Application of highly sensitive saturation labeling to the analysis of differential protein expression in infected ticks from limited samples

<p>Abstract</p> <p>Background</p> <p>Ticks are vectors of pathogens that affect human and animal health worldwide. Proteomics and genomics studies of infected ticks are required to understand tick-pathogen interactions and identify potential vaccine antigens to control pathogen transmission. One of the limitations for proteomics research in ticks is the amount of protein that can be obtained from these organisms. In the work reported here, individual naturally-infected and uninfected <it>Rhipicephalus </it>spp. ticks were processed using a method that permits simultaneous extraction of DNA, RNA and proteins. This approach allowed using DNA to determine pathogen infection, protein for proteomics studies and RNA to characterize mRNA levels for some of the differentially expressed proteins. Differential protein expression in response to natural infection with different pathogens was characterized by two-dimensional (2-D) differential in gel electrophoresis (DIGE) saturation labeling in combination with mass spectrometry analysis. To our knowledge, this is the first report of the application of DIGE saturation labeling to study tick proteins.</p> <p>Results</p> <p>Questing and feeding <it>Rhipicephalus </it>spp. adult ticks were collected in 27 farms located in different Sicilian regions. From 300 collected ticks, only 16 were found to be infected: <it>R. sanguineus </it>with <it>Rickettsia conorii </it>and <it>Ehrlichia canis</it>; <it>R. bursa </it>with <it>Theileria annulata</it>; and <it>R. turanicus </it>with <it>Anaplasma ovis</it>. The proteomic analysis conducted from a limited amount of proteins allowed the identification of host, pathogen and tick proteins differentially expressed as a consequence of infection.</p> <p>Conclusion</p> <p>These results showed that DIGE saturation labeling is a powerful technology for proteomics studies in small number of ticks and provided new information about the effect of pathogen infection in ticks.</p

Perinatal exposure to 5-metoxytryptamine, behavioural-stress reactivity and functional response of 5-HT1A receptors in the adolescent rat

Serotonin is involved in a wide range of physiological and patho-physiological mechanisms. In particular, 5-HT1A receptors are proposed to mediate stress-adaptation. The aim of this research was to investigate in adolescent rats: first, the consequences of perinatal exposure to 5- metoxytryptamine (5MT), a 5-HT1/5-HT2 serotonergic agonist, on behavioural-stress reactivity in elevated plus maze, open field and forced swim tests; secondly, whether the behavioural effects induced by perinatal exposure to 5MT on open field and forced swim tests were affected by the selective 5-HT1A receptor agonist LY 228729, a compound able to elicit a characteristic set of motor behaviours on these experimental models, and by the co-administration of the selective and silent 5-HT1A antagonist WAY 100635. Results indicate that a single daily injection of 5MT to, pregnant dams from gestational days 12 to 21 (1 mg/kg s.c.), and to the pups from postnatal days 2 to 18 (0.5 mg kg s.c.), induce in the adolescent rat offspring: an increase in the percentage of entries and time spent on the open arms in the elevated plus maze; a reduction in locomotor activity and rearing frequency, and an increase in the time spent on the central areas in the open field test; a decrease in immobility and an increase in swimming in the forced swim test. Acute administration of LY 228729 (1.5 mg/kg s.c.) strongly decreases rearing frequency and increases peripheral activity in the open field test, and decreases immobility and increases swimming in the forced swim test both in perinatally vehicle and 5MT-exposed offspring. Co-administration of WAY 100635 (0.25 mg/kg s.c.) abolishes the effects exerted by LY 228729. These results suggest that, in the adolescent rat, perinatal exposure to 5MT enhances the stress-related adaptive behavioural responses, presumably through a predominant action on presynaptic 5-HT1A receptors and does not deteriorate the functional response of 5-HT1A receptors to selective agonist and antagonist compounds

