Torsades de pointes (TdP) ventricular tachycardia typically occurs in the setting of early afterdepolarizations; it contributes to arrhythmias and sudden death in congenital and acquired heart disease. Window L-type Ca2+ current (ICaL) has a central role in the arrhythmogenesis and may be particularly important under beta-adrenergic stimulation. We studied the properties of ICaL in myocytes from the dog with chronic atrioventricular block (cAVB) that has cardiac hypertrophy and an increased susceptibility to TdP. Peak ICaL densities at baseline (K+ - and Na+ -free solutions, 10 mmol l(-1) [EGTA]pip) in cAVB were comparable to control, but inactivation was shifted to the right, resulting in a larger window current area in cAVB. Under beta-adrenergic stimulation, the window current area was increased and shifted to the left, but less so in cAVB (maximum at -27 mV, versus -32 mV in control). ICaL during a step to -35 mV showed a transient reduction immediately after the peak. Test steps to 0 mV, simultaneous recording of [Ca2+]i and manipulation of sarcoplasmic reticulum (SR) Ca2+ release showed that this resulted from inhibition and fast recovery of ICaL with SR Ca2+ release. The extent of this dynamic modulation was larger in cAVB than in control (23 +/- 2% of the initially available current, versus 13 +/- 3%; P<0.05). Early afterdepolarizations (EADs) in cAVB myocytes under beta-adrenergic stimulation typically occurred in the window current voltage range and after decline of [Ca2+]i. In conclusion, in cAVB, the larger window current, its rightward shift and enhanced dynamic modulation by SR Ca2+ release may contribute to an increased incidence of EADs in cAVB under beta-adrenergic stimulation.

Perforated whole-cell configuration of patch clamp was used to determine the contribution of the electrogenic Na+/HCO3- cotransport (NBC) on the shape of the action potential in cat ventricular myocytes. Switching from Hepes to HCO3- buffer at constant extracellular pH (pH(o)) hyperpolarized resting membrane potential (RMP) by 2.67 +/- 0.42 mV (n = 9, P < 0.05). The duration of action potential measured at 50% of repolarization time (APD50) was 35.8 +/- 6.8% shorter in the presence of HCO3- than in its absence (n = 9, P < 0.05). The anion blocker SITS prevented and reversed the HCO3- -induced hyperpolarization and shortening of APD. In addition, no HCO3- -induced hyperpolarization and APD shortening was observed in the absence of extracellular Na+. Quasi-steady-state currents were evoked by 8 s duration voltage-clamped ramps ranging from -130 to +30 mV. A novel component of SITS-sensitive current was observed in the presence of HCO3-. The HCO3- -sensitive current reversed at -87 +/- 5 mV (n = 7), a value close to the expected reversal potential of an electrogenic Na+/HCO3- cotransport with a HCO3-:Na+ stoichiometry ratio of 2: 1. The above results allow us to conclude that the cardiac electrogenic Na+/HCO3- cotransport has a relevant influence on RMP and APD of cat ventricular cells.

Severe acute respiratory syndrome (SARS) is a highly contagious and lethal disease caused by a new type of coronavirus, SARS-associated coronavirus (SARS-CoV). Currently, there is no efficient treatment and prevention for this disease. We constructed recombinant adenoviral vectors that can express shRNAs, which inhibited the expression of SARS-CoV genes effectively in mammalian cells.

Since antibodies currently constitute the most rapidly growing class of human therapeutics, the high-yield production of recombinant antibodies and antibody fragments is a real challenge. Using as model a monoclonal antibody directed against the human prion protein that we prepared previously and tested for its therapeutic value, we describe here experimental conditions allowing the production of large quantities (up to 35 mg/l of bacterial culture) of correctly refolded and totally functional single chain fragment variable (scFv). These quantities were sufficient to characterize the binding properties of this small recombinant fragment through in vitro and ex vivo approaches. Interestingly, this scFv retains full binding capacity for its antigen, i.e. the human prion protein, when compared with the corresponding Fab or whole antibody, and recognizes soluble, solid-phase-adsorbed, and membrane-bound prion protein. This strongly suggests that from the mAb cloning step to the refolding of the recombinant fragment, each stage is well controlled, leading to almost 100% functional scFv. These results are of interest not only in view of possible immunotherapy for prion diseases, but also more generally in emphasizing the great promise of these small recombinant molecules in the context of targeted therapies.

