An Innovative Phase I Trial Design Allowing for the Identification of Multiple Potential Maximum Tolerated Doses with Combination Therapy of Targeted Agents

Treatment for cancer often involves combination therapies used both in medical practice and clinical trials. Korn and Simon listed three reasons for the utility of combinations: 1) biochemical synergism, 2) differential susceptibility of tumor cells to different agents, and 3) higher achievable dose intensity by exploiting non-overlapping toxicities to the host. Even if the toxicity profile of each agent of a given combination is known, the toxicity profile of the agents used in combination must be established. Thus, caution is required when designing and evaluating trials with combination therapies. Traditional clinical design is based on the consideration of a single drug. However, a trial of drugs in combination requires a dose-selection procedure that is vastly different than that needed for a single-drug trial. When two drugs are combined in a phase I trial, an important trial objective is to determine the maximum tolerated dose (MTD). The MTD is defined as the dose level below the dose at which two of six patients experience drug-related dose-limiting toxicity (DLT). In phase I trials that combine two agents, more than one MTD generally exists, although all are rarely determined. For example, there may be an MTD that includes high doses of drug A with lower doses of drug B, another one for high doses of drug B with lower doses of drug A, and yet another for intermediate doses of both drugs administered together. With classic phase I trial designs, only one MTD is identified. Our new trial design allows identification of more than one MTD efficiently, within the context of a single protocol. The two drugs combined in our phase I trial are temsirolimus and bevacizumab. Bevacizumab is a monoclonal antibody targeting the vascular endothelial growth factor (VEGF) pathway which is fundamental for tumor growth and metastasis. One mechanism of tumor resistance to antiangiogenic therapy is upregulation of hypoxia inducible factor 1α (HIF-1α) which mediates responses to hypoxic conditions. Temsirolimus has resulted in reduced levels of HIF-1α making this an ideal combination therapy. Dr. Donald Berry developed a trial design schema for evaluating low, intermediate and high dose levels of two drugs given in combination as illustrated in a recently published paper in Biometrics entitled “A Parallel Phase I/II Clinical Trial Design for Combination Therapies.” His trial design utilized cytotoxic chemotherapy. We adapted this design schema by incorporating greater numbers of dose levels for each drug. Additional dose levels are being examined because it has been the experience of phase I trials that targeted agents, when given in combination, are often effective at dosing levels lower than the FDA-approved dose of said drugs. A total of thirteen dose levels including representative high, intermediate and low dose levels of temsirolimus with representative high, intermediate, and low dose levels of bevacizumab will be evaluated. We hypothesize that our new trial design will facilitate identification of more than one MTD, if they exist, efficiently and within the context of a single protocol. Doses gleaned from this approach could potentially allow for a more personalized approach in dose selection from among the MTDs obtained that can be based upon a patient’s specific co-morbid conditions or anticipated toxicities.

Development of HIF-1α/HIF-1β heterodimerization inhibitors using a novel bioluminescence reporter assay system for in vitro high throughput screening and in vivo imaging

Tumor growth often outpaces its vascularization, leading to development of a hypoxic tumor microenvironment. In response, an intracellular hypoxia survival pathway is initiated by heterodimerization of hypoxia-inducible factor (HIF)-1α and HIF-1β, which subsequently upregulates the expression of several hypoxia-inducible genes, promotes cell survival and stimulates angiogenesis in the oxygen-deprived environment. Hypoxic tumor regions are often associated with resistance to various classes of radio- or chemotherapeutic agents. Therefore, development of HIF-1α/β heterodimerization inhibitors may provide a novel approach to anti-cancer therapy. To this end, a novel approach for imaging HIF-1α/β heterodimerization in vitro and in vivo was developed in this study. Using this screening platform, we identified a promising lead candidate and further chemically derivatized the lead candidate to assess the structure-activity relationship (SAR). The most effective first generation drug inhibitors were selected and their pharmacodynamics and anti-tumor efficacy in vivo were verified by bioluminescence imaging (BLI) of HIF-1α/β heterodimerization in the xenograft tumor model. Furthermore, the first generation drug inhibitors, M-TMCP and D-TMCP, demonstrated efficacy as monotherapies, resulting in tumor growth inhibition via disruption of HIF-1 signaling-mediated tumor stromal neoangiogenesis.

