Investigation of I -compounds (newly discovered indigenous DNA modifications) by different approaches

I-compounds are newly discovered covalent DNA modifications detected by the $\sp{32}$P-postlabeling assay. They are age-dependent, tissue-specific and sex-different. The origin(s), chemistry and function(s) of I-compounds are unknown. The total level of I-compounds in 8-10 month old rat liver is 1 adduct in 10$\sp7$ nucleotides, which is not neglectable. It is proposed that I-compounds may play a role in spontaneous tumorigenesis and aging.^ In the present project, I-compounds were investigated by several different approaches. (1) Dietary modulation of I-compounds. (2) Comparison of I-compounds with persistent carcinogen DNA adducts and 5-methylcytosine. (3) Strain differences of I-compounds in relation to organ site spontaneous tumorigenesis. (4) Effects of nongenotoxic hepatocarcinogenes on I-compounds.^ It was demonstrated that the formation of I-compounds is diet-related. Rats fed natural ingredient diet exhibited more complex I-spot patterns and much higher levels than rats fed purified diet. Variation of major nutrients (carbohydrate, protein and fat) in the diet, produced quantitative differences in I-compounds of rat liver and kidney DNAs. Physiological level of vitamin E in the diet reduced intensity of one I-spot compared with vitamin E deficient diet. However, extremely high level of vitamin E in the diet gave extra spot and enhanced the intensities of some I-spots.^ In regenerating rat liver, I-compounds levels were reduced, as carcinogen DNA adducts, but not 5-methylcytosine, i.e. a normal DNA modification.^ Animals with higher incidences of spontaneous tumor or degenerative diseases tended to have a lower level of I-compounds.^ Choline devoid diet induced a drastic reduction of I-compound level in rat liver compared with choline supplemented diet. I-compound levels were reduced after multi-doses of carbon tetrachloride (CCl$\sb4$) exposure in rats and single dose exposure in mice. An inverse relationship was observed between I-compound level and DNA replication rate. CCl$\sb4$-related DNA adduct was detected in mice liver and intensities of some I-spots were enhanced 24 h after a single dose exposure.^ The mechanisms and explanations of these observations will be discussed. I-compounds are potentially useful indicators in carcinogenesis studies. ^

Molecular mechanisms of prostacyclin receptor signaling: The intracellular domains in G protein coupling

To better understand the mechanisms of how the human prostacyclin receptor (1P) mediates vasodilation and platelet anti-aggregation through Gs protein coupling, a strategy integrating multiple approaches including high resolution NMR experiments, synthetic peptide, fluorescence spectroscopy, molecular modeling, and recombinant protein was developed and used to characterize the structure/function relationship of important segments and residues of the IP receptor and the α-subunit of the Gs protein (Gαs). The first (iLP1) and third (iLP3) intracellular loops of the IP receptor, as well as the Gαs C-terminal domain, relevant to the Gs-mediated IP receptor signaling, were first identified by observation of the effects of the mini gene-expressed corresponding protein segments in HEK293 cells which co-expressed the receptor and Gαs. Evidence of the IP iLP1 domain interacted with the Gαs C-terminal domain was observed by fluorescence and NMR spectroscopic studies using a constrained synthetic peptide, which mimicked the IP iLP1 domain, and the synthetic peptide, which mimicked Gαs C-terminal domain. The solution structural models and the peptide-peptide interaction of the two synthetic protein segments were determined by high resolution NMR spectroscopy. The important residues in the corresponding domains of the IP receptor and the Gαs predicted by NMR chemical shift mapping were used to guide the identification of their protein-protein interaction in cells. A profile of the residues Arg42 - Ala48 of the IP iLP1 domain and the three residues Glu392 ∼ Leu394 of the Gαs C-terminal domain involved in the IP/Gs protein coupling were confirmed by recombinant proteins. The data revealed an intriguing speculation on the mechanisms of how the signal of the ligand-activated IP receptor is transmitted to the Gs protein in regulating vascular functions and homeostasis, and also provided substantial insights into other prostanoid receptor signaling. ^