Evolutionarily conserved Dmrt1 encodes a transcriptional regulator that is expressed exclusively in the gonads and is required for testis differentiation. Here we report that four transcripts of the mouse Dmrt1 were generated in developing gonads and adult testis by alternative splicing. Dmrt1 a encodes the known protein with 374 amino acids. Dmrt1 b, Dmrt1 c, and Dmrt1 d encode predicted proteins with 212, 257, and 194 amino acids, respectively. Dmrt1 a2 and Dmrt1 a3 have anterior alternative polyadenylation signals in 3'UTR than the known Dmrt1 transcript (Dmrt1 a1). Dmrt1 a2 lacks 18 nucleic acids in 3'UTR. Interestingly, Dmrt1 b lacks exon5, Dmrt1 c lacks exon3, and Dmrt1 d lacks both exon1 and exon2 which encode DM domain. RT-PCR showed that all of Dmrt1 transcripts were only detectable in adult testis. However, during gonadogenesis, the multiple alternatively spliced transcripts all had gonad-specific and sexually dimorphic expression profiles. Northern blot and real time fluorescent quantitative RT-PCR further indicated that the expression of Dmrt1 a was dominantly higher than those of Dmrt1 b, c, and d, although they showed a similar up and down pattern of expression during embryo development. The expression of Dmrt1 transcripts climbed up to a climax at 13.5dpc in both male and female gonad. Afterwards, expression in male gonad decreased to a low level and maintained, whereas female one reduced rapidly until undetectable in adult ovary. These results provide new insight into roles of regulation at level of splicing of the Dmrt1 in governing sex differentiation.

In previous in vivo voltammetry studies, acute nicotine administration increased striatal dopamine clearance. The current study aimed to determine whether nicotine also increases [(3)H]dopamine uptake across the time course of the previous voltammetry studies and whether dopamine transporter trafficking to the cell surface mediates the nicotine-induced augmentation of dopamine clearance in striatum. Rats were administered nicotine (0.32 mg/kg, s.c.); striatal synaptosomes were obtained 5, 10, 40 or 60 min later. Nicotine increased (25%) the V(max) of [(3)H]dopamine uptake at 10 and 40 min. To determine whether the increase in V(max) was due to an increase in dopamine transporter density, [(3)H]GBR 12935 (1-(2-[bis(4-fluorophenyl)methoxy]ethyl)-4-(3-phenylpropyl)piperazine dihydrochloride) binding was performed using rat striatal membranes; no differences were found between nicotine and saline-control groups at 5, 10 or 40 min post-injection, indicating that nicotine did not increase striatal dopamine transporter density; however, [(3)H]GBR 12935 binding assays determine both cell surface and intracellular dopamine transporter. Changes in cellular dopamine transporter localization in striatum were determined using biotinylation and subfractionation approaches; no differences between nicotine and saline-control groups were observed at 10 and 40 min post-injection. These results suggest that the nicotine-induced increase in dopamine uptake and clearance in striatum may occur via a trafficking-independent mechanism.

Progesterone participates in the regulation of several reproductive functions in birds through its interaction with intracellular progesterone receptors (PR) which exhibit two isoforms (PR-A and PR-B) with different function and regulation. We determined the content of PR isoforms in several brain regions of immature (1.5 months-old), mature (12 months-old) and aged (48 months-old) male and female chickens by Western blot analysis. PR isoforms protein content changed in tissue-and age-specific manner in the brain of both male and female chickens. In female animals PR-B content was higher in the hypothalamus of mature animals than in other ages while PR-A content increased in the cerebellum of aged animals. No significant changes were observed in PR isoforms content in the cortex and the tectum of female animals. In contrast, in male animals PR-B content was higher in the hypothalamus of aged animals. The cerebellum, tectum and cortex of male animals did not present significant changes in PR isoforms content. In all studied regions of female animals PR-B was the predominant isoform, whereas, in males a significant predominance of PR-B was only found in the hypothalamus of aged animals. These results demonstrate that PR isoforms content presents a sexual dimorphism in the brain of chickens that changes in tissue- and age-specific manner, and suggest that these variations contribute to the regulation of progesterone actions in brain throughout the lifespan of male and female chickens.

