Epigenetic mechanisms in pubertal brain maturation.

Puberty is a critical period of development during which the reemergence of gonadotropin-releasing hormone secretion from the hypothalamus triggers a cascade of hormone-dependent processes. Maturation of specific brain regions including the prefrontal cortex occurs during this window, but the complex mechanisms underlying these dynamic changes are not well understood. Particularly, the potential involvement of epigenetics in this programming has been under-examined. The epigenome is known to guide earlier stages of development, and it is similarly poised to regulate vital pubertal-driven brain maturation. Further, as epigenetic machinery is highly environmentally responsive, its involvement may also lend this period of growth to greater vulnerability to external insults, resulting in reprogramming and increased disease risk. Importantly, neuropsychiatric diseases commonly present in individuals during or immediately following puberty, and environmental perturbations including stress may precipitate disease onset by disrupting the normal trajectory of pubertal brain development via epigenetic mechanisms. In this review, we discuss epigenetic processes involved in pubertal brain maturation, the potential points of derailment, and the importance of future studies for understanding this dynamic developmental window and gaining a better understanding of neuropsychiatric disease risk.

A diagnostic study of Echinococcus multilocularis in red foxes (Vulpes vulpes) from Great Britain.

Alveolar echinococcosis is caused by a parasitic tapeworm Echinococcus multilocularis and is a serious disease with high fatality in humans. The definitive primary host is the red fox (Vulpes vulpes) but domestic animals (dogs and to a lesser extent cats) as well as several genera of rodents can also be infected with the parasite. There is, to date, no evidence of indigenous cases of E. multilocularis in Great Britain (GB) but in most of continental Europe the parasite is considered to be endemic and/or slowly spreading. All pet dogs entering the United Kingdom (UK) under the pet travel scheme (PETS) are therefore currently treated with an anthelmintic effective against Echinococcus spp. Surveillance of red foxes is required to demonstrate disease freedom and maintain this regulation to prevent further geographical spread of the parasite to free areas within the EU. A study of 588 wild red foxes collected from across Great Britain (GB) between October 1999 and November 2000 found no Echinococcus spp. This report describes a further study of GB foxes collected predominately during 2005 and 2006. Fox faecal samples (n=384) were examined for both E. multilocularis and Echinococcus granulosus using an egg isolation procedure followed by PCR method, based on published primer sets. A non-specific primer set that amplifies Taenia spp. as well as Mesocestoides, Dipylidium and Diphyllobothrium was also included in the assay to validate the test procedure as these parasites are expected to be more common in wild fox populations. All faecal samples tested negative for both E. multilocularis and E. granulosus but results for approximately 35% of the samples indicated the presence of Taenia spp. or other closely related cestodes. This data contributes to the evidence that suggests that E. multilocularis is not present in mainland Britain and justifies the requirement for ongoing surveillance to demonstrate disease freedom.

Report of Trichinella spiralis in a red fox (Vulpes vulpes) in Northern Ireland.

No systematic studies of the occurrence of Trichinella in wildlife have been carried out in Northern Ireland (NI) in recent years, and the last reports of trichinellosis in livestock and human outbreaks in NI date back to 1979 and 1945, respectively. In this study, covering the period 2003/2004 and 2007/2008, a total of 443 red foxes (Vulpes vulpes) were collected throughout the country and screened for trichinellosis using a modified muscle digest method. One examined animal was found to be infected with larvae from Trichinella spiralis, indicating a national prevalence in NI of Trichinella in foxes of 0.2%. This prevalence compares well to the findings reported from the bordering Republic of Ireland [Rafter, P., Marucci, G., Brangan, P., Pozio, E., 2005. Rediscovery of Trichinella spiralis in red foxes (Vulpes vulpes) in Ireland after 30 years of oblivion. J. Infect. 50, 61-65] and could be a further indication for a sylvatic Trichinella life cycle existing independently from the domestic cycle.

Detection and surveillance for animal trichinellosis in GB.

