Harmonization service and global library of models to support country-driven global information on salt-affected soils.

Global distribution of salt-affected soils (SAS) has remained at about 1 billion hectares in the literature over the years despite changes in climate, sea levels, and land use patterns which influence the distribution. Lack of periodic update of input soil data, data gaps, and inconsistency are part of the reasons for constant SAS distribution in the literature. This paper proposes harmonization as a suitable alternative for managing inconsistent data and minimizing data gaps. It developed a new harmonization service for supporting country-driven global SAS information update. The service contains a global library of harmonization models for harmonizing inconsistent soil data. It also contains models for identifying gaps in SAS database and for showing global distribution where harmonization of available data is needed. The service can be used by countries to develop national SAS information and update global SAS distribution. Its data availability index is useful in identifying countries without SAS data in the global database, which is a convenient way to identify countries to mobilize when updating global SAS information. Its application in 27 countries showed that the countries have more SAS data than they currently share with the global databases and that most of their data require SAS harmonization.

Y-box binding protein from Schistosoma mansoni: interaction with DNA and RNA.

A Schistosoma mansoni homologue of the human Y-box binding protein (SMYB1), as well as truncated proteins containing its N-terminal Cold Shock Domain (CSD) or its C-terminal domain (TAIL) were cloned into the p-MAL-c2 expression vector and produced in Escherichia coli. In order to characterize the interactions of these proteins to an inverted CCAAT motif present in a number of gene promoters, their binding to DNA was measured by Electrophoretic Mobility Shift Assays. SMYB1 bound to single- and double-stranded DNA containing the CCAAT motif and could bind also to RNA. The truncated CSD and TAIL domain proteins bound to dsDNA and RNA, but exhibited distinct binding patterns. Protein-DNA interaction was also investigated in vivo, using the Yeast One-Hybrid System. The plasmid constructs were GSTTRI, a DNA fragment composed of three copies of the CCAAT motif of the S. mansoni glutathione S-transferase gene promoter and four oligonucleotides spanning different regions of the S. mansoni p14 gene promoter. None of the yeast clones transformed with the above plasmids was able to grow in selective medium or to activate the transcription of the HIS3 reporter gene, suggesting that SMYB1 could not interact with these promoters in vivo.

Cis-acting elements, CArG-, E-, CCAAT- and TATA-boxes may be involved in sexually regulated gene transcription in Schistosoma mansoni.

Schistosomes undergo various morphological and metabolic changes during their development, reflected in a finely tuned regulation of protein and/or gene expression. The mechanisms involved in the control of gene expression during the development of the parasite are not understood. Two actin genes had been previously cloned and observed to be differentially expressed during the maturation of the parasite. The SmAct gene contains four putative cis-regulatory elements (TATA-, CCAAT-, E- and CArG-boxes). Our objective was to investigate in greater detail the expression pattern of two actin genes and verify if the binding of nuclear proteins to the promoter elements of SmAct correlated with the expression profile observed. We detected little variation in the expression of actin genes during the first seven days of schistosomula culture in vitro. However, we observed significantly higher levels of expression in males compared to female adults. CArG and CCAAT elements bound to a greater extent and formed distinct complexes with male in comparison to female nuclear extracts. In contrast, female extracts bound weakly to the E-box probe while no binding was observed with male extracts. Taken together these results describe cis-acting elements that appear to be involved in sexually regulated gene expression in Schistosoma mansoni.

Cis-acting elements, CArG-, E-, CCAAT- and TATA-boxes may be involved in sexually regulated gene transcription in Schistosoma mansoni

Schistosomes undergo various morphological and metabolic changes during their development, reflected in a finely tuned regulation of protein and/or gene expression. The mechanisms involved in the control of gene expression during the development of the parasite are not understood. Two actin genes had been previously cloned and observed to be differentially expressed during the maturation of the parasite. The SmAct gene contains four putative cis-regulatory elements (TATA-, CCAAT-, E- and CArG-boxes). Our objective was to investigate in greater detail the expression pattern of two actin genes and verify if the binding of nuclear proteins to the promoter elements of SmAct correlated with the expression profile observed. We detected little variation in the expression of actin genes during the first seven days of schistosomula culture in vitro. However, we observed significantly higher levels of expression in males compared to female adults. CArG and CCAAT elements bound to a greater extent and formed distinct complexes with male in comparison to female nuclear extracts. In contrast, female extracts bound weakly to the E-box probe while no binding was observed with male extracts. Taken together these results describe cis-acting elements that appear to be involved in sexually regulated gene expression in Schistosoma mansoni

Lack of DNA methylation in Schistosoma mansoni.