Regulation of p53 antiproliferative function by transactivation domain phosphorylation at threonine 18 and serine 20

We investigated the induction and physiological role of Thr18 and Ser20 phosphorylation of p53 in response to DNA damage caused by treatment with ionizing (IR) or ultraviolet (UV) radiation. Polyclonal antibodies specifically recognizing phospho-Thr18 and phospho-Ser20 were used to detect p53 phosphorylation in vivo. Analyses of five wild-type (wt) p53 containing cell lines revealed lineage specific differences in phosphorylation of Thr18 and Ser20 after treatment with IR or UV. Importantly, the phosphorylation of p53 at Thr18 and Ser20 correlated with induction of the p53 downstream targets p21Waf1/Cip1 (p21) and Mdm-2, suggesting a transactivation enhancing role for Thr18 and Ser20 phosphorylation. Whereas Thr18 phosphorylation appears to abolish side-chain hydrogen bonding between Thr18 and Asp21, Ser20 phosphorylation may introduce charge attraction between Ser20 and Lys24. Both of these interactions could contribute to stabilizing α-helical conformation within the p53 transactivation domain. Mutagenesis-derived phosphorylation mimicry of p53 at Thr18 and Ser20 by Asp substitution (p53T18D/S20D) altered transactivation domain conformation and significantly reduced the interaction of p53 with the transactivation repressor Mdm-2. Mdm-2 interaction was also reduced with p53 containing a single site Asp substitution at Ser20 (p53S20D) and with the Thr18/Asp21 hydrogen bond disrupting p53 mutants p53T18A, p53T18D and p53D21A. In contrast, no direct effect was observed on the interaction of p53T18A, p53T18D and p53D21A with the basal transcription factor TAF II31. However, prior incubation of p53T18A, p53T18D and p53D21A with Mdm-2 modulated TAFII31 interaction, suggesting Mdm-2 blocks the accessibility of p53 to TAFII31. Consistently, p53-null cells transfected with p53S20D and p53T18A, p53T18D and p53D21A demonstrated enhanced endogenous p21 expression; transfection with p53T18D/S20D most significantly enhanced p21 and fas/APO-1 (fas ) expression. Expression of p53T18A, p53T18D and p53D21A in p53/Mdm-2-double null cells exhibited no discernible differences in p21 expression. Cell proliferation was also significantly curtailed in p53-null cells transfected with p53T18D/S20D relative to cells transfected with wt p53. We conclude the irradiation-induced phosphorylation of p53 at Thr18 and Ser20 alters the α-helical conformation of its transactivation domain. Altered conformation reduces direct interaction with the transrepressor Mdm-2, enhancing indirect recruitment of the basal transcription factor TAFII31, facilitating sequence-specific transactivation function resulting in proliferative arrest. ^

Biochemical and genetic analysis of two protein kinases JNKK2 and MEKK3

Mitogen-activated protein kinase (MAPK) cascades are conserved eukaryotic signaling modules consisting of a MAPK, a MAPKK and a MAP3K. MAPK cascades are involved in many cellular responses including proliferation, differentiation, apoptosis, stress and immune responses. ^ The first part of this thesis describes the cloning and biochemical analysis of JNKK2, a member of MAPKK gene family. Our results demonstrate that JNKK2 is a specific JNK activator and activates the JNK-dependent signal transduction pathway in vivo by inducing c-Jun and ATF2-mediated gene expression. We also found that JNKK2 is specifically activated by a MAP3K MEKK2 through formation of MEKK2-JNKK2-JNK1 triple complex module. JNKK2 is likely to mediate specific upstream signals to activate JNK cascade. ^ The second part of this thesis describes biochemical and gene disruption analysis of MEKK3, a member of MAP3K gene family. We showed that overexpression of MEKK3 strongly activates both JNK and p38 MAPKs but only weakly activates ERK. MEKK−/− embryos die at about embryonic day (E) 11. MEKK3−/− embryos displayed defects in blood vessel development in the yolk sacs, and in the myocardium and endocardium development at E9.5. The angiogenesis in the head, intersomitic region and placenta was also abnormal. These results demonstrate that MEKK3, a member of MAP3K MEKK/STE11 subgene family, is essential for early embryonic cardiovascular development. Furthermore, it was found that disruption of MEKK3 did not alter the expression of vascular endothelial growth factor-1 (VEGF-1), angiopoietin-1, -2 and their respective receptors Flt-1, Flk-1, Tie-1, Tie-2. Finally, MEKK3 was shown to activate myocyte-specific enhancer factor 2C (MEF2C), a crucial transcription factor for early embryonic cardiovascular development through the p38 MAPK cascade, suggesting that MEF2C is one of the key targets of the MEEKK3 signaling pathway during early embryonic cardiovascular development. ^