The zoonotic disease trichinellosis is considered one of the re-emerging diseases with surveillance and control methods constantly gaining more importance worldwide. Recent change in European Union (EU) legislation introduces Trichinella-free production, and the possibility of risk-based monitoring for Trichinella in pigs. This has increased the role of wildlife surveillance programmes and their impact on protecting human health as well as highlighted the need for harmonised surveillance protocols and test methods for these infections. A modified digest method, based on the EU reference method for Trichinella testing of pig meat, was used to screen foxes present in Great Britain (England, Scotland and Wales) for trichinellosis. The method was validated using batched pools of 10 g foreleg muscle from up to 20 foxes (maximum amount 200 g). The method gave an average trichinae recovery rate of 71% for spiked samples. Assuming this recovery rate applies to all contaminated samples, then the test sensitivity would be 70% for all tissue samples with 0.1 trichinae per 10 g of foreleg muscle, 99.9% for samples with 1 trichinae per 10 g, and 100% for samples with 2 or more trichinae per 10 g. In two separate studies, conducted between 1999 to 2001 (Smith et al., 2003) and 2003 to 2007, over 3500 wild foxes have been screened for Trichinella with negative results. In the second study reported here, foxes were collected from locations throughout Great Britain using a stratified sampling method based on fox population densities. All work was conducted in compliance with appropriate quality assurance systems, latterly under ISO 9001. Results to date indicate the national prevalence of trichinellosis in foxes is <0.001 based on a 10 g individual sample size, an infection level of 1 larva per gram (l pg) and 95% confidence interval. This, together with no reports of trichinellosis in domesticated pigs, suggests that Britain can be considered a region of negligible risk of trichinellosis.

Type I phosphatidylinositol 4-phosphate 5-kinase directly interacts with ADP-ribosylation factor 1 and is responsible for phosphatidylinositol 4,5-bisphosphate synthesis in the golgi compartment.

Phosphatidylinositol (PtdIns) 4,5-bisphosphate is involved in many aspects of membrane traffic, but the regulation of its synthesis is only partially understood. Golgi membranes contain PI 4-kinase activity and a pool of phosphatidylinositol phosphate (PIP), which is further increased by ADP-ribosylation factor 1 (ARF1). COS7 cells were transfected with alpha and beta forms of PI 4-kinase, and only membranes from COS7 cells transfected with PI 4-kinase beta increased their content of PIP when incubated with ARF1. PtdIns(4, 5)P(2) content in Golgi membranes was nonexistent but could be increased to a small extent upon adding either cytosol or Type I or Type II PIP kinases. However, when ARF1 was present, PtdIns(4,5)P(2) levels increased dramatically when membranes were incubated in the presence of cytosol or Type I, but not Type II, PIP kinase. To examine whether ARF1 could directly activate Type I PIP 5-kinase, we used an in vitro assay consisting of phosphatidycholine-containing liposomes, ARF1, and PIP 5-kinase. ARF1 increased Type I PIP 5-kinase activity in a guanine nucleotide-dependent manner, identifying this enzyme as a direct effector for ARF1.

Cell damage-induced conformational changes of the pro-apoptotic protein Bak in vivo precede the onset of apoptosis.

Investigation of events committing cells to death revealed that a concealed NH2-terminal epitope of the pro-apoptotic protein Bak became exposed in vivo before apoptosis. This occurred after treatment of human Jurkat or CEM-C7A T-lymphoma cells with the mechanistically disparate agents staurosporine, etoposide or dexamethasone. The rapid, up to 10-fold increase in Bak-associated immunofluorescence was measured with epitope-specific monoclonal antibodies using flow cytometry and microscopy. In contrast, using a polyclonal antibody to Bak, immunofluorescence was detected both before and after treatment. There were no differences in Bak protein content nor in subcellular location before or after treatment. Immunofluorescence showed Bcl-xL and Bak were largely associated with mitochondria and in untreated cells they coimmunoprecipitated in the presence of nonioinic detergent. This association was significantly decreased after cell perturbation suggesting that Bcl-xL dissociation from Bak occurred on exposure of Bak's NH2 terminus. Multiple forms of Bak protein were observed by two dimensional electrophoresis but these were unchanged by inducers of apoptosis. This indicated that integration of cellular damage signals did not take place directly on the Bak protein. Release of proteins, including Bcl-xL, from Bak is suggested to be an important event in commitment to death.