Schistosoma mansoni genomic DNA from male and female adult worms was subjected to restriction by the isoschizomeric endonucleases HpaII and MspI, which display different sensitivities with respect to cytosine methylation. The digested DNA was hybridized with 13 S. mansoni probes. Southern blot analysis showed that there were no observable differences in the restriction patterns of the two isoschizomers and that the patterns were identical in male and female parasites. Adenine methylation was also ruled out since no differences were observed with DpnI, Sau3A1, or MboI restriction enzymes. The methylation-dependent restriction endonuclease McrBC, which cleaves DNA containing methylcytosine and will not cleave unmethylated DNA, did not digest S. mansoni genomic DNA. These results demonstrate that the genome of adult S. mansoni is not methylated.

Evaluation of Schistosoma mansoni retinoid X receptor (SmRXR1 and SmRXR2) activity and tissue distribution.

Recently, we reported the identification of cDNA's encoding retinoid X receptor (RXR) homologues in Schistosoma mansoni. RXRs are known to be involved in the regulation of genes important for homeostasis and development. Previous studies indicated that SmRXR1 plays a role in the regulation of the female-specific gene, p14. Herein, we report that SmRXR2 also binds to cis-elements present in the p14 upstream region when evaluated in yeast reporter strains. SmRXR2 shows a pattern of recognition of cis-sequences present in the p14 gene upstream region different than SmRXR1. However, the SmRXR2 C (DNA binding) domain binds promiscuously in electrophoretic mobility shift assays to cis-elements of the p14 upstream region. The SmRXRs differ in their ability to activate transcription. The N-terminal A/B domain of SmRXR1 is necessary and sufficient for autonomous transcription activation function (AF) in yeast. SmRXR2 does not exhibit an equivalent autonomous AF. SmRXR1 and SmRXR2 fail to dimerize when investigated both in the yeast two-hybrid system and in immunoprecipitation experiments. In situ hybridization experiments using paraffin sections of adult worms demonstrate that SmRXR1 and SmRXR2 exhibit both common and unique cell type distribution which indicates that SmRXR1 and SmRXR2 both play a role in regulating gene expression in certain cells, yet each plays a distinct role in modulating the expression of genes in other cell types. Both SmRXR1 and SmRXR2 localize to vitelline cells. These studies provide a solid basis for improving our understanding of RXRs and their importance in female-specific gene regulation.

Identification and functional characterization of a member of the PUR-alpha family from Schistosoma mansoni.

Schistosoma mansoni p14 gene encodes an eggshell precursor that is expressed only in vitelline cells of mature female worms in response to a male stimulus. The upstream region of the p14 gene contains several potential cis-acting regulatory sequences. We used the upstream region of the p14 gene as bait in a yeast-one-hybrid screen of a S. mansoni cDNA library to identify interacting proteins. We report the identification and characterization of a cDNA (S. mansoni PUR-alpha (SmPUR-alpha)) encoding a protein homologous to single-stranded DNA transcription activator PUR-alpha, that binds to the p14 upstream region and activates transcription of the HIS3 reporter gene in yeast. SmPUR-alpha has a predicted molecular mass of 30 kDa and shares an overall homology of 63% with mammalian PUR-alpha. The DNA binding domain of SmPUR-alpha is highly conserved. We show by gel shift assays that GST-SmPUR-alpha binds to oligonucleotides comprising the p14 upstream region. SmPUR-alpha binds preferentially to single-stranded DNA and also binds RNA. Unlike the mammalian homologue, SmPUR-alpha exhibits little specificity for the PUR element GGn, but shows strong preference for a sequence containing alternating pyrimidines. Our data support that SmPUR-alpha is a single-copy gene and through reverse transcriptase-polymerase chain reaction and in situ hybridization, we show that SmPUR-alpha is constitutively transcribed in many cell types and thus likely plays a role as a general transcription activator in schistosomes.

Comparison between site-specific DNA binding proteins of male and female Schistosoma mansoni.

Several amplicons with approximately 120 bp each, obtained from the upstream domain of Schistosoma mansoni female-specific gene F-10, were coupled to Dynabeads M-280 streptavidin. The beads were used as a matrix for affinity purification of nuclear proteins obtained from mixed populations of adult worms. A protein of approximately 12 kDa, bound to the DNA in a sequence-independent manner. In contrast, when the DNA matrix was narrowed down to smaller synthetic oligonucleotides, bearing sequences corresponding to the TATA box and the CAAT box, band-shift assays revealed that different nuclear proteins from either adult male or female worms formed complexes with the DNA adduct. In order to characterise the bound proteins, the same oligonucleotides were UV cross-linked to the male and female protein extracts. Whilst the band shift experiments showed that the proteins from each sex produced a distinct mobility pattern when the TATA box sequences were tested and a similar one when the CAAT box sequences were added to the proteins, UV cross-linking experiments revealed clear qualitative differences between both, male and female proteins and also between the proteins binding to the two motifs. These results are compatible with a model in which the differential expression of the F-10 gene might depend on individual sub-sets of proteins.