Modulation of cytochrome P450 enzyme activity and PAH -induced skin carcinogenesis by naturally occurring coumarins

The present study was designed to determine the potential anticarcinogenic activity of naturally occurring coumarins and their mechanism of action. The results indicated that several naturally occurring coumarins including bergamottin, coriandrin, imperatorin, isopimpinellin, and ostruthin, to which humans are routinely exposed in the diet, were effective inhibitors and/or inactivators of CYP1A1-mediated ethoxyresorufin-O-dealkylase (EROD) or CYP2B1-mediated pentoxyresorufin-O-dealkylase (PROD) in mouse liver microsomes. In addition, bergamottin and corandrin were also found to be inhibitors of purified human P450 1A1 in vitro. Further studies with coriandrin revealed that this compound was a mechanism-based inactivator of P450 1A1 and covalently bound to the P450 1A1 apoprotein. In cultured mouse keratinocytes, bergamottin and coriandrin effectively inhibited the B(a) P metabolism and significantly decreased covalent binding of B(a) P and DMBA to keratinocyte DNA and anti-diol-epoxide-DNA adducts derived from both B(a) P and DMBA in keratinocytes. The data from in vivo experiments showed that bergamottin and coriandrin were potent inhibitors of covalent binding of B (a) P to epidermal DNA and the formation of (+) anti BPDE-DNA adduct, whereas imperatorin and isopimpinellin were more potent inhibitors of covalent binding of DMBA to epidermal DNA. The ability of coumarins to inhibit covalent binding of B (a) P to DNA in mouse epidermis was positively correlated with their inhibitory effect P450 1A1 in vitro, while the inhibitory effect of coumarins on covalent binding of DMBA to epidermal DNA was positively correlated with their inhibitory effects on P450 2B1 and negatively to their inhibitory activity toward P450 1A1. The data from tumor experiments indicated that bergamottin, ostruthin, and coriandrin inhibited tumor initiation by B (a) P in a two-stage carcinogenesis protocol. Bergamottin was most effective in this regard and produced a dose dependent inhibition of papilloma formation in these experiments. In addition, imperatorin was an effective inhibitor of skin tumorigenesis induced by DMBA in SENCAR mouse skin using both a two-stage and a complete carcinogenesis protocol. At dose levels higher than those effective against DMBA, imperatorin also inhibited tumor initiation by B (a) P. The results to date demonstrate that several naturally occurring coumarins possess the ability to block tumor initiation and tumorigenesis by PAHs such as B (a) P and DMBA through inhibition of the P450s involved in the metabolic activation of these hydrocarbons. A working model for the involvement of specific P450s in the metabolic activation of these two PAHs was proposed. ^

An integrated molecular biology and computational approach to CYP1A1 expression in human brain

