The functional and structural interactions between v-{\it mos\/} proteins and cell cycle control elements

The v-mos gene of Moloney murine sarcoma virus (Mo-MuSv) encodes a serine/threonine protein kinase capable of inducing cellular transformation. The c-mos protein is an important cell cycle regulator that functions during meiotic cell division cycles in germ cells. The overall function of c-mos in controlling meiosis is becoming better understood but the role of v-mos in malignant transformation of cells is largely unknown.^ In this study, v-mos protein was shown to be phosphorylated by M phase kinase in vitro and in vivo. The kinase activity and neoplastic transforming ability of v-mos is positively regulated by the phosphorylation. Together with the earlier finding of activation of M phase kinase by c-mos, these results raise the possibility of mutual regulation between M phase kinase and mos kinases.^ In addition to its functional interaction with the M phase kinase, the v-mos protein was shown to be present in the same protein complex with a cyclin-dependent kinase (cdk). In addition, an antibody that recognizes the cdk proteins was shown to co-precipitate the v-mos proteins in the interphase and mitotic cells transformed by p85$\sp{\rm gag-mos}$. Cdk proteins have been shown to be associated with nonmitotic cyclins which are potential oncogenes. The perturbation of cdk kinase or the activation of non-mitotic cyclins as oncogenes by v-mos could contribute directly to v-mos induced cellular transformation. v-mos proteins were also shown to interact with tubulin and vimentin, the essential components of microtubules and type IV intermediate filaments, respectively. The organizations of both microtubules and intermediate filaments are cell cycle-regulated. These results suggest that the v-mos kinase could be directly involved in inducing morphological changes typically seen in transformed cells.^ The interactions between the v-mos protein and these cell cycle control elements in regards to v-mos induced neoplastic transformation are discussed in detail in the text. ^

Molecular mechanisms of prostatic carcinoma growth and progression

Prostate cancer represents the most commonly diagnosed malignancies in American men and is the second leading cause of male cancer deaths. The overall objectives of this research were designed to understand the cellular and molecular mechanisms of prostatic carcinoma growth and progression. This dissertation was divided into two major parts: (1) to clone and characterize soluble factor(s) associated with bone that may mediate prostatic carcinoma growth and progression; (2) to investigate the roles of extracellular matrix in prostatic carcinogenesis.^ The propensity of prostate cancer cells to metastasize to the axial skeleton and the subsequent osteoblastic reactions observed in the bone indicate the possible reciprocal cellular interaction between prostate cancer cells and the bone microenvironment. To understand the molecular and cellular basis of this interaction, I focused on the identification and cloning of soluble factor(s) from bone stromal cells that may exert direct mitogenic action on cultured prostate cells. A novel BPGF-1 gene expressed specifically by bone and male accessory sex organs (prostate, seminal vesicles, and coagulating gland) was identified and cloned.^ The BPGF-1 was identified and cloned from a cDNA expression library prepared from a human bone stromal cell line, MS. The conditioned medium (CM) of this cell line contains mitogenic materials that induce human prostate cancer cell growth both in vivo and in vitro. The cDNA expression library was screened by an antibody prepared against the mitogenic fraction of the CM.^ The cloned BPGF-1 cDNA comprises 3171 nucleotides with a single open reading frame of 1620 nucleotides encoding 540 amino acids. The BPGF-1 gene encodes two transcripts (3.3 and 2.5 kb) with approximately equal intensity in human cells and tissues, but only one transcript (2.5 kb) in rat and mouse tissues. Southern blot analysis of human genomic DNA revealed a single BPGF-1 gene. The BPGF-1 gene is expressed predominantly in bone and seminal vesicles, but at a substantially lower level in prostate. Polyclonal antibodies generated from synthetic peptides that correspond to the nucleotide sequences of the cloned BPGF-1 cDNA reacted with a putative BPGF-1 protein with an apparent molecular weight of 70 kDa. The conditioned media isolated from COS cells transfected with BPGF-1 cDNA stimulated the proliferation and increased the anchorage-independent growth of prostate epithelial cells. These findings led us to hypothesize that BPGF-1 expression in relevant organs, such as prostate, seminal vesicles, and bone, may lead to local prostate cancer growth, metastasis to the seminal vesicles, and subsequently dissemination to the skeleton.^ To assess the importance of extracellular matrix in prostatic carcinogenesis, the role of extracellular matrix in induction of rat prostatic carcinoma growth in vivo was evaluated. NbE-1, a nontumorigenic rat prostatic epithelial cell line, was induced to form carcinoma in athymic nude hosts by coinjecting them with Matrigel and selected extracellular matrix components. Induction of prostatic tumor formation by laminin and collagen IV was inhibited by their respective antibodies. Prostatic epithelial cells cloned from the tumor tissues were found to form tumors in athymic nude hosts in the absence of exogenously added extracellular matrix. These results suggest that extracellular matrix induce irreversibly prostatic epithelial cells that behave distinctively different from the parental prostatic epithelial cell line. ^

