A porphyrin dye with monoexponential fluorescence intensity and anisotropy decay behavior in spherical micelles.
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Journal Article↗
Biomedical subjects
Werner Kunz
Publications and source records attributed to Werner Kunz.
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↗
Micelle and solvent relaxation in aqueous sodium dodecylsulfate solutions.
Dielectric spectra have been measured for aqueous sodium dodecylsulfate (SDS) solutions up to 0.1 mol L-1 at 25 degrees C over the frequency range 0.005 < or = nu GHz-1 < or = 89. The spectra exhibit two relaxation processes at approximately 0.03 GHz and 0.2 GHz associated with the presence of micelles in addition to the dominant solvent relaxation process at approximately 18 GHz and a small contribution at approximately 1.8 GHz due to H2O molecules hydrating the micelles. Detailed analysis reveals that the micelles bind 20 water molecules per SDS unit, but not as strongly as trimethylalkylammonium halide surfactants do. The relaxation times and amplitudes of both micelle relaxation processes can be simultaneously analysed with the theory of Grosse, yielding the effective volume of a SDS unit in the micelle and the lateral diffusion coefficient of the bound counterions. The findings of this investigation fully corroborate recent molecular dynamics simulations on structure and dynamics of SDS micelles.
Journal Article↗
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↗
Biooxidation of n-hexanol by alcohol oxidase and catalase in biphasic and micellar systems without solvent.
Alcohol oxidase from Pichia pastoris together with catalase from bovine liver was used to oxidize n-hexanol to hexanal. For this purpose, an aqueous buffer solution was mixed with large amounts of hexanol by simple agitation, yielding a biphasic system, or by adding the nonionic surfactant Brij 35. Initial velocities and reaction yields after 24 h were measured as a function of various parameters such as the amounts of enzymes, hexanol, or surfactant. High enzymatic activity was determined for hexanol concentrations of between 20 mass% and 80 mass% without using any additional organic solvent. The homogenization of the biphasic systems with the help of Brij 35 did not yield a significant improvement of the bioconversion, which would justify the use of surfactants.
Alcohol Oxidoreductases↗
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↗
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↗
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↗
When is a parasite species a species?
Regrettably, 140 years after the publication of Darwin's Origin of Species, we face the grotesque situation that we still do not know what is a species whose origin Darwin wanted to explain. A generally applicable species definition is not available. Is there a basic unit of biodiversity above the level of individuals? Do we try to define something that does not exist in reality? The strong potential for the evolution of genetic variability in parasites together with the importance of species diagnosis for applied fields of parasite research make biodiversity research a key role in parasitology. Frequent occurrence of sympatric speciation, clonal reproduction, selfing, sib mating or parthenogenesis imply exceptional conditions for the evolution of gene pool diversities in parasites.
Animals↗
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↗