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Christoph G Grevelding

Publications and source records attributed to Christoph G Grevelding.

16 recordsLinked to original sources
PubMed2006

Herbimycin A suppresses mitotic activity and egg production of female Schistosoma mansoni.

The eggs of the endoparasite Schistosoma are the causative agent of schistosomiasis, an important disease of humans, which is endemic in (sub-) tropical regions. The absence of a vaccine with sufficient protective qualities and increasing resistance to approved and established drugs like praziquantel, justify the exploration of novel ways to fight schistosomes. Our strategy is based on interference with the sexual maturation of the female. Prerequisites for gonad development in adult females are a continuous pairing contact with the male and significantly increased mitotic activity. In this study we show that the male governs sexual maturation of the female, as the separation of couples causes a clear reduction of female mitotic activity and, consequently, egg production. We demonstrate that treatment of schistosomes with Herbimycin A, an inhibitor of protein tyrosine kinases (PTKs), mimics the separation of couples as the drug blocks mitotic activity and egg production of paired females. However, the synthesis of the eggshell precursor protein p14 is elevated. Furthermore, we show for the first time in invertebrates that Herbimycin A decreases tyrosine phosphorylation and PTK stability in schistosomes. Summarised, our data provide evidence that PTKs have key functions in regulating gonad development, eggshell gene expression and, consequently, egg production. Therefore, we suggest envisaging schistosome PTKs as novel targets for strategies to combat schistosomiasis.

Animals↗
PubMed2005

The effect of a mutagen (N-methyl-N-nitro-N-nitrosoguanidine) on cultured cells from adult Schistosoma japonicum.

The transforming effect of N-methyl-N-nitro-N-nitrosoguanidine (MNNG) on cultured cells from Schistosoma japonicum (S. japonicum) was studied using mono-factor and orthogonal tests. Under the influence of MNNG, cultured cells grew well, and cell survival time was more than 246 days in low-serum medium. When treated with 3 mug/ml MNNG for 48 h, the number of dividing cells increased significantly as determined by bright-field and scanning electron microscopy (SEM). Under these conditions, abundant microvilli, ruffles, microridges, papillae and blebs were observed on the surface of the induced cells. Treatment with MNNG may overcome existing limitations to get continually proliferating schistosome cells and open the possibility to immortalize isolated cells.

Animals↗
PubMed2005

Biolistic transformation of Schistosoma mansoni with 5' flanking regions of two peptidase genes promotes tissue-specific expression.

The gene-regulatory elements controlling peptidase expression in Schistosoma mansoni are unknown. A genomic DNA library was constructed from which 5' flanking fragments of the cathepsins F (SmCF; 649 bp) and B2 (SmCB2; 810 bp) peptidase genes were isolated. These were cloned into a GFP-expression vector for biolistic transformation of schistosomes. Both fragments promoted expression of GFP that was localised in the gut for SmCF and tegument for SmCB2, consistent with previous immunochemical data. Promoter-deletion of the SmCF gene indicated the importance of one or more transcription factor binding sites in the first 169 bp for both GFP-expression and its tissue specificity.

Animals↗
PubMed2004

Schistosoma.

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Animals↗
PubMed2004

The Schistosoma mansoni Src kinase TK3 is expressed in the gonads and likely involved in cytoskeletal organization.

Cytoplasmic protein tyrosine kinases of the Src family play a pivotal role in the regulation of cellular processes including proliferation and differentiation. Among other functions, Src kinases are involved in regulating the cell architecture. In an approach to identify protein tyrosine kinases from the medically important parasite Schistosoma mansoni, we isolated the TK3 gene by degenerate primer PCR and cDNA library screening. Sequencing of the complete cDNA and data-base analyses indicated that TK3 is a Src family kinase. Its predicted size of 71 kDa was confirmed by Western blot analysis. Southern blot analysis showed that TK3 is a single-copy gene, and Northern blot and RT-PCR experiments indicated its expression in both sexes and throughout development. Localization studies by in situ hybridization and immunolocalization revealed that TK3 is predominantly expressed in the reproductive organs such as the testes of the male and the ovary as well as the vitellarium of the female. Its enzymatic activity was confirmed by functional analyses. In transient transfection experiments with HEK293 cells, TK3 phosphorylated the well-known Src-kinase substrate p130 Cas, an intracellular scaffolding protein. Yeast two-hybrid screenings in a heterologous invertebrate system identified dAbi, vinculin and tubulin as binding partners, representing molecules that fulfill functions in the cell architecture of many organisms. These findings suggest that TK3 may play a role in signal transduction pathways organizing the cytoskeleton in the gonads of schistosomes.

