Prostate cancer (PCa) is one of the major public health problems in Western countries. Recently, the TMPRSS2:ERG gene fusion, which results in the aberrant expression of the transcription factor ERG, ...has been shown to be the most common gene rearrangement in PCa. Previous studies have determined the contributions of this fusion in PCa disease initiation and/or progression in vitro and in vivo. In this study on TMPRSS2:ERG regulation in PCa, we used an androgen receptor and TMPRSS2:ERG fusion double-negative PCa cell model: PC3c. In three cell clones with different TMPRSS2:ERG expression levels, ectopic expression of the fusion resulted in significant induction of cell migration and invasion in a dose-dependent manner. In agreement with this phenotype, high-throughput microarray analysis revealed that a set of genes, functionally associated with cell motility and invasiveness, were deregulated in a dose-dependent manner in TMPRSS2:ERG-expressing cells. Importantly, we identified increased MMP9 (Metalloproteinase 9) and PLXNA2 (Plexin A2) expression in TMPRSS2:ERG-positive PCa samples, and their expression levels were significantly correlated with ERG expression in a PCa cohort. In line with these findings, there was evidence that TMPRSS2:ERG directly and positively regulates MMP9 and PLXNA2 expression in PC3c cells. Moreover, PLXNA2 upregulation contributed to TMPRSS2:ERG-mediated enhancements of PC3c cell migration and invasion. Furthermore, and importantly, PLXNA2 expression was upregulated in metastatic PCa tumors compared with localized primary PCa tumors. This study provides novel insights into the role of the TMPRSS2:ERG fusion in PCa metastasis.
Two ELISA assays were developed to test the reactivity of soluble sHLAs with anti-HLA class I mAbs of IgG or IgM isotype. A panel of 40 different alleles of sHLA antigens was produced using 57 ...lymphoblastoid B-cell lines, which had been generated from class-I-phenotyped PBLs. Using 14 mAbs, the expression of 13 different sHLA antigens (sHLA-A2, -A3, -A11, -A24, -A29, -B7, -B8, -B13, -B14, -B27, -B44, -B57, and -B58) by 43 different cell lines was confirmed. In addition, the expected absence of these alleles from the culture supernatant of 11 cell lines was confirmed. Cross-reactivities of mAbs observed by microlymphocytotoxicity assays were also detected by ELISA. The results of this extensive analysis confirmed previous results demonstrating that sHLA class I typing by ELISA correlated with HLA class I cell typing by microlymphocytotoxicity 1-3. Furthermore, additional information about the fine specificities of two mAbs was obtained. An anti-B27/44 IgM mAb appeared to react only with sHLA-B44 but not with sHLA-B27; mAb CR11-351, previously reported to react with HLA-A2, 28, bound also to sHLA-A1, -3, -11, and -A24.
The precise molecular mechanisms underlying the switch between the two developmental stages of
Toxoplasma gondii, and the metabolic adaptations occurring during this stage conversion are poorly ...understood. Because inhibitors of mitochondrial respiration are known to trigger differentiation from tachyzoite into bradyzoite stages, we believe that some of the switch components may be sought in the regulation of central carbohydrate metabolism. We have previously described a cDNA encoding a bradyzoite-specific enolase,
ENO1. We now report the isolation and characterization of another enolase-encoding cDNA (
ENO2) that is expressed preferentially in the tachyzoite stage. The deduced amino acid sequences of ENO1 and ENO2 share 73.65 % identity. They both display significant homologies to plant enolases with the presence of two plant-like peptide insertions, a pentapeptide EWGW(Y)C(S) and a dipeptide EK (or DK). We demonstrate that deletions of the ENO1 pentapeptide motif on its own or together with the dipeptide reduce drastically the affinity for the 2PGA substrate, suggesting that the evolutionary acquisition of these peptides in enolases of land plants and apicomplexan parasites contribute a specific function to their enzymatic activities.
T. gondii ENO1 and ENO2 were also expressed as active recombinant enzymes in
Escherichia coli. While ENO1 and ENO2 display similar
K
m values, the pure tachyzoite-specific enzyme (ENO2) has a threefold specific activity at
V
max compared with that of the bradyzoite-specific enolase (ENO1). Moreover, immunoblot analyses performed using polyclonal antibodies raised against the recombinant enzymes revealed that the native enolase in tachyzoite and bradyzoite are also antigenically distinct. Taken together, our results indicate that the differences witnessed between the two activities may be instrumental in maintaining glycolysis in pace with the distinct stage-specific requirements of carbohydrate metabolism.