Insights into transcription enhancer factor 1 (TEF-1) activity from the solution structure of the TEA domain.

Transcription enhancer factor 1 is essential for cardiac, skeletal, and smooth muscle development and uses its N-terminal TEA domain (TEAD) to bind M-CAT elements. Here, we present the first structure of TEAD and show that it is a three-helix bundle with a homeodomain fold. Structural data reveal how TEAD binds DNA. Using structure-function correlations, we find that the L1 loop is essential for cooperative loading of TEAD molecules on to tandemly duplicated M-CAT sites. Furthermore, using a microarray chip-based assay, we establish that known binding sites of the full-length protein are only a subset of DNA elements recognized by TEAD. Our results provide a model for understanding the regulation of genome-wide gene expression during development by TEA/ATTS family of transcription factors.

Morphoproteomic analysis reveals an overexpressed and constitutively activated phospholipase D1-mTORC2 pathway in endometrial carcinoma.

The mammalian target of rapamycin (MTOR) assembles into two distinct complexes: mTOR complex 1 (mTORC1) is predominantly cytoplasmic and highly responsive to rapamycin, whereas mTOR complex 2 (mTORC2) is both cytoplasmic and nuclear, and relatively resistant to rapamycin. mTORC1 and mTORC2 phosphorylatively regulate their respective downstream effectors p70S6K/4EBP1, and Akt. The resulting activated mTOR pathways stimulate protein synthesis, cellular proliferation, and cell survival. Moreover, phospholipase D (PLD) and its product, phosphatidic acid (PA) have been implicated as one of the upstream activators of mTOR signaling. In this study, we investigated the activation status as well as the subcellular distribution of mTOR, and its upstream regulators and downstream effectors in endometrial carcinomas (ECa) and non-neoplastic endometrial control tissue. Our data show that the mTORC2 activity is selectively elevated in endometrial cancers as evidenced by a predominant nuclear localization of the activated form of mTOR (p-mTOR at Ser2448) in malignant epithelium, accompanied by overexpression of nuclear p-Akt (Ser473), as well as overexpression of vascular endothelial growth factor (VEGF)-A isoform, the latter a resultant of target gene activation by mTORC2 signaling via hypoxia-inducible factor (HIF)-2alpha. In addition, expression of PLD1, one of the two major isoforms of PLD in human, is increased in tumor epithelium. In summary, we demonstrate that the PLD1/PA-mTORC2 signal pathway is overactivated in endometrial carcinomas. This suggests that the rapamycin-insensitive mTORC2 pathway plays a major role in endometrial tumorigenesis and that therapies designed to target the phospholipase D pathway and components of the mTORC2 pathway should be efficacious against ECa.

The effects of proteasome inhibition on angiogenesis and autophagy in human prostate cancer cells