Subolesin expression in response to pathogen infection in ticks

<p>Abstract</p> <p>Background</p> <p>Ticks (Acari: Ixodidae) are vectors of pathogens worldwide that cause diseases in humans and animals. Ticks and pathogens have co-evolved molecular mechanisms that contribute to their mutual development and survival. Subolesin was discovered as a tick protective antigen and was subsequently shown to be similar in structure and function to akirins, an evolutionarily conserved group of proteins in insects and vertebrates that controls NF-kB-dependent and independent expression of innate immune response genes. The objective of this study was to investigate subolesin expression in several tick species infected with a variety of pathogens and to determine the effect of subolesin gene knockdown on pathogen infection. In the first experiment, subolesin expression was characterized in ticks experimentally infected with the cattle pathogen, <it>Anaplasma marginale</it>. Subolesin expression was then characterized in questing or feeding adult ticks confirmed to be infected with <it>Anaplasma</it>, <it>Ehrlichia</it>, <it>Rickettsia</it>, <it>Babesia </it>or <it>Theileria </it>spp. Finally, the effect of subolesin knockdown by RNA interference (RNAi) on tick infection was analyzed in <it>Dermacentor variabilis </it>males exposed to various pathogens by capillary feeding (CF).</p> <p>Results</p> <p>Subolesin expression increased with pathogen infection in the salivary glands but not in the guts of tick vector species infected with <it>A. marginale</it>. When analyzed in whole ticks, subolesin expression varied between tick species and in response to different pathogens. As reported previously, subolesin knockdown in <it>D. variabilis </it>infected with <it>A. marginale </it>and other tick-borne pathogens resulted in lower infection levels, while infection with <it>Francisella tularensis </it>increased in ticks after RNAi. When non-tick-borne pathogens were fed to ticks by CF, subolesin RNAi did not affect or resulted in lower infection levels in ticks. However, subolesin expression was upregulated in <it>D. variabilis </it>exposed to <it>Escherichia coli</it>, suggesting that although this pathogen may induce subolesin expression in ticks, silencing of this molecule reduced bacterial multiplication by a presently unknown mechanism.</p> <p>Conclusions</p> <p>Subolesin expression in infected ticks suggested that subolesin may be functionally important for tick innate immunity to pathogens, as has been reported for the akirins. However, subolesin expression and consequently subolesin-mediated innate immunity varied with the pathogen and tick tissue. Subolesin may plays a role in tick innate immunity in the salivary glands by limiting pathogen infection levels, but activates innate immunity only for some pathogen in the guts and other tissues. In addition, these results provided additional support for the role of subolesin in other molecular pathways including those required for tissue development and function and for pathogen infection and multiplication in ticks. Consequently, RNAi experiments demonstrated that subolesin knockdown in ticks may affect pathogen infection directly by reducing tick innate immunity that results in higher infection levels and indirectly by affecting tissue structure and function and the expression of genes that interfere with pathogen infection and multiplication. The impact of the direct or indirect effects of subolesin knockdown on pathogen infection may depend on several factors including specific tick-pathogen molecular interactions, pathogen life cycle in the tick and unknown mechanisms affected by subolesin function in the control of global gene expression in ticks.</p

Effect of Acetaldehyde on CRF release form incubated hypothalamic explants

In the past, Acetaldehyde (ACD), the main metabolite of ethanol (ETOH), was mainly studied for its toxic and adverse effects (1). However, recently, ACD was reported to determine behavioural and neurochemical effects, following ETOH assumption in rodents (2, 3). Indeed, ACD enhances dopamine levels in nucleus accumbens, stimulates beta-endorphin release from hypothalamic cells, mediating alcohol reinforcing effects (4). Since little is known about the effects of ACD on other central neuropeptides, in this research we aimed to investigate ACD influence on hypothalamic corticotropin-releasing factor (CRF) release. In order to ascertain this hypothesis, different doses of ACD (1, 10, 3x10 microM) have been administered in incubated hypothalamic explants and CRF concentration have been detected by a radioimmunoassay. Our results show that, following 20 min of incubation at 37°C, ACD is able to significantly increase hypothalamic CRF release in a dose-dependent manner (p<0.05). This effect could be exerted by ACD itself or by other mediators; anyway this could explain the well-known stimulating effect induced by EtOH on ACTH release (5). Further experiments are needed to clarify the mechanisms underlying ACD effect on central neurotransmission. 1. Glud. E. Q J Stud Alcohol. 1949 Sep; 10(2):185-97. 2. Webb et all. Alcohol Clin Exp Res. 2002 May; 26(5):695-704 3. Zimatkin et all Alcohol Clin Exp Res. 2001 Jul; 25(7):982-8. 4. Rodd-Henrichs et al Pharmacol Biochem Behav 72 (1-2):55 64 5. Lee S. et all Endocrinology 145 (10): 4470- 4479

Analysis of Ki-Ras mutations in stage I rectal carcinomas and respective regional lymph nodes.

In this work we show that the percentage of Ki-RAS mutations in codons 12 and 13 in rectal cancer are sensibly lower than in colon cancer, providing further evidence that these two kinds of tumors should be considered two different entities. Moreover, we show that the detection in regional lymph nodes of the same mutation of primary tumor might represent an indicator of lymph nodes metastasis in rectal carcinoma not detected in routine histologic examination