CELLULAR AND DEVELOPMENTAL FUNCTIONS OF THE XENOPUS ARVCF-CATENIN:KAZRIN COMPLEX

Xenopus ARVCF (xARVCF), a member of p120-catenin subfamily, binds cadherin cytoplasmic domains to enhance cadherin metabolic stability, or when dissociated, modulates Rho-family GTPases. We previously found that xARVCF binds directly to Xenopus KazrinA (xKazrinA), a widely expressed, conserved protein that bears little homology to established protein families. xKazrinA is also known to influence keratinocyte proliferation-differentiation and cytoskeletal activity. In my study, I first evaluated the expression pattern of endogenous Kazrin RNA and protein in Xenopus embryogenesis as well as in adult tissues. We then collaboratively predicted the helical structure of Kazrin’s coiled-coil domain, and I obtained evidence of Kazrin’s dimerization/oligomerization. In considering the intracellular localization of the xARVCF-catenin:xKazrin complex, I did not resolve xKazrinA in a larger ternary complex with cadherin, nor did I detect its co-precipitation with core desmosomal components. Instead, screening revealed that xKazrinA binds spectrin. This suggested a potential means by which xKazrinA localizes to cell-cell junctions, and indeed, biochemical assays confirmed a ternary xARVCF:xKazrinA:xβ2-spectrin complex. Functionally, I demonstrated that xKazrin stabilizes cadherins by negatively modulating the RhoA small-GTPase. I further revealed that xKazrinA binds to p190B RhoGAP (an inhibitor of RhoA), and enhances p190B’s association with xARVCF. Supporting their functional interaction in vivo, Xenopus embryos depleted of xKazrin exhibited ectodermal shedding, a phenotype that could be rescued with exogenous xARVCF. Cell shedding appeared to be caused by RhoA activation, which consequently altered actin organization and cadherin function. Indeed, I was capable of rescuing Kazrin depletion with ectopic expression of p190B RhoGAP. In addition, I obtained evidence that xARVCF and xKazrin participate in craniofacial development, with effects observed upon the neural crest. Finally, I found that xKazrinA associates further with delta-catenin and p0071-catenin, but not with p120-catenin, suggesting that Kazrin interacts selectively with additional members of the p120-catenin sub-family. Taken together, my study supports Kazrin’s essential role in development, and reveals KazrinA’s biochemical and functional association with ARVCF-catenin, spectrin and p190B RhoGAP.