The proteasome degrades approximately 80% of intracellular proteins to maintain homeostasis. Proteasome inhibition is a validated therapeutic strategy, and currently, proteasome inhibitor bortezomib is FDA approved for the treatment of MM and MCL. Specific pathways affected by proteasome inhibition have been identified, but mechanisms of the anti-tumor effects of proteasome inhibition are not fully characterized and cancer cells display marked heterogeneity in terms of their sensitivity to proteasome inhibitor induced cell death. ^ The antitumor effects of proteasome inhibition involve suppression of tumor angiogenesis and vascular endothelial growth factor (VEGF) expression, but the mechanisms involved have not been clarified. In this dissertation I investigated the mechanisms underlying the effects of two proteasome inhibitors, bortezomib and NPI-0052, on VEGF expression in human prostate cancer cells. I found that proteasome inhibitors selectively downregulated hypoxia inducible factor 1alpha (HIF-1α) protein and its transcriptional activity to inhibit VEGF expression. Mechanistic studies demonstrated that proteasome inhibitors mediate the induction of the unfolded protein response (UPR) and that downregulation of HIF-1α is caused by eukaryotic translation initiation factor 2α (eIF2α) phosphorylation and translation repression. Importantly, I showed that proteasome inhibitors activated the UPR in some cells but not in others. My observation may have implications for the design of combination regimens that are based on exploiting proteasome inhibitor-induced ER stress.^ Although proteasome inhibitors have shown modest activity on prostate cancer, there is general consensus that no single agent is likely to have significant activity in prostate cancer. In the second part of this dissertation I attempted to exploit the effects of proteasome inhibition on the UPR to design a combination therapy that would enhance cancer cell death. Autophagy is a lysosome dependent degradation pathway that functions to eliminate long-lived protein and subcellular structures. Targeting autophagy has been shown to inhibit tumors in preclinical studies. I found that inhibition of autophagy with chloroquine or 3-methyladenine enhanced proteasome inhibitor induced cell death and the effects were associated with increased intracellular stress as marked by aggresome formation. Multiple cancers appear to be resistant to proteasome inhibition treatment alone. The implications of synergy for the combined inhibition of autophagy and the proteasome would likely apply to other cancers aside from prostate cancer. ^

Hypoxia-induced transcriptional regulation through chromatin modifications and NC2 function

In response to tumor hypoxia, specific genes that promote angiogenesis, proliferation, and survival are induced. Globally, I find that hypoxia induces a mixed pattern of histone modifications that are typically associated with either transcriptional activation or repression. Furthermore, I find that selective activation of hypoxia-inducible genes occurs simultaneously with widespread repression of transcription. I analyzed histone modifications at the core promoters of hypoxia-repressed and -activated genes and find that distinct patterns of histone modifications correlate with transcriptional activity. Additionally, I discovered that trimethylated H3-K4, a modification generally associated with transcriptional activation, is induced at both hypoxia-activated and repressed genes, suggesting a novel pattern of histone modifications induced during hypoxia. ^ In order to determine the mechanism of hypoxia-induced widespread repression of transcription, I focused my studies on negative cofactor 2 (NC2). Previously, we found that hypoxia-induced repression of the alpha-fetoprotein (AFP) gene occurs during preinitiation complex (PIC) assembly, and I find that NC2, an inhibitor of PIC assembly, is induced during hypoxia. Moreover, I find that the beta subunit of NC2 is essential for hypoxia-mediated repression of AFP, as well as the widespread repression of transcription observed during hypoxia. Previous data in Drosophila and S. cerevisiae indicate that NC2 functions as either an activator or a repressor of transcription. The mechanism of NC2-mediated activation remains unclear; although, Drosophila NC2 function correlates with specific core promoter elements. I tested if NC2 activates transcription in mammalian cells using this core promoter-specific model as a guide. Utilizing site-specific mutagenesis, I find that NC2 function in mammalian cells is not dependent upon specific core promoter elements; however, I do find that mammalian NC2 does function in a gene-specific manner as either an activator or repressor of transcription during hypoxia. Furthermore, I find that binding of the alpha subunit of NC2 specifically correlates with NC2-mediated transcriptional activation. NC2α and NC2β are both required for NC2-mediated transcriptional activation; whereas, NC2β alone is required for hypoxia-induced transcriptional repression. Together, these data indicate that hypoxia mediates changes in gene expression through both chromatin modifications and NC2 function. ^

Measurement of isotopic uranium in water for compliance monitoring by liquid scintillation counting with alpha/beta discrimination

