Amygdala protein kinase C epsilon regulates corticotropin-releasing factor and anxiety-like behavior

Corticotropin-releasing factor (CRF), its receptors, and signaling pathways that regulate CRF expression and responses are areas of intense investigation for new drugs to treat affective disorders. Here, we report that protein kinase C epsilon (PKCɛ) null mutant mice, which show reduced anxiety-like behavior, have reduced levels of CRF messenger RNA and peptide in the amygdala. In primary amygdala neurons, a selective PKCɛ activator, ψɛRACK, increased levels of pro-CRF, whereas reducing PKCɛ levels through RNA interference blocked phorbol ester-stimulated increases in CRF. Local knockdown of amygdala PKCɛ by RNA interference reduced anxiety-like behavior in wild-type mice. Furthermore, local infusion of CRF into the amygdala of PKCɛ^−/− mice increased their anxiety-like behavior. These results are consistent with a novel mechanism of PKCɛ control over anxiety-like behavior through regulation of CRF in the amygdala.     

Conversion of bovine pancreatic DNase I to a repair endonuclease with a high selectivity for abasic sites.

Bovine pancreatic deoxyribonuclease I (DNase I) is a nuclease of relatively low specificity which interacts with DNA in the minor groove. No contacts are made between the protein and the major groove of the nucleic acid. DNase I is structurally homologous to exonuclease III, a DNA-repair enzyme with multiple activities. One of the main differences between the two enzymes is the presence of an additional alpha-helix in exonuclease III, in a position suggestive of interaction with the major groove of DNA. Recombinant DNA techniques have been used to add 14 amino acids, comprising the 10 amino acids of the exonuclease III alpha-helix flanked by a glycine rich region, to DNase I. The polypeptide has been inserted after serine 174, an amino acid on the surface of DNase I corresponding to the location of the extra alpha-helix in exonuclease III. The recombinant protein, DNase-exohelix, has been purified and its catalytic activities towards DNA investigated. The recombinant protein demonstrated a high selectivity for endonucleolytic cleavage at abasic sites in DNA, a property of exonuclease III but not native DNase I. Thus the insertion of 14 amino acids at Ser174, converts DNase I to an exonuclease III-like enzyme with DNA-repair properties.     

DNA methylation changes in murine breast adenocarcinomas allow the identification of candidate genes for human breast carcinogenesis

Epigenetic inactivation due to aberrant promoter methylation is a key process in breast tumorigenesis. Murine models for human breast cancer have been established for nearly every important human oncogene or tumor suppressor gene. Mouse-to-human comparative gene expression and cytogenetic profiling have been widely investigated for these models; however, little is known about the conservation of epigenetic alterations during tumorigenesis. To determine if this key process in human breast tumorigenesis is also mirrored in a murine breast cancer model, we mapped cytosine methylation changes in primary adenocarcinomas and paired lung metastases derived from the polyomavirus middle T antigen mouse model. Global changes in methylcytosine levels were observed in all tumors when compared to the normal mammary gland. Aberrant methylation and associated gene silencing was observed for Hoxa7, a gene that is differentially methylated in human breast tumors, and Gata2, a novel candidate gene. Analysis of HOXA7 and GATA2 expression in a bank of human primary tumors confirms that the expression of these genes is also reduced in human breast cancer. In addition, HOXA7 hypermethylation is observed in breast cancer tissues when compared to adjacent tumor-free tissue. Based on these studies, we present a model in which comparative epigenetic techniques can be used to identify novel candidate genes important for human breast tumorigenesis, in both primary and metastatic tumors.     

In vitro characterization of a gamma-secretase radiotracer in mammalian brain

Inhibition of gamma-secretase is a potential therapeutic target for Alzheimer's disease (AD). The present studies have characterized the in vitro properties of a radiolabeled small molecule gamma-secretase inhibitor, [3H]compound D (Yan et al., 2004, J. Neurosci.24, 2942-2952) in mammalian brain. [3H]Compound D was shown to bind with nanomolar affinity (Kd = 0.32-1.5 nM) to a single population of saturable sites in rat, rhesus and human brain cortex homogenates, the density of binding sites ranging from 4 to 7 nM across the species. Competition studies with a structurally diverse group of gamma-secretase inhibitors with a wide range of binding affinities showed that the binding affinities of these compounds correlated well with their ability to inhibit gamma-secretase in vitro. Autoradiographic studies showed that the specific binding of [3H]compound D was widely distributed throughout adult rat, rhesus and normal human brain. There did not appear to be any difference in distribution of [3H]compound D specific binding sites in AD cortex compared with control human cortex as measured using tissue section autoradiography, nor any correlation between gamma-secretase binding and plaque burden as measured immunohistochemically. [3H]compound D is a useful tool to probe the expression and pharmacology of gamma-secretase in mammalian brain.     

