Browsing by Subject "inhibitor"

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Gene Expression Profiling in Hepatic Tissue of Newly Weaned Pigs Fed Pharmacological Zinc and Phytase Supplemented Diets
(2008-09) Martinez-Montemayor, Michelle M.; Hill, Gretchen M.; Raney, Nancy E.; Rilington, Valencia D.; Tempelman, Robert J.; Link, Jane E.; Wilkinson, Christopher P.; Ramos, Antonio M.; Ernst, Catherine W.; Wilkinson, Christopher P.
Zinc (Zn) is an essential trace element. However, Zn bioavailability from commonly consumed plants may be reduced due to phytic acid. Zn supplementation has been used to treat diarrheal disease in children, and in the U.S. swine industry at pharmacological levels to promote growth and fecal consistency, but underlying mechanisms explaining these beneficial effects remain unknown. Moreover, adding supplemental phytase improves Zn bioavailability. Thus, we hypothesized that benefits of pharmacological Zn supplementation result from changes in gene expression that could be further affected by supplemental phytase. The goal of this study was to investigate the effects of feeding newly weaned pigs dietary Zn (150, 1,000, or 2,000 mg Zn/kg) as Zn oxide with or without phytase [500 phytase units (FTU)/kg] for 14 d on hepatic gene expression. Liver RNA from pigs fed 150, 1,000, or 2,000 mg Zn/kg, or 1,000 mg Zn/kg with phytase (n = 4 per treatment) was reverse transcribed and examined using the differential display reverse transcription polymerase chain reaction technique. Liver RNA from pigs fed 150 or 2,000 mg Zn/kg ( n = 4 per treatment) was also evaluated using a 70-mer oligonucleotide microarray. Results: Expressed sequence tags for 61 putatively differentially expressed transcripts were cloned and sequenced. In addition, interrogation of a 13,297 element oligonucleotide microarray revealed 650 annotated transcripts (FDR <= 0.05) affected by pharmacological Zn supplementation. Seven transcripts exhibiting differential expression in pigs fed pharmacological Zn with sequence similarities to genes encoding GLO1, PRDX4, ACY1, ORM1, CPB2, GSTM4, and HSP70.2 were selected for confirmation. Relative hepatic GLO1 (P < 0.0007), PRDX4 (P < 0.009) and ACY1 (P < 0.01) mRNA abundances were confirmed to be greater in pigs fed 1,000 (n = 8) and 2,000 (n = 8) mg Zn/kg than in pigs fed 150 (n = 7) mg Zn/kg. Relative hepatic HSP70.2 (P < 0.002) mRNA abundance was confirmed to be lower in pigs fed 2,000 mg Zn/kg than in pigs fed 150 or 1,000 mg Zn/kg. Conclusion: Results suggest that feeding pharmacological Zn (1,000 or 2,000 mg Zn/kg) affects genes involved in reducing oxidative stress and in amino acid metabolism, which are essential for cell detoxification and proper cell function.
Identifying Novel Inhibitors of RpFabG in Typhus-inducing Rickettsia prowazekii
(2019) Panatpur, Aparna; Beckham, Josh
Epidemic typhus is a rickettsial disease that is contracted via ticks and lice found on the flying squirrel. The disease is caused by Rickettsia prowazekii, an intracellular, gram-negative coccobacillus. The biosynthetic pathways of Rickettsia prowazekii and its host are intertwined. Therefore, the ideal antipathogenic drugs would not target a protein that is found within the biochemical pathway of the human host. There is a type II fatty acid synthase pathway that is unique to Rickettsia prowazekii, which can be distinguished from the multienzyme type I fatty- acid synthase pathway used in humans. Fab G 3-ketoacyl-(acyl-carrier-protein) reductase (RpFabG) is a protein that is specific to this type II pathway. Hence, this research is focused on finding a small molecule drug that inhibits RpFabG. The coding DNA sequence for the RpFabG protein was previously cloned into a pNICJBsa4 plasmid, which was transformed into BL21(DE3 Escherichia coli and Dh5α competent cells. The transformed bacteria were cultured in LB media, and the cells were harvested. The expressed protein was purified via Ni-NTA affinity chromatography, made possible by a His6 tag on the vector. Gel electrophoresis was performed to determine the purity of the obtained protein sample; due to indication of slight contamination, gel filtration fast protein liquid chromatography was run on a concentrated protein sample. Genetic Optimization of Ligand Docking (GOLD is a molecular docking software package that was used to rank potential inhibitors of RpFabG according to binding strength; ethyl acetoacetate (EAA and acetoacyl coenzyme A (AAC) were determined to have high binding strengths and thus determined to be strong potential inhibitors. An enzyme assay was run to determine the functionality of the enzyme, with EAA and AAC as substrates. AAC successfully decreased the enzymatic activity of RpFabG, suggesting its potential as a novel drug. DSF assay results were equivocal, indicating that inhibition assays should be run on AAC to further assess its potential as an inhibitor.
