Recombinant NhSAG1 ELISA: a sensitive and specific assay for detecting antibodies against Neospora hughesi in equine serum

Neospora hughesi is a recently identified cause of equine protozoal myeloencephalitis. However, the significance of this parasite is poorly understood. An enzyme-linked immunosorbent assay (ELISA) with a recombinant form of the N. hughesi 29-kDa surface antigen (rNhSAG1) was developed for serodiagnosis of equine N. hughesi infections. Parallel ELISA analysis showed that animals immunized or infected with N. hughesi exhibited greater antibody reactivity with rNhSAG1 than with the Neospora caninum homolog, rNcSAG1. The rNhSAG1 ELISA showed 94.4% sensitivity and 95.0% specificity when compared with N. hughesi western blot results for 1,006 samples. The N. hughesi seroprevalence was 3.4% for the 1,917 samples tested by ELISA, which is less than earlier reports. Importantly, western blot analysis of ELISA-positive sera revealed only 18 true seropositive samples for an even lower seroprevalence of 0.9%. These results imply that Neospora spp. infections are uncommon in horses. The sensitivity and specificity exhibited by the rNhSAG1 ELISA suggest that it has a potential use for serodiagnosis of N. hughesi infection in equids. Furthermore, the high-throughput capability of the ELISA will allow for screening large sample sets, which should provide a better understanding of N. hughesi epidemiology.     

In vivo expression of a Cicer arietinum beta-galactosidase in potato tubers leads to a reduction of the galactan side-chains in cell wall pectin

We report the generation of Solanum tuberosum transformants expressing Cicer arietinum betaIII-Gal. betaIII-Gal is a beta-galactosidase able to degrade cell wall pectins during cell wall loosening that occurs prior to cell elongation. cDNA corresponding to the gene encoding this protein was identified among several chickpea beta-galactosidase cDNAs, and named CanBGal-3. CanBGal-3 cDNA was expressed in potato under the control of the granule-bound starch synthase promoter. Three betaIII-Gal transformants with varying levels of expression were chosen for further analysis. The transgenic plants displayed no significant altered phenotype compared to the wild type. However, beta-galactanase and beta-galactosidase activities were increased in the transgenic tuber cell walls and this affected the potato tuber pectins. A reduction in the galactosyl content of up to 50% compared to the wild type was observed in the most extreme transformant, indicating a reduction of 1,4-beta-galactan side-chains, as revealed by analysis with LM5 specific antibodies. Our results confirm the notion that the pectin-degrading activity of chickpea betaIII-Gal reported in vitro also occurs in vivo and in other plants, and confirm the involvement of betaIII-Gal in the cell wall autolysis process. An increase in the homogalacturonan content of transgenic tuber cell walls was also observed by Fourier transform infrared spectroscopy (FTIR) analysis.     

Ensiling characteristics, nutrient composition, and in situ ruminal and whole tract degradability of brown midrib and leafy corn silage

A study was conducted to compare the ensiling characteristics, chemical composition, and the ruminal and total tract nutrient degradabilities of leafy (Cargill F227) and brown midrib (Mycogen TMF94) corn silage hybrids. Corn was grown in Saint-Jean-sur-Richelieu, Quebec, Canada, harvested at a target 350 g kg(-1) dry matter (DM) content, and ensiled in mini-silos for 0, 2, 4, 8, 16, and 45 d. Two non-lactating Holstein cows fitted with ruminal and proximal duodenal cannulae were used to determine ruminal and whole tract nutrient degradability. Forage from both hybrids went through a rapid fermentation with a sharp decline in pH during the first 2 d of ensiling, pH in both silage being less than 4.0 after 45 d. Lactic acid concentration was however greater for leafy than brown midrib corn. Chemical analysis of silage after 45 d of ensiling revealed that hybrids differed in their composition. Compared to leafy corn, brown midrib corn had lower neutral detergent fiber (NDF), acid detergent fiber (ADF), acid detergent lignin (ADL), crude protein (CP), and neutral detergent and acid detergent insoluble proteins, but higher starch and net energy of lactation (NEL) values. Results of the in situ incubation experiment indicated that compared to leafy corn brown midrib corn had greater ruminal DM (64 vs. 54%), CP (73 vs. 71%), and NDF (32 vs. 24%) degradabilities. Brown midrib corn silage also had greater DM ruminal (53 vs. 48%) and total tract (67 vs. 61%) digestibilities, as well as greater NDF ruminal (34 vs. 25%), intestinal (10 vs. 8%), and total tract (43 vs. 33%) digestibilities. Type of corn hybrid will thus greatly affect silage chemical composition and nutrient digestibility.     