DEVELOPMENTAL AND CELLULAR FUNCTIONS OF DELTA-CATENIN

Catenins have diverse and powerful roles in embryogenesis, homeostasis or disease progression, as best exemplified by the well-known beta-catenin. The less studied delta-catenin likewise contains a central Armadillo-domain. In common with other p120 sub-class members, it acts in a variety of intracellular compartments and modulates cadherin stability, small GTPase activities and gene transcription. In mammals, delta-catenin exhibits neural specific expression, with its knock-out in mice correspondingly producing cognitive defects and synaptic dysfunctions. My work instead employed the amphibian, Xenopus laevis, to explore delta-catenin’s physiological functions in a distinct vertebrate system. Initial isolation and characterization indicated delta-catenin’s expression in Xenopus. Unlike the pattern observed for mammals, delta-catenin was detected in most adult Xenopus tissues, although enriched in embryonic structures of neural fate as visualized using RNA in-situ hybridization. To determine delta-catenin’s requirement in amphibian development, I employed anti-sense morpholinos to knock-down gene products, finding that delta-catenin depletion results in developmental defects in gastrulation, neural crest migration and kidney tubulogenesis, phenotypes that were specific based upon rescue experiments. In biochemical and cellular assays, delta-catenin knock-down reduced cadherin levels and cell adhesion, and impaired activation of RhoA and Rac1, small GTPases that regulate actin dynamics and morphogenetic movements. Indeed, exogenous C-cadherin, or dominant-negative RhoA or dominant-active Rac1, significantly rescued delta-catenin depletion. Thus, my results indicate delta-catenin’s essential roles in Xenopus development, with contributing functional links to cadherins and Rho family small G proteins. In examining delta-catenin’s nuclear roles, I identified delta-catenin as an interacting partner and substrate of the caspase-3 protease, which plays critical roles in apoptotic as well as non-apoptotic processes. Delta-catenin’s interaction with and sensitivity to caspase-3 was confirmed using assays involving its cleavage in vitro, as well as within Xenopus apoptotic extracts or mammalian cell lines. The cleavage site, a highly conserved caspase consensus motif (DELD) within Armadillo-repeat 6 of delta-catenin, was identified through peptide sequencing. Cleavage thus generates an amino- (1-816) and carboxyl-terminal (817-1314) fragment each containing about half of the central Armadillo-domain. I found that cleavage of delta-catenin both abolishes its association with cadherins, and impairs its ability to modulate small GTPases. Interestingly, the carboxyl-terminal fragment (817-1314) possesses a conserved putative nuclear localization signal that I found is needed to facilitate delta-catenin’s nuclear targeting. To probe for novel nuclear roles of delta-catenin, I performed yeast two-hybrid screening of a mouse brain cDNA library, resolving and then validating its interaction with an uncharacterized KRAB family zinc finger protein I named ZIFCAT. My results indicate that ZIFCAT is nuclear, and suggest that it may associate with DNA as a transcriptional repressor. I further determined that other p120 sub-class catenins are similarly cleaved by caspase-3, and likewise bind ZIFCAT. These findings potentially reveal a simple yet novel signaling pathway based upon caspase-3 cleavage of p120 sub-family members, facilitating the coordinate modulation of cadherins, small GTPases and nuclear functions. Together, my work suggested delta-catenin’s essential roles in Xenopus development, and has revealed its novel contributions to cell junctions (via cadherins), cytoskeleton (via small G proteins), and nucleus (via ZIFCAT). Future questions include the larger role and gene targets of delta-catenin in nucleus, and identification of upstream signaling events controlling delta-catenin’s activities in development or disease progression.

A study in the molecular and cellular functions of GSK3beta in prostate adenocarcinoma

Kinases are part of a complex network of signaling pathways that enable a cell to respond to changes in environmental conditions in a regulated and coordinated way. For example, Glycogen Synthase Kinase 3 beta (GSK3β) modulates conformational changes, protein-protein interaction, protein degradation, and activation of unique domains in proteins that transduce signals from the extracellular milieu to the nucleus. ^ In this project, I investigated the expression and function that GSK3β exhibits in prostate cells. The capacity of GSK3β to regulate two transcription factors (JUN and CREB), which are known to be inversely utilized in prostate tumor cells, was measured. JUN/AP1 is constitutively activated in PC-3 cells; whereas, CREB/CRE activity is ∼20 fold less than the former. GSK3β overexpression obliterates JUN/AP1 activity. With respect to CREB GSK3β increases CREB/CRE activity. Cellular levels of active GSK3β can determine whether JUN or CREB is preferentially active in the PC-3s. Theoretically, in response to a particular cellular context or stimulus, a cell may coordinate JUN and CREB function by regulating GSK3β.^ A comparison of various prostate cell lines showed that active GSK3β is less expressed in normal prostate epithelial cells than in tumor cells. Differentially expressed active (GSK3β) may correlate with progression of prostate carcinoma. If a known marker associated with carcinoma of the prostate could be shown to be regulated by GSK3β then, further study of GSK3β may lead to a better understanding of both possible prevention of the disease and improved therapy for advanced stages. ^ The androgen receptor (AR) is an intriguing phosphoprotein whose regulation is potentially determined by a variety of kinases. One of these is (GSK3β) I found that (GSK3β) is a regulator of the androgen receptor in both the unliganded and liganded states. It can inhibit AR function as measured by reporter assays. Also, GSK3β associates with the AR at the DNA binding domain because deletion constructs expressing either the n-terminus or the c-terminus (both having the DBD in common) immunoprecipitated with GSK3β. Increased understanding of how GSK3β functions in prostate cancer would provide clues into how (1) certain signal pathways are coordinated and (2) the androgen receptor may be regulated. ^