Progress in the search for histidine acid phosphatase inhibitors in Francisella tularensis
(2014-04) Pu, Ruoyi; Robertus, Jon D.
Histidine acid phosphatase (hap) is an important enzyme in the bacteria Francisella Tularensis that is responsible for the cleavage of phosphate groups from phosphorylated proteins. Here, we report on the process for discovering potential inhibitors of the hap enzyme. A sample of the active enzyme was obtained through cloning of the DNA sequence and expression and purification of the enzyme. Using the GOLD program, virtual screening was performed on a crystal structure of the hap enzyme found in the Protein Database (PDB) and two compounds from the top results were ordered from ChemBridge. Three compounds, the two found from virtual screening and a third random compound, were tested for inhibition using a spectrophotometric assay. Out of the three compounds, only compound 5852635 showed potential inhibition of the hap enzyme after enzyme inhibition assays. Further work can now be done on compound 5852635 to determine whether it will consistently inhibit the hap enzyme and the IC50 of this compound. Also, more virtual screening work can be done to find other compounds which inhibit the hap enzyme.
Study on Inhibition Mechanism of Polymer Parts in Selective Inhibition Sintering Process
(University of Texas at Austin, 2015) Nouri, Hadis; Khoshnevis, Behrokh
The selective inhibition sintering (SIS) process is an additive manufacturing technique that builds parts based on powder sintering. In this process parts are made upon deposition of a solution, called inhibitor, at the boundary profile. The inhibitor prevents polymer particles to coalescence under heat exposure on each layer. The inhibited boundary profile acts as a sacrificial mold that can be easily removed, leaving a chemically pure part. One of the influential factors in fabrication resolution by SIS is the proper selection of an inhibitor. The best inhibition mechanism results in ease of separation of the part from undesired regions while maintaining part accuracy. In this paper, we investigate a framework for selecting an appropriate inhibitor for the process. Different experiments have been performed and inhibition theory has been studied on polyamide (PA) samples. Specifically, as an alternative to exhaustive experiments on solutions and polymers, a hybrid method based on crystallization is proposed to characterize the effectiveness of the inhibitor. Differential Scanning Calorimetry (DSC) tests are used to study changes in thermal properties of the samples. It is found that the sintering period increases as crystallinity of the polymer decreases. Results show that polymer samples exposed to sodium hypochlorite solution has longer sintering periods. Design of experiments has been used to study shrinkage behavior of polymer samples through bulk sintering.
Targeting a Critical Enzyme to Combat Bubonic Plague
(2016) Vasudevan, Anita; Beckham, Josh
Yersinia pestis is the bacterium that causes plague infection, which continues to be prevalent in rural areas of the world. Moreover, because pneumonic plague has a high mortality rate even with antibiotic therapy, aerosolized Y. pestis is a powerful bioterrorism weapon. As antibiotic-resistant strains of Y. pestis appear, novel therapeutic targets are increasingly needed to combat this disease. YopH is a phosphatase in Y. pestis that is necessary for infection and was therefore considered a novel therapeutic target for plague. The gene for YopH was synthesized via primer overlap PCR, cloned into the pNIC-Bsa4 plasmid, and expressed in E. coli. The enzyme was purified using Ni-NTA affinity chromatography and FPLC gel filtration. Virtual screening in GOLD and ICM was used to dock large chemical libraries into the active site of an X-ray crystallography structure of YopH in order to identify a compound that can serve as an inhibitor of this enzyme, thereby treating plague infection. Top-scoring ligands were identified and purchased for testing via biochemical assays. The inhibition activity of each ligand was measured, and one compound, of 4-[(2-bromo-4-methylphenyl)amino]-4-oxo-2-butenoic acid (Chembridge ID 5303660), showed potential as an inhibitor. Kinetic experiments and differential scanning fluorimetry suggested that this compound may not be a competitive inhibitor of YopH. YopH was crystallized, but crystals formed in clustered star-like patterns which were not ideal for diffraction. Future experiments will focus on optimizing crystals. The ultimate goal is cocrystallizing YopH with the compound of interest in order to further characterize this relationship and determine if the compound may serve as a novel therapeutic for plague.