Subcellular localisation of VEGF in different pituitary cells. Changes of its expression in oestrogen induced prolactinomas

Summary Vascular endothelial growth factor (VEGF) is an important angiogenic factor in the pituitary gland. The objective of this study was to unveil the VEGF subcellular localisation in different pituitary cell types and to evaluate changes in its expression at different time intervals after oestrogen stimulation. A relevant feature demonstrated was the identification of this cytokine in the nucleus and cytoplasm of lactotrophs, somatotrophs and gonadotrophs, as well as in follicle-stellate cells of male rats. Oestrogen treatment increased the number of VEGF immunopositive cells and its expression detected differentially by western blot in both nucleus and cytoplasm of pituitary cells when compared to the control. At ultrastructural level VEGF appeared associated with nucleolus and euchromatin involving a possible internal autocrine loop. In lactotrophs, the predominant cell of the tumour, VEGF was immunodetected in RER, Golgi complex, and vesicular organelles, supporting further the association with an auto-paracrine effect exerted by VEGF. The nucleus/cytoplasm ratio of VEGF revealed a prevalent accumulation of VEGF in the cytoplasm. The presence of VEGF in the nucleus may probably be associated with a translocation to this cell compartment. This study demonstrated a cytoplasmic and nuclear immunolocalisation of VEGF in normal and tumoural adenohypophyseal cells. In the course of prolactinoma development, the oestrogen stimulated VEGF expression in tumoural cells, promoting a vascular adaptation which contributes to growth and progression of the tumour.     

Enhanced activation of Akt and extracellular-regulated kinase pathways by simultaneous occupancy of Gq-coupled 5-HT2A receptors and Gs-coupled 5-HT7A receptors in PC12 cells

The most commonly prescribed antidepressants, the serotonin (5-HT) selective reuptake inhibitors, increase 5-HT without targeting specific receptors. Yet, little is known about the interaction of multiple receptor subtypes expressed by individual neurons. Specifically, the effect of increases in cAMP induced by Gs-coupled 5-HT receptor subtypes on the signaling pathways modulated by other receptor subtypes has not been studied. We have, therefore, examined the activation of the extracellular-regulated kinase (ERK) and Akt pathways by Gs-coupled 5-HT7A receptors and Gq-coupled 5-HT2A receptors, which are co-expressed in discrete brain regions. Agonists for both receptors were found to activate ERK and Akt in transfected PC12 cells. 5-HT2A receptor-mediated activation of the two pathways was found to be Ca2+-dependent. In contrast, 5-HT7A receptor-mediated activation of Akt required increases in both [cAMP] and intracellular [Ca2+], while activation of ERK was inhibited by Ca2+. The activation of ERK and Akt stimulated by simultaneous treatment of cells with 5-HT2A and 5-HT7A receptor agonists was found to be at least additive. Cell-permeable cAMP analogs mimicked 5-HT7A receptor agonists in enhancing 5-HT2A receptor-mediated activation of ERK and Akt. A role was identified for the cAMP-guanine exchange factor, Epac, in this augmentation of ERK, but not Akt, activation. Our finding of enhanced activation of neuroprotective Akt and ERK pathways by simultaneous occupancy of 5-HT2A and 5-HT7A receptors may also be relevant to the interaction of other neuronally expressed Gq- and Gs-coupled receptors.     