The role of specific microbial Ags in the induction of experimental inflammatory bowel disease is poorly understood. Oral infection of susceptible C57BL/6 mice with Toxoplasma gondii results in a ...lethal ileitis within 7-9 days postinfection. An immunodominant Ag of T. gondii (surface Ag 1 (SAG1)) that induces a robust B and T cell-specific response has been identified and a SAG1-deficient parasite (Deltasag1) engineered. We investigated the ability of Deltasag1 parasite to induce a lethal intestinal inflammatory response in susceptible mice. C57BL/6 mice orally infected with Deltasag1 parasites failed to develop ileitis. In vitro, the mutant parasites replicate in both enterocytes and dendritic cells. In vivo, infection with the mutant parasites was associated with a decrease in the chemokine and cytokine production within several compartments of the gut-associated cell population. RAG-deficient (RAG1(-/-)) mice are resistant to the development of the ileitis after T. gondii infection. Adoptive transfer of Ag-specific CD4(+) effector T lymphocytes isolated from C57BL/6-infected mice into RAG(-/-) mice conferred susceptibility to the development of the intestinal disease. In contrast, CD4(+) effector T lymphocytes from mice infected with the mutant Deltasag1 strain failed to transfer the pathology. In addition, resistant mice (BALB/c) that fail to develop ileitis following oral infection with T. gondii were rendered susceptible following intranasal presensitization with the SAG1 protein. This process was associated with a shift toward a Th1 response. These findings demonstrate that a single Ag (SAG1) of T. gondii can elicit a lethal inflammatory process in this experimental model of pathogen-driven ileitis.
TgMIC6, TgMIC7, TgMIC8 and TgMIC9 are members of a novel family of transmembrane proteins localized in the micronemes of the protozoan parasite Toxoplasma gondii. These proteins contain multiple ...epidermal growth factor-like domains, a putative transmembrane spanning domain and a short cytoplasmic tail. Sorting signals to the micronemes are encoded in this short tail. We established previously that TgMIC6 serves as an escorter for two soluble adhesins, TgMIC1 and TgMIC4. Here, we present the characterization of TgMIC6 and three additional members of this family, TgMIC7, -8 and -9. Consistent with having sorting signals localized in its C-terminal tail, TgMIC6 exhibits a classical type I membrane topology during its transport along the secretory pathway and during storage in the micronemes. TgMIC6 is processed at the N-terminus, probably in the trans-Golgi network, and the cleavage site has been precisely mapped. Additionally, like other members of the thrombospondin-related anonymous protein family, TgMIC2, TgMIC6 and TgMIC8 are proteolytically cleaved near their C-terminal domain upon discharge by micronemes. We also provide evidence that TgMIC8 escorts another recently described soluble adhesin, TgMIC3. This suggests that the existence of microneme protein complexes is not an exception but rather the rule. TgMIC6 and TgMIC8 are expressed in the rapidly dividing tachyzoites, while TgMIC7 and TgMIC9 genes are predominantly expressed in bradyzoites, where they presumably also serve as escorters.
Women empowerment is an important issue of socioeconomic development of a country. However, in Benin where the situation of women remains worrying, the question of empowerment is very little tackled ...by research. Accordingly, this paper analyses the empowerment of rural women in the central part of Benin. Based on a random sample of 190 rural women and using empowerment indices, the study shows that only 19% of the women enjoy a decision-making autonomy whereas 41% of them are free of mobility. About the leadership, the most of the women can neither integrate nor leading any rural organisation without the consent of their husband, although they clearly enjoy a financial autonomy. The results also show that there is a significant relationship between decision-making autonomy and freedom of mobility. In the same way, decision-making autonomy is correlated with the marital status of women. Due to the influence of social norms and customs on the current situation of the women in the study area, one can suppose that any boosting of the empowerment process is subject to a major change in these factors.