ADP-ribosylation-factor-regulated phospholipase D activity localizes to secretory vesicles and mobilizes to the plasma membrane following N-formylmethionyl-leucyl-phenylalanine stimulation of human neutrophils.

Phospholipase D (PLD) is responsible for the hydrolysis of phosphatidylcholine to produce phosphatidic acid and choline. Human neutrophils contain PLD activity which is regulated by the small GTPases, ADP-ribosylation factor (ARF) and Rho proteins. In this study we have examined the subcellular localization of the ARF-regulated PLD activity in non-activated neutrophils and cells 'primed' with N-formylmethionyl-leucyl-phenylalanine (fMetLeuPhe). We report that PLD activity is localized at the secretory vesicles in control cells and is mobilized to the plasma membrane upon stimulation with fMetLeuPhe. We conclude that the ARF-regulated PLD activity is translocated to the plasma membrane by secretory vesicles upon stimulation of neutrophils with fMetLeuPhe in inflammatory/priming doses. We propose that this relocalization of PLD is important for the subsequent events occurring during neutrophil activation.

ADP-ribosylation factor 1-regulated phospholipase D activity is localized at the plasma membrane and intracellular organelles in HL60 cells.

ADP-ribosylation factor (ARF), a small GTPase required for vesicle formation, has been identified as an activator of phospholipase D (PLD), thus implying that PLD is localized at intracellular organelles. HL60 cells were prelabelled with [14C]acetate for 72 h and, after disruption, fractionated on a linear sucrose gradient. ARF1-regulated PLD activity in each fraction was assessed by measurement of phosphatidylethanol production. Two peaks of activity were identified, coincident with markers for Golgi/endoplasmic reticulum/granules (endomembranes) and plasma membrane respectively. Analysis of the fractions using exogenous phosphatidylcholine as substrate confirmed the presence of ARF1-dependent PLD activity in endomembranes and plasma membrane, and also identified an additional activity in the cytosol. In formyl-Met-Leu-Phe-stimulated cells, PLD activity as assessed by phosphatidylethanol formation was also associated with both the plasma membrane and endomembranes. Since ARF1-regulated PLD activity requires phosphatidylinositol 4,5-bisphosphate (PIP2), the distributions of inositol lipids and the kinases responsible for lipid phosphorylation were examined. PIP2 was highly enriched at the plasma membrane, whereas phosphatidylinositol (PI) and phosphatidylinositol 4-phosphate (PI4P), the precursors for PIP2 synthesis, were found predominantly at endomembranes. The distribution of PI 4-kinase and PI4P 5-kinase activities confirmed the plasma membrane as the major site of PIP2 production. However, endomembranes possessed substantial PI 4-kinase activity and some PI4P 5-kinase activity, illustrating the potential for PIP2 synthesis. It is concluded that:(1) ARF1-regulated PLD activity is localized at endomembranes and the plasma membrane, (2) PIP2 is available at both membrane compartments to function as a cofactor for ARF-regulated PLD, and (3) in intact cells, formyl-Met-Leu-Phe stimulates PLD activity at endomembranes as well as plasma membrane.

Didecanoyl phosphatidylcholine is a superior substrate for assaying mammalian phospholipase D.

Phospholipase D (PLD) activity in crude or solubilized membranes from mammalian tissues is difficult to detect with the current assay techniques, unless a high radioactive concentration of substrate and/or long incubation times are employed. Generally, the enzyme has to be extracted and partially purified on one column before easy detection of activity. Furthermore, PLD activity in cultured cells can only be detected by the available assay techniques in the presence of guanosine 5'-[gamma-thio]-triphosphate (GTP[S]) and a cytosolic factor [usually ADP-ribosylation factor (Arf)]. In this paper we report that the use of didecanoyl phosphatidylcholine (C10-PC) in mammalian PLD assays considerably increases the detection limit. C10-PC was compared with the commonly used dipalmitoyl phosphatidylcholine (C16-PC) as a substrate for PLD activity from membranes of human neutrophils, human placenta and pig brain, and from placental cytosol. C10-PC was superior to C16-PC by a factor of 2-28 depending on assay conditions and tissue, and it allowed the detection of GTP[S]-and Arf-stimulated PLD activity without addition of phosphatidylinositol 4,5-bisphosphate.