Diversity of functional genes of methanogens, methanotrophs and sulfate reducers in deep-sea hydrothermal environments

To contribute to the identification of methanogens, methanotrophs and sulfate-reducing bacteria (SRB) in microbial communities from the 13 degrees N (East Pacific Rise) and Rainbow (Mid-Atlantic Ridge) hydrothermal vent fields, we investigated the diversity of mcrA, pmoA and dsrAB genes sequences. Clone libraries were obtained using DNA isolated from fragments of diffuse vents, sediment and in situ samplers. The clones were categorized by restriction fragment length polymorphism, and representatives of each group were sequenced. Sequences were related to that of hyperthermophilic (order Methanopyrales and family Methanocaldococcaceae), thermophilic and mesophilic (family Methanococcaceae) methanogens, thermophilic (proposed genus 'Methylothermus') and mesophilic type I methanotrophs, and hyperthermophilic (order Archaeoglobales), thermophilic (order Thermodesulfobacteriales) and mesophilic (family Desulfobulbaceae) SRB. Several of the obtained sequences were distantly related to the genes of cultivated organisms, providing evidence of the existence of novel lineages in the three functional groups. This study provides for the first time an insight into the diversity of several functional genes of deep-sea hydrothermal system microorganisms.     

Functional characterization of an LCCL-lectin domain containing protein family in Plasmodium berghei

Using bioinformatic, proteomic, immunofluorescence, and genetic cross methods, we have functionally characterized a family of putative parasite ligands as potential mediators of cell-cell interactions. We name these proteins the Limulus clotting factor C, Coch-5b2, and Lgl1 (LCCL)-lectin adhesive-like protein (LAP) family. We demonstrate that this family is conserved amongst Plasmodium spp. It possesses a unique arrangement of adhesive protein domains normally associated with extracellular proteins. The proteins are expressed predominantly, though not exclusively, in the mosquito stages of the life cycle. We test the hypothesis that these proteins are surface proteins with 1 member of this gene family, lap1, and provide evidence that it is expressed on the surface of Plasmodium berghei sporozoites. Finally, through genetic crosses of wild-type Pblap1+ and transgenic Pblap1- parasites, we show that the null phenotype previously reported for sporozoite development in a Pblap1- mutant can be rescued within a heterokaryotic oocyst and that infectious Pblap1 sporozoites can be formed. The mutant is not rescued by coparasitization of mosquitoes with a mixture Pblap1+ and Pblap1- homokaryotic oocysts.     

Distribution dynamics of a great bustard metapopulation throughout a decade: influence of conspecific attraction and recruitment

Dispersing individuals can use conspecifics as indicators of habitat quality and aggregate at traditionally occupied sites, leaving other favourable patches unoccupied. Here we test the predictions of the conspecific-based habitat selection hypothesis on a Spanish great bustard (Otis tarda) metapopulation, currently fragmented due to recent human-induced habitat changes. The number of birds had increased by 23% between 1988 and 1998, but not consistently among leks. Leks that were large in 1988 increased, while those that were small decreased, which suggests that dispersing individuals used the numbers of conspecifics as cues for breeding-site selection. Moreover, leks with high productivity increased, while those with low productivity decreased. Finally, lek distribution was markedly stable throughout the decade, with no establishment of new leks, and suitable habitat patches remained unoccupied, as predicted by the conspecific attraction hypothesis. These results were corroborated by a simulation model which incorporated natal dispersal rates between leks as obtained through radio-tracking of 15 birds that survived throughout their 4-year dispersal period. In conclusion, in spite of the apparent increase in total numbers throughout the decade, both conspecific attraction and local differences in reproductive success contributed to a more aggregated distribution, increasing the species' vulnerability to local catastrophes, and the risks of reduced genetic diversity and extinction of small leks.     

Simultaneous EEG-fMRI during a Working Memory Task: Modulations in Low and High Frequency Bands

Background

EEG studies of working memory (WM) have demonstrated load dependent frequency band modulations. FMRI studies have localized load modulated activity to the dorsolateral prefrontal cortex (DLPFC), medial prefrontal cortex (MPFC), and posterior parietal cortex (PPC). Recently, an EEG-fMRI study found that low frequency band (theta and alpha) activity negatively correlated with the BOLD signal during the retention phase of a WM task. However, the coupling of higher (beta and gamma) frequencies with the BOLD signal during WM is unknown.