A simple and inexpensive method is described for analysis of uranium (U) activity and mass in water by liquid scintillation counting using $\alpha$/$\beta$ discrimination. This method appears to offer a solution to the need for an inexpensive protocol for monitoring U activity and mass simultaneously and an alternative to the potential inaccuracy involved when depending on the mass-to-activity conversion factor or activity screen.^ U is extracted virtually quantitatively into 20 ml extractive scintillator from a 1-$\ell$ aliquot of water acidified to less than pH 2. After phase separation, the sample is counted for a 20-minute screening count with a minimum detection level of 0.27 pCi $\ell\sp{-1}$. $\alpha$-particle emissions from the extracted U are counted with close to 100% efficiency with a Beckman LS6000 LL liquid scintillation counter equipped with pulse-shape discrimination electronics. Samples with activities higher than 10 pCi $\ell\sp-1$ are recounted for 500-1000 minutes for isotopic analysis. Isotopic analysis uses events that are automatically stored in spectral files and transferred to a computer during assay. The data can be transferred to a commercially available spreadsheet and retrieved for examination or data manipulation. Values for three readily observable spectral features can be rapidly identified by data examination and substituted into a simple formula to obtain $\sp{234}$U/$\sp{238}$U ratio for most samples. U mass is calculated by substituting the isotopic ratio value into a simple equation.^ The utility of this method for the proposed compliance monitoring of U in public drinking water supplies was field tested with a survey of drinking water from Texas supplies that had previously been known to contain elevated levels of gross $\alpha$ activity. U concentrations in 32 samples from 27 drinking water supplies ranged from 0.26 to 65.5 pCi $\ell\sp{-1}$, with seven samples exceeding the proposed Maximum Contaminant Level of 20 $\mu$g $\ell\sp{-1}$. Four exceeded the proposed activity screening level of 30 pCi $\ell\sp{-1}$. Isotopic ratios ranged from 0.87 to 41.8, while one sample contained $\sp{234}$U activity of 34.6 pCi $\ell\sp{-1}$ in the complete absence of its parent, $\sp{238}$U. U mass in the samples with elevated activity ranged from 0.0 to 103 $\mu$g $\ell\sp{-1}$. A limited test of screening surface and groundwaters for contamination by U from waste sites and natural processes was also successful. ^

Crystal structure of the alpha(6)beta(6) holoenzyme of propionyl-coenzyme A carboxylase.

Propionyl-coenzyme A carboxylase (PCC), a mitochondrial biotin-dependent enzyme, is essential for the catabolism of the amino acids Thr, Val, Ile and Met, cholesterol and fatty acids with an odd number of carbon atoms. Deficiencies in PCC activity in humans are linked to the disease propionic acidaemia, an autosomal recessive disorder that can be fatal in infants. The holoenzyme of PCC is an alpha(6)beta(6) dodecamer, with a molecular mass of 750 kDa. The alpha-subunit contains the biotin carboxylase (BC) and biotin carboxyl carrier protein (BCCP) domains, whereas the beta-subunit supplies the carboxyltransferase (CT) activity. Here we report the crystal structure at 3.2-A resolution of a bacterial PCC alpha(6)beta(6) holoenzyme as well as cryo-electron microscopy (cryo-EM) reconstruction at 15-A resolution demonstrating a similar structure for human PCC. The structure defines the overall architecture of PCC and reveals unexpectedly that the alpha-subunits are arranged as monomers in the holoenzyme, decorating a central beta(6) hexamer. A hitherto unrecognized domain in the alpha-subunit, formed by residues between the BC and BCCP domains, is crucial for interactions with the beta-subunit. We have named it the BT domain. The structure reveals for the first time the relative positions of the BC and CT active sites in the holoenzyme. They are separated by approximately 55 A, indicating that the entire BCCP domain must translocate during catalysis. The BCCP domain is located in the active site of the beta-subunit in the current structure, providing insight for its involvement in the CT reaction. The structural information establishes a molecular basis for understanding the large collection of disease-causing mutations in PCC and is relevant for the holoenzymes of other biotin-dependent carboxylases, including 3-methylcrotonyl-CoA carboxylase (MCC) and eukaryotic acetyl-CoA carboxylase (ACC).