Optimising Seagrass Conservation for Ecological Functions

Ecosystems - Animals are central to numerous ecological processes that shape the structure and function of ecosystems. It follows that species that are strongly linked to specific functions can...     

RIC-3 Exclusively Enhances the Surface Expression of Human Homomeric 5-Hydroxytryptamine Type 3A (5-HT3A) Receptors Despite Direct Interactions with 5-HT3A, -C, -D, and -E Subunits

Although five 5-hydroxytryptamine type 3 (5-HT3) subunits (A–E) have been cloned, knowledge on the regulation of their assembly is limited. RIC-3 has been identified as a chaperone specific for the pentameric ligand-gated nicotinic acetylcholine and 5-HT₃ receptors. Therefore, we examined the impact of RIC-3 on differently composed 5-HT₃ receptors with the focus on 5-HT3C, -D, and -E subunits. The influence of RIC-3 on these receptor subtypes is supported by the presence of RIC3 mRNA in tissues expressing at least one of the subunits 5-HT3C, -D, and -E. Furthermore, immunocytochemical studies on transfected mammalian cells revealed co-localization in the endoplasmic reticulum and direct interaction of RIC-3 with 5-HT3A, -C, -D, and -E. Functional and pharmacological characterization was performed using HEK293 cells expressing 5-HT3A or 5-HT3A + 5-HT3B (or -C, -D, or -E) in the presence or absence of RIC-3. Ca²⁺ influx analyses revealed that RIC-3 does not influence the 5-HT concentration-response relationship on 5-HT₃A receptors but leads to differential increases of 5-HT-induced maximum response (E_max) on cells expressing different subunits. Increases of E_max were due to analogously enhanced B_max values for binding of the 5-HT₃ receptor antagonist [³H]GR65630. The observed enhanced cell surface expression of the tested 5-HT3 subunit combinations correlated with the increased surface expression of 5-HT3A as determined by flow cytometry. In conclusion, we showed that RIC-3 can interact with 5-HT3A, -C, -D, and -E subunits and predominantly enhances the surface expression of homomeric 5-HT₃A receptors in HEK293 cells. These data implicate a possible role of RIC-3 in determining 5-HT₃ receptor composition in vivo.     

Deep Brain Stimulation Imposes Complex Informational Lesions

Deep brain stimulation (DBS) therapy has become an essential tool for treating a range of brain disorders. In the resting state, DBS is known to regularize spike activity in and downstream of the stimulated brain target, which in turn has been hypothesized to create informational lesions. Here, we specifically test this hypothesis using repetitive joint articulations in two non-human Primates while recording single-unit activity in the sensorimotor globus pallidus and motor thalamus before, during, and after DBS in the globus pallidus (GP) GP-DBS resulted in: (1) stimulus-entrained firing patterns in globus pallidus, (2) a monophasic stimulus-entrained firing pattern in motor thalamus, and (3) a complete or partial loss of responsiveness to joint position, velocity, or acceleration in globus pallidus (75%, 12/16 cells) and in the pallidal receiving area of motor thalamus (ventralis lateralis pars oralis, VLo) (38%, 21/55 cells). Despite loss of kinematic tuning, cells in the globus pallidus (63%, 10/16 cells) and VLo (84%, 46/55 cells) still responded to one or more aspects of joint movement during GP-DBS. Further, modulated kinematic tuning did not always necessitate modulation in firing patterns (2/12 cells in globus pallidus; 13/23 cells in VLo), and regularized firing patterns did not always correspond to altered responses to joint articulation (3/4 cells in globus pallidus, 11/33 cells in VLo). In this context, DBS therapy appears to function as an amalgam of network modulating and network lesioning therapies.     

An overview of malaria transmission from the perspective of Amazon Anopheles vectors

In the Americas, areas with a high risk of malaria transmission are mainly located in the Amazon Forest, which extends across nine countries. One keystone step to understanding the Plasmodium life cycle in Anopheles species from the Amazon Region is to obtain experimentally infected mosquito vectors. Several attempts to colonise Ano- pheles species have been conducted, but with only short-lived success or no success at all. In this review, we review the literature on malaria transmission from the perspective of its Amazon vectors. Currently, it is possible to develop experimental Plasmodium vivax infection of the colonised and field-captured vectors in laboratories located close to Amazonian endemic areas. We are also reviewing studies related to the immune response to P. vivax infection of Anopheles aquasalis, a coastal mosquito species. Finally, we discuss the importance of the modulation of Plasmodium infection by the vector microbiota and also consider the anopheline genomes. The establishment of experimental mosquito infections with Plasmodium falciparum, Plasmodium yoelii and Plasmodium berghei parasites that could provide interesting models for studying malaria in the Amazonian scenario is important. Understanding the molecular mechanisms involved in the development of the parasites in New World vectors is crucial in order to better determine the interaction process and vectorial competence.