Molecular and cytological analysis of seed dormancy in Arabidopsis thaliana

Seed dormancy is defined as the failure of an intact viable seed to complete germination under favorable conditions. Many higher plants express seed dormancy to optimize the distribution of germination over time in a population of seeds. Seed dormancy can prevent preharvest sprouting as well as the untimely germination during winter. During the last decades several physiological investigations were performed on seed dormancy. But even though the role of plant hormones is known as well as the effect of some chemicals and the environment, the knowledge about the underlying molecular mechanisms remains fragmentary. This thesis describes two approaches to gain more insight in the molecular mechanisms. At the one hand, the chromatin structure during seed maturation and dormancy was investigated. While a difference of chromatin structure of dormant and non-dormant seeds was not measurable, indications were found that embryo nuclei reduce their volume during seed maturation. This could be related with the acquisition of desiccation tolerance during seed maturation. In addition, the gene responsible for the dormancy phenotype of the reduced dormancy 2 (rdo2) mutant was cloned and identified as a TFIIS transcription elongation factor. The encoded protein shows strong conservation with the yeast homologue, but has a plant specific function, as it cannot complement the phenotype of the yeast mutant. In yeast the TFIIS protein is known to interact physically with the RNA polymerase II complex to release it from transcriptional arrest. RDO2 is expressed in all tissues, but highest in seeds. The transcript level increases during seed maturation and reaches a maximum in mature seeds. Stable transformants expressing an RDO2-YFP fusion protein express fluorescence in the nuclei, which is consistent with the annotated function of RDO2 as a TFIIS protein. Quantitative Real Time PCR revealed, that the transcript level of DOG1, a key dormancy gene, is reduced in rdo2 mutant seeds during maturation compared to Ler. This would at least partially explain the reduced dormancy of the rdo2 mutant. RDO2 is the second cloned gene after HUB1 that links transcription regulation to dormancy. It is probable, that during seed maturation, when the water content of the seeds is reduced and the nuclei shrink, transcription elongation becomes less efficient, therefore a transcription elongation factor like RDO2 is increasingly required

Identification of the Arabidopsis REDUCED DORMANCY 2 Gene Uncovers a Role for the Polymerase Associated Factor 1 Complex in Seed Dormancy

The life of a plant is characterized by major phase transitions. This includes the agriculturally important transitions from seed to seedling (germination) and from vegetative to generative growth (flowering induction). In many plant species, including Arabidopsis thaliana, freshly harvested seeds are dormant and incapable of germinating. Germination can occur after the release of dormancy and the occurrence of favourable environmental conditions. Although the hormonal control of seed dormancy is well studied, the molecular mechanisms underlying the induction and release of dormancy are not yet understood

Non-pathogenic trypanosomatid protozoa as a platform for protein research and production

All currently existing eukaryotic protein expression systems are based on autonomous life forms. To exploit the potential practical benefits associated with parasitic organisms we have developed a new protein expression system based on Leishmania tarentolae (Trypanosomatidae), a protozoan parasite of lizards. To achieve strong transcription, the genes of interest were integrated into the small subunit ribosomal RNA gene. Expression levels obtained were up to 30mg of recombinant protein per liter of suspension culture and increased linearly with the number of integrated gene copies. To assess the system's potential for production of post-translationally modified proteins, we have expressed human erythropoietin in L. tarentolae. The recombinant protein isolated from the culture supernatants was biologically active, natively processed at the N-terminus, and N-glycosylated. The N-glycosylation was exceptionally homogenous, with a mammalian-type biantennary oligosaccharide and the Man(3)GlcNAc(2) core structure accounting for >90% of the glycans present. L. tarentolae is thus the first described biotechnologically useful unicellular eukaryotic organism producing biantennary fully galactosylated, core-alpha-1,6-fucosylated N-glycans. (C) 2002 Elsevier Science (USA). All rights reserved

Non-pathogenic trypanosomatid protozoa as a platform for protein research and production

All currently existing eukaryotic protein expression systems are based on autonomous life forms. To exploit the potential practical benefits associated with parasitic organisms we have developed a new protein expression system based on Leishmania tarentolae (Trypanosomatidae), a protozoan parasite of lizards. To achieve strong transcription, the genes of interest were integrated into the small subunit ribosomal RNA gene. Expression levels obtained were up to 30mg of recombinant protein per liter of suspension culture and increased linearly with the number of integrated gene copies. To assess the system's potential for production of post-translationally modified proteins, we have expressed human erythropoietin in L. tarentolae. The recombinant protein isolated from the culture supernatants was biologically active, natively processed at the N-terminus, and N-glycosylated. The N-glycosylation was exceptionally homogenous, with a mammalian-type biantennary oligosaccharide and the Man(3)GlcNAc(2) core structure accounting for >90% of the glycans present. L. tarentolae is thus the first described biotechnologically useful unicellular eukaryotic organism producing biantennary fully galactosylated, core-alpha-1,6-fucosylated N-glycans. (C) 2002 Elsevier Science (USA). All rights reserved

Evaluation of the Diagnostic Value of Contrast-Enhanced Voiding Urosonography with Regard to the Further Therapy Regime and Patient Outcome—A Single-Center Experience in an Interdisciplinary Uroradiological Setting