Modeling survival and the development of second primary tumors among Hodgkin's disease patients

Hodgkin's disease (HD) is a cancer of the lymphatic system. Survivors of HD face varieties of consequent adverse effects, in which secondary primary tumors (SPT) is one of the most serious consequences. This dissertation is aimed to model time-to-SPT in the presence of death and HD relapses during follow-up.^ The model is designed to handle a mixture phenomenon of SPT and the influence of death. Relapses of HD are adjusted as a covariate. Proportional hazards framework is used to define SPT intensity function, which includes an exponential term to estimate explanatory variables. Death as a competing risk is considered according to different scenarios, depending on which terminal event comes first. Newton-Raphson method is used to estimate the parameter estimates in the end.^ The proposed method is applied to a real data set containing a group of HD patients. Several risk factors for the development of SPT are identified and the findings are noteworthy in the development of healthcare guidelines that may lead to the early detection or prevention of SPT.^

Characterization of cellular and molecular functions of the p21 -activated kinase, Shk1, in the fission yeast, Schizosaccharomyces pombe

The p21-activated kinase, Shk1, is an essential serine/threonine kinase required for normal cell polarity, proper mating response, and hyperosmotic stress response, in the fission yeast, Schizosaccharomyces pombe. This study has established a novel role for Shk1 as a microtubule regulator in fission yeast and, in addition, characterized a potential biological substrate of Shk1. Cells defective in Shk1 function were found to exhibit malformed interphase and mitotic microtubules, are hypersensitive to the microtubule disrupting drug thiabendazole (TBZ), and are cold sensitive for growth. Microtubule disruption by TBZ results in a significant reduction of Shk1 kinase activity, which is restored after cells are released from the drug, thus providing a correlation between Shk1 kinase activity and active microtubule polymerization. Consistent with a role for Shk1 as a microtubule regulator, GFP-Shk1 fusion proteins localize to interphase microtubules and mitotic microtubule spindles. Furthermore, loss of Tea1, a presumptive microtubule regulator in fission yeast, exacerbates the growth and microtubule defects of cells deficient in Shk1 function, and results in illicit Shk1 localization. Moreover, loss of the Cdc2 inhibitory kinase Wee1, which has been implicated as a mediator of the Shk1 pathway, leads to significant microtubule defects. Intriguingly, Wee1 protein levels are markedly reduced both by partial loss of Shk1 function and by treatment with TBZ. These results suggest that Shk1 is required for proper regulation of microtubule dynamics in fission yeast and may interact with Tea1 and Wee1 in this regulatory process. ^ To further understand Shk1 function in fission yeast, a yeast two-hybrid screen for proteins that interact with the Shk1 catalytic domain was performed. This screen led to the identification of a novel protein, Skb10 (for S&barbelow;hk1 k&barbelow;inase b&barbelow;inding protein 10). Coprecipitation experiments demonstrated that Skb10 associates with Shk1 in S. pombe cells. (Abstract shortened by UMI.) ^

Genetic and functional analyses of cell division proteins FtsZ and FtsA in Escherichia coli

