Stage-specific alternative splicing of the heat-shock transcription factor during the life-cycle of Schistosoma mansoni.

Stage-specific alternative splicing of the heat-shock transcription factor of Schistosoma mansoni (SmHSF) generates isoforms with structural diversity that may modulate the activity of SmHSF at different life-stages, and thus may regulate the expression of different genes at different developmental stages. RT-PCR, cloning and DNA-sequence analyses showed stage-specific alternative splicing inside the DNA-binding domain (DBD) involving introns I1 and I2, and beyond the DBD involving introns I4a and I7. Retention of introns I2 and I4 would inactivate SmHSF since they contain termination codons. Retention of intron I1 would add 11 amino acids inside the DBD and may change the DNA-binding specificity of SmHSF; intron I7 would add 13 amino acids to the effector region of HSF. Retention of introns was more pronounced in cercariae (larval stage living in water) than in adult worms (parasitic form in mammals). The isoforms were expressed in bacteria, but functional evaluation was not feasible, because only the isoform lacking introns was soluble while isoforms with introns were insoluble. However, stage-specific alternative splicing that changed HSF function in vivo was evidenced in intact cercariae. The cercarial SmHSF mRNA was enriched with introns I2 and I4a that contain termination codons. Therefore, translation of the SmHSF mRNA was impaired, and the SmHSF protein was undetectable. Consequently, the HSP70 gene could not be transcribed, and the HSP70 mRNA was missing. Alternative splicing was observed for short DNA segments (33-45 bp) bound by splice signals, located in the coding region. These are not bona fida exons since they are not flanked by introns. Yet, they are not regular introns since they are often found in mature mRNA. Alternative splicing of these DNA segments caused structural diversity that could modulate the function of the gene product.

Differences in DNA-sequence recognition between the DNA-binding domain fragment and the full-length molecule of the heat-shock transcription factor of schistosome.

Binding and inhibition studies reveal that the DNA-binding domain (DBD) fragment and the full-length molecule of the heat-shock transcription factor of schistosome (SmHSF) differ in DNA sequence recognition. SmHSF does not recognize the ideal HSE consensus sequence (nGAAnnTTCnnGAAn) but recognizes a variant HSE that contains nGTAn instead of nGAAn in the third pentamer. The DBD reacts efficiently with the ideal HSE sequence and with lower affinity with the variant HSE sequence. These findings suggest that elements inside and outside the DBD contribute to the DNA-binding specificity of HSF.

Characterization of an insulin receptor-related receptor in Biomphalaria glabrata embryonic cells.

Tyrosine kinase receptors play a key role in the communication of cells with their environment. Growth hormone receptors, such as insulin receptors, are involved in the regulation of cell growth, differentiation and metabolism in multicellular organisms. Insulin-related peptides and members of the insulin receptor subfamily have been described in a wide variety of invertebrates, including freshwater molluscs. In this paper, we describe the metabolic effect of insulin on a mollusc cell line (Bge) derived from embryos of the snail Biomphalaria glabrata. Using a PCR strategy, we have cloned from Bge cells a cDNA encoding a protein (BgIR) homologous to, and exhibiting all of the typical features of insulin receptors. Northern blot analysis confirmed the expression of BgIR in B. glabrata snails and suggested its wide distribution in the snail body. Bge cells have been shown to provide the environmental conditions necessary for the in vitro development of the sporocysts of Schistosoma mansoni, a trematode parasite that uses B. glabrata as an intermediate host. The possible implication of BgIR in the activating and proliferating processes observed in Bge cells during their coculture with S. mansoni larvae is discussed.

Transient expression assays with the proximal promoter of a newly characterized actin gene from the oyster Crassostrea gigas.

We undertook the characterization of an actin gene and its proximal promoter in the oyster Crassostrea gigas. A complete actin cDNA was identified, sequenced and its amino acid sequence deduced. Comparative analysis showed a high homology with actin of other species and that this gene is closer to the cytoplasmic form of actins than to the muscle type. A probe derived from the 5'-untranslated region of the cDNA was then used to isolate the actin gene from a genomic library. The gene was sequenced and shown to contain a single 643 bp intron. A 1670 bp fragment upstream from the open reading frame was isolated and sequenced. This upstream region displays typical features of actins such as a serum response element (CarG box). This fragment was cloned into the promoterless vector pGL3-basic and the resulting construct was transfected into cells of dissociated oyster heart primary cultures. Its capacity to express the luciferase in this in vitro homologous system was monitored and showed high expression levels. This is the first complete actin sequence reported so far for the oyster C. gigas and its promoter is the first available among bivalves.

Identification of NF-AT-like transcription factor in Schistosoma mansoni: its possible involvement in the antiparasitic action of cyclosporin A.

Cyclosporin A (CsA) has been found to exert potent anti-parasite activity against a wide range of protozoan and helminth parasites. In schistosomes, evidence has been accumulated to propose that the drug damages parasites by mechanisms independent of its immunosuppressive properties. Moreover, the absence of correlation between anti-schistosomal properties and inhibition of peptidyl-prolyl cis-trans isomerase activity of cyclophilins (CsA receptors) for various drug analogs, argued against a direct implication of cyclophilins in the lethal effect of CsA. We describe, in S. mansoni, the existence of NF-AT-like transcription factors, a protein family already characterized by its sensitivity to CsA. The observation that CsA treatment of S. mansoni larvae inhibited the expression of the Sm28GST protein and the characterization of a functional NF-AT-like site in the gene encoding this protein, provide new insights in the understanding of the antischistosomal effect of CsA. Our results also support the hypothesis that the regulatory function of NF-AT-like proteins might be responsible for parasite development and survival in the host and open new perspectives in studies of helminth biology.