LINKAGE RELATIONSHIPS AMONG PROTEIN-CODING LOCI IN FISHES OF THE GENUS XIPHOPHORUS

Inbred strains of three species of fishes of the genus Xiphophorus (platyfish and swordtails) were crossed to produce intra- and interspecific F(,1) hybrids, which were then backcrossed to one or both parental stocks. Backcross hybrids were used for the analysis of segregation and linkage of 33 protein-coding loci (whose products were visualized by starch gel electrophoresis) and a sex-linked pigment pattern gene. Segregation was Mendelian for all loci with the exception of one instance of segregation distortion. Six linkage groups of enzyme-coding loci were established: LG I, ADA --6%-- G(,6)PD --24%-- 6PGD; LG II, Est-2 --27%-- Est-3 --0%-- Est-5 --23%-- LDH-1 --16%-- MPI; LG III, AcPh --38%-- G(,3)PD-1 (GUK-2 --14%-- G(,3)PD-1 is also in LG III, but the position of GUK-2 with respect to AcPh has not yet been determined); LG IV, GPI-1 --41%-- IDH-1; LG V, Est-1 --38%-- MDH-2; and LG VI, P1P --7%-- UMPK-1 (P1P is a plasma protein, very probably transferrin).^ Sex-specific recombination appeared absent in LG II and LG IV locus pairs; significantly higher male recombination was demonstrated in LG I but significantly higher female recombination was detected in LG V. Only one significant population-specific difference in recombination was detected, in the G(,6)PD - 6PGD region of LG I; the notable absence of such effects implies close correspondence of the genomes of the species used in the study. Two cases of possible evolutionary conservation of linkage groups in fishes and mammals were described, involving the G(,6)PD - 6PGD linkage in LG I and the cluster of esterase loci in LG II. One clear case of divergence was observed, that of the linkage of ADA in LG I. It was estimated that a minimum of (TURN)50% of the Xiphophorus genome was marked by the loci studied. Therefore, the prior probability that a new locus will assort independently from the markers already established is estimated to be less than 0.5. A maximum of 21 of the 24 pairs of chromosomes could be marked with at least one locus.^ Only the two LG V loci showed a significant association with a postulated gene controlling the severity of a genetically controlled melanoma caused by abnormal proliferation of macromelanophore pigment pattern cells. The independence of melanotic severity from all other informative markers implies that one or at most a few major genes are involved in control of melanotic severity in this system. ^

AGROBACTERIUM VIRB10 CONTRIBUTIONS TO TYPE IV SUBSTRATE SECRETION, T-PILUS BIOGENESIS, AND OUTER MEMBRANE PORE FORMATION

The VirB/D4 type IV secretion system (T4SS) of Agrobacterium tumefaciens functions to transfer substrates to infected plant cells through assembly of a translocation channel and a surface structure termed a T-pilus. This thesis is focused on identifying contributions of VirB10 to substrate transfer and T-pilus formation through a mutational analysis. VirB10 is a bitopic protein with several domains, including a: (i) cytoplasmic N-terminus, (ii) single transmembrane (TM) α-helix, (iii) proline-rich region (PRR), and (iv) large C-terminal modified β-barrel. I introduced cysteine insertion and substitution mutations throughout the length of VirB10 in order to: (i) test a predicted transmembrane topology, (ii) identify residues/domains contributing to VirB10 stability, oligomerization, and function, and (iii) monitor structural changes accompanying energy activation or substrate translocation. These studies were aided by recent structural resolution of a periplasmic domain of a VirB10 homolog and a ‘core’ complex composed of homologs of VirB10 and two outer membrane associated subunits, VirB7 and VirB9. By use of the substituted cysteine accessibility method (SCAM), I confirmed the bitopic topology of VirB10. Through phenotypic studies of Ala-Cys insertion mutations, I identified “uncoupling” mutations in the TM and β-barrel domains that blocked T-pilus assembly but permitted substrate transfer. I showed that cysteine replacements in the C-terminal periplasmic domain yielded a variety of phenotypes in relation to protein accumulation, oligomerization, substrate transfer, and T-pilus formation. By SCAM, I also gained further evidence that VirB10 adopts different structural states during machine biogenesis. Finally, I showed that VirB10 supports substrate transfer even when its TM domain is extensively mutagenized or substituted with heterologous TM domains. By contrast, specific residues most probably involved in oligomerization of the TM domain are required for biogenesis of the T-pilus.

