This research examined the impacts of water quality, phosphate-based corrosion inhibitors and pipe wall exposure on free chlorine (HOCl)/chloramine (NH2Cl) degradation and haloacetic acid (HAA)/N-nitrosodimethylamine (NDMA) formation in simulated distribution system water mains and household plumbing at bench-scale and pilot scale. In bench-scale bottle tests, the reactivity of fresh/pre-corroded pipe materials with HOCl/NH2Cl in decreasing order was: ductile iron, copper, lead. The addition of phosphate-based corrosion inhibitors generally increased HOCl/NH2Cl degradation for fresh iron coupons, but decreased HOCl/NH2Cl decay only for fresh copper coupons. Generally, these corrosion inhibitors did not impact HAA formation. Copper corrosion products, including Cu(II), Cu2O, CuO and Cu2(OH)2CO3, catalyzed HAA and NDMA formation. For HAAs, copper catalysis increased with increasing pH from 6.6 to 8.6 and/or increasing concentrations of these copper corrosion products. Interactions of copper with natural organic matter (NOM), likely by complexation, and the subsequent increase in the reactivity of NOM were proposed to be the primary reason for the increased HAA formation.NDMA formation increased with increasing Cu(II) concentrations, DMA concentrations, alkalinity and hardness but was inhibited by the presence of NOM. The transformation of NH2Cl to dichloramine (NHCl2) and complexation of copper with DMA were proposed to be involved in elevating the formation of NDMA at pH 7.0. Finally, in pilot-scale modified pipe loop tests, copper catalysis of NDMA formation was confirmed, especially under laminar flow conditions, and iron was shown to possibly catalyze NDMA formation under turbulent conditions. Orthophosphate increased the catalytic effects of iron but decreased copper catalysis on NDMA formation by either modifying the properties of the iron-associated suspended particles or reducing the dissolved metal concentrations. Orthophosphate increased chloramine decay when in contact with iron, likely by promoting nitrite formation, but orthophosphate decreased chloramine decay for copper and lead by reducing the availability of metal corrosion products.

It is well known boronic acids have its unique chemistry and related applications in organic synthesis. The boronic acid functionally group also plays very important roles in medicinal chemistry and chemical biology. For example, boronic acids have been developed as potential therapeutic agents, chemical biology tools. All these applications are directly related to the unique electronic and chemical properties of the boronic acid group. Herein, several application of boronic acids have been studied: 1) several groups of compounds were found as bacterial quorum sensing inhibitors; 2) a boronate compound was developed as a probe for detecting reactive oxygen species (ROS); and 3) boronic acid-modified aptamers can be used for glycoprotein recognition.

SecA is a central component of the general secretion system that is essential for growth and virulence of bacteria. A series of fluorescein analogs were tested against ATPase activities of Escherichia coli SecA. Rose Bengal (RB) and Erythrosin B are potent inhibitors abolishing the activities of three forms of SecA ATPase with IC50 in µM range. Both inhibit SecA intrinsic ATPase with two mechanisms depending on ATP concentrations, indicating they influence the two non-identical nucleotide binding sites differently. RB shows different inhibitory effects against three forms of SecA ATPase activities, suggesting that the inhibition is related to the conformation of SecA. RB with IC50 at sub-µM level is the most potent inhibitor of SecA ATPases and SecA-dependent protein translocation to date. The fluorescein analogs inhibit intrinsic ATPase of Bacillus subtilis SecA similarly, and also exhibit antibacterial effects in E. coli and B. subtilis. Our findings indicate the value of fluorescein analogs as probes for mechanistic studies of SecA and the potential development of new SecA-targeted antimicrobial agents. A series of SecA derivatives with truncated C-terminus within the first long α-helix of the helix-bundle extending the ATPase catalytic domain of N68 was analyzed. These SecA variants interact with lipids, and those containing the C-terminal portion of the long α-helix starting at residues #639 form the ring-like structure in liposomes, indicating the critical domains for forming the protein-conducting channel. The presence and length of the C-domain influence the response to RB of NBDII mutants and C-terminal truncates of SecA. Thus this region may interact with the inhibitors and is involved in the structure and regulation of SecA ATPase activity. B. subtilis SecA was analyzed for interspecies comparison. Despite sharing high homology, this SecA homolog cannot complement E. coli mutants with SecA defect. Phospholipids do not stimulate ATPase activities of B. subtilis SecA, but induce its conformational changes, leading to the lipid-specific domains and ring-like structures similar to E. coli SecA. These pore-ring structures may represent part of the protein-conducting channels. Therefore, the potential structural roles of SecA in the protein translocation machinery may be universal in both Gram-negative and Gram-positive bacteria.

