The opportunistic pathogen P. aeruginosa is the leading cause of morbidity and mortality in cystic fibrosis patients. During chronic infection of the cystic fibrosis lung, P. aeruginosa undergoes conversion to a mucoid phenotype, constitutively producing the exopolysaccharide alginate, composed of the uronic acids D-mannuronate and L-guluronate. This alginate production contributes significantly to virulence in the cystic fibrosis lung. Evidence suggests that the acetylation state of the mannuronate component of the alginate influences the ability of components of the immune system to phagocytose the organism. To garner new and relevant information regarding the mechanism of alginate acetylation in Pseudomonas aeruginosa, a variety of approaches were undertaken. Analysis of the alginate produced by algX, algG, and algK alginate biosynthesis mutants revealed that the small oligouronides they produced were unacetylated. This strongly supports the hypothesis that the mannuronates are acetylated in periplasm, and that a polymer of at least some specific size is required. While three alginate biosynthesis gene products (AlgI, AlgJ, and AlgF) have been shown to be involved in alginate acetylation, another gene in the cluster, algX, shares 30% identity with one of them and thus generates speculation as to its potential involvement in the process. To test this possibility, an algX mutant was complemented with a plasmid carrying a mutation at a conserved residue shown to be required for alginate acetylation in the homologous protein. Analysis of alginate from this construct suggested that AlgX is not involved in alginate acetylation. To determine if changes in levels of alginate acetylation are accomplished at the transcriptional level, transcript levels of several alginate biosynthesis genes in different media were determined by real-time PCR. As qRT-PCR had not been previously performed on any of the alginate biosynthesis genes, this yielded important information about the transcription of the operon. In addition, beta-galactosidase assays on upstream regions of several biosynthesis genes identified two previously unrecognized promoters, one upstream of algG and one upstream of algI. The remaining approach was to examine protein interactions of AlgF, the protein product of one of the three acetylation genes. 2-D redox SDS-PAGE gels indicated that disulfide bonding may be important for interactions with this protein. While mass spectrometry was unable to identify the binding partners of AlgF, efforts are ongoing to create a mutation in the P. aeruginosa genome that changes the cysteine residue in AlgF to a serine residue. This would be a definitive method for determining the importance of disulfide bonding in AlgF.

Background: The evaluation process for listing a patient on the liver transplant list is complicated and involves multiple consultations from various specialists, as well as extensive imaging and physiological studies. Although there are data on the outcomes of those listed, we know little about those that are denied listing. This research project will identify the reasons for liver transplant listing denial and predictors of death following denial for this challenging group of patients. Methods: Data from all patients (n=1,500) evaluated for a liver transplant from 1997 to 2007 by the Department of Gastroenterology, Hepatology, and Nutrition located at Virginia Commonwealth University Health System’s (VCUHS) Hume-Lee Transplant Program were reviewed to identify patients denied listing (n=350). Simple descriptive characteristics were generated and the reasons for denial were assessed. The Social Security Death Index was used to determine and/or confirm mortality and multiple logistic regression was conducted to determine the predictors of death following denial of transplant listing. Results: The majority of the denied patients were white males and the mean age was 50.9, SE= 0.542). The primary liver disease diagnosis for those denied listing was Hepatitis C Virus (HCV) (33.6%). Study participants whose primary diagnosis was ethyl alcohol abuse or hepatocellular carcinoma had greater odds of dying after not being listed when compared to those diagnosed with HCV; however, these findings were not statistically significant. The majority of participants were denied listing for Hepatic-related (38.8%), psychosocial-related (21.7%), and cardiac-related (15.7%) reasons. Men were two times more likely to die after denial than women (OR= 2.18, CI= 1.03, 4.62). Patients with a MELD score less than 30 were less likely to die after being denied listing compared to those with MELD scores 31 to 40. The risk of dying after denial was not statistically different for patients who were denied listing for hepatic-related and cardiac-related reasons compared to subjects who were denied for cancer. Conclusions: Our findings have clear implications for the future of transplant medicine and raise additional questions. The analysis shows men, those 51 years of age and older and patients with MELD scores between 31 and 40 are more likely to die after not being listed for transplant. We did not find significant evidence that those with particular primary liver disease diagnoses were more likely to die following denial for listing. Other studies taking into account the population of patients that are listed as well as those denied listing are necessary in order to understand the patho-physiological mechanisms so that patient-specific therapies may be developed if appropriate.