The protozoan parasite Toxoplasma gondii differentially expresses two distinct enolase isoenzymes known as ENO1 and ENO2, respectively. To understand differential gene expression during tachyzoite to ...bradyzoite conversion, we have characterized the two T.gondii enolase promoters. No homology could be found between these sequences and no TATA or CCAAT boxes were evident. The differential activation of the ENO1 and ENO2 promoters during tachyzoite to bradyzoite differentiation was investigated by deletion analysis of 5′-flanking regions fused to the chloramphenicol acetyltransferase reporter followed by transient transfection. Our data indicate that in proliferating tachyzoites, the repression of ENO1 involves a negative distal regulatory region (nucleotides −1245 to −625) in the promoter whereas a proximal regulatory region in the ENO2 promoter directs expression at a low level. In contrast, the promoter activity of ENO1 is highly induced following the conversion of tachyzoites into resting bradyzoites. The ENO2 promoter analysis in bradyzoites showed that there are two upstream repression sites (nucleotides −1929 to −1067 and −456 to −222). Furthermore, electrophoresis mobility shift assays demonstrated the presence of DNA-binding proteins in tachyzoite and bradyzoite nuclear lysates that bound to stress response elements (STRE), heat shock-like elements (HSE) and other cis-regulatory elements in the upstream regulatory regions of ENO1 and ENO2. Mutation of the consensus AGGGG sequence, completely abolished protein binding to an oligonucleotide containing this element. This study defines the first characterization of cis-regulatory elements and putative transcription factors involved in gene regulation of the important pathogen T.gondii.
The parasite Toxoplasma gondii expresses a 55 kDa protein or TgDRE that belongs to a novel family of proteins characterized by the presence of three domains, a human splicing factor 45-like motif ...(SF), a glycine-rich motif (G-patch), and a RNA recognition motif (RRM). The two latter domains are mainly known as RNA-binding domains, and their presence in TgDRE, whose partial DNA repair function was demonstrated, suggests that the protein could also be involved in the RNA metabolism. In this work, we characterized the structure and function of the different domains by using single or multidomain proteins to define their putative role. The SF45-like domain has a helical conformation and is involved in the oligomerization of the protein. The G-patch domain, mainly unstructured on its own as well as in the presence of the SF upstream and RRM downstream domains, is able to bind small RNA oligonucleotides. We also report the structure determination of the RRM domain from the NMR data. It adopts a classical βαββαβ topology consisting of a four-stranded β sheet packed against two α helices but does not present the key residues for the RNA interaction. In contrast, our analysis shows that the RRM of TgDRE is not only unable to bind small RNA oligonucleotides but it also shares the protein−protein interaction characteristics with two unusual RRMs of the U2AF heterodimeric splicing factor. The presence of both RNA- and protein-binding domains seems to indicate that TgDRE could also be involved in RNA metabolism.
We have identified three novel Toxoplasma gondii proteins showing close structural similarity to molecules of the SAG1 family, a group of glycosylphosphatidylinositol-anchored surface antigens ...expressed by the invasive stages of T. gondii. The novel proteins, denominated SAG5A, SAG5B and SAG5C, are encoded by tandemly arrayed and tightly clustered genes containing no introns. The 367 amino acid-long SAG5B and SAG5C are 97.5% identical to each other, whereas SAG5A (362 amino acids) consists of a C-terminal domain sharing 98% identity with SAG5B and SAG5C, and an N-terminal domain whose identity to the other SAG5 polypeptides is only 42%. Expression analysis of the T. gondii strains RH (virulent) and 76 K (avirulent) showed that all members of the SAG5 cluster are transcribed in T. gondii tachyzoites and bradyzoites. However, immunoblot studies on the RH strain revealed that the synthesis of SAG5A does not occur in tachyzoites and is possibly controlled at the post-transcriptional level. On the contrary, SAG5B and SAG5C were detected by immunoblot in tachyzoite lysates and found to migrate in the 40-45 kDa range under reducing conditions or at approximately 34 kDa under unreduced conditions. Triton X-114 partitioning of tachyzoite protein lysates treated with phosphatidylinositol-specific phospholipase C indicated that SAG5B and SAG5C are glycosylphosphatidylinositol-anchored membrane-associated molecules. Consistently, immunofluorescence analysis of transformed tachyzoites over-expressing SAG5B or SAG5C showed that these molecules are targeted to the parasite surface. The characterisation of the SAG5 locus sheds further light on the complex repertoire of SAG1-related genes in T. gondii, that now comprises 14 highly homologous members and five distantly related genes belonging to the SAG2 family.
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