Reagents that bind proteins are applicable in biology for detection of molecules, perturbation of signaling pathways and development of small-molecule pharmaceuticals. Protein ligands interact with proteins, inhibiting or altering their function. They are isolated from combinatorial libraries to interact with a specific target, using selection techniques such as phage display or yeast-two-hybrid assay. For the latter, one inconvenience is the detection of false positives, which can be solved by screening pools containing the samples to be tested, instead of individual samples. Samples are distributed in the pools following a pooling design. The PI-deconvolution pooling design was developed to screen cDNA libraries using the yeast-two-hybrid assay, which are smaller in size than protein ligand combinatorial libraries. Modifications to the PI-deconvolution screening technique were developed to adapt it to the screening of protein ligand combinatorial libraries using the yeast-two-hybrid assay. Every spot of the array containing the combinatorial library was randomly pooled. However, the yeast-two-hybrid assay loses sensitivity when strains are pooled. As PI-deconvolution requires detecting every interaction, we determined the optimal amount of library members that can be pooled in a spot, and the optimal number of replicates to ensure the detection of an interaction.<p> The yeast-two-hybrid assay was used to perform a screening of a combinatorial library with seven domains of BCR-ABL, which were pooled according to PI-deconvolution. BCR-ABL is a chimeric protein with unregulated kinase activity that is responsible for chronic myelogenous leukemia. The scaffold used in the combinatorial library was an engineered intein that forms lariat peptides. After a screening of this library was performed, positive interactions were detected in 775 spots of the arrays that contained 1432 positive hits. Only 53 spots were deconvoluted. The coding sequences of the lariat peptides were determined for 23 lariat peptides interacted with the GEF domain of BCR, and for ABL, two with the FABD domain, one with the SH1 domain, and one with the SH3 domain. Finally, a &beta;-galactosidase assay was performed to assess the affinity of the lariat peptides for their target.<p> The isolated lariat peptides are potential inhibitors of BCR-ABL that can have therapeutic potential. This study will improve other screenings of combinatorial libraries with the yeast-two-hybrid assay.

The pharmacological development of non-steroidal contraceptives has yet to be achieved. Arresting oocyte maturation without blocking ovulation has been evaluated using different inhibitors of the phosphodiesterase 3A (PDE3A). Unfortunately, PDE3A is also expressed in the heart and blood vessels, and inhibition of PDE3A in oocytes can produce cardiovascular side effects. We reviewed the literature on available PDE3 inhibitors and selected cilostazol (CLZ), which is an FDA approved therapeutic. CLZ has the ability to decrease cellular adenosine uptake and consequently antagonizes side effects of PDE3A inhibition in vital organs. CLZ inhibited oocyte meiotic maturation in vitro. CLZ has more degenerative impact on arrested oocytes than matured oocytes, indicating that prolonged meiotic arrest of oocytes is harmful. Administration of CLZ any time from 9h before the ovulatory stimulus to 4h after the stimulus resulted in ovulation of immature oocytes. Controlling CLZ dose, time of CLZ administration, and time of oocyte collection resulted in ovulation of oocytes at different meiotic stages. Oral administrations of CLZ in naturally cycling mice were also observed to block pregnancy whereas remating of those previously treated females resulted in normal offspring and litter sizes. Therefore, CLZ does not only have a wide margin of contraception but also is reversible. Ovulated immature oocytes were observed to have higher rates of advanced chromatin configuration and cortical granule distribution, normal spindle and chromosomal organization, maturation, and in vitro fertilization (IVF) than ovarian immature oocytes. Ovulated metaphase I oocytes that were matured in vitro or in vivo had higher IVF rates than ovulated mature oocytes. Ovulated germinal vesicle (GV) oocytes that were in vitro matured also showed higher IVF rates but when in vivo matured, they had lower IVF rates than ovulated mature oocytes because of the high degeneration and low fertilization rates associated with in vivo maturation of GV oocytes. In summary, CLZ merits further evaluation as a non-steroidal contraceptive and is capable of producing oocytes of various meiotic stages with advanced developmental features.

No description available.