Investigation of the deregulation of interleukin-6 in two non-Hodgkin's lymphoma cell lines

Increased serum interleukin-6 (IL-6) is a poor prognostic factor for patients with lymphoma. This may be related to the fact that IL-6 has been shown to be an autocrine and paracrine growth factor for lymphoma cells. We have investigated the regulation of IL-6 in two lymphoma cell lines which produce IL-6 as an autocrine growth factor. The cell lines, LY3 and LY12, were established from two patients with non-Hodgkin's lymphoma. One patient had diffuse large cell lymphoma (LY3), whereas the other had small noncleaved cell lymphoma (LY12). There was no rearrangement or amplification of the IL-6 gene, but we detected IL-1 alpha and TNF production in addition to IL-6. We investigated the effect of inhibitors of IL-1 and TNF on IL-6 production in LY3 and LY12. Our results show that IL-6 production is mainly secondary to endogenous IL-1 production in LY3 cells, however LY12 cells produce IL-6 via a different mechanism since neither anti-IL-1 nor anti-TNF significantly inhibited IL-6 production.^ Transfection of LY12 cells with wildtype and mutant IL-6 promoter-chloramphenicol acetyl transferase constructs, showed increased activity of a trans-acting factor that binds to the NF-kB motif. Therefore, we determined whether there were abnormalities in members of the NF-kB family of transcription factors, such as p65, p50, p52/lyt-10 or rel, which bind to kB motifs. We found increased expression of the p52/lyt-10 transcription factor and activation of the NF-kB pathway in LY12. However, expression of p50, p65 and rel was not increased in LY12 cells. Future investigations could be aimed at determining the effect of inhibitors of NF-kB on IL-6 production. ^

GENETIC ORGANIZATION OF THE STRUCTURAL GENES FOR NITRATE REDUCTASE (TN5)

Nitrate reductase in Escherichia coli is a membrane-bound anaerobic enzyme that is repressed by oxygen and induced by nitrate. The genetic organization of the structural genes for the two larger subunits of nitrate reductase ((alpha) and (beta)) was determined by immunoprecipitation analysis of the formation of these proteins in nitrate reductase-deficient mutants resulting from transposon Tn5 mutagenesis. The results suggested that the genes encoding the (alpha) and (beta) subunits (narG and H) were arranged in an operon with transcription in the direction promoter(--->)(alpha)(--->)(beta). Segments of the chromosome containing the Tn5 inserts from several of the mutants were cloned into plasmid pBR322 and the positions of the transposons determined by restriction mapping. The Tn5 insertion sites were localized on two contiguous EcoRI fragments spanning about 6.6 kilobases of DNA. The narI gene (proposed to encode the (gamma) subunit) was positioned immediately downstream from the (beta)-gene (narH) by Southern analysis of Tn10 insertions into the narI locus. A Tn10 insertion into the narK locus, proposed to encode a nitrate-sensitive repressor of other anaerobic enzymes, was located about 1.5 kilobases upstream from the narGHI operon promoter. The narL locus, proposed to encode a nitrate-sensitive positive regulator of the narGHI operon and known to be genetically linked to the other nar genes, was demonstrated to lie outside a 19.3-kilobase region of the chromosome which encompasses the other nar genes. The physical limit of the narGHI promoter was defined by studying the effect of Tn5 insertions into a hybrid plasmid containing the functional operon. The points of origin of the coding regions for the (alpha) and (beta) genes were deduced by alignment of the chromosomal map of Tn5 insertion sites with the sizes of (alpha) and (beta) subunit fragments produced by plasmids carrying these Tn5 inserts in the nar operon. The coding region for the (alpha) subunit (143,000 daltons) begins about 250 nucleotides downstream from the deduced limit of the promoter region and includes about 4.0 kilobases of DNA; the region encoding (beta) (60,000 daltons) lies immediately downstream from the (alpha)-gene and is approximately 1.6 kilobases in length. The adjacent region encoding the (gamma) subunit (19,000 daltons) is approximately 0.5 kilobase in length. ^