The cytochromes P450 comprise a superfamily of heme-containing mono-oxygenases. These enzymes metabolize numerous xenobiotics, but also play a role in metabolism of endogenous compounds. The P450 1A1 enzyme generally metabolizes polycyclic aromatic hydrocarbons, and its expression can be induced by aryl hydrocarbon receptor (AhR) activation. CYP1A1 is an exception to the generality that the majority of CYPs demonstrate highest expression in liver; CYP1Al is present in numerous extrahepatic tissues, including brain. This P450 has been observed in two forms, wildtype (WT) and brain variant (BV), arising from alternatively spliced mRNA transcripts. The CYP1A1 BV mRNA presented an exon deletion and was detected in human brain but not liver tissue of the same individuals. ^ Quantitative PCR analyses were performed to determine CYP1A1 WT and BV transcript expression levels in normal, bipolar disorder or schizophrenic groups. In our samples, we show that CYP1A1 BV mRNA, when present, is found alongside the full-length form. Furthermore, we demonstrate a significant decrease in expression of CYP1A1 in patients with bipolar disorder or schizophrenia. The expression level was not influenced by post-mortem interval, tissue pH, age, tobacco use, or lifetime antipsychotic medication load. ^ There is no indication of increased brain CYP1A1 expression in normal smokers versus non-smokers in these samples. We observed slightly increased CYP1A1 expression only in bipolar and schizophrenic smokers versus non-smokers. This may be indicative of complex interactions between neuronal chemical environments and AhR-mediated CYP1A1 induction in brain. ^ Structural homology modeling demonstrated that P450 1A1 BV has several alterations to positions/orientations of substrate recognition site residues compared to the WT isoform. Automated substrate docking was employed to investigate the potential binding of neurological signaling molecules and neurotropic drugs, as well as to differentiate specificities of the two P450 1A1 isoforms. We consistently observed that the BV isoform produced energetically favorable substrate dockings in orientations not observed for the same substrate in the WT isoform. These results demonstrated that structural differences, namely an expanded substrate access channel and active site, confer greater capacity for unique compound docking positions suggesting a metabolic profile distinct from the wildtype form for these test compounds. ^

Molecular mechanisms of Ca$\sp{2+}$ signal transduction from cardiac troponin C to troponin I

$\rm Ca\sp{2+}$-dependent exposure of an N-terminal hydrophobic region in troponin C (TnC) is thought to be important for the regulation of contraction in striated muscle. To study these conformational changes in cardiac troponin (cTnC), the $\varepsilon$C and $\varepsilon$H chemical shifts for all 10 Met residues in cTnC were sequence-specific assigned on NMR spectra using a combination of two dimensional NMR techniques and site-directed mutagenesis. The assigned methyl-Met chemical shifts were used as structural markers to monitor conformational changes induced by $\rm Ca\sp{2+}.$ The results showed that binding of $\rm Ca\sp{2+}$ to the regulatory site in the N-domain induced large changes in the $\varepsilon$H and $\varepsilon$C chemical shifts of Met 45, Met 80, Met 81 in the predicted N-terminal hydrophobic region, but had no effect on the chemical shifts of Met residues located in the C-domain. These results suggest that the $\rm Ca\sp{2+}$-dependent functions of cTnC are mainly through N-terminal domain of cTnC.^ To further define the molecular mechanism by which TnC regulates muscle contraction, single Cys residues were engineered at positions 45, 81, 84 or 85 in the N-terminal hydrophobic region of cTnC to provide sites for attachment of specific blocking groups. Blocking groups were coupled to these Cys residues in cTnC mutants and the covalent adducts were tested for activity in TnC-extracted myofibrils. Covalent modification of cTnC(C45) had no effect on maximal myofibril ATPase activity. Greatly decreased myofibril ATPase activity resulted when the peptide or biotin was conjugated to residue 81 in cTnC(C81), while less inhibition resulted from covalent modification of cTnC(C84) or cTnC(C85). The results suggest that limited sites of the N-terminal hydrophobic region in cTnC are important for transducing the $\rm Ca\sp{2+}$ signal to troponin I (TnI) and are sensitive to modification, while other regions are less important or can adapt to steric hindrances introduced by bulky blocking groups.^ Although the exposed TnI interaction site in the N-terminal hydrophobic region of TnC is crucial for function of TnC, other regions in the N-domain of TnC may also participate in transducing the $\rm Ca\sp{2+}$ signal and conferring the maximal activation of actomyosin ATPase. The interactions between the B-/C-helices of cTnC and cTnI were characterized using a combination of site-directed mutagenesis, fluorescence and covalent modification. The results suggest that the $\rm Ca\sp{2+}$-dependent interactions of the B-/C-helices of cTnC with TnI may be required for the maximal activation of muscle contraction. ^