Biomphalaria glabrata embryonic cells express a protein with a domain homologous to the lectin domain of mammalian selectins.

We have cloned from Biomphalaria glabrata, the intermediate host of the helminth parasite Schistosoma mansoni, a 36-kDa apparent-molecular-mass molecule (BgSel) that shares sequence identity with selectins of the cell-adhesion-molecule superfamily. BgSel exhibited in its C-terminal part a putative C-type lectin domain similar to the selectin lectin domain. Using antibodies to the recombinant BgSel protein, we demonstrated the presence of BgSel in snail hemocytes as well as in the cell line derived from B. glabrata embryos (Bge). Anti-BgSel antibodies specifically recognized a 79-kDa component in Bge-cell-secreted products that was supposed to represent the native form of BgSel, as was confirmed after glycosidase treatment. Lectins are known to be implicated in recognition mechanisms participating in humoral and cellular immunity in molluscs. The possible role of BgSel in the interaction between sporocysts and Bge cells, particularly in the in vitro model of sporocyst development dependent on Bge cell factors, remains to be determined.

Cloning of the SmSPO-1 gene preferentially expressed in sporocyst during the life cycle of the parasitic helminth Schistosoma mansoni1.

Schistosomes are parasitic helminths with a complex life cycle in human and snail hosts. They express stage-specific genes that conceivably determine distinct properties of the parasite at different developmental stages. Here we report the stage-specific gene SmSPO-1, which is preferentially expressed in sporocysts residing in the snail host. The cDNA and the gene were cloned and sequenced. The cDNA, from cap site to the poly(A) addition site, is 498 bp long. It encodes a protein of 117 amino acids with a hydrophobic signal peptide of 18 residues, indicating that SmSPO-1 is a secreted or a membranal protein. In the gene the cDNA is split into four exons spread over 2.1 kb of chromosomal DNA.

Conservation and divergence of NF-Y transcriptional activation function.

The CCAAT-binding protein NF-Y is involved in the regulation of a variety of eukaryotic genes and is formed in higher eukaryotes by three subunits NF-YA/B/C. We have characterized NF-Y of the trematode parasite Schistosoma mansoni and studied the structure and the function of the SMNF-YA subunit. In this work, we present the cloning and sequence analysis of the B subunit of the parasite factor. SMNF-YB contains the conserved HAP-3 homology domain but the remaining part of the protein was found to be highly divergent from all other species. We demonstrated by transfections of GAL4 fusion constructs, that mouse NF-YB does not contain activation domains while the C-terminal part of SMNF-YB has transcriptional activation potential. On the other hand, the N-terminal parts of SMNF-YA and mouse NF-YA were shown to mediate transactivation; the integrity of a large 160 amino acid glutamine-rich domain of NF-YA was required for this function and an adjacent serine- and threonine-rich domain was necessary for full activity in HepG2, but redundant in other cell types. Transactivation domains identified in SMNF-YB are also rich in serine and threonine residues. Our results indicate that serine/threonine-richsequences from helminth parasites potentiate trans-cription and that such structures have diverged during evolution within the same transcription factor.

Characterization of an intracellular receptor for activated protein kinase C (RACK) from the mollusc Biomphalaria glabrata, the intermediate host for Schistosoma mansoni.

A receptor for activated protein kinase C (RACK) was characterized from the mollusc Biomphalaria glabrata, the intermediate host for the human parasite Schistosoma mansoni. This protein was shown to possess structural and functional characteristics of other RACK proteins from various cells and organisms. Its ability to bind mammalian PKCs also confirmed the conservation of PKC and RACK interactive domains throughout evolution. Results of immunolocalization indicated the presence of Bg RACK in the cytoplasm of mollusc hemocytes and B. glabrata embryonic (Bge) cells with a more intense staining around the nucleus. These results are in agreement with the association of RACK proteins with cytoskeletal elements.

Snail control strategies for reduction of schistosomiasis transmission.

As intermediate hosts, molluscs play a major role in the transmission of schistosomes; they are the sites of an intense multiplication of parasites. Thus, snail control strategies are considered a priority for the reduction of schistosomiasis transmission. Here, Vinca Lardans and Colette Dissous review the efficacy of environmental management and the use of molluscicides and biological agents to control snail populations. They then describe the development of diagnostic tests, based on the detection of parasite antigens or specific parasite DNA sequences in snail tissues, to detect the early infection of snails. Finally, they discuss progress in studying the molecular basis of susceptibility and resistance phenotypes, and the possible application of the genetic manipulation of molluscs.

Nucleotide and deduced amino acid sequences of Biomphalaria glabrata actin cDNA.

The complete nucleotide sequence of Biomphalaria glabrata actin has been cloned by PCR amplification and screening of a cDNA library of Biomphalaria glabrata. The comparison of the deduced amino acid sequence with other actins suggests that a cytoskeletal form of the protein has been cloned.

Polyclonal B lymphocyte activation induced by mouse hepatitis virus A59 infection.

Mouse hepatitis viruses (MHV) diversely affect immune responses, depending on the viral strain and the mouse genetic background. Here, we studied the effect of MHV-A59 infection on B cell responses of 129/Sv and CBA mice. Our results indicate that in these strains, MHV-A59 induces spleen cell activation that leads to enlargement of the spleen without structural alteration. Infection triggers production by B lymphocytes of large amounts of immunoglobulin G2a, mostly without viral specificity. This polyclonal immunoglobulin production is dependent on the presence of functional T helper cells. This polyclonal B lymphocyte activation induced by MHV-A59 infection can have pathological implications, such as the enhancement of concomitant autoimmune reactions.