Amino Acid Sequence↗
PubMed2004

Cytological and biochemical evidence for a gonad-preferential interplay of SmFKBP12 and SmTbetaR-I in Schistosoma mansoni.

In eukaryotes, FK506-binding proteins with a molecular weight of 12 kDa (FKBP12s) influence a variety of signal transduction pathways that regulate cell division, differentiation, and ion homeostasis. Amongst these, TGFbeta signaling and calcineurin (CN) phosphatase activity is modulated by FKBP12 via binding to TGFbeta-family type I receptors (TbetaR-Is) or to the CN subunit A, respectively. In this work, we demonstrate the tissue-specific expression of the Schistosoma mansoni FKBP12 homologue (SmFKBP12) in the gonads of female parasites as well as in the tegument of both genders. Components of the TGFbeta pathway have been characterized in schistosomes and their roles in mediating host-parasite or male-female interactions proposed. We show that a schistosome TGFbeta-family type I receptor (SmTbetaR-I, SmRK-1) is expressed in the female gonads, suggesting that SmFKBP12 may regulate its activity in this tissue. This hypothesis is supported by yeast two-hybrid analyses showing a direct binding of SmFKBP12 and SmTbetaR-I, which was specifically inhibited by the drug FK506. Our data provide the first evidence for the activity of a transmembrane receptor in the vitellarium of schistosome females and indicate that FKBP12-meditated regulation of the TGFbeta pathway is evolutionarily conserved in a primitive metazoan such as Schistosoma. Furthermore, we show that the schistosome CN (SmCN) is not expressed in the female gonads, but co-localizes with SmFKBP12 only in the tegument. From these data we conclude an SmFKBP12/SmTbetaR-I, but not an SmCN/SmFKBP12 interplay in the female gonads.

Animals↗
PubMed2003

Transplantation of in vitro-generated Schistosoma mansoni mother sporocysts into Biomphalaria glabrata.

Specific studies on schistosome gene functions require both access to the parasite stages, preferably the larvae, and to complete the life cycle. In the present study, we investigated whether short-term in vitro cultivation of sporocysts and surgical transplantation into snails could be combined to produce cercariae. Miracidia were maintained in vitro in the presence of Biomphalaria glabrata embryonic (Bge) cells or, alternatively, in Bge-cell-conditioned medium. The transformation of miracidia to mother sporocysts was observed in both cases. Two day-old sporocysts were transplanted into the cephalopedal sinus of recipient snails. Transplantation efficiencies varied between 16% and 43%, depending on the culture of the sporocysts in terms of the number of cercariae producing snails. Cercariae recovered from these snails were used to successfully infect hamsters, demonstrating that short term in vitro-generated sporocysts undergo normal cercariogenesis following transplantation. This combination of in vitro cultivation and transplantation may be useful for novel experimental approaches to investigate the genes involved in larval development or host-parasite molecular interactions.

Animals↗
PubMed2003

The uptake of Texas Red-BSA in the excretory system of schistosomes and its colocalisation with ER60 promoter-induced GFP in transiently transformed adult males.

The excretory system of schistosomes has focused some attention during the last years since accumulating evidence suggests that it plays an important role in the host-parasite interaction. Signalling molecules such as phosphatases, but also proteases have been localised in the excretory system. To some extent, however, localisation studies are limited by the fact that sections of fixed specimens are used. In this study, we tested the fluorescent molecules FITC-dextran and Texas Red-BSA for their ability to enter the excretory system of living Schistosoma mansoni males. It is demonstrated that the dyes selectively stain the excretory tubules which are widely distributed along the worm body. This finding was used to investigate whether the staining of worms with Texas Red-BSA can help to localise transgene activity in worms which were transiently transformed by particle bombardment. A vector was used for transformation which contained the green fluorescent protein gene, under the control of the regulatory elements of the cysteine protease ER60 gene. After transformation and staining, confocal laser scanning microscopy revealed that ER60-induced green fluorescent protein activity colocalises with Texas Red-BSA in the excretory tubules. The results suggest a role for ER60 during the host-parasite interaction. Furthermore, the colocalisation approach introduced here opens further perspectives to characterise gene-expression profiles in this parasite.