Characterization of NARJ: A molecular chaperone involved in assembly of nitrate reductase

The major goal of this work was to define the role of accessory protein, NARJ, in assembly of nitrate reductase which is a membrane-bound multisubunit enzyme that can catalyze the reduction of nitrate to nitrite under anaerobic growth in E. coli. Nitrate reductase is encoded by the nar GHJI operon under control of the narG promoter. The purified nitrate reductase is composed of three subunits: $\alpha,\ \beta,$ and $\gamma.$ The NARJ protein which is encoded by the third gene (narJ) is not found to be associated with any of the purified preparations of the enzyme, but is required for active nitrate reductase. In this study the product of the narJ gene was identified. NARJ appeared to be produced at a reduced level, compared to the other proteins encoded by the nar operon. Since NARJ could not be overexpressed to a level for an efficient purification, NARJ was expressed and purified as a recombinant protein with polyhistidine tag. The recombinant protein NARJ-6His could functionally replace native NARJ. Purified NARJ-6His is a dimeric protein which contains no identifiable cofactors or unique secondary structure. NARJ was localized in the cytoplasm, and was not associated with nitrate reductase in the membrane. In vivo NARJ activated the $\alpha\beta$ complex and stabilized the $\alpha$ subunit against protease degradation. In the absence of the membrane-bound $\gamma$ subunit, NARJ formed an intermediate complex with $\alpha\beta$ in the cytosol. Based on these studies, NARJ fits the formal definition of a molecular chaperone. It appears to be required only for the biogenesis of nitrate reductase and, therefore, is defined as a private chaperone specifically involved in the assembly of nitrate reductase system. ^

Characterization of osteopontin charge forms produced by osteoblasts

Osteopontin (OPN) is a highly-phosphorylated extracellular matrix protein localized in bone, kidney, placenta, T-lymphocytes, macrophages, smooth muscle of the vascular system, milk, urine, and plasma. In ROS 17/2.8 osteoblast-like osteosarcoma cells, 1,25-dihydroxyvitamin D3 [1,25(OH)2D 3] regulates OPN at the transcriptional level resulting in increased steady state mRNA levels and increased production of OPN protein, maximal at 48 hours. Using ROS 17/2.8 cells as an osteoblast model, OPN was purified from culture medium after three hour treatments of either vehicle (ethanol) or 1,25(OH)2D3 via barium citrate precipitation followed by immunoaffinity chromatography. ^ Here, further evidence of regulation of OPN by 1,25(OH)2D 3 at the posttranslational level is presented. Prior to the up-regulation of OPN at the transcriptional level, 1,25(OH)2D3 induces a shift in OPN isoelectric point (pI) detected on two-dimensional gels from pI 4.6 to pI 5.1. Loading equal amounts of [32P]-labeled OPN recovered from ROS 17/2.8 cells exposed to 1,25(OH)2D3 or vehicle alone for three hours reveals that the shift from pI 4.6 to 5.1 is the result of reduced phosphorylation. Using structural analogs to 1,25(OH) 2D3, analog AT [25-(OH)-16-ene-23-yne-D3], which triggers Ca2+ influx through voltage sensitive Ca2+ channels but does not bind to the vitamin D receptor, mimicked the OPN pI shift while analog BT [1,25(OH)2-22-ene-24-cyclopropyl-D 3], which binds to the vitamin D receptor but does not allow Ca 2+ influx, did not. Inclusion of the Ca2+ channel blocker nifedipine also blocks the charge shift conversion of OPN. Further analysis of the signaling pathway initiated by 1,25(OH)2D3 reveals that inhibition of the cyclic 3′,5′ -adenosine monophosphate-dependent kinase, protein kinase A, or inhibition of the cyclic 3′,5′-guanine monophosphate-dependent kinase, protein kinase G, also prevents the charge shift conversion. ^ Isolation of OPN from rat femurs and tibiae provides evidence for the existence of these two OPN charge forms in vivo, evidenced by differential migration on isoelectric focusing gels and sodium dodecyl sulfate-polyacrylamide gels. Peptide sequencing of rat long bone fractions revealed the presence of a presumed dentin specific protein, dentin matrix protein-1 (DMP-1). Western blot analysis confirmed the existence of DMP-1 in these fractions. ^ Using the OPN charge forms in functional assays, it was determined that the charge forms have differential roles in both cell surface and mineralization functions. In cell attachment assays and Ca2+ influx assays using PC-3 prostate cancer cells, the pI 5.1 charge form of OPN was found to permit binding and increase intracellular Ca2+ concentrations of PC-3 cells. The increase in intracellular Ca2+ concentration was found to be integrin αvβ3-dependent. In mineralization assays, the pI 4.6 charge form of OPN promoted hydroxyapatite formation, while the pI 5.1 charge form had improved Ca2+ binding ability. ^ In conclusion, these findings suggest that 1,25(OH) 2D3 regulates OPN not only at the transcriptional level, but also plays a role in determination of the OPN phosphorylation state. The latter involves a short term (less than three hours) treatment and is associated with membrane-initiated Ca2+ influx. Functional assays utilizing the two OPN charge forms reveal the dependence of OPN post-translational state on its function. ^

