Identification of three novel Toxoplasma gondii rhoptry proteins

The rhoptries are key secretory organelles from apicomplexan parasites that contain proteins involved in invasion and modulation of the host cell. Some rhoptry proteins are restricted to the posterior bulb (ROPs) and others to the anterior neck (RONs). As many rhoptry proteins have been shown to be key players in Toxoplasma invasion and virulence, it is important to identify, understand and characterise the biological function of the components of the rhoptries. In this report, we identified putative novel rhoptry genes by identifying Toxoplasma genes with similar cyclical expression profiles as known rhoptry protein encoding genes. Using this approach we identified two new rhoptry bulb (ROP47 and ROP48) and one new rhoptry neck protein (RON12). ROP47 is secreted and traffics to the host cell nucleus, RON12 was not detected at the moving junction during invasion. Deletion of ROP47 or ROP48 in a type II strain did not show major influence in in vitro growth or virulence in mice.     

The effect of forest type on throughfall deposition and seepage flux: a review

Converting deciduous forests to coniferous plantations and vice versa causes environmental changes, but till now insight into the overall effect is lacking. This review, based on 38 case studies, aims to find out how coniferous and deciduous forests differ in terms of throughfall (+stemflow) deposition and seepage flux to groundwater. From the comparison of coniferous and deciduous stands at comparable sites, it can be inferred that deciduous forests receive less N and S via throughfall (+stemflow) deposition on the forest floor. In regions with relatively low open field deposition of atmospheric N (<10 kg N ha⁻¹ year⁻¹), lower NH₄⁺ mean throughfall (+stemflow) deposition was, however, reported under conifers compared to deciduous forest, while in regions with high atmospheric N pollution (>10 kg N ha⁻¹ year⁻¹), the opposite could be concluded. The higher the open field deposition of NH₄⁺, the bigger the difference between the coniferous and deciduous throughfall (+stemflow) deposition. Furthermore, it can be concluded that canopy exchange of K⁺, Ca²⁺ and Mg²⁺ is on average higher in deciduous stands. The significantly higher stand deposition flux of N and S in coniferous forests is reflected in a higher soil seepage flux of NO₃⁻, SO₄²⁻, K⁺, Ca²⁺, Mg²⁺ and Al(III). Considering a subset of papers for which all necessary data were available, a close relationship between throughfall (+stemflow) deposition and seepage was found for N, irrespective of the forest type, while this was not the case for S. This review shows that the higher input flux of N and S in coniferous forests clearly involves a higher seepage of NO₃⁻ and SO₄²⁻ and accompanying cations K⁺, Ca²⁺, Mg²⁺ and Al(III) into the groundwater, making this forest type more vulnerable to acidification and eutrophication compared to the deciduous forest type.     

Unravelling enzymatic discoloration in potato through a combined approach of candidate genes,QTL, and expression analysis

Enzymatic discoloration (ED) of potato tubers was investigated in an attempt to unravel the underlying genetic factors. Both enzyme and substrate concentration have been reported to influence the degree of discoloration and as such this trait can be regarded as polygenic. The diploid mapping population C × E, consisting of 249 individuals, was assayed for the degree of ED and levels of chlorogenic acid and tyrosine. Using this data, Quantitative Trait Locus (QTL) analysis was performed. Three QTLs for ED have been found on parental chromosomes C3, C8, E1, and E8. For chlorogenic acid a QTL has been identified on C2 and for tyrosine levels, a QTL has been detected on C8. None of the QTLs overlap, indicating the absence of genetic correlations between these components underlying ED, in contrast to earlier reports in literature. An obvious candidate gene for the QTL for ED on Chromosome 8 is polyphenol oxidase (PPO), which was previously mapped on chromosome 8. With gene-specific primers for PPO gene POT32 a CAPS marker was developed. Three different alleles (POT32-1, -2, and -3) could be discriminated. The segregating POT32 alleles were used to map the POT32 CAPS marker and QTL analysis was redone, showing that POT32 coincides with the QTL peak. A clear correlation between allele combinations and degree of discoloration was observed. In addition, analysis of POT32 gene expression in a subset of genotypes indicated a correlation between the level of gene expression and allele composition. On average, genotypes having two copies of allele 1 had both the highest degree of discoloration as well as the highest level of POT32 gene expression.     

