Project description:Organoids were established from patients with ovarian cancer. DNA was extracted from organoids and primary tissue to investigate whether organoids retain the same genomic abnormalities and disease-associated features.
Project description:Organoids were established from patients with ovarian cancer. DNA was extracted from organoids and primary tissue to investigate whether organoids retain the same genomic abnormalities and disease-associated features.
Project description:Tissue for normal colonic stem cell isolation was obtained via colectomy from a colorectal cancer patient. It was shown that the resected tissue and the in vitro-cultured organoids grown from tissue-derived adult stem cells do not harbor chromosomal alterations. For that purpose, genomic DNA from resected mucosa or genomic DNA from adult stem cell-derived organoids grown in a 3-dimensional culture system was compared to white blood cell genomic DNA obtained from the same individual. Furthermore, in vitro organoids which were cultured and serially passaged for several weeks did not acquire chromosomal alterations. Analysis of chromosomal stability of colonic tissue or in vitro-cultured organoids via comparison with blood cells derived from the same individual.
Project description:A total of 3 independently derived organoids were compared to patient tumor samples and patient normal samples to determine if the tumor-derived organoids represent the genomic landscape of the tumor from which they were derived.
Project description:Kynureninase is a member of a large family of catalytically diverse but structurally homologous pyridoxal 5'-phosphate (PLP) dependent enzymes known as the aspartate aminotransferase superfamily or alpha-family. The Homo sapiens and other eukaryotic constitutive kynureninases preferentially catalyze the hydrolytic cleavage of 3-hydroxy-l-kynurenine to produce 3-hydroxyanthranilate and l-alanine, while l-kynurenine is the substrate of many prokaryotic inducible kynureninases. The human enzyme was cloned with an N-terminal hexahistidine tag, expressed, and purified from a bacterial expression system using Ni metal ion affinity chromatography. Kinetic characterization of the recombinant enzyme reveals classic Michaelis-Menten behavior, with a Km of 28.3 +/- 1.9 microM and a specific activity of 1.75 micromol min-1 mg-1 for 3-hydroxy-dl-kynurenine. Crystals of recombinant kynureninase that diffracted to 2.0 A were obtained, and the atomic structure of the PLP-bound holoenzyme was determined by molecular replacement using the Pseudomonas fluorescens kynureninase structure (PDB entry 1qz9) as the phasing model. A structural superposition with the P. fluorescens kynureninase revealed that these two structures resemble the "open" and "closed" conformations of aspartate aminotransferase. The comparison illustrates the dynamic nature of these proteins' small domains and reveals a role for Arg-434 similar to its role in other AAT alpha-family members. Docking of 3-hydroxy-l-kynurenine into the human kynureninase active site suggests that Asn-333 and His-102 are involved in substrate binding and molecular discrimination between inducible and constitutive kynureninase substrates.