Barriers and facilitators to nurses' acceptance of and implications for injury rates of an IV catheter safety device

This study has the purpose of determining the barriers and facilitators to nurses' acceptance of the Johnson and Johnson Protectiv®* Plus IV catheter safety needle device and implications for needlestick injuries at St. Luke's Episcopal Hospital, Houston, Texas. A one-time cross-sectional survey of 620 responding nurses was conducted by this researcher during December, 2000. The study objectives were to: (1) describe the perceived (a) organizational and individual barriers and facilitators and (b) acceptance of implementation of the IV catheter device; (2) examine the relative importance of these predictors; (3) describe (a) perceived changes in needlestick injuries after implementation of the device; (b) the reported incidence of injuries; and (c) the extent of underreporting by nurses; and (4) examine the relative importance of (a) the preceding predictors and (b) acceptance of the device in predicting perceived changes in needlestick injuries. Safety climate and training were evaluated as organizational factors. Individual factors evaluated were experience with the device, including time using it and frequency of use, and background information, including nursing unit, and length of time as a nurse in this hospital and in total nursing career. The conceptual framework was based upon the safety climate model. Descriptive statistics and multiple and logistic regression were utilized to address the study objectives. ^ The findings showed widespread acceptance of the device and a strong perception that it reduced the number of needlesticks. Acceptance was notably predicted by adequate training, appropriate time between training and device use, solid safety climate, and short length of service, in that order. A barrier to acceptance was nurses' longtime of use of previous needle technologies. Over four-fifths of nurses were compliant in always using the device. Compliance had two facilitators: length of time using device and, to a lesser extent, safety climate. Rates of compliance tended to be lower among nurses in units in which the device was frequently used. ^ High quality training and an atmosphere of caring about nurse safety stand out as primary facilitators that other institutions would need to adopt in order to achieve maximum success in implementing safety programs involving utilization of new safety devices. ^

Polybrominated diphenyl ethers: Potential human exposure through household dust and dryer lint

This MPH thesis consists of (1) literature review of the relatively new synthetic persistent organic pollutants (POP), polybrominated diphenyl ethers (PBDEs), a type of flame retardant posing a potential public health hazard, (2) Presentation of data on PBDE levels in dryer lint from Dallas, TX and Hamburg, Germany. ^ PBDEs are used as additive fire retardants in plastics, polyurethane foam and electronic equipment to reduce flammability and thus save life and property. PBDEs have been widely used beginning in the 1970s. They resemble polychlorinated biphenyls (PCBs) in structure and toxicity. PBDEs are found in environmental sediments, sludges, and wildlife and even in human blood, milk and tissues. ^ PBDEs, due to their lipophilicity, accumulate in fat and other tissues and biomagnify up the food chain, with increasing concentrations. Animal studies have suggested potential health effects including thyroid disruption, permanent learning and memory impairment, fetal malformations, developmental neurotoxicity and, at high doses, possibly cancer. ^ PBDE levels are increasing in blood and breast milk in North America, but PBDEs intake unlike PCBs appears to be not primarily through food; food PBDE levels in the U.S. are not markedly higher than in Europe yet U.S. human blood and milk levels are much higher. For this reason various exposure pathways including PBDE contaminated dust and air have been studied to better characterize routes of PBDE intake into humans. ^ The scientific literature on PBDE levels in household dust reports higher PBDE concentration in dust than that found in dryer lint; levels in the U.S are elevated compared to other countries with congeners such as BDE 47, 99, 100 and 209 predominating. The United Kingdom has elevated BDE 209 due to high usage of Deca commercial mixture. These studies suggest that indoor PBDE contamination through household dust could be a potential source of PBDE exposure and body burden especially in young children. ^ PBDE levels in dryer lint from U.S ranged from 321 to 3073 ng/g (Mean: 1138 ng/g, Median: 803 ng/g) and from Germany were from 330 to 2069 ng/g (Median: 71ng/g, Mean: 361 ng/g). High median levels in U.S samples indicate contamination of lint with PBDEs although the source of the PBDEs in lint may be from dryer electrical components or air deposition onto clothes, lint may be one source of PBDE exposure to humans. ^