Receptor tyrosine kinase HER2-mediated signaling pathways in metastasis and angiogenesis

Metastasis, the major cause of morbidity and mortality in most cancers, is a highly organized and organ-selective process. The receptor tyrosine kinase HER2 enhances tumor metastasis, however, its role in homing to metastatic organs is poorly understood. The chemokine receptor CXCR4 has recently been shown to mediate the malignant cancer cells to specific organs. Here we show that HER2 enhances the expression of CXCR4 by increasing CXCR4 protein synthesis and inhibiting its degradation. We also observed significant correlation between HER2 and CXCR4 expression in human breast tumor tissues, and an association between CXCR4 expression and a poor overall survival rate in patients with breast cancer. Furthermore, we found that CXCR4 is required for HER2-induced invasion, migration, and adhesion activities in vitro . Finally we established stable transfectants using retroviral RNA interference to inhibit CXCR4 expression and showed that the CXCR4 is required for HER2-mediated lung metastasis in vivo. These results provide a plausible mechanism for HER2-mediated breast tumor metastasis and homing to metastatic organs, and establish a functional link between the receptor tyrosine kinase HER2 and the chemokine receptor CXCR4 signaling pathways. ^ The HER2 overexpression activates PI-3K/Akt pathways and plays an important role in mediating cell survival and tumor development. Hypoxia inducible factors (HIF) are the key regulator for angiogenesis and energy metabolism, and thereby enhance tumor growth and metastasis. HIF activation occurs in the majority of human cancers, including the HER2 overexpressing cancer cells. Previous reports suggested that increased PI-3K/Akt may activate HIF pathway in various tumors, but the detail mechanism is still not completely understood. Here we found that HER2/PI-3K/Akt pathway induces HIF-1α activation, which is independent of hypoxia, but relatively weaker than hypoxic stimulation. This phenomenon was further observed in Akt knock out mouse embryonic fibroblast cells. The PI-3K/Akt pathway does not affect HIF-1α binding with its E3 ligase VHL, but enhances the binding affinity between HIF-1α and β unit. Furthermore, we found Akt phosphorylates HIF-1β at serine 271 and further regulated HIF transcriptional activity. Our findings provided one mechanism that HER2 induce HIF activation via Akt to promote angiogenesis, and this process is independent on hypoxia, which may have implications in the oncogenic activity of HER2 and PI-3K/Akt pathway. ^

Regulation of chromatin structure, Smad binding and AFP expression by the Forkhead box transcription factor: Foxa1

Transcription factors must be able to access their DNA binding sites to either activate or repress transcription. However, DNA wrapping and compaction into chromatin occludes most binding sites from ready access by proteins. Pioneer transcription factors are capable of binding their DNA elements within a condensed chromatin context and then reducing the level of nucleosome occupancy so that the chromatin structure is more accessible. This altered accessibility increases the probability of other transcription factors binding to their own DNA binding elements. My hypothesis is that Foxa1, a ‘pioneer’ transcription factor, activates alpha-fetoprotein (AFP) expression by binding DNA in a chromatinized environment, reducing the nucleosome occupancy and facilitating binding of additional transcription factors.^ Using retinoic-acid differentiated mouse embryonic stem cells, we illustrate a mechanism for activation of the tumor marker AFP by the pioneer transcription factor Foxa1 and TGF-β downstream effector transcription factors Smad2 and Smad4. In differentiating embryonic stem cells, binding of the Foxa1 forkhead box transcription factor to chromatin reduces nucleosome occupancy and levels of linker histone H1 at the AFP distal promoter. The more accessible DNA is subsequently bound by the Smad2 and Smad4 transcription factors, concurrent with activation of transcription. Chromatin immunoprecipitation analyses combined with siRNA-mediated knockdown indicate that Smad protein binding and the reduction of nucleosome occupancy at the AFP distal promoter is dependent on Foxa1. In addition to facilitating transcription factor binding, Foxa1 is also associated with histone modifications related to active gene expression. Acetylation of lysine 9 on histone H3, a mark that is associated active transcription, is dependent on Foxa1, while methylation of H3K4, also associated with active transcription, is independent of Foxa1. I propose that Foxa1 potentiates a region of chromatin to respond to Smad proteins, leading to active expression of AFP.^ These studies demonstrate one mechanism whereby a transcription factor can alter the accessibility of additional transcription factors to chromatin, by altering nucleosome positions. Specifically, Foxa1 exposes DNA so that Smad4 can bind to its regulatory element and activate transcription of the tumor-marker gene AFP.^