In the current model for bacterial cell division, the FtsZ protein forms a ring that marks the division plane, creating a cytoskeletal framework for the subsequent action of other essential division proteins such as FtsA and ZipA. The putative protein complex ultimately generates the division septum. The essential cell division protein FtsZ is a functional and structural homolog of eukaryotic tubulin, and like tubulin, FtsZ hydrolyzes GTP and self-assembles into protein filaments in a strictly GTP-dependent manner. FtsA shares sequence similarity with members of the ATPase superfamily that include actin, but its actual function remains unknown. To test the division model and elucidate functions of the division proteins, this dissertation primarily focuses on the analysis of FtsZ and FtsA in Escherichia coli. ^ By tagging with green fluorescent protein, we first demonstrated that FtsA also exhibits a ring-like structure at the potential division site. The localization of FtsA was dependent on functional FtsZ, suggesting that FtsA is recruited to the septum by the FtsZ ring. In support of this idea, we showed that FtsA and FtsZ directly interact. Using a novel E. coli in situ assay, we found that the FtsA-FtsZ interaction appears to be species-specific, although an interspecies interaction could occur between FtsA and FtsZ proteins from two closely related organisms. In addition, mutagenesis of FtsA revealed that no single domain is solely responsible for its septal localization or interaction with FtsZ. To explore the function of FtsA, we purified FtsA protein and demonstrated that it has ATPase activity. Furthermore, purified FtsA stimulates disassembly of FtsZ polymers in a sedimentation assay but does not affect GTP hydrolysis of FtsZ. This result suggests that in the cell, FtsA may function similarly in regulating dynamic instability of the FtsZ ring during the cell division process. ^ To elucidate the structure-function relationship of FtsZ, we carried out thorough genetic and functional analyses of the mutagenized FtsZ derivatives. Our results indicate that the conserved N-terminal domain of FtsZ is necessary and sufficient for FtsZ self-assembly and localization. Moreover, we discovered a critical role for an extreme C-terminal domain of FtsZ that consists of only 12 residues. Truncated FtsZ derivatives lacking this domain, though able to polymerize and localize, are defective in ring formation in vivo as well as interaction with FtsA and ZipA. Alanine scanning mutagenesis of this region pinpointed at least five residues necessary for the function of FtsZ. Studies of protein levels and protein-protein interactions suggested that these residues may be involved in regulating protein stability and/or FtsZ-FtsA interactions. Interestingly, two of the point mutants exhibited dominant-negative phenotypes. ^ In summary, results from this thesis work have provided additional support for the division machinery model and will contribute to a better understanding of the coordinate functions of FtsA and FtsZ in the cell division process. ^

Biochemical and genetic characterization of the Saccharomyces cerevisiae Hsp110 molecular chaperone Sse1

The Ssel/Hsp110 molecular chaperones are a poorly understood subgroup of the Hsp70 chaperone family. Hsp70 can refold denatured polypeptides via a carboxyl-terminal peptide binding domain (PBD), which is regulated by nucleotide cycling in an amino-terminal ATPase domain. However, unlike Hsp70, both Sse1 and mammalian Hsp110 bind unfolded peptide substrates but cannot refold them. To test the in vivo requirement for interdomain communication, SSE1 alleles carrying amino acid substitutions in the ATPase domain were assayed for their ability to complement sse1Δ phenotypes. Surprisingly, all mutants predicted to abolish ATP hydrolysis complemented the temperature sensitivity of sse1Δ, whereas mutations in predicted ATP binding residues were non-functional. Remarkably, the two domains of Ssel when expressed in trans functionally complement the sse1Δ growth phenotype and interact by coimmunoprecipitation analysis, indicative of a novel type of interdomain communication. ^ Relatively little is known regarding the interactions and cellular functions of Ssel. Through co-immunoprecipitation analysis, we found that Ssel forms heterodimeric complexes with the abundant cytosolic Hsp70s Ssa and Ssb in vivo. Furthermore, these complexes can be efficiently reconstituted in vitro using purified proteins. The ATPase domains of Ssel and the Hsp70s were found to be critical for interaction as inactivating point mutations severely reduced interaction efficiency. Ssel stimulated Ssal ATPase activity synergistically with the co-chaperone Ydj1 via a novel nucleotide exchange activity. Furthermore, FES1, another Ssa nucleotide exchange factor, can functionally substitute for SSE1/2 when overexpressed, suggesting that Hsp70 nucleotide exchange is the fundamental role of the Sse proteins in yeast, and by extension, the Hsp110 homologs in mammals. ^ Cells lacking SSE1 were found to accumulate prepro-α-factor, but not the cotranslationally imported protein Kar2, similar to mutants in the Ssa chaperones. This indicates that the interaction between Ssel and Ssa is functionally significant in vivo. In addition, sse10 cells are compromised for cell wall strength, likely a result of decreased Hsp90 chaperone activity with the cell integrity MAP kinase SIC. Taken together, this work established that the Hsp110 family must be considered an essential component of Hsp70 chaperone biology in the eukaryotic cell.^