This paper studied the effect of neurosteroid dehydroepiandrosterone sulfate on spontaneous glutamate release in the prelimbic cortex by using electrophysiological and biochemical methods combined with a pharmacological approach and made some comparisons with those in the hippocampus. The results showed that dehydroepiandrosterone sulfate increased the frequency of miniature excitatory postsynaptic currents in the prelimbic cortex and hippocampus; sigma-1 receptor antagonist partially blocked the effect of dehydroepiandrosterone sulfate in the prelimbic cortex, but completely blocked it in the hippocampus; D1 receptor antagonist, adenylyl cyclase inhibitor and protein kinase A inhibitor completely blocked the effect of dehydroepiandrosterone sulfate in the prelimbic cortex; dehydroepiandrosterone sulfate increased the activity of protein kinase A in the prelimbic cortex and hippocampus; the effect of dehydroepiandrosterone sulfate on protein kinase A was completely blocked by sigma-1 receptor antagonist in the hippocampus, but was partially blocked in the prelimbic cortex; interestingly, here again, the effect of dehydroepiandrosterone sulfate on protein kinase A was completely blocked by D1 receptor antagonist in the prelimbic cortex. These results suggest that dehydroepiandrosterone sulfate promotes presynaptic glutamate release in the prelimbic cortex via activation of D1 and sigma-1 receptors.

We evaluated the effects of different levels of the potent environmental toxicant and teratogen, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), on molar development in mice in six inbred strains, all with TCDD responsive Ahr alleles.

TomatEST is a secondary database integrating expressed sequence tag (EST)/cDNA sequence information from different libraries of multiple tomato species. Redundant EST collections from each species are organized into clusters (gene indices). A cluster consists of one or multiple contigs. Multiple contigs in a cluster represent alternatively transcribed forms of a gene. The set of stand-alone EST sequences (singletons) and contigs, representing all the computationally defined 'Transcript Indices', are annotated according to similarity versus protein and RNA family databases. Sequence function description is integrated with the Gene Ontologies and the Enzyme Commission identifiers for a standard classification of gene products and for the mapping of the expressed sequences onto metabolic pathways. Information on the origin of the ESTs, on their structural features, on clusters and contigs, as well as on functional annotations are accessible via a user-friendly web interface. Specific facilities in the database allow Transcript Indices from a query be automatically classified in Enzyme classes and in metabolic pathways. The 'on the fly' mapping onto the metabolic maps is integrated in the analytical tools. The TomatEST database website is freely available at http://biosrv.cab.unina.it/tomatestdb.

Diesel exhaust particle (DEP) has been identified as a class 2A human carcinogen and closely related to the increased incidence of respiratory allergy, cardiopulmonary morbidity and mortality, and risk of lung cancer. However, the molecular mechanisms of DEP mutagenicity/carcinogenicity are still largely unknown. In the present study, we focused on the mutagenicity of DEPs in human-hamster hybrid (A(L)) cells and evaluated the role of cell-particle interaction in mediating mutagenic process. We found that DEPs formed micron-sized aggregates in the medium and located mainly in large cytoplasmic vacuoles of cells by 24h treatment. The cellular granularity was increased by DEP treatment in a dose-dependent manner. DEPs resulted in a dose-dependent increase of mutation yield at CD59 locus in A(L) cells, while inflicting minimal cytotoxicity. There was a more than two-fold increase of mutation yield at CD59 locus in A(L) cells exposed to DEPs at a dose of 50mug/ml. Such induction was significantly reduced by concurrent treatment with phagocytosis inhibitors, cytochalasin B and ammonium chloride (p<0.05). These results provided direct evidence that DEPs was mutagenic in mammalian cells and that cell-particle interaction played an essential role in the process.

The carbohydrate response element (ChoRE) is a cis-acting sequence found in the promoters of genes induced transcriptionally by glucose. The ChoRE is composed of two E box-like motifs that are separated by 5 bp and is recognized by two basic helix-loop-helix/leucine zipper (bHLH/LZ) proteins, ChREBP and Mlx, which heterodimerize to bind DNA. In this study, we demonstrate that two ChREBP/Mlx heterodimers interact to stabilize binding to the tandem E box-like motifs in the ChoRE. Based on a model structure that we generated of ChREBP/Mlx bound to the ChoRE, we hypothesized that intermolecular interactions between residues within the Mlx loop regions of adjacent heterodimers are responsible for stabilizing the complex. We tested this hypothesis by preparing Mlx variants in which the loop region was replaced with that of another family member or mutated at several key residues. These Mlx variants retained their ability to bind to a single perfect E-box motif as a heterodimer with ChREBP, but no longer bound to the ChoRE nor supported glucose responsive activity. In summary, our results support a model in which the loop regions of Mlx play an important functional role in mediating the coordinate binding of ChREBP/Mlx heterodimers to the ChoRE.