Structure and biosynthesis of a pyruvate -dependent enzyme---phosphatidylserine decarboxylase from {\it Escherichia coli\/}

Phosphatidylserine decarboxylase of E. coli, a cytoplasmic membrane protein, catalyzes the formation of phosphatidylethanolamine, the principal phospholipid of the organism. The activity of the enzyme is dependent on a covalently bound pyruvate (Satre and Kennedy (1978) J. Biol. Chem. 253, 479-483). This study shows that the enzyme consists of two nonidentical subunits, $\alpha$ (Mr = 7,332) and $\beta$ (Mr = 28,579), with the pyruvate prosthetic group in amide linkage to the amino-terminus of the $\alpha$ subunit. Partial protein sequence and DNA sequence analysis reveal that the two subunits are derived from a proenzyme ($\pi$ subunit, Mr = 35,893) through a post-translational event. During the conversion of the proenzyme to the $\alpha$ and $\beta$ subunits, the peptide bond between Gly253-Ser254 is cleaved, and Ser254 is converted to the pyruvate prosthetic group at the amino-terminus of the $\alpha$ subunit (Li and Dowhan (1988) J. Biol. Chem. 263, 11516-11522).^ The proenzyme cannot be detected in cells carrying either single or multiple copies of the gene (psd), but can be observed in a T7 RNA polymerase/promoter and transcription-translation system. The cleavage of the wild-type proenzyme occurs rapidly with a half-time on the order of 2 min. Changing of the Ser254 to cysteine (S254C) or threonine (S254T) slows the cleavage rate dramatically and results in mutants with a half-time for processing of around 2-4 h. Change of the Ser254 to alanine (S254A) blocks the cleavage of the proenzyme. The reduced processing rate with the mutations of the proenzyme is consistent with less of the functional enzyme being made. Mutants S254C and S254T produce $\sim$15% and $\sim$1%, respectively, of the activity of the wild-type allele, but can still complement a temperature-sensitive mutant of the psd locus. Neither detectable activity nor complementation is observed by mutant S254A. These results are consistent with the hydroxyl-group of the Ser254 playing a critical role in the cleavage of the peptide bond Gly253-Ser254 of the pro-phosphatidylserine decarboxylase, and support the mechanism proposed by Snell and co-workers (Recsei and Snell (1984) Annu. Rev. Biochem. 53, 357-387) for the formation of the prosthetic group of pyruvate-dependent decarboxylases. ^

Self -management behavior in patients with cystic fibrosis

Self-management is being promoted in cystic fibrosis (CF). However, it has not been well studied. Principal aims of this research were (1) to evaluate psychometric properties of a CF disease status measure, the NIH Clinical Score; (2) to develop and validate a measure of self-management behavior, the SMQ-CF scale, and (3) to examine the relation between self-management and disease status in CF patients over two years.^ In study 1, NIH Clinical Scores for 200 patients were used. The scale was examined for internal consistency, interrater reliability, and content validity using factor analysis. The Cronbach's alpha (.81) and interrater reliability (.90) for the total scale were high. General scale items were less reliable. Factor analysis indicated that most of the variance in disease status is accounted for by Factor 1 which consists of pulmonary disease items.^ The SMQ-CF measures the performance of CF self-management. Pilot testing was done with 98 CF primary caregivers. Internal consistency reliability, social desirability bias, and content validity using factor analysis were examined. Internal consistency was good (alpha =.95). Social desirability correlation was low (r =.095). Twelve factors identified were consistent with conceptual groupings of behaviors. Around two hundred caregivers from two CF centers were surveyed and multivariate analysis of variance was used to assess construct validity. Results confirmed expected relations between self-management, patient age, and disease status. Patient age accounted for 50% and disease status 18% of the variance in the SMQ-CF scale.^ It was hypothesized that self-management would positively affect future disease status. Data from 199 CF patients (control and education intervention groups) were examined. Models of hypothesized relations were tested using LISREL structural equation modeling. Results indicated that the relations between baseline self-management and Time 1 disease status were not significant. Significant relations were observed in self-management behaviors from time 1 to time 2 and patterns of significant relations differed between the two groups.^ This research has contributed to refinements in the ability to measure self-management behavior and disease status outcomes in cystic fibrosis. In addition, it provides the first steps in exploratory behavioral analysis with regard to self-management in this disease. ^

