to 2-dimensional monolayers when deprived of agents that stimulate cAMP/PKA signaling. This morphogenetic event is driven by the activation of cell-on-substrate locomotility, providing a convenient assay for events that regulate the initiation of locomotion. In this system, the extracellular signal regulated kinase (ERK) pathway became activated as follicles converted to monolayer, as demonstrated by immunoblotting for activation-specific phosphorylation and nuclear accumulation of ERK. Inhibition of ERK activation using the drug PD98059 effectively prevented cells from beginning to migrate. PD98059 inhibited cell spreading, actin filament reorganization and the assembly of focal adhesions, cellular events that mediate the initiation of thyroid cell locomotility. Akt (PKB) signaling was also activated during follicle-to-monolayer conversion and the phosphoinositide 3-kinase (PI3-kinase) inhibitor, wortmannin, also blocked the initiation of cell movement. Wortmannin did not, however, block activation of ERK signaling. These findings, therefore, identify the ERK and PI3-kinase signaling pathways as important stimulators of thyroid cell locomotility. These findings are incorporated into a model where the initiation of thyroid cell motility constitutes a morphogenetic checkpoint regulated by coordinated changes in stimulatory (ERK, PI3-kinase) and tonic inhibitory (cAMP/PKA) signaling pathways. Cell Motil. Cytoskeleton 49:93-103, 2001. (C) 2001 Wiley-Liss, Inc.

Microtubule integrity is essential for apical polarization and epithelial morphogenesis in the thyroid

In this study, we examined the contribution of microtubules to epithelial morphogenesis in primary thyroid cell cultures. Thyroid follicles consist of a single layer of polarized epithelial cells surrounding a closed compartment, the follicular lumen. Freshly isolated porcine thyroid cells aggregate and reorganize to form follicles when grown in primary cultures. Follicular reorganization is principally a morphogenetic process that entails the assembly of biochemically distinct apical and basolateral membrane domains, delimited by tight junctions. The establishment of cell surface polarity during folliculogenesis coincided with the polarized redistribution of microtubules, predominantly in the developing apical poles of cells. Disruption of microtubule integrity using either colchicine or nocodazole caused loss of defined apical membrane domains, tight junctions and follicular lumina. Apical membrane and tight junction markers became randomly distributed at the outer surfaces of aggregates. In contrast, the basolateral surface markers, E-cadherin and Na+,K+-ATPase, remained correctly localized at sites of cell-cell contact and at the free surfaces of cell aggregates. These findings demonstrate that microtubules play a necessary role in thyroid epithelial morphogenesis. Specifically, microtubules are essential to preserve the correct localization of apical membrane components within enclosed cellular aggregates, a situation that is also likely to pertain where lumina must be formed from solid aggregates of epithelial precursors. (C) 2001 Wiley-Liss, Inc.