Arabinosylguanine: Metabolism, action, and lineage-specific cytotoxicity in human leukemia cells

9-β-D-arabinosylguanine (ara-G), an analogue of deoxyguanosine, has demonstrated T-lymphoblast selective anti-leukemia activity both in vitro and in vivo in cell lines and primary cells and in phase I investigations. The present work was initiated to identify factors that result in this selectivity. ^ The cytotoxicity of ara-G is manifest only after its phosphorylation. Experiments using cell lines transfected to overexpress specific nucleoside kinases demonstrated that the phosphorylation of ara-G to its monophosphate is by both cytoplasmic deoxycytidine kinase and mitochondria) deoxyguanosine kinase. Ara-G monophosphate is converted to its 5′-triphosphate (ara-GTP) in cells by these kinases and then incorporated into DNA. Mechanistic studies demonstrated that incorporation of ara-GTP into DNA was a necessary event for the induction of cell death. ^ Pharmacokinetic and pharmacodynamic studies utilizing three human acute leukemia cell lines, CEM (T-lymphoblastic), Raji (B-lymphoblastic), and ML-1 (myeloid) were performed. CEM cells were most sensitive to ara-G-induced inhibition of colony formation, accumulated ara-GTP at a faster rate and to a greater degree than either Raji or ML-1, but incorporated the lowest number of ara-G molecules into DNA. The position of incorporation was internal and similar in all cell lines. The terminal elimination phase of ara-GTP was >24 h and similar in these cells. Comparisons between inhibition of colony formation and ara-GTP incorporation into DNA demonstrated that while within a cell line there was correlation among these parameters, between cell lines there was no relationship between number of incorporated ara-G molecules and ara-G(TP)-mediated toxicity suggesting that there were additional factors. ^ The expression of membrane bound Fas and Fast was unchanged in all cell lines. In contrast, there was a 2-fold increase in soluble Fast, which was found exclusively in CEM cells. Ara-G-mediated apoptosis in CEM occurred from all phases of the cell cycle and was abrogated partially by Fas antagonist antibodies. These data suggest that Fas-mediated cell death due to the liberation of sFasL may be responsible for the hypersensitivity to ara-G manifested by immature T-cells such as CEM. The role of Fas in ara-G induced death of acute T-lymphoblastic leukemia cells during therapy needs to be tested. ^

Studies of MEKK2 activation mechanisms and its biological role in LPS/TLR4 signaling