Microtubule poisons have proven to be effective in the treatment of a variety of malignancies. Although taxol-based derivatives promote microtubule stabilization, there is continuing interest in compounds that, like colchicines, act as microtubule destabilizing agents. Previous work from this laboratory showed that the novel microtubule poison, JG- 03-14, was active against breast tumor cells, promoting autophagic cell death. In the current work, we studied the influence of JG-03-14 on p53 wild type HCT116 colon carcinoma cells. A crystal violet sensitivity assay indicated that JG-03-14 induced growth inhibition, with 75% suppression of growth evident at a concentration of 500 nM. Time course studies of drug effects on cell viability indicated that JG-03-14 also produced cell killing. FACS analysis demonstrated that the HCT-116 cells arrested in the G2/M stage; furthermore, there was evidence of a hyperdiploid population that would be consistent with failure of the cells to divide despite completion of DNA synthesis. Finally, there was evidence of a small sub G0/G1cell population, indicating that the cells were not dying primarily by apoptosis, and suggesting that JG-03-14 induces an alternative mode of cell death. In contrast to cell shrinkage and nuclear fragmentation that was evident after treatment with taxol ( a positive control for apoptosis), DAPI staining of HCT-116 cells treated with JG-03-14 showed intact and enlarged nuclei, again consistent with the absence of apoptosis. Furthermore, there was no evidence of mitotic catastrophe (micronuclei in binucleated cells). Based on previous studies in MCF-7 and MDA-MB231 cell lines that demonstrated a substantial population of autophagic cells, HCT-116 cells were subjected to staining with acridine orange and monodansylcadaverine after treatment with JG-03-14. While control cells tended to show a single large autophagic vesicle closely associated with the cell nucleus, treatment with JG-03-14 resulted in extensive distribution of small acidic vesicles within the cytoplasm, indicative of autophagy. GFP-LC3 transfected cells incubated with JG-03-14 showed punctuated patterns that were also consistent with the promotion of autophagy. Finally, activation of the DNA damage response pathway was ruled out by the lack of induction of p53 and p21 in cells treated with JG-03-14. In summary, our studies indicate that the JG-03-14 induces both growth arrest and autophagic cell death in HCT116 colon carcinoma cells. The possibility of an alternative mode of cell death induced by JG-03-14 makes it a potentially usefull candidate as a chemotherapeutic drug that could be used to treat cancers resistant to apoptosis. Our result also suggested that JG-03-14 failed to induce bone marrow toxicity adding to its potential for clinical use.

Ribosomes form the core of the protein biosynthesis machinery and are essential to life. Ribosome biogenesis is a complex cellular process involving transcription of rRNA, pre-rRNA processing, rRNA modification and simultaneous assembly of ribosomal proteins. RNA nucleotide modification is observed in all domains of life. While there is enormous conservation of ribosome structure, very few post-transcriptional rRNA modifications have been conserved throughout evolution. A notable example of such rare conservation is the dimethylation of two adjacent adenosines in the 3’-terminal helix, a highly conserved region of the small subunit rRNA. Enzymes that carry out these dimethylations are equally conserved and are collectively known as the KsgA/Dim1 family of methyltransferases. The first member of the family, KsgA, was identified in E. coli as the determinant for resistance to the aminoglycoside antibiotic Kasugamycin. Orthologs have since been described in organisms of wide spread evolutionary origins as well as in eukaryotic cellular organelles, thus underscoring the unprecedented conservation of this family of enzymes and the resultant rRNA modification. The higher evolutionary orthologs of KsgA have adopted secondary roles in ribosome biogenesis in addition to their dimethyltransferase role. The eukaryotic ortholog, Dim1, is essential for proper processing of the primary rRNA transcript. Recently, KsgA has been speculated to function as a late stage ribosome biogenesis factor and a ΔksgA genotype in E. coli has been linked to cold sensitivity and altered ribosomal profiles. This report focuses on the biochemical characterization of KsgA and its interaction with the 30S subunit. We have established the salt conditions required for optimal KsgA methyltransferase activity while confirming that KsgA recognizes a translationally inactive conformation of 30S subunit in vitro. Our study of the functional conservation of KsgA/Dim1 enzymes in the bacterial system revealed that KsgA and the evolutionarily higher orthologs could recognize a common ribosomal substrate. This indicates that the recognition elements of both, the protein and the small subunit, have remained largely unchanged during the course of evolution. Finally, based on our site directed mutagenesis and biochemical studies, we report that KsgA binds to structural components of 16S rRNA other than the helix containing the target nucleosides.