Animals↗
PubMed2003

A stress-responsive glutathione S-transferase confers resistance to oxidative stress in Caenorhabditis elegans.

Previous studies demonstrated that the Caenorhabditis elegans GST-p24 is upregulated at the steady state mRNA level in response to oxidative stress. A transcriptional upregulation was confirmed in the current study by analyzing Ce-GST-p24 promoter-reporter constructs in transgenic C. elegans strains CL2166 and CL3166. The transgenic strain BL1, which overexpresses the Ce-GST-p24 enzyme (as a GFP fusion protein controlled by its own promoter), was generated to investigate the function of this enzyme in vivo. Stress experiments with BL1 demonstrated an increased resistance to intracellularly induced oxidative stress, as compared to wild type. The consequences of a decrease in the Ce-GST-p24 enzyme concentration were examined by RNAi-treatment of BL1 C. elegans to silence both the endogene and the transgene Ce-GST-p24 and by the analysis of the K08F4.7 homozygous deletion mutant. In both cases, the reduced Ce-GST-p24 enzyme level resulted in a significant decrease in the stress resistance of the nematodes. These results clearly demonstrate a direct correlation between the concentration of Ce-GST-p24 and the resistance to oxidative stress. We have demonstrated for the first time that manipulation of the expression of a single GST can modulate the organismal response to oxidative stress. The enzymatic activity of this detoxification enzyme was examined with various substrates, giving emphasis to lipid peroxidation products. The Ce-GST-p24 was also localized in BL1 C. elegans by confocal laser-scanning microscopy, revealing a wide-spread distribution profile.

Animals↗
PubMed2002

A novel Syk-family tyrosine kinase from Schistosoma mansoni which is preferentially transcribed in reproductive organs.

The complete coding deoxyribonucleic acid for a novel tyrosine kinase (TK) of the human parasite Schistosoma mansoni has been cloned and characterized. The molecule was designated TK4. The sequence predicts a translation product of about 140 kDa containing two Src homology 2 domains and a tyrosine kinase domain. Data base analyses indicate that TK4 belongs to the Syk family of TKs which has not been identified in schistosomes or other Acoelomata yet. The presence of a member of the Syk family in this phylum supports previous findings demonstrating that TK subclasses were established early in evolution. Although Northern blot and reverse transcription polymerase chain reaction analyses show transcription of TK4 in larval stages and adult schistosomes of both genders, TK4 is more abundantly transcribed in males. In situ hybridization data demonstrate the gender-independent occurrence of TK4 transcripts in parenchymatic cells. Significant signals were detected in the oocytes of the female and in the spermatocytes of the male suggesting that TK4, among other functions, may play a role in germ cell development. This is an unexpected finding considering that Syk-family TKs of invertebrates and vertebrates described so far are not involved in the differentiation of the gonads.

Amino Acid Sequence↗
PubMed2002

Characterisation of the cysteine protease ER60 in transgenic Schistosoma mansoni larvae.

Proteinases have been found to play important roles in parasites. They are involved in developmental processes and facilitate invasion of host tissues as well as the digestion of host molecules for nutrition. The cysteine protease ER60 from Schistosoma mansoni, originally characterised in adults to be expressed in excretory organs, was analysed in larval stages. Transcripts were found in miracidia, in vitro generated mother sporocysts and cercariae. After cloning the promoter and terminator of the ER60 gene, a transformation vector was constructed containing the green fluorescent protein reporter gene flanked by the regulatory elements. The ER60-green fluorescent protein vector was used for transfection experiments of COS-7 cells demonstrating the functionality of the promoter in the heterologous system. To analyse the expression pattern of ER60-green fluorescent protein in larval S. mansoni, in vitro generated mother sporocysts were transformed by particle bombardment, a method which allows gene transfer into schistosomes. Molecular analyses demonstrated transcription and translation of the transgene. Furthermore, confocal laser scanning microscopy revealed ER60-induced green fluorescent protein fluorescence within the larvae. Inside primary sporocysts, tissue-specific activity was localised in the gland cells, protonephridia and several cytons. These results suggest that ER60 is expressed in the ES system of larvae and, amongst other functions, may play a role in penetration and migration processes.