Rust-red grain beetle,Cryptolestes ferrugineus, and flat grain beetle,Cryptolestes pusillus: Antennal and behavioral responses to synthetic components of their aggregation pheromones.

Antennal and behavioral responses of the rust-red grain beetle,Cryptolestes ferrugineus, and the flat grain beetle,C. pusillus, to synthetic samples of the macrocyclic lactones reported to comprise their aggregation pheromones were investigated. Electroantennogram (EAG) recordings were obtained successfully from both species for the first time. Females of both species showed larger EAGs than males. The EAGs ofC. ferrugineus showed a high degree of specificity for conspecific aggregation pheromone components;C. pusillus showed much less specificity. Behavioral tests were conducted using two-choice pitfall bioassays. Separation of the results into the two effects of activity stimulation and direction finding showed that both effects contributed to the overall response, although sometimes to different extents. The strain ofC. pusillus studied responded equally well to both components of its pheromone, whereas it had been reported previously that only one was active, the other acting as a Synergist and eliciting no response when tested alone. With both species, behavioral response was elicited with a single lactone, suggesting that it might not be necessary to use both components for field use. Particularly surprising was thatC. pusillus showed a greater response to the pheromone components ofC. ferrugineus than to its own. Aeration of the two species and thermal desorption of the collected volatiles confirmed production of the expected lactones, and aeration of authentic lactones showed that the response was not due to the C.ferrugineus pheromone components being markedly more volatile. This response, which seems to be an actual preference, is the first to be discovered among the cucujid beetles and encourages optimism that a practical lure for various species may not need to be as complex as originally feared.

Antigens in whooping cough vaccine and antibody levels induced by vaccination of children.

The content of 3 antigens--filamentous haemagglutinin, lymphocytosis-promoting factor, and serotype-specific agglutinogens (fimbriae)--was determined in the current UK whole-cell whooping cough vaccine. Antibodies to these antigens and to outer membrane proteins and lipopolysaccharide of Bordetella pertussis were measured in the serum of unvaccinated children and children who had received 1, 2, or 3 doses of the vaccine. Children who had received one dose of vaccine had varied low antibody titres. Children who had received two or three doses had significantly higher antibody titres to all the antigens tested, as did some unvaccinated children with no history of whooping cough. This study shows that filamentous haemagglutinin, lymphocytosis promoting factor, and outer membrane proteins are immunogenic constituents of the whole-cell vaccine. Their inclusion in a subcellular vaccine would not involve novel antigens.

Studies on protective immunity to aerosol challenge with Legionella pneumophila.

Guinea pigs exposed to 1, 2 or 3 sub-lethal aerosol infections with L. pneumophila developed ELISA serum antibodies after each infection, but were not protected against a lethal aerosol challenge. They died earlier than untreated control animals, though with the same acute exudative bronchopneumonia. Lung bacterial counts were lower in the immunized animals. The extent of pulmonary lesions increased with each successive sublethal infection and lymphoid cell infiltration was prominent after the second. Animals immunized with serotype-specific antigen developed serum antibodies, but were also not protected against lethal aerosol challenge and died earlier than controls.

Rabbit nasopharyngeal colonization by Bordetella pertussis: the effects of immunization on clearance and on serum and nasal antibody levels.

Two Bordetella pertussis antigen preparations, outer membrane protein (OMP) and filamentous haemagglutinin (FHA), and a standard vaccine were used to immunize rabbits, and the effects on nasopharyngeal colonization by the organism were determined. Antibodies were measured in serum and in nasal washes by ELISA before and after challenge of the rabbits with 10(6) bacteria of strain M2. Recoveries of B. pertussis in nasal washes were used to assess colonization, which in controls persisted for at least 65 days. Some rabbits of all the immunized groups showed enhanced clearance, but there was no correlation between the elimination of B. pertussis and serum antibodies to OMP, FHA, lipopolysaccharide, lymphocytosis-promoting factor or agglutinogen 3. In contrast, nasal IgA antibody to FHA showed significant inverse correlation with bacterial persistence. Such antibody was induced by the OMP preparation as well as by FHA, but to different extents depending on the immunization schedule and adjuvant used.