In sea urchin development, cell fate specification appears by the 60-cell stage embryo when several embryonic territories are recognized: the small micromeres, the large micromeres which will generate primary mesenchyme cells, the vegetal2 layer that will give rise to pigment cells, immunocytes, and muscle cells, the vegetal1 layer, as well as the oral and aboral ectoderm. A Delta-Notch signaling event is required for the differential specification of mesodermal cells that will give rise to secondary mesenchyme cells (SMCs). SMCs produce four cell types: pigment cells, blastocoelar cells, circumesophageal muscle cells, and coelomic pouch cells. Pigment cells are the first to be specified. During primary invagination at the gastrula stage, eight pigment cell progenitors delaminate from the archenteron into the blastocoel. By the pluteus stage, approximately 30 pigment cells are embedded in the ectoderm. Pigment cells produce echinochrome, a napthoquinone pigment. Previously, several genes in the sea urchin embryo were isolated that are expressed specifically in pigment cell precursors during the blastula stage. The goal of this research was to characterize a subset of these genes, which are highly similar to: the polyketide synthase gene (Pks), a sulfotransferase gene (Sult), three different members of the flavin-containing monooxygenase gene family (Fmo), and the transcription factor glial cells missing (Gcm). Polyketide synthases (PKSs) are a large family of multifunctional proteins mainly found in bacteria, fungi, and plants. They are responsible for the biosynthesis of a variety of polyketide compounds including antibiotics and mycotoxins. In the sea urchin, SpPks is required for echinochrome biosynthesis. Flavin-containing monooxygenases (FMOs) are NADPH-dependent flavoproteins mainly found in bacteria, plants, and higher metazoan. They are responsible for catalyzing the oxidation of several compounds including the detoxification of xenobiotics and activation of numerous metabolites. It is known that SpFmo1 is required for echinochrome biosynthesis. Sulfotransferases are found from bacteria through higher eukaryotes. These enzymes catalyze the sulfate conjugation of several substrates resulting in either compound detoxification or bioactivation.

In recent years, tyrosine kinase inhibitors have been widely used for the treatment of certain tumors as so-called targeted therapy. Many studies are concerned with their metabolism and the role of enzymes in the biotransformation process, but very little is known about the impact of tyrosine kinase inhibitors on the expression and activity of biotransformation enzymes. Nevertheless modification of the expression and activity of enzymes may cause adverse interactions of co-administered drugs and their negative impact on the human body. This diploma thesis studies the effect of tyrosine kinase inhibitors vandetanib and lenvatinib and cytotoxic alkaloid ellipticine on biotransformation enzymes in a rat model organism in vivo. The aim was to characterize the effect of the investigated compounds on gene expression, protein expression and activity of cytochromes P450 (CYP) 1A1, 1A2 and 1B1 and flavin-containing monooxygenases FMO1 and FMO3 in renal and hepatic microsomes. Microsomes and RNA were isolated from kidneys of control rats and the pretreated rats. Western blot and immunodetection was used to compare the protein expression levels of studied enzymes in kidney and liver. By reverse transcription, cDNA was prepared from isolated RNA and used as a template for quantitative PCR to compare the...

The flavin-containing monooxygenases (FMO / E.C.1.14.13.8) are microsomal NADPH and oxygen-dependent flavoprotein enzymes that catalyze the oxidation of a wide variety of xenobiotics, including drugs and environmental toxicants. Nucleophiles containing nitrogen, sulfur, phosphorus and selenium heteroatoms are the substrates of FMO. Bovine liver microsomal FMO enzyme activity was characterized using methimazole as substrate, which is a highly specific substrate for FMO. From 12 different bovine liver samples, microsomes were prepared and the average specific activity of bovine liver microsomal FMO was found to be 2.37 &amp / #61617 / 0.30 nmol/min/mg (Mean &amp / #61617 / SE, n=12). The rate of reaction was linear up to 0.5 mg of bovine liver microsomal protein. The maximum FMO enzyme activity was detected at 37 &amp / #61616 / C and at pH 8.0. Effects of detergents / Triton X-100 and Emulgen 913, on FMO activity were determined and found that enzyme activity increased by the addition of either detergent at all concentrations (0.1%-1.0%). The apparent Vmax and Km values of bovine liver microsomal FMO for methimazole substrate were found as 1.23 nmol/min/mg and 0.11 mM, respectively. Thermostability of bovine liver microsomal FMO was studied at four different temperatures / 24 &amp / #61616 / C, 37 &amp / #61616 / C, 50 &amp / #61616 / C and 65 &amp / #61616 / C. The incubation time required for the complete loss of enzyme activity was 5 minutes at 65 &amp / #61616 / C, 10 minutes at 50 &amp / #61616 / C and 6.5 hours at 37 &amp / #61616 / C. 68 % of the activity was still detectable at the end of 53 hours at 24 &amp / #61616 / C. Bovine liver microsomal activity towards two drug substrates, imipramine and chlorpromazine, was also determined and found to be 3.73 and 3.75 nmol NADPH oxidized/min/mg, respectively. Effects of two drug substrates, imipramine and chlorpromazine, on bovine liver microsomal FMO-catalyzed methimazole oxidation activity was also studied and found that they inhibit FMO activity at all concentrations studied. Modulation of bovine liver microsomal FMO activity was studied using three different heavy metal ions / Ni+2, Cd+2 and Hg+2. At all other concentrations studied for each heavy metal ion and at all substrate methimazole concentrations (0.1, 0.2, 0.5, 1.0 mM), FMO-catalyzed methimazole oxidation activity decreased compared to control activity. KI values for Ni+2, Cd+2 and Hg+2 were found to be 0.5 mM, 0.085 mM, 4.6 &amp / #61549 / M, respectively. From the Dixon plot, the pattern of inhibition for three heavy metal ions was observed to be noncompetitive.