Two prognostic factors in breast cancer: Novel functions of EGFR and beta-catenin

In this study, we demonstrated the novel functions of two important prognostic markers in breast cancer, EGFR and b -catenin in proliferation and/or other transformation phenotype. ^ First we demonstrated that EGFR could be detected in the nucleus in highly proliferating tissues, including primary breast cancer samples and a breast cancer cell line. We found that EGFR contained a strong transactivation domain, complexed with an AT-rich consensus DNA sequence and activated promoters containing this sequence, including cyclin D1 promoter. Therefore, EGFR may function as a transcription factor to activate genes required for highly proliferating activity such as cyclin D1 in breast cancer. ^ In the second part of this study, we identified b -catenin as an important prognostic factor in breast cancer. We found that cyclin D1 was one of the genes regulated by b -catenin in breast cancer cells. The transactivation activity of b -catenin correlated significantly with cyclin D1 expression in both breast cancer cell lines and in breast cancer patient samples, in which high b -catenin activity correlated with poor prognosis of the patients. Moreover, blockage of b -catenin activity significantly inhibited transformation phenotypes in breast cancer cells. Therefore, our results indicate that b -catenin can be involved in breast cancer formation and/or progression and may serve as a target for breast cancer therapy. ^

FUNCTIONS OF DEADENYLATION FACTORS IN MRNA DECAY AND MRNA PROCESSING BODY FORMATION

Most newly synthesized messenger RNAs possess a 5’ cap and a 3’ poly(A) tail. The process of poly(A) tail shortening, also termed deadenylation, is important for post-transcriptional gene regulation, because deadenylation not only leads to mRNA translational inhibition but also is the first step of major mRNA degradation. Translationally inhibited mRNAs can be stored and/or degraded in dynamic cytoplasmic foci termed mRNA processing bodies, or P bodies, which are conserved in eukaryotes. To shed new light on the mechanisms of P body formation and P body functions, I focused on the link between deadenylation factors and P bodies. I found that the two major deadenylation complexes, Pan3-Pan2 and Ccr4-Caf1, can both be enriched in P bodies. The deadenylase activity of the Ccr4-Caf1 complex is prerequisite for P body formation. Pan3, but not the deadenylase Pan2, is essential for P body formation. While the C-terminal domain of Pan3 is important for interaction with Pan2, Pan3 N-terminal domain is important for Pan3 to form cytoplasmic foci colocalizing with P bodies and to promote mRNA decay. Interestingly, Pan3 N-terminal domain may be phosphorylated to regulate Pan3 localization and functions. Aside from the functions of the two deadenylation complexes in P bodies, I also studied all reported human P body proteins as a whole using bioinformatics. This effort not only has generated a comprehensive picture of the functions of and interactions among human P body proteins, but also has predicted proteins that may regulate P body formation and/or functions. In summary, my study has established a direct link between mRNA deadenylation and P body formation and has also led to new hypotheses to guide future research on how P body dynamics are controlled.