Specificity of somatostatin receptor and G -protein interactions as characterized by somatostatin receptor subtype specific antibodies

The neuropeptide somatostatin is a widely distributed general inhibitor of endocrine, exocrine, gastrointestinal and neural functions. The biological actions of somatostatin are initiated by interaction with high affinity, plasma membrane somatostatin receptors (sst receptors). Five sst receptor subtypes have been cloned and sequence analysis shows they are all members of the G protein coupled receptor superfamily. The G proteins play a pivotal role in sst receptor signal transduction and the specificity of somatostatin receptor-G protein coupling defines the possible range of cellular responses. However, the data for endogenous sst receptor and G protein coupling is very limited, and even when it is available, the sst receptor subtypes involved in G protein coupling and signal transduction are unknown due to the expression of multiple sst receptor subtypes in target cell lines or tissues of somatostatin.^ In an effort to characterize each individual sst receptor subtypes, antisera against unique C-terminal regions of different sst receptor subtypes have been developed in our lab. In this report, antisera made against the sst1, sst2A and sst4 receptors are characterized. They are highly specific to their corresponding receptors and efficiently immunoprecipitate the sst receptors. Using these antibodies, the cell lines expressing these sst receptor subtypes were identified with both immunoprecipitation and Western blot methods. The development of sst receptor subtype specific antibodies make it possible to determine the specificity of the sst receptor subtype and G protein coupling in target cells or tissues expressing multiple sst receptors, two questions were addressed by this thesis: (1) whether different cellular environments affect receptor subtype and G protein coupling; (2) whether different sst receptors couple to different G proteins in similar cellular environments.^ Taken together our findings, both sst1 and sst2A receptors couple with G$\alpha\sb{\rm i1},$ G$\alpha\sb{\rm i2}$ and G$\alpha\sb{\rm i3}$ in CHO cells, G$\alpha\sb{\rm i2}$ and G$\alpha\sb{\rm i3}$ in GH$\sb4$C$\sb1$ cells. Further, sst2A receptors couple with G$\alpha\sb{\rm i1},$ G$\alpha\sb{\rm i2}$ and G$\alpha\sb{\rm i3}$ in AR4-2J cells while sst4 receptors couple with G$\alpha\sb{\rm i2}$ and G$\alpha\sb{\rm i3}$ in CHO cells. Therefore, the G protein coupling of the same sst receptors in different cell lines is basically similar in that they all couple with multiple $\alpha$-subunits of the G$\rm \sb{i}$ proteins, suggesting cellular environment has little effect on receptor and G protein coupling. Moreover, different sst receptors have similar G protein coupling specificities in the same cell line, suggesting components other than receptor and G$\alpha$ subunits in the signal transduction pathways may contribute to specific functions of each sst receptor subtype. This series of experiments represent a novel approach in dissecting signal transduction pathways and may have general application in the field. Furthermore, this is the first systematic study of sst receptor subtype and G protein $\alpha$-subunit interaction in both transfected cells and in normal cell lines. The information generated will be very useful in our understanding of sst receptor signal transduction pathways and in directing future sst receptor research. ^