Background and Objectives: Vesicoureteral reflux (VUR) describes a common pediatric anomaly in pediatric urology with a prevalence of 1–2%. In diagnostics, in addition to the gold standard of voiding cystourethrography (VCUG), contrast-enhanced urosonography (ceVUS) offers a radiation-free procedure, which, despite its advantages, is not yet widely used. In the present single-center study, subsequent therapeutic procedures and outcomes after ceVUS of 49 patients were investigated. The aim of the study is to investigate the efficacy of ceVUS with the intention of broader clinical implementation. Materials and Methods: Between 2016 and 2020, 49 patients were retrospectively included and received a ceVUS to evaluate VUR. With a distribution of 47:2 (95.9%), a clear female predominance was present. The age of the patients varied between 5 months and 60 years at the time of ceVUS. All examinations were all performed and subsequently interpreted by a single experienced radiologist (EFSUMB level 3). Results: Compared to intraoperative findings, ceVUS shows a sensitivity of 95.7% with a specificity of 100%. Allergic reactions to the contrast medium could not be observed. Conclusion: With its high sensitivity and intraoperative validation, ceVUS offers an excellent alternative to VCUG, the gold standard in the diagnosis of VUR. In addition, ceVUS is a radiation-free examination method with a low risk profile that offers an exceptional diagnostic tool in the diagnostic clarification of recurrent urinary tract infections with the suspected diagnosis of VUR and should also be included in the consideration of a diagnosis next to the established VCUG, especially in younger children

Evaluation of the Diagnostic Value of Contrast-Enhanced Voiding Urosonography with Regard to the Further Therapy Regime and Patient Outcome—A Single-Center Experience in an Interdisciplinary Uroradiological Setting

Background and Objectives: Vesicoureteral reflux (VUR) describes a common pediatric anomaly in pediatric urology with a prevalence of 1–2%. In diagnostics, in addition to the gold standard of voiding cystourethrography (VCUG), contrast-enhanced urosonography (ceVUS) offers a radiation-free procedure, which, despite its advantages, is not yet widely used. In the present single-center study, subsequent therapeutic procedures and outcomes after ceVUS of 49 patients were investigated. The aim of the study is to investigate the efficacy of ceVUS with the intention of broader clinical implementation. Materials and Methods: Between 2016 and 2020, 49 patients were retrospectively included and received a ceVUS to evaluate VUR. With a distribution of 47:2 (95.9%), a clear female predominance was present. The age of the patients varied between 5 months and 60 years at the time of ceVUS. All examinations were all performed and subsequently interpreted by a single experienced radiologist (EFSUMB level 3). Results: Compared to intraoperative findings, ceVUS shows a sensitivity of 95.7% with a specificity of 100%. Allergic reactions to the contrast medium could not be observed. Conclusion: With its high sensitivity and intraoperative validation, ceVUS offers an excellent alternative to VCUG, the gold standard in the diagnosis of VUR. In addition, ceVUS is a radiation-free examination method with a low risk profile that offers an exceptional diagnostic tool in the diagnostic clarification of recurrent urinary tract infections with the suspected diagnosis of VUR and should also be included in the consideration of a diagnosis next to the established VCUG, especially in younger children

Tyrosine 146 of the Human Na⁺/Taurocholate Cotransporting Polypeptide (NTCP) Is Essential for Its Hepatitis B Virus (HBV) Receptor Function and HBV Entry into Hepatocytes

Na+/taurocholate cotransporting polypeptide (NTCP, gene symbol SLC10A1) is a hepatic bile acid uptake carrier participating in the enterohepatic circulation of bile acids. Apart from its transporter function, NTCP acts as the high-affinity liver-specific receptor for the hepatitis B virus (HBV), which attaches via its preS1-peptide domain of the large surface protein to NTCP, subsequently leading to endocytosis of the virus/NTCP-receptor complex. Although the process of NTCP-dependent HBV infection of hepatocytes has received much attention over the last decade, the precise molecular sites of the virus/NTCP interaction have not been fully identified. Inspection of the primary protein sequence of human NTCP revealed 139YIYSRGIY146 as a highly conserved tyrosine-rich motif. To study the role of Y139, Y141 and Y146 amino acids in NTCP biology, the aforementioned residues were substituted with alanine, phenylalanine or glutamate (mimicking phosphorylation) using site-directed mutagenesis. Similar to wt NTCP, the Y139A, Y141A, Y146A, Y141F, Y146F, and Y146E mutants were expressed at the plasma membrane of HEK293 cells and exhibited intact bile acid transport function. Y146A, Y146E, and Y146F demonstrated transport kinetics comparable to wild-type NTCP with Km values of 57.3–112.4 µM and Vmax values of 6683–7579 pmol/mg protein/min. Only Y141E was transport deficient, most likely due to an intracellular accumulation of the mutant protein. Most importantly, Y146A and Y146E mutation completely abrogated binding of the viral preS1-peptide to NTCP, while the Y146F mutant of NTCP showed some residual binding competence for preS1. Consequently, the NTCP mutants Y146A and Y146E, when expressed in HepG2 hepatoma cells, showed complete loss of susceptibility for in vitro HBV infection. In conclusion, tyrosine 146, and to some extent tyrosine 141, both belonging to the tyrosine-rich motif 139YIYSRGIY146 of human NTCP, are newly identified amino acid residues that play an essential role in the interaction of HBV with its receptor NTCP and, thus, in the process of virus entry into hepatocytes