Animals↗
PubMed2002

HSP70-controlled GFP expression in transiently transformed schistosomes.

Among the parasitic helminths schistosomes are of high medical and economic importance. Despite of the world-wide relevance of this parasite, very little is known about the cellular mechanisms controlling its development and concerning the host-parasite interaction. Within the last decade a great effort has been made in this blood fluke to identify genes which play important roles during these processes. However, molecular analysis was limited by the fact, that neither function nor regulation of candidate genes could be investigated in this organism due to the lack of transformation protocols. Here, we present the strategy of ballistic gene transfer to introduce and characterize transgenes in different schistosome life stages. As a transformation vector, the heat shock protein 70 (hsp70) gene promoter and terminator from Schistosoma mansoni were cloned and fused to the green fluorescent protein (GFP) reporter gene. In a first attempt, the hsp70--GFP vector was successfully tested in a eukaryotic cell line. Thereafter, adult male schistosomes and sporocysts were transformed with this vector, and GFP expression was demonstrated using molecular and microscopical methods. PCR, reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot analyses confirmed the presence, transcription and translation of the transgene in adults. Confocal laser scanning microscopy revealed GFP-activity at various sites along the surface of the worms after hs induction and within sporocysts. These results suggest diverse roles for hsp70 during the development of schistosomes. Furthermore, the results demonstrate the feasibility of this method and open the perspective to analyze a variety of molecular functions in schistosomes.

Animals↗
PubMed2002

Quantification of DNA synthesis in multicellular organisms by a combined DAPI and BrdU technique.

The development of a novel method to detect and quantify mitotic activity in multicellular organisms is reported. The method is based on the combinatorial use of 4',6-diamidino-2-phenylindole (DAPI) as a dye for the specific staining of DNA and the thymidine analog 5-bromo-2'-deoxyuridine (BrdU) as a marker for DNA synthesis. It is shown that on nitrocellulose filters, the amount of DNA can be determined by DAPI as a prerequisite for the subsequent quantification of mitotic activity by BrdU. As a model system to prove the applicability of this technique, the blood fluke Schistosoma mansoni has been used. It is demonstrated that the DNA synthesis rate is higher in adult female schistosomes than in adult males. Furthermore, dimethyl sulfoxide, a widely used solvent for many mitogens and inhibitors of mitosis, has no influence on mitotic activity in adult schistosomes.

Animals↗
PubMedDate not supplied

Schistosomiasis and the molecular biology of the male-female interaction of S. mansoni.

Parasitic helminths of the genus Schistosoma are the causative agents of schistosomiasis, an infectious disease affecting humans and animals. For humans, it is one of the most prevalent parasitemias in the world, second behind malaria. Estimates of the World Health Organisation (WHO) indicate that more than 200 million people live in endemic areas (WHO, Fact Sheet No 115). With respect to animals, a number of 530 million cattle is estimated to live in endemic territories of Africa and Asia. In the last two decades ambitious efforts have been made to develop an effective vaccine against schistosomes, but without resounding success. In addition, there is a pressing need to develop new anthelmintics due to the potential emerging resistance against the commonly used drug praziquantel. Therefore, the understanding of essential physiological or developmental processes of schistosome biology and attempts to intervene in these processes may open new ways to control the parasite. Towards this end, one possibility is to study the unusual biology of schistosomes. These digenean parasites differ from other parasitic flukes by living in the blood vessels. Furthermore, schistosomatids are the only bisexual family of the class trematoda. A nearly unique phenomenon in nature is that a continuous pairing-contact is essential for the development of the reproductive organs of the female, an aspect for the possible design of novel control strategies.

Animals↗