MEKK2 is an evolutionarily conserved mitogen-activated protein kinase (MAPK) kinase kinase (MAP3K) that controls the MAPK and IKK-NF-κB pathways. The MAPK and IKK pathways are intracellular signaling networks that are crucial for the Toll-like receptor (TLR) mediated innate immunity, cellular stress and many other physiological responses. Members of the MAP3K family are central to the activation of these processes. However, the molecular mechanisms underlying stimuli-mediated MAP3K activation remain largely unknown. In this study, we identified a key phosphoserine residue, Ser-519 in MEKK2, and its equivalent site Ser-526 in MEKK3 within their activation loop whose phosphorylation are essential for their optimal activation. Mutation of this regulatory serine to an alanine severely impaired MEKK2 activation and MEKK2 signaling to its downstream targets. To demonstrate that physiological stimuli induce this serine phosphorylation, we generated an antibody that specifically recognizes the phosphorylated serine residue. We found that many, but not all, of the MAPK agonists, including the TLR ligands, growth factors, cytokines and cellular stresses, induced this regulatory serine phosphorylation in MEKK2, suggesting an involvement of MEKK2 in the activation of the MAPK cascade leading to different cellular responses. We further investigated the specific role of MEKK2 in LPS/TLR4 signaling by using MEKK2−/− mice. We found that MEKK2 was selectively required for LPS-induced ERK1/2 activation, but not JNK, p38 or NF-κB activation. We also found that MEKK2 was involved in TLR4 dependent induction of proinflammatory cytokines and LPS-induced septic shock. In conclusion, we identified a key regulatory serine residue in the activation loop of MEKK2 whose phosphorylation is a key sensor of receptor- and cellular stress-mediated signals. We also demonstrated that MEKK2 is crucial for TLR4-mediated innate immunity. ^

The contribution of A3 adenosine receptor signaling to pulmonary inflammation and mucus secretion in the mouse

Adenosine has been implicated in chronic lung diseases such as asthma and COPD. Most physiological actions of adenosine are mediated through G-protein coupled adenosine receptors. Four subtypes of adenosine receptors have been identified, A1, A2A, A2B, and A 3. However, the specific roles of the various adenosine receptors in processes central to asthma and COPD are not well understood in part due to the lack of adequate animal models that examine the effect of adenosine on the development of lung disease. In this study we have investigated the expression and function of the A3 adenosine receptor in pulmonary eosinophilia and mucus production/secretion in adenosine deaminase (ADA)-deficient mice in which adenosine levels are elevated. ADA-deficient mice develop features of asthma and COPD, including lung eosinophilia and mucus hyperplasia in association with elevated lung adenosine levels. The A3 receptor was found to be expressed in eosinophils and mucus producing cells in the airways of ADA-deficient. Disruption of A3 receptor signaling in ADA-deficient mice by genetic removal of the receptor or treatment with MRS 1523, a selective A3 adenosine receptor antagonist, prevented airway eosinophilia and mucus production. Although eosinophils were decreased in the airways of ADA-deficient mice with disrupted A3 receptor signaling, elevations in circulating and lung interstitial eosinophils persisted, suggesting signaling through the A3 receptor is needed for the migration of eosinophils into the airways. Further examination of the role of the A3 receptor in mucus biology demonstrated that the A3 receptor is neither required nor is overexpression of the receptor in clara cells sufficient for mucus production in naive mice. Transgenic overexpression of the A3 receptor did elucidate a role for the A3 receptor in the secretion of mucus into the airways of ovalbumin challenged mice. These findings identify an important role for the A3 adenosine receptor in regulating lung eosinophilia and mucus secretion in inflammatory lung diseases. Therefore, the A3 adenosine receptor may represent a novel therapeutic target for the treatment and prevention of asthma. ^

Molecular mechanisms by which p53/LSD1 and ERalpha/Trim24 complexes mediate gene regulation

In this dissertation, I identify two molecular mechanisms by which transcription factors cooperate with their co-regulators to mediate gene regulation. In the first part, I demonstrate that p53 directly recruits LSD1, a histone demethylase, to AFP chromatin to demethylate methylated H3K4 and actively mediate transcription repression. Loss of p53 and LSD1 interaction at chromatin leads to derepression of AFP in hepatic cells. In the second part, I reveal that Trim24 functions as an important co-activator in ERα-mediated gene activation in response to estrogen stimulation. Trim24 is recruited by ligand-bound ERα to chromatin and stabilizes ERα-chromatin interactions by binding to histone H3 via its PHD finger, which preferentially recognizes unmethylated H3K4. ^