Drug abuse and addiction are considered to be a result, at least in part, of the rewarding effects produced by increasing dopamine levels. 5-HT3 serotonin receptors have been shown to indirectly affect dopamine levels. Therefore, the effect of the 5-HT3 receptor partial agonist, MD-354, on the actions of psychomotor stimulants was analyzed in mouse locomotor activity assays to determine whether MD-354 is working through a 5-HT3 receptor agonist or antagonist mode of action. Studies with (+)amphetamine and (+)methamphetamine in combination with MD-354 indicated MD-354 is either devoid of action or is behaving similar to the 5-HT3 receptor antagonist, ondansetron. This effect could be occurring centrally; however peripheral effects can not be discounted. In combination with cocaine, MD-354 behaved similar to the 5-HT3 receptor agonist, SR 57227A, known to act both centrally and peripherally. This difference between central and peripheral effects could account for the different modes of action observed with MD-354. Studies also involved synthesis of potentially brain-penetrant carbamate analogs of MD-354, and QSAR to assist in validating a 5-HT3 receptor agonist pharmacophore.

Francisella tularensis was a facultative intracellular pathogen and a gram-negative coccobacillus which has been categorized by the CDC as a potential class A select agent due to its highly infectious properties and high mortality rates. Francisella tularensis was also responsible for the zoonotic disease tularemia, which was usually transmitted by arthropod vectors or via contact with infected animals. Francisella tularensis subspecies novicida has been used by many researchers in genetic pathogenesis experiments to try to elucidate genes responsible for virulence factors. One of these virulence factors was a capsular material which has been thought to be involved in either increasing pathogenicity or infectivity of this organism upon engulfment by its principal host cell, the macrophage. There were many potential genetic loci which may be involved in this biosynthetic process of encapsulation. One such locus has excellent homology to the capsule biosynthesis operon of Bacillus anthracis, which, under certain conditions, creates a polyglutamic acid capsule (PGA). A transposon mutation in the amide ligase (capB) in LVS has a reduced virulence in murine infection models. I wished to investigate whether Francisella novicida was capable of producing such a capsule and under which environmental conditions this capsule was made. I have created a site-directed mutant of the capB gene in Francisella novicida U112 using targeted mutagenesis via PCR SOEing and have introduced this mutation via electroporation of a suicide vector. I have tested our mutant against preimmune serum treatments and have shown reduced viability as well as a reduced capacity for replication inside RAW 264.7 murine macrophages. I assayed for production of a PGA capsule via immunodot blot and electron microscopy as well as analysis by mass spectrophotometry of capsular extracts. I also tested various media constituents and different environmental conditions to determine which external stimuli may contribute to PGA capsule biosynthesis as well as regulatory changes in transcript levels of this operon.

The propensity for seizures in patients with epilepsy is due to underlying cortical hyperexcitability, the mechanisms of which are poorly understood. Particularly difficult to treat are patients with developmental malformations of cortex. Using the freeze-lesion rat model of one such malformation, polymicrogyria, we identified, in lesioned cortex, alterations in specific interneuron subpopulations that may promote hyperexcitability. Previous studies demonstrate increased excitatory input to the paramicrogyral region. An increase in the frequency of spontaneous excitatory postsynaptic currents (sEPSCs) recorded from pyramidal cells has also been shown. We report an increase in sEPSCs recorded from one subtype of interneuron, the low threshold-spiking interneuron (LTS), while sEPSCs in the fast-spiking (FS) interneuron remain unchanged. Distributed equally to pyramidal cells and interneurons, extra excitatory afferents should simply increase overall activity level but maintain the balance of excitation and inhibition. Selective changes in one or more interneuron subpopulations could allow inhibition to appear unchanged, while permitting problematic alterations in inhibitory circuitry. In what appears to be a morphological division of labor, interneurons with intralaminar orientations are typically characterized as FS, while intracolumnar orientations are associated with LTS cells. These cells are clearly distinguished by a combination of visual identification and electrophysiological and intrinsic properties. We report that these characteristics are unchanged in lesioned cortex, indicating that the malformation is not responsible for intrinsic alteration of the cell types. However, some firing properties demonstrate slight differences that may, in cooperation with the altered level of input, amplify the pro-epileptogenic changes in circuitry. Finally, we also report that there is anomalous expression of metabotropic glutamate receptors (mGluR) in malformed cortex. Our data show that the expression of mGluR5, normally causing no functional response in control cortex, contributes to the activation of interneurons in paramicrogyral (PMG) cortex. These findings provide new insight to the mechanisms of cortical hyperexcitability and identify a possible target for future pharmacological intervention.