Risk of Second Malignant Neoplasms Following Proton Arc Therapy and Volumetric Modulated Arc Therapy for Prostate Cancer

The risk of second malignant neoplasms (SMNs) following prostate radiotherapy is a concern due to the large population of survivors and decreasing age at diagnosis. It is known that parallel-opposed beam proton therapy carries a lower risk than photon IMRT. However, a comparison of SMN risk following proton and photon arc therapies has not previously been reported. The purpose of this study was to predict the ratio of excess relative risk (RRR) of SMN incidence following proton arc therapy to that after volumetric modulated arc therapy (VMAT). Additionally, we investigated the impact of margin size and the effect of risk-minimized proton beam weighting on predicted RRR. Physician-approved treatment plans were created for both modalities for three patients. Therapeutic dose was obtained with differential dose-volume histograms from the treatment planning system, and stray dose was estimated from the literature or calculated with Monte Carlo simulations. Then, various risk models were applied to the total dose. Additional treatment plans were also investigated with varying margin size and risk-minimized proton beam weighting. The mean RRR ranged from 0.74 to 0.99, depending on risk model. The additional treatment plans revealed that the RRR remained approximately constant with varying margin size, and that the predicted RRR was reduced by 12% using a risk-minimized proton arc therapy planning technique. In conclusion, proton arc therapy was found to provide an advantage over VMAT in regard to predicted risk of SMN following prostate radiotherapy. This advantage was independent of margin size and was amplified with risk-optimized proton beam weighting.

Identification and phenotypic characterization of a second collagen adhesin, Scm, and genome-based identification and analysis of 13 other predicted MSCRAMMs, including four distinct pilus loci, in Enterococcus faecium.

Attention has recently been drawn to Enterococcus faecium because of an increasing number of nosocomial infections caused by this species and its resistance to multiple antibacterial agents. However, relatively little is known about the pathogenic determinants of this organism. We have previously identified a cell-wall-anchored collagen adhesin, Acm, produced by some isolates of E. faecium, and a secreted antigen, SagA, exhibiting broad-spectrum binding to extracellular matrix proteins. Here, we analysed the draft genome of strain TX0016 for potential microbial surface components recognizing adhesive matrix molecules (MSCRAMMs). Genome-based bioinformatics identified 22 predicted cell-wall-anchored E. faecium surface proteins (Fms), of which 15 (including Acm) had characteristics typical of MSCRAMMs, including predicted folding into a modular architecture with multiple immunoglobulin-like domains. Functional characterization of one [Fms10; redesignated second collagen adhesin of E. faecium (Scm)] revealed that recombinant Scm(65) (A- and B-domains) and Scm(36) (A-domain) bound to collagen type V efficiently in a concentration-dependent manner, bound considerably less to collagen type I and fibrinogen, and differed from Acm in their binding specificities to collagen types IV and V. Results from far-UV circular dichroism measurements of recombinant Scm(36) and of Acm(37) indicated that these proteins were rich in beta-sheets, supporting our folding predictions. Whole-cell ELISA and FACS analyses unambiguously demonstrated surface expression of Scm in most E. faecium isolates. Strikingly, 11 of the 15 predicted MSCRAMMs clustered in four loci, each with a class C sortase gene; nine of these showed similarity to Enterococcus faecalis Ebp pilus subunits and also contained motifs essential for pilus assembly. Antibodies against one of the predicted major pilus proteins, Fms9 (redesignated EbpC(fm)), detected a 'ladder' pattern of high-molecular-mass protein bands in a Western blot analysis of cell surface extracts from E. faecium, suggesting that EbpC(fm) is polymerized into a pilus structure. Further analysis of the transcripts of the corresponding gene cluster indicated that fms1 (ebpA(fm)), fms5 (ebpB(fm)) and ebpC(fm) are co-transcribed, a result consistent with those for pilus-encoding gene clusters of other Gram-positive bacteria. All 15 genes occurred frequently in 30 clinically derived diverse E. faecium isolates tested. The common occurrence of MSCRAMM- and pilus-encoding genes and the presence of a second collagen-binding protein may have important implications for our understanding of this emerging pathogen.

The use of second-generation antipsychotics and the changes in physical growth in children and adolescents with perinatally acquired HIV.