Dual role of interleukin-12 on ultraviolet -induced immune suppression

Skin cancer is the most prevalent form of neoplasia, with over one million newcases diagnosed this year. UV radiation is a ubiquitous environmental agent that induces skin cancer. In addition to its carcinogenic effect, UV radiation also suppresses cell-mediated immune responses. This immune suppression is not only observed at the site of irradiation, but UV radiation also induces systemic immune suppression. Since UV radiation has a limited ability to penetrate the skin, the question of the mechanism of this systemic immune suppression arises. A number of studies have suggested that UV radiation induce systemic effects through the production of immunoregulatory cytokines, such as IL-4 and IL-10. These cytokines affect the immune response by altering systemic antigen presentation, specifically by suppressing the activation of Th1 cells while allowing the activation of Th2 cells. Because IL-12 is an important regulator of Th1 cell activation, we tested the hypothesis that administration of IL-12 could overcome UV-induced immune suppression. ^ The studies presented here are divided into dime specific aims. In the first specific aim, the ability of IL-12 to overcome UV-induced immune suppression was examined. IL-12 could overcome UV-induced immune suppression as well as prevent the generation of and neutralize the activity of preformed suppressor cells induced by UV radiation. In the second specific aim, the mechanism by which IL-12 overcomes UV-induced immune suppression was examined. IL-12 overcame UV-induced immune suppression by blocking the production of immunoregulatory cytokines such as IL-4, IL-10 and TNF-α. In the third specific aim, the effect of UV radiation on antigen presentation was investigated. UV radiation was found to decrease the production of biologically active IL-12. In addition, UV also increased the production of IL-12p40 homodimer, an antagonist of IL-12p70 heterodimer. This result suggests that IL-12 may have a dual role in the immune suppression induced by, UV radiation. On one hand the biologically active IL-12p70 heterodimer blocks UV-induced immune suppression. In contrast, IL-12p40 homodimer may mediate the suppressive effect of UV radiation. This paradox indicates that IL-12 may have a greater regulatory role in the immune response than was previously suspected. ^

Medical advice, diabetes self-management, and health outcomes of a multi-ethnic population from the National Health and Nutrition Examination Survey 2007--2008

Diabetes self-management, an essential component of diabetes care, includes weight control practices and requires guidance from providers. Minorities are likely to have less access to quality health care than White non-Hispanics (WNH) (American College of Physicians-American Society of Internal Medicine, 2000). Medical advice received and understood may differ by race/ethnicity as a consequence of the patient-provider communication process; and, may affect diabetes self-management. ^ This study examined the relationships among participants’ report of: (1) medical advice given; (2) diabetes self-management, and; (3) health outcomes for Mexican-Americans (MA) and Black non-Hispanics (BNH) as compared to WNH (reference group) using data available through the National Health and Nutrition Examination Survey (NHANES) for the years 2007–2008. This study was a secondary, single point analysis. Approximately 30 datasets were merged; and, the quality and integrity was assured by analysis of frequency, range and quartiles. The subjects were extracted based on the following inclusion criteria: belonging to either the MA, BNH or WNH categories; 21 years or older; responded yes to being diagnosed with diabetes. A final sample size of 654 adults [MA (131); BNH (223); WNH (300)] was used for the analyses. The findings revealed significant statistical differences in medical advice reported given. BNH [OR = 1.83 (1.16, 2.88), p = 0.013] were more likely than WNH to report being told to reduce fat or calories. Similarly, BNH [OR = 2.84 (1.45, 5.59), p = 0.005] were more likely than WNH to report that they were told to increase their physical activity. Mexican-Americans were less likely to self-monitor their blood glucose than WNH [OR = 2.70 (1.66, 4.38), p<0.001]. There were differences among ethnicities for reporting receiving recent diabetes education. Black, non-Hispanics were twice as likely to report receiving diabetes education than WNH [OR = 2.29 (1.36, 3.85), p = 0.004]. Medical advice reported given and ethnicity/race, together, predicted several health outcomes. Having recent diabetes education increased the likelihood of performing several diabetes self-management behaviors, independent of race. ^ These findings indicate a need for patient-provider communication and care to be assessed for effectiveness and, the importance of ongoing diabetes education for persons with diabetes.^