Methodology

In 16 healthy adult subjects, we first investigated EEG-BOLD signal correlations for theta (5–7 Hz), alpha1 (8–10), alpha2 (10–12 Hz), beta1 (13–20), beta2 (20–30 Hz), and gamma (30–40 Hz) during the retention period of a WM task with set size 2 and 5. Secondly, we investigated whether load sensitive brain regions are characterised by effects that relate frequency bands to BOLD signals effects.

Principal Findings

We found negative theta-BOLD signal correlations in the MPFC, PPC, and cingulate cortex (ACC and PCC). For alpha1 positive correlations with the BOLD signal were found in ACC, MPFC, and PCC; negative correlations were observed in DLPFC, PPC, and inferior frontal gyrus (IFG). Negative alpha2-BOLD signal correlations were observed in parieto-occipital regions. Beta1-BOLD signal correlations were positive in ACC and negative in precentral and superior temporal gyrus. Beta2 and gamma showed only positive correlations with BOLD, e.g., in DLPFC, MPFC (gamma) and IFG (beta2/gamma). The load analysis revealed that theta and—with one exception—beta and gamma demonstrated exclusively positive load effects, while alpha1 showed only negative effects.

Conclusions

We conclude that the directions of EEG-BOLD signal correlations vary across brain regions and EEG frequency bands. In addition, some brain regions show both load sensitive BOLD and frequency band effects. Our data indicate that lower as well as higher frequency brain oscillations are linked to neurovascular processes during WM.     

The role of caveolae and caveolin 1 in calcium handling in pacing and contraction of mouse intestine

In mouse intestine, caveolae and caveolin‐1 (Cav‐1) are present in smooth muscle (responsible for executing contractions) and in interstitial cells of Cajal (ICC; responsible for pacing contractions). We found that a number of calcium handling/dependent molecules are associated with caveolae, including L‐type Ca²⁺ channels, Na⁺‐Ca²⁺ exchanger type 1 (NCX1), plasma membrane Ca²⁺ pumps and neural nitric oxide synthase (nNOS), and that caveolae are close to the peripheral endo‐sarcoplasmic reticulum (ER‐SR). Also we found that this assemblage may account for recycling of calcium from caveolar domains to SR through L‐type Ca ⁺ channels to sustain pacing and contractions. Here we test this hypothesis further comparing pacing and contractions under various conditions in longitudinal muscle of Cav‐1 knockout mice (lacking caveolae) and in their genetic controls. We used a procedure in which pacing frequencies (indicative of functioning of ICC) and contraction amplitudes (indicative of functioning of smooth muscle) were studied in calcium‐free media with 100 mM ethylene glycol tetra‐acetic acid (EGTA). The absence of caveolae in ICC inhibited the ability of ICC to maintain frequencies of contraction in the calcium‐free medium by reducing recycling of calcium from caveolar plasma membrane to SR when the calcium stores were initially full. This recycling to ICC involved primarily L‐type Ca²⁺ channels; i.e. pacing frequencies were enhanced by opening and inhibited by closing these channels. However, when these stores were depleted by block of the sarco/endoplasmic reticulum Ca²⁺‐ATPase (SERCA) pump or calcium release was activated by carbachol, the absence of Cav‐1 or caveolae had little or no effect. The absence of caveolae had little impact on contraction amplitudes, indicative of recycling of calcium to SR in smooth muscle. However, the absence of caveolae slowed the rate of loss of calcium from SR under some conditions in both ICC and smooth muscle, which may reflect the loss of proximity to store operated Ca channels. We found evidence that these channels were associated with Cav‐1. These changes were all consistent with the hypothesis that a reduction of the extracellular calcium associated with caveolae in ICC of the myenteric plexus, the state of L‐type Ca²⁺ channels or an increase in the distance between caveolae and SR affected calcium handling.