Second-generation antipsychotics (SGAs) are increasingly prescribed to treat psychiatric symptoms in pediatric patients infected with HIV. We examined the relationship between prescribed SGAs and physical growth in a cohort of youth with perinatally acquired HIV-1 infection. Pediatric AIDS Clinical Trials Group (PACTG), Protocol 219C (P219C), a multicenter, longitudinal observational study of children and adolescents perinatally exposed to HIV, was conducted from September 2000 until May 2007. The analysis included P219C participants who were perinatally HIV-infected, 3-18 years old, prescribed first SGA for at least 1 month, and had available baseline data prior to starting first SGA. Each participant prescribed an SGA was matched (based on gender, age, Tanner stage, baseline body mass index [BMI] z score) with 1-3 controls without antipsychotic prescriptions. The main outcomes were short-term (approximately 6 months) and long-term (approximately 2 years) changes in BMI z scores from baseline. There were 236 participants in the short-term and 198 in the long-term analysis. In linear regression models, youth with SGA prescriptions had increased BMI z scores relative to youth without antipsychotic prescriptions, for all SGAs (short-term increase = 0.192, p = 0.003; long-term increase = 0.350, p < 0.001), and for risperidone alone (short-term = 0.239, p = 0.002; long-term = 0.360, p = 0.001). Participants receiving both protease inhibitors (PIs) and SGAs showed especially large increases. These findings suggest that growth should be carefully monitored in youth with perinatally acquired HIV who are prescribed SGAs. Future research should investigate the interaction between PIs and SGAs in children and adolescents with perinatally acquired HIV infection.

Ultrasound feedback and motivational interviewing targeting smoking cessation in the second and third trimesters of pregnancy.

INTRODUCTION: Cigarette smoking during pregnancy is associated with poor maternal and child health outcomes. Effective interventions to increase smoking cessation rates are needed particularly for pregnant women unable to quit in their first trimester. Real-time ultrasound feedback focused on potential effects of smoking on the fetus may be an effective treatment adjunct, improving smoking outcomes. METHODS: A prospective randomized trial was conducted to evaluate the efficacy of a smoking cessation intervention consisting of personalized feedback during ultrasound plus motivational interviewing-based counseling sessions. Pregnant smokers (N = 360) between 16 and 26 weeks of gestation were randomly assigned to one of three groups: Best Practice (BP) only, Best Practice plus ultrasound feedback (BP+US), or Motivational Interviewing-based counseling plus ultrasound feedback (MI+US). Assessments were conducted at baseline and end of pregnancy (EOP). RESULTS: Analyses of cotinine-verified self-reported smoking status at EOP indicated that 10.8% of the BP group was not smoking at EOP; 14.2% in the BP+US condition and 18.3% who received MI+US were abstinent, but differences were not statistically significant. Intervention effects were found conditional upon level of baseline smoking, however. Nearly 34% of light smokers (< or =10 cigarettes/day) in the MI+US condition were abstinent at EOP, followed by 25.8% and 15.6% in the BP+US and BP conditions, respectively. Heavy smokers (>10 cigarettes/day) were notably unaffected by the intervention. DISCUSSION: Future research should confirm benefit of motivational interviewing plus ultrasound feedback for pregnant light smokers and explore mechanisms of action. Innovative interventions for pregnant women smoking at high levels are sorely needed.