Purpose: To evaluate the physical activity patterns of diabetic adults by the type of treatment they received Method: The study used secondary data collected by the National Health and Nutrition Examination Survey (NHANES) from 2001 to 2006. NHANES is a continuous study which measures the health and nutritional status of non-institutionalized citizens in the United States. To be eligible for the study, participants had to be an adult 18 years or older who responded during the interview phase of the survey that they had diabetes. Participants with any missing data pertaining to the variables were excluded. After exclusions, the final size of the study population was 957. The type of treatment was defined as: insulin only, oral antidiabetic medication only, or neither. Physical activity was defined according to the guidelines set forth by the American Diabetes Association. A logistic model was used to assess the association between the type of treatment and regular physical activity. All data analyses were performed using SAS 9.1. Results: Overall, only 28.2% of the study participants were involved in regular physical activity. With respect to the type of treatment they received, a majority of the participants (69.9%) took oral antidiabetic medication, while 23.1% used insulin. Only 7.1% didn’t take antidiabetic medication or insulin. Most of the study participants were either overweight or obese (86.1%). In relation to diabetes treatment type, the frequency of taking oral antidiabetic medication among those who were diagnosed with diabetes when they were 40 years of age or older was greater (76.9%) than the frequency of insulin use (64.9%) . Among this segment of the population, 81.6% didn’t use insulin or oral antidiabetic medication. Study participants who had diabetes for five years or less were more likely to take oral antidiabetic medication only, with 47.7% taking oral antidiabetic medication compared to 33.2% taking insulin. The crude odds ratio for insulin treatment and physical activity was 0.72 (CI, 0.32-1.61) while the crude odds ratio for treatment consisting of oral antidiabetic medication and physical activity was 0.61 (CI, 0.31-1.21). After adjusting for confounding, the odds of being physically active for patients on insulin treatment was 0.62 (CI, 0.28-1.39), and for those on oral antidiabetic medication the odds of being physically active was 0.53 (CI, 0.27-1.08), indicating that there was no statistical significance between either treatment group and physical activity participation. Conclusions: Although not statistically significant, the prevalence of regular physical activity was highest (37.3%) among diabetic individuals who used neither insulin nor oral antidiabetic medication to control their diabetes, while 26.7% of participants who used oral antidiabetic drugs and 30% of participants who used insulin exercised regularly. However, the failure to participate in physical activity is a common problem among all diabetics, irrespective of group distinctions. Thus, all diabetics should be encouraged to participate in physical activity to reduce future complications.

CD23 has long been appreciated to be a natural, negative regulator of IgE synthesis. This understanding is due in part to animal models in which CD23 deficient or CD23 transgenic animals display exacerbated or reduced IgE levels respectively. Interestingly, CD23 is susceptible to proteolytic cleavage from the cell surface. When this occurs, CD23 loses its regulatory capability and the solubilized form can lead to pro-inflammatory events through its cytokinergic activity on macrophages. Thus, targeting this specific cleavage would be beneficial to the control of allergic disease by stabilizing CD23 at the cell surface. Inhibitor studies performed by our group as well as others indicate that the enzyme responsible for CD23 ectodomain shedding is a hydroxamate-sensitive metalloproteinase. Through collaboration with the Blobel group, we analyzed various ADAM KO mouse embryonic fibroblasts (MEFs) and found no involvement of ADAMs 8,9,12,15,17,19, and 33 in CD23 shedding, however we did find a role for ADAM10. Using ADAM10 KO MEFs and ADAM10 specific inhibitors, we discovered that ADAM10 is indeed the CD23 cleaving enzyme or “sheddase”. Thus, developing strategies that would target ADAM10 could have an effect on sCD23 release and IgE production. In the CNS, signaling through the kainate receptor (KAR) by glutamate causes an increase in ADAM10 expression. Human B cells were found to express a GluK2 containing kainate receptor and its activation increased ADAM10 expression which is in agreement with KAR activation in the CNS. Although glutamate is considered a neurotransmitter, it signals in the periphery and elevated levels are associated with certain immune disorders. A significant corresponding increase in sCD23 release is observed as well. Remarkably, this activation induced a strong increase in B cell proliferation, IgG, and IgE production and these events can be reversed through the use of NS102, a specific KAR antagonist. Thus, we report for the first time the unique presence on B cells of a neurotransmitter receptor and that activation of this receptor could serve as a novel mechanism for enhancing B cell activation and Ig production. This enhancement and control thereof has implications for allergic and autoimmune diseases. Lastly, the CD23-ADAM10 axis was examined in a non-allergic disease state, B cell chronic lymphocytic leukemia (BCLL). BCLL is characterized by a large accumulation of CD23+ cells and high levels of soluble CD23 in the sera. After further analysis, we show that ADAM10 is indeed over-expressed in BCLL and could account for the high levels seen in this patient population. Furthermore, specifically targeting ADAM10 resulted in reduced soluble CD23 release, reduced proliferation, and enhanced apoptosis induction. Taken together the novel finding that ADAM10 is involved in CD23 shedding allows for targeted therapeutic intervention of both atopic and non-atopic disease states.

