The homeostatic mechanism for the respiratory system involves the tight control of expression of several gene products. One such gene is a member of the multi-substrate oxidase system called cytochrome P450 (P450). Pulmonary P450s may be involved in the tissue-specific detoxification, and/or bio-activation of chemical agents. Additionally, pulmonary cytochrome P450s, particularly the human iso-form 4B1, could play an important role in the development and homeostasis of the pulmonary tissue by removing androgens that cause deleterious effects on lung maturation. Human cytochrome P450 4B1 (CYP 4B1) is a predominant cytochrome P450 activity in the lung. CYP 4B1 protein is reported to bio-activate some pulmonary toxicants. Therefore it may mediate chemical-induced damage to that organ. However, the high expression levels of CYP 4B1 gene products in the lung (approximately 80% of the total P450 activity) may suggest the involvement of the iso-form in normal physiological functions for pulmonary tissue. On the other hand, the results of this and other laboratories have shown an apparent species-specific difference in the catalytic activity of 4B1 protein in man versus rodents. For instance, the rodent iso-form is potent at activating pro-carcinogens and devoid of androgen hydrolyzing activities towards androgen metabolism. The human 4B1 protein has higher 6-beta-testosterone hydroxylase activity while the rodent iso-form is devoid of this action. Furthermore, the manner in which the 4B1 gene is regulated appears to be species-specific. Therefore, my studies have been directed toward further defining the genetic mechanism of action that regulates the human 4B1 gene expression levels. My research objectives have been divided into three specific aims: to determine the presence or absence of cis-acting elements located in the 3.0 Kbp upstream fragment of the human 4B1 gene; to determine the DNA structure of the 4.5 Kbp fragment that overlaps and lies further upstream to the 3.0 Kbp regulatory region of the human 4B1 gene; and to determine the actions of a contiguous 6.5 Kbp fragment containing the entire putative upstream region of 4B1 gene on transcription of the luciferase reporter gene in human lung cells. The results of my studies show the presence of multiple promoter elements in addition to the TATA-box that are contained in the CYP 4B 13.0 Kbp upstream fragment. Nucleotide sequence analysis of the 4.5 Kbp upstream fragment that contains 1.0 Kbp overlapping fragment with the 3.0 Kbp fragment based on restriction mapping confirms that it is indeed part of the contiguous region of the CYP 4B1 gene. A number of consensus DNA regulatory motifs have been identified. (Abstract shortened by UMI.)

Phagocytes, neutrophils and macrophages, are part of the non-specific host defense system in control bacterial infection by the phagocytosis and killing of these microbes through free radical-dependent and free radical-independent mechanisms. In contrast, endothelial cells, in addition to their role in maintenance of homeostasis, have classically been perceived to play a supportive role in host immune response by releasing chemoattractants that recruit phagocytes to the site of infection. Recent studies have, however, demonstrated that the endothelium is capable of responding to stimulation by cytokines as part of their activation responses in host immunity. This dissertation explores the role of endothelia as effector cells in host response. The findings from this dissertation demonstrated that endothelial cells cultured on three dimensional GelfoamRTM are activated by penicillin G to phagocytosis and kill S. aureus. Even though O2-· and NO· are known to exhibit microbicidal activity, it appears that these free radicals do not play an integral part in the observed killing of S. aureus. To further investigate the host defense role of NO·, primary cultures of endothelial cells were transduced with retroviral vector encoding NOS II gene. Upon transduction, these cells released NO· at a constant flux over a long period of time. This provided an excellent model to study the antimicrobial activity of NO· at cellular fluxes without the complications of controlling the rate of NO· from a NO·-releasing compound or those associated with cytokine treatment. When infected with either S. aureus or E. coli, NOS II transduced endothelial cells, producing NO·, phagocytosed both bacteria. However, only E. coli was sensitive to NO· dependent bacterial killing. Taken together, these studies reveal the antimicrobial roles played by endothelial cells upon activation. The mechanisms utilized by these cells include both free radical-dependent and free radical-independent pathways.

The concept that many enzymes and factors involved in mammalian DNA replication function together as an organized multiprotein complex has been supported by an ever-increasing body of evidence. In this study, a discrete high molecular weight multiprotein complex containing DNA polymerase alpha was identified by a native Western blotting technique. An enrichment of this complex was seen at each step in its purification. The integrity of this complex was maintained after ion-exchange chromatography and sucrose gradients sedimentation. We have designated this complex the DNA synthesome. The DNA synthesome was further purified by electroeluting this complex from a native polyacrylamide gel. We have verified that the electroeluted synthesome is capable to support all aspects of DNA replication in vitro. Furthermore, SDS-PAGE analysis of the electroeluted DNA synthesome revealed the presence of at least 25 major polypeptides with molecular weights ranging from 20 kD to 240 kD. In addition, using Western blot and enzymatic analysis, we have shown that this complex contains replication essential proteins DNA polymerase delta, proliferating cell nuclear antigen (PCNA), replication protein A (RP-A), and topoisomerases I and II. Taken together, our evidence suggests that the DNA synthesome represents the fundamental DNA replication unit the human cell. We also validated the use of the synthesome as an in vitro model for studying mechanism of action of anticancer drugs that directly affect cellular DNA synthesis. The effects of gemcitabine (dFdC) and araC on in vitro SV40 DNA synthesis mediated by the DNA synthesome was compared with that of on intact cell DNA synthesis. Our results showed that dFdC is a more potent inhibitor of intact cell DNA synthesis and in vitro SV40 DNA replication than araC. Although dFdCTP is more potent than araCTP at inhibiting in vitro SV40 DNA synthesis, there is no significant difference between the inhibitory effect of these two drugs on the activity of the MCF7 synthesome associated DNA polymerases alpha and delta. Our results also suggested that the decrease in the synthesome mediated in vitro SV40 DNA synthesis by dFdCTP and araCTP is primarily through inhibition of the synthesome associated DNA polymerase alpha activity.