Dietary intake patterns and relationships to polybrominated diphenyl ether (PBDE) and phthalate body burden

Polybrominated diphenyl ethers (PBDEs) and phthalates are chemicals of concern because of high levels measured in people and the environment as well as the demonstrated toxicity in animal studies and limited epidemiological studies. Exposure to these chemicals has been associated with a range of toxicological outcomes, including developmental effects, behavioral changes, endocrine disruption, effects on sexual health, and cancer. Previous research has shown that both of these classes of chemicals contaminate food in the United States and worldwide. However, how large a role diet plays in exposure to these chemicals is currently unknown. To address this question, an exploratory analysis of data collected as part of the 2003-04 National Health and Nutrition Examination Survey (NHANES) was conducted. Associations between dietary intake (assessed by 24-hour dietary recalls) for a range of food types (meat, poultry, fish, and dairy) and levels PBDEs and phthalate metabolites were analyzed using multiple linear regression modeling. Levels of individual PBDE congeners 28, 47, 99, 100 as well as total PBDEs were found to be significantly associated with the consumption of poultry. Metabolites of di-(2-ethylhexyl) phthalate (DEHP) were found to be associated with the consumption of poultry, as well as with an increased consumption of fat of animal origin. These results, combined with results from previous studies, suggest that diet is an important route of intake for both PBDEs and phthalates. Further research needs to be conducted to determine the sources of food contamination with these toxic chemicals as well as to describe the levels of contamination of US food in a large, representative sample.^

Analysis of Agrobacterium tumefaciens VirB6 and VirB9 of the VirB /D4 type IV secretion system

Agrobacterium tumefaciens uses the VirB/D4 type IV secretion system (T4SS) to translocate oncogenic DNA (T-DNA) and protein substrates to plant cells. Independent of VirD4, the eleven VirB proteins are also essential for elaboration of a conjugative pilus termed the T pilus. The focus of this thesis is the characterization and analysis of two VirB proteins, VirB6 and VirB9, with respect to substrate translocation and T pilus biogenesis. Observed stabilizing effects of VirB6 on other VirB subunits and results of protein-protein interaction studies suggest that VirB6 mediates assembly of the secretion machine and T pilus through interactions with VirB7 and VirB9. Topology studies support a model for VirB6 as a polytopic membrane protein with a periplasmic N terminus, a large internal periplasmic loop, five transmembrane segments, and a cytoplasmic C terminus. Topology studies and Transfer DNA immunoprecipitation (TrIP) assays identified several important VirB6 functional domains: (i) the large internal periplasmic loop mediates interaction of VirB6 with the T-DNA, (ii) the membrane spanning region carboxyl-terminal to the large periplasmic loop mediates substrate transfer from VirB6 to VirB8, and (iii) the terminal regions of VirB6 are required for substrate transfer to VirB2 and VirB9. To analyze structure-function relationships of VirB9, the phenotypic consequences of dipeptide insertion mutations were characterized. Substrate discriminating mutations were shown to selectively export the oncogenic T-DNA and VirE2 to plant cells or a mobilizable IncQ plasmid to bacterial cells. Mutations affecting VirB9 interactions with VirB7 and VirB10 were localized to the C- and N- terminal regions respectively. Additionally, “uncoupling” mutations identified in VirB11 and VirB6 that block T pilus assembly, but not substrate transfer to recipient cells, were also identified in VirB9. These results in conjunction with computer analysis establish that VirB9, like VirB6, is also composed of distinct regions or domains that contribute in various ways to secretion channel activity and T pilus assembly. Lastly, in vivo immunofluorescent studies suggest that VirB9 localizes to the outer membrane and may play a role similar to that of secretion/ushers of types II and III secretion systems to facilitate substrate translocation across this final bacterial barrier. ^