We describe PReMod, a new database of genome-wide cis-regulatory module (CRM) predictions for both the human and the mouse genomes. The prediction algorithm, described previously in Blanchette et al. (2006) Genome Res., 16, 656-668, exploits the fact that many known CRMs are made of clusters of phylogenetically conserved and repeated transcription factors (TF) binding sites. Contrary to other existing databases, PReMod is not restricted to modules located proximal to genes, but in fact mostly contains distal predicted CRMs (pCRMs). Through its web interface, PReMod allows users to (i) identify pCRMs around a gene of interest; (ii) identify pCRMs that have binding sites for a given TF (or a set of TFs) or (iii) download the entire dataset for local analyses. Queries can also be refined by filtering for specific chromosomal regions, for specific regions relative to genes or for the presence of CpG islands. The output includes information about the binding sites predicted within the selected pCRMs, and a graphical display of their distribution within the pCRMs. It also provides a visual depiction of the chromosomal context of the selected pCRMs in terms of neighboring pCRMs and genes, all of which are linked to the UCSC Genome Browser and the NCBI. PReMod: http://genomequebec.mcgill.ca/PReMod.

The NAR online Molecular Biology Database Collection is a public resource that contains links to the databases described in this issue of Nucleic Acids Research, previous NAR database issues, as well as a selection of other molecular biology databases that are freely available on the web and might be useful to the molecular biologist. The 2007 update includes 968 databases, 110 more than the previous one. Many databases that have been described in earlier issues of NAR come with updated summaries, which reflect recent progress and, in some instances, an expanded scope of these databases. The complete database list and summaries are available online on the Nucleic Acids Research web site http://nar.oxfordjournals.org/.

The human FMR1 gene contains a CGG repeat in its 5' untranslated region. The repeat length in the normal population is polymorphic (5-55 CGG repeats). Lengths beyond 200 CGGs (full mutation) result in the absence of the FMR1 gene product, FMRP, through abnormal methylation and gene silencing. This causes Fragile X syndrome, the most common inherited form of mental retardation. Elderly carriers of the premutation, defined as a repeat length between 55 and 200 CGGs, can develop a progressive neurodegenerative syndrome: Fragile X-associated tremor/ataxia syndrome (FXTAS). In FXTAS, FMR1 mRNA levels are elevated and it has been hypothesised that FXTAS is caused by a pathogenic RNA gain-of-function mechanism. We have developed a knock in mouse model carrying an expanded CGG repeat (98 repeats), which shows repeat instability and displays biochemical, phenotypic and neuropathological characteristics of FXTAS. Here, we report further repeat instability, up to 230 CGGs. An expansion bias was observed, with the largest expansion being 43 CGG units and the largest contraction 80 CGG repeats. In humans, this length would be considered a full mutation and would be expected to result in gene silencing. Mice carrying long repeats ( approximately 230 CGGs) display elevated mRNA levels and decreased FMRP levels, but absence of abnormal methylation, suggesting that modelling the Fragile X full mutation in mice requires additional repeats or other genetic manipulation.

An automated algorithm has been developed to segment stripped (non-brain tissue excluded) T1-weighted MRI brain volumes into left and right cerebral hemispheres and cerebellum+brainstem. The algorithm, which uses the Graph Cuts technique, performs a fully automated segmentation in approximately 30 s following pre-processing. It is robust and accurate and has been tested on datasets from two scanners using different field strengths and pulse sequences. We describe the Graph Cuts algorithm and compare the results of Graph Cuts segmentations against "gold standard" manual segmentations and segmentations produced by three popular software packages used by neuroimagers: BrainVisa, CLASP, and SurfRelax.

The variation resources within the University of California Santa Cruz Genome Browser include polymorphism data drawn from public collections and analyses of these data, along with their display in the context of other genomic annotations. Primary data from dbSNP is included for many organisms, with added information including genomic alleles and orthologous alleles for closely related organisms. Display filtering and coloring is available by variant type, functional class or other annotations. Annotation of potential errors is highlighted and a genomic alignment of the variant's flanking sequence is displayed. HapMap allele frequencies and linkage disequilibrium (LD) are available for each HapMap population, along with non-human primate alleles. The browsing and analysis tools, downloadable data files and links to documentation and other information can be found at http://genome.ucsc.edu/.