Transcriptional regulation and functional analysis of the Wilms' tumor gene WT1

The Wilms' tumor gene, WT1, encodes a zinc finger transcription factor which functions as a tumor suppressor. Defects in the WT1 gene can result in the development of nephroblastoma. WT1 is expressed during development, primarily in the metanephric kidney, the mesothelial lining of the abdomen and thorax, and the developing gonads. WT1 expression is tightly regulated and is essential for renal development. The WT1 gene encodes a protein with a proline-rich N-terminus which functions as a transcriptional repressor and C-terminus contains 4 zinc fingers that mediate DNA binding. WT1 represses transcription from a number of growth factors and growth factor receptors. WT1 mRNA undergoes alternative splicing at two sites, resulting in 4 mRNA species and polypeptide products. Exon 5, encoding 17 amino acids is alternatively spliced, and is located between the transcriptional repression domain and the DNA binding domain. The second alternative splice is the terminal 9 nucleotides of zinc finger 3, encoding the tripeptide Lys-Thr-Ser (KTS). The presence or absence of KTS within the zinc fingers of WT1 alters DNA binding.^ I have investigated transcriptional regulation of WT1, characterizing two means of repressing WT1 transcription. I have cloned a transcriptional silencer of the WT1 promoter which is located in the third intron of the WT1 gene. The silencer is 460 bp in length and contains an Alu repeat. The silencer functions in cells of non-renal origin.^ I have found that WT1 protein can autoregulate the WT1 promoter. Using the autoregulation of the WT1 promoter as a functional assay, I have defined differential consensus DNA binding motifs of WT1 isoforms lacking and containing the KTS tripeptide insertion. With these refined consensus DNA binding motifs, I have identified two additional targets of WT1 transcriptional repression, the proto-oncogenes bcl-2 and c-myc.^ I have investigated the ability of the alternatively spliced exon 5 to influence cell growth. In cell proliferation assays, isoforms of WT1 lacking exon 5 repress cell growth. WT1 isoforms containing exon 5 fail to repress cell growth to the same extent, but alter the morphology of the cells. These experiments demonstrate that the alternative splice isoforms of WT1 have differential effects on the function of WT1. These findings suggest a role for the alternative splicing of WT1 in metanephric development. ^

Functions and intramolecular interactions of ribosomal RNA in decoding of termination codons

Two regions in the 3$\prime$ domain of 16S rRNA (the RNA of the small ribosomal subunit) have been implicated in decoding of termination codons. Using segment-directed PCR random mutagenesis, I isolated 33 translational suppressor mutations in the 3$\prime$ domain of 16S rRNA. Characterization of the mutations by both genetic and biochemical methods indicated that some of the mutations are defective in UGA-specific peptide chain termination and that others may be defective in peptide chain termination at all termination codons. The studies of the mutations at an internal loop in the non-conserved region of helix 44 also indicated that this structure, in a non-conserved region of 16S rRNA, is involved in both peptide chain termination and assembly of 16S rRNA.^ With a suppressible trpA UAG nonsense mutation, a spontaneously arising translational suppressor mutation was isolated in the rrnB operon cloned into a pBR322-derived plasmid. The mutation caused suppression of UAG at two codon positions in trpA but did not suppress UAA or UGA mutations at the same trpA positions. The specificity of the rRNA suppressor mutation suggests that it may cause a defect in UAG-specific peptide chain termination. The mutation is a single nucleotide deletion (G2484$\Delta$) in helix 89 of 23S rRNA (the large RNA of the large ribosomal subunit). The result indicates a functional interaction between two regions of 23S rRNA. Furthermore, it provides suggestive in vivo evidence for the involvement of the peptidyl-transferase center of 23S rRNA in peptide chain termination. The $\Delta$2484 and A1093/$\Delta$2484 (double) mutations were also observed to alter the decoding specificity of the suppressor tRNA lysT(U70), which has a mutation in its acceptor stem. That result suggests that there is an interaction between the stem-loop region of helix 89 of 23S rRNA and the acceptor stem of tRNA during decoding and that the interaction is important for the decoding specificity of tRNA.^ Using gene manipulation procedures, I have constructed a new expression vector to express and purify the cellular protein factors required for a recently developed, realistic in vitro termination assay. The gene for each protein was cloned into the newly constructed vector in such a way that expression yielded a protein with an N-terminal affinity tag, for specific, rapid purification. The amino terminus was engineered so that, after purification, the unwanted N-terminal tag can be completely removed from the protein by thrombin cleavage, yielding a natural amino acid sequence for each protein. I have cloned the genes for EF-G and all three release factors into this new expression vector and the genes for all the other protein factors into a pCAL-n expression vector. These constructs will allow our laboratory group to quickly and inexpensively purify all the protein factors needed for the new in vitro termination assay. (Abstract shortened by UMI.) ^