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by beta-amyloid (Aβ) aggregation/oligomerization, biometal dyshomeostasis, oxidative stress, and neuroinflammation. The multifactorial nature of AD may indicate the therapeutic potential of multifunctional ligands that tackle various risk factors simultaneously as effective AD-modifying agents. This notion is further supported by the fact that while numerous AD-modifying agents targeting one single risk factor have been developed and a number of them entered clinical trials, none of them has been successfully approved by the FDA. Furthermore, neuronal cell membrane/lipid rafts (CM/LR) have been demonstrated to associate with all the indicated risk factors, indicating that this relationship can be exploited therapeutically to design strategically distinct multifunctional ligands by incorporating CM/LR anchorage into molecular design. With the long-term goal of developing multifunctional ligands to slow or stop the progression of AD, recently we have embarked on the development of bivalent multifunctional Aβ oligomerization inhibitors (BMOIs) as potential AD-modifying agents. These BMAOIs contain curcumin as the multifunctional moiety and cholesterol as the CM/LR anchorage moiety linked by a spacer to co-target AβOs, CM/LR, and oxidative stress. The hypothesis of the BMAOI strategy is that BMAOIs will anchor/target the multifunctional AβO inhibitor moiety inside, or in the vicinity of, CM/LR in which Aβ oligomerization, Aβ/biometal interaction and oxidative stress occur to efficiently interfere with these processes. In support of this hypothesis, proof-of-concept of the BMAOIs strategy has been reached through our preliminary studies. Our results demonstrated that: 1) BMAOIs containing curcumin as the multifunctional AβO inhibitor and cholesterol as CM/LR anchor primarily localize to CM/LR while curcumin does not; 2) BMAOIs with optimal spacer length efficiently inhibit the production of intracellular AβOs and protect MC65 cells from AβO-induced cell death (EC50~3 µM) while curcumin exhibits no significant activity; 3) these active BMAOIs retain curcumin’s antioxidant and metal complexation properties. Our preliminary studies also demonstrated the critical roles of spacer length and connectivity in the molecular design of BMAOIs and one lead compound was identified for further structural modification and optimization. Furthermore, this lead compound was shown to cross the blood-brain barrier (BBB) in a preliminary in vivo study as well as bind to Aβ plaques. Taken together, these results clearly reach the proof-of-concept of BMAOIs and confirm the rationale of designing BMAOIs to develop potential AD-modifying agents. In this thesis, we continued the exploration and validation of the BMAOI strategy by designing and biological characterizing a series of BMAOIs containing cholesterylamine as the CM/LR anchorage moiety and curcumin as the multifunctional moiety. Ten BMAOIs with the spacer length of 15, 17, 19, 21, and 23 atoms were designed and synthesized. Initially, these BMAOIs were tested for the neuroprotective activity against the AβO-induced cytotoxicity in human neuroblastoma MC65 cells. Then, Western blot analysis was performed for active BMAOIs to confirm the association of neuroprotection and suppression of AβOs. Furthermore, active BMAOIs were examined for antioxidant and metal complexation properties. Finally, Aβ plaque binding was examined using transgenic AD mice brain sections. Our results demonstrated that the same spacer length but different connectivity are preferred in this new series of BMAOIs for neuroprotective activity as that of the lead compound from cholesterol series. Moreover, the neuroprotection activity is closely associated with the inhibition of AβOs as demonstrated by Western blot analysis. In addition, the active BMAOIs retain the antioxidant and biometal binding properties of curcumin. More importantly, the binding affinity to the Aβ plaques was again confirmed for the new BMAOIs containing cholesterylamine. In summary, the design and characterization of the new series BMAOIs further confirmed the rationale of BMAOI strategy and their potential to lead to a new direction in development of effective AD-modifying and treatment agents.