Central regulatory pathways promoting stress adaptation utilize various neurotransmitters/neuropeptides, such as urocortin 1 (Ucn1) and neuropeptide Y (NPY). Ucn1 is abundantly expressed in the nonpreganglionic Edinger-Westphal nucleus (npEW), where it is codistributed with NPY-immunoreactive (ir) terminals. A special role for both neuropeptides has been postulated in stress adaptation. Using double-labeling immunohistochemistry, we observed close appositions between NPY-ir terminals and neurons immunoreactive for Ucn1 in the rat, as well as in the human npEW. Therefore, we hypothesized that NPY might control the activity of Ucn1-positive neurons in the npEW. To test this hypothesis, NPY was injected into the lateral cerebral ventricle of rats, resulting in a strong activation of npEW Ucn1 neurons as revealed by Fos immunohistochemistry. Ucn1 mRNA was also upregulated in the npEW 2 hours after the injection of NPY. In a search for the type of NPY receptor that mediates this NPY-induced recruitment of npEW-Ucn1 cells, we found that the great majority of Ucn1 cells exhibited NPY Y5 receptor immunoreactivity, and only a few of the Ucn1 cells coexpressed the Y1 receptor. We concluded that NPY, via NPY Y5 and to a lesser extent via the Y1 receptors, exerts a stimulatory action on Ucn1 cells in the npEW. Further studies are currently in progress to elucidate the significance of this NPY-Ucn1 interaction in the npEW.

The nitric oxide (NO)-cGMP pathway is implicated in modulation of visual information processing in the retina. Despite numerous functional studies of this pathway, information about the retinal distribution of the major downstream effector of NO, soluble guanylyl cyclase (sGC), is very limited. In the present work, we have used immunohistochemistry and multiple labeling to determine the distribution of sGC in rat retina. sGC was present at high levels in inner retina but barely detectable in outer retina. Photoreceptors and horizontal cells, as well as Müller cells, were immunonegative, whereas retinal ganglion cells exhibited moderate staining for sGC. Strong immunostaining was found in subpopulations of bipolar and amacrine cells, but staining was weak in rod bipolar cells, and AII amacrine cells were immunonegative. Double labeling of sGC with neuronal nitric oxide synthase showed that the two proteins are generally located in adjacent puncta in inner plexiform layer, implying paracrine interactions. Our results suggest that the NO-cGMP pathway modulates the neural circuitry in inner retina, preferentially within the cone pathway.

The hypothalamic paraventricular nucleus (PVN) coordinates major neuroendocrine and behavioral mechanisms, particularly responses to homeostatic challenges. Parvocellular and magnocellular PVN neurons are richly innervated by pituitary adenylate cyclase-activating polypeptide (PACAP) axons. Our recent functional observations have also suggested that PACAP may be an excitatory neuropeptide at the level of the PVN. Nevertheless, the exact localization of PACAP-producing neurons that project to the PVN is not understood. The present study examined the specific contribution of various brain areas sending PACAP innervation to the rat PVN by using iontophoretic microinjections of the retrograde neuroanatomical tracer cholera toxin B subunit (CTb). Retrograde transport was evaluated from hypothalamic and brainstem sections by using multiple labeling immunofluorescence for CTb and PACAP. PACAP-containing cell groups were found to be retrogradely labeled from the PVN in the median preoptic nucleus; preoptic and lateral hypothalamic areas; arcuate, dorsomedial, ventromedial, and supramammillary nuclei; ventrolateral midbrain periaqueductal gray; rostral and midlevel ventrolateral medulla, including the C1 catecholamine cell group; nucleus of the solitary tract; and dorsal motor nucleus of vagus. Minor PACAP projections with scattered double-labeled neurons originated from the parabrachial nucleus, pericoeruleus area, and caudal regions of the nucleus of the solitary tract and ventrolateral medulla. These observations indicate a multisite origin of PACAP innervation to the PVN and provide a strong chemical neuroanatomical foundation for interaction between PACAP and its potential target neurons in the PVN, such as parvocellular CRH neurons, controlling physiologic responses to stressful challenges and other neuroendocrine or preautonomic PVN neurons.