The PI3K-PKB/Akt-PTEN signal transduction pathway orchestrates a variety of fundamental cell processes and its deregulation is implicated in several human diseases, including cancer. While the importance of this pathway to many cellular functions is well established, the mechanisms leading to context-specific physiological outcomes in response to a variety of stimuli remain largely unknown. Spatial restriction of signaling events is one of the means to coordinate specific cellular responses. To investigate the subcellular locus-specific roles of the major PI3K effector PKB/Akt in various cell processes, I have devised a novel experimental system employing cellular compartment-directed PKB/Akt pseudosubstrate inhibitors. The work herein describes the development and characterization of the localized PKB/Akt pseudosubstrate inhibitor system and its application to investigate potential locus-specific functions in established PKB/Akt-regulated cellular processes. Subcellular compartment-restricted PKB/Akt inhibition in the 3T3L1 adipocyte differentiation model revealed that nuclear and plasma membrane, but not cytoplasmic, PKB/Akt activity is required for terminal adipocyte differentiation. Nuclear and plasma membrane pools of PKB/Akt were found to contribute to distinct stages of adipocyte differentiation, revealing that PKB/Akt activity impacts multiple points of this program. The localized PKB/Akt pseudosubstrate inhibitor system was also utilized to investigate the importance of distinct subcellular pools of PKB/Akt in breast epithelial cells. MCF-10A human breast epithelial cells can be grown in three-dimensional culture to form acinar structures that recapitulate in vivo mammary glandular architecture. Expression of the plasma membrane PKB/Akt inhibitor during cell growth in three-dimensional culture severely impaired acinar formation. On the other hand, expression of the nuclear PKB/Akt inhibitor during acinar development resulted in the formation of large, misshapen, multi-acinar structures. Assessment of the migratory capacity of MCF-10A cells upon localized PKB/Akt inhibition revealed that nuclear PKB/Akt inhibition promoted, while plasma membrane PKB/Akt inhibition impaired, MCF-10A cell migration. The development of locus-specific PKB/Akt inhibitors represents the first attempt to prioritize the targets of this kinase based on their subcellular localization. This work and its immediate extensions will further our understanding of the biology of PKB/Akt, a multi-tasking kinase with profound roles in development, cellular and organismal homeostasis and disease.

The ErbB family of receptors are key regulators of growth, differentiation, migration and survival of epithelial cells. CI-1033 is an irreversible pan-ErbB tyrosine kinase inhibitor that has the ability to inhibit EGFR function but has shown limited therapeutic efficacy. Lovastatin targets the activity of HMG-CoA reductase, the rate-limiting step in the mevalonate pathway. In this study, the ability of lovastatin to potentiate the cytotoxic effects of CI-1033 was evaluated. The combination of lovastatin and CI-1033 exhibited some cooperative cytotoxic activity in a squamous cell carcinoma–derived cell line. This combination resulted in enhanced cell death by induction of a potent apoptotic response. Furthermore, this drug combination inhibited EGF-induced EGFR autophosphorylation and activation of the downstream signaling effectors, ERK and AKT. These findings suggest that combining lovastatin and tyrosine kinase inhibitors may represent a novel combinational therapeutic approach in squamous cell carcinoma of the head and neck.

Fluoxetine (FLX), the active ingredient of Prozac™, is a member of the selective serotonin reuptake inhibitor (SSRI) class of anti-depressant drugs and is present in aquatic environments worldwide. Previous studies reported that FLX is an endocrine disruptor in fish, bioconcentrating in tissues including the brain. Evidence implicates that serotonin influences the activity of the hypothalamo-pituitary-interrenal (HPI) stress axis, thus exposure to FLX may disrupt the teleost stress response. This study examined in vitro cortisol production in rainbow trout (Oncorhynchus mykiss) head kidney/interrenal cells exposed to FLX and 14C-pregnenolone metabolism in head kidney microsome preparations of FLX-exposed trout. Results indicated that cells exposed in vitro to increasing concentrations of FLX had lower cortisol production and cell viability (versus control) and microsomes isolated from trout exposed to 54 μg/L FLX had higher pregnenolone metabolism versus those of control and low FLX-exposed (0.54 μg/L) trout.

<p>The burden of hypertension and subsequent in Tibet is quite profound and disproportionate when compared to other Chinese populations. Thus, there has a recent impetus to focus on low-cost sustainable health interventions to ameliorate this tremendous burden. Factors of compliance of first-line low dose hypertensive medications are not known in semi-nomadic Tibetan herdsmen at high altitude.</p><p>A retrospective analysis of a de-identified database for a single blinded equal allocation randomized control trial for a dietary reduced sodium salt substitute completed in 2009 using STATA 11.2 (STATA INC. College Station, TX) and logistic regression was performed. Patients were recruited from two townships at 4300 m altitude and northwest of Lhasa, the regional capital. Eligibility criteria included: age 40 years and older, with hypertension (&#8805; 140mmHg / &#8805; 90 mmHg) , enrollment in salt substitute trial, and prescription of hypertensive medication. Primary outcome was compliance to medication at three and six months of follow-up. Factor variables included and adjusted for included: sex, age, blood pressure, township, class of medication, and trial arm assignment.</p><p>The overall rate of non-compliance was 33.0% (38/115) after three months and 12.9% (28/217) after six months. After three months follow-up patients with Stage I and Stage II hypertension were at an adjusted odds ratio of 0.03(95%CI: 0.002-0.70) and 0.13(95%CI: 0.012-1.37) times lower odds of non-compliance when compared patients with only isolated systolic hypertension, (p=0.028 and 0.089, respectively). Furthermore, at six months of follow-up patients prescribed combination pharmacologic therapy had an adjusted odds ratios of 0.20 (95%CI: 0.05-0.81) times lower odds than those patients on diuretic only, p =0.023.</p> / Thesis