Molecular characterisation of a NADH ubiquinone oxidoreductase subunit 5 from Schistosoma mansoni and inhibition of mitochondrial respiratory chain function by testosterone.

Complementary DNA, encoding the mitochondrial enzyme NADH-ubiquinone oxidoreductase subunit 5 (SmND5) of the human parasite Schistosoma mansoni was isolated by screening a S. mansoni cDNA library with a human androgen receptor (hAR) cDNA probe. The complete nucleotide and deduced aminoacid sequences of SmND5 were determined. Southern blot analysis revealed the occurrence of a single copy gene for SmND5 and by means of RT-PCR, it was shown that sex- and stage-specific expression of SmND5 occurred. In order to establish a functional relationship between the mitochondrial enzyme and the androgen receptor, the effects of testosterone were compared to those of classical respiratory chain inhibitors, using adult schistosome and beef heart submitochondrial particles. Physiological concentrations of testosterone were able to inhibit the maintenance of proton gradient across the mitochondrial membranes, as well as ATP synthesis. The steroid was found to be cytotoxic to the larvae, but not to adult schistosomes. A model is proposed to explain the observed in vivo testosterone-related differences in worm burdens, in experimental chronic infections.

Schistosoma mansoni: susceptibility differences between male and female mice can be mediated by testosterone during early infection.

In murine Schistosoma mansoni infections, fewer adult worms develop in male than in female mice infected with the same number of cercariae. To evaluate a potential role for testosterone in this phenomenon, testosterone levels were manipulated in groups of CBA/J mice that were then infected and monitored for survival rates, worm burdens, organomegaly, and egg production. By 16 weeks of infection, more than 80% of mice in groups with low levels of testosterone (untreated females, castrated males, or carrier-treated castrates) were dead, while less than 40% of those in groups with high levels of testosterone (sham-castrated males, testosterone-treated castrates, or testosterone-treated female mice) succumbed to infection. The mean number of worms recovered from mice in the low testosterone level groups was comparable among groups, and significantly greater than that from those in high-testosterone-level groups. The degree of organomegaly observed correlated strongly with worm burden, but the number of hepatic eggs per female worm did not differ significantly between groups. When male mice were castrated or sham-castrated 5 weeks after S. mansoni infection, no significant differences in host survival occurred. Furthermore, female mice treated with testosterone demonstrated reduced worm burdens if the testosterone was given 10 days prior to infection but not if the testosterone was given 10 days or 5 weeks after infection. Thus, the host sex bias observed in parallel-infected male and female mice appears to be related to the presence of male gonadal tissue or testosterone early in infection, during the development of immature schistosomules.

Characterization of a HMG2-like protein from Schistosoma mansoni.

An HMG2-like protein was purified from nuclear extracts of adult Schistosoma mansoni. Investigation of the amino acid composition of the schistosome HMG2-like protein showed that glutamic acid, glycine, aspartic acid and lysine were the most abundant. Carbohydrate analysis showed that the HMG2-like protein presented a low degree of glycosylation, galactose or glucose being the major monosaccharide constituent. Incubation of live schistosomes with 32P followed by isolation of nuclear proteins showed that the HMG-2 like protein could be phosphorylated. Partial sequence analysis of cyanogen bromide peptides revealed the occurrence of a phosphorylation consensus motif. The schistosome HMG2-like protein was found to bind preferentially to single-stranded DNA. The results suggest that the major non-histone S. mansoni nuclear protein belongs to the HMG family.

Extraction and partial characterization of non-histone nuclear proteins of Schistosoma mansoni.

A pool of nuclear proteins from adult worms of Schistosoma mansoni was analyzed for amino acid composition and found to be compatible with high mobility group (HMG) proteins. One of the schistosome HMG proteins was identified as HMG 2 by one-dimensional and two-dimensional PAGE. Stage-specific differences in the HMG-like protein composition were encountered when adult worms were compared to schistosomula, the larval form. Immobilization of the adult male and female nuclear proteins onto nitrocellulose, followed by hybridization against 32P-F-10, a schistosome sex specific gene encoding a major egg shell protein, revealed distinct banding patterns. On the other hand, a synthetic oligonucleotide, derived from the 3' untranslated end of the F-10 gene and possibly containing one regulatory element of the gene, bound mainly to male low MW proteins.