GxxxG motif of severe acute respiratory syndrome coronavirus spike glycoprotein transmembrane domain is not involved in trimerization and is not important for entry

Recently, a paper was published in which it was proposed that the GxxxG motif of the severe acute respiratory syndrome (SARS) coronavirus spike (S) protein transmembrane domain plays a vital role in oligomerization of the protein (E. Arbely, Z. Granot, I. Kass, J. Orly, and I. T. Arkin, Biochemistry 45:11349-11356, 2006). Here, we show that the GxxxG motif is not involved in SARS S oligomerization by trimerization analysis of S GxxxG mutant proteins. In addition, the capability of S to mediate entry of SARS S-pseudotyped particles overall was affected moderately in the mutant proteins, also arguing for a nonvital role for the GxxxG motif in SARS coronavirus entry.     

Glutathione Protects Lactococcus lactis against Acid Stress

Previously we showed that glutathione (GSH) can protect Lactococcus lactis against oxidative stress (Y. Li et al., Appl. Environ. Microbiol. 69:5739-5745, 2003). In the present study, we show that the GSH imported by L. lactis subsp. cremoris SK11 or produced by engineered L. lactis subsp. cremoris NZ9000 can protect both strains against a long-term mild acid challenge (pH 4.0) and a short-term severe acid challenge (pH 2.5). This shows for the first time that GSH can protect a gram-positive bacterium against acid stress. During acid challenge, strain SK11 containing imported GSH and strain NZ9000 containing self-produced GSH exhibited significantly higher intracellular pHs than the control. Furthermore, strain SK11 containing imported GSH had a significantly higher activity of glyceraldehyde-3-phosphate dehydrogenase than the control. These results suggest that the acid stress resistance of starter culture can be improved by selecting L. lactis strains capable of producing or importing GSH.     

Predicting future community composition from random soil seed bank sampling – evidence from a drained lake bottom

Questions: What is the accuracy and reliability of the commonly used random soil sampling methodology for predicting seedling density, species richness and composition of the emerging seedling community? Location: Lake Kraenepoel, western Belgium. Methods: We compared density, species composition and observed and rarefactioned species richness of the seedling community emerging on a soft water lake bed exposed after drainage with the seedling community germinating in the laboratory from random soil samples in the same plots. Results: Seedling density did not differ between the two methods and there was a significant correlation between seedling density on the exposed lake bed and in the soil samples. This indicates that future seedling density can be reliably predicted based on soil sampling, in particular for the most abundant species. The most frequently occurring and abundant species among the seedlings in the soil samples were also the most frequent and abundant species germinating on the exposed lake bed. In contrast, species richness was much higher on the exposed lake bed than in the soil samples, and this difference was still significant for annual species after correction for differences in sampling intensity by rarefaction. We found no correlation between the number of species retrieved by the two methods. Although seedlings of rare and target species emerged on the lake bed, random soil sampling clearly failed to detect seeds of most of these species. Conclusions: Random soil sampling at a commonly used intensity and using the standard germination conditions can accurately predict future total seedling density and the density of the most abundant species. However, the method is not reliable for predicting the probability of establishment of populations of uncommon species. When executing a seed bank study, sampling intensity and germination conditions need to be adapted to the nature and the level of detail of the research question to be answered.     

Online biochemical detection of glutathione-S-transferase P1-specific inhibitors in complex mixtures

A high-resolution screening (HRS) technology is described, which couples 2 parallel enzyme affinity detection (EAD) systems for substrates and inhibitors of rat cytosolic glutathione-S-transferases (cGSTs) and purified human GST P1 to gradient reversed-phase high-performance liquid chromatography (HPLC). The cGSTs and GST P1 EAD systems were optimized and validated first in flow injection analysis (FIA) mode, and optimized values were subsequently used for HPLC mode. The IC(50) values of 8 ligands thus obtained online agreed well with the IC(50) values obtained with microplate reader-based assays. For ethacrynic acid, an IC(50) value of 1.8 +/- 0.4 microM was obtained with the cGSTs EAD system in FIA mode and 0.8 +/- 0.6 microM in HPLC mode. For ethacrynic acid with the GST P1 EAD system, IC(50) values of 6.0 +/- 2.9 and 3.6 +/- 2.8 microM were obtained in FIA and HPLC modes, respectively. An HRS GST EAD system, consisting of both the cGSTs and the GST P1 EAD system in HPLC mode in parallel, was able to separate complex mixtures of compounds and to determine online their individual affinity for cGSTs and GST P1. Finally, a small library of GST inhibitors, synthesized by reaction of several electrophiles with glutathione (GSH), was successfully screened with the newly developed parallel HRS GST EAD system. It is concluded that the present online gradient HPLC-based HRS screening technology offers new perspectives for sensitive and simultaneous screening of general cGSTs and specific GST P1 inhibitors in mixtures.