Student Number : 9306227A - PhD thesis - School of Molecular and Cell Biology - Faculty of Science / It is well documented that N-capping motifs are stabilising local motifs for -helices. N-capping motifs have been identified within hGST A1-1 at the N-terminal ends of -helix 9 and helix 6. The conservational role of these two motifs in protein stability, folding and function was investigated. -Helix 9 is a unique structural feature to class Alpha GSTs that is important for its catalytic functioning. This amphipathic helix is highly dynamic, where upon ligand binding at the active-site, the delocalised C-terminal region becomes immobilised to form a structured helix forming a “lid” over the active-site. The specific role of the Asp N-cap motif toward the stability and dynamics of helix 9 was determined by substituting the Asp-209 for a Gly. ANS binding and urea-induced activity studies showed that by removing the N-cap motif of helix 9 in hGST A1-1, the helix 9 is destabilised rendering a less hydrophobic binding site compared to that in the wild-type. The helical content of the peptide, corresponding to helix 9 in the C-terminal region of hGST A1-1 (208 -222), decreased significantly upon the removal of the N-cap motif. The explanation for the conservation of the Asp N-cap residue can be found in its stabilising role of the C-terminal region of class Alpha GSTs. This stabilising role was however less apparent in context of the protein compared to that in the peptide. Majority of the atomic contacts owing to the stability of helix 9 appear to be governed by non-local tertiary interactions rather than local interactions, such as the N-cap motif. These tertiary interactions are likely to include short and long range contacts between residues on the surface of the protein that are already known to contribute towards the stability of the C-terminal region. In this study, the ligand displacement-studies and the molecular docking results strongly suggest that 8-aniline-1-napthalene sulfonate binds at the H-site in hGST A1-1. The N-capping motif of helix 6 identified in class Alpha GSTs is located within the core of domain 2. This motif is a common feature found amongst almost all GST-like proteins and is thought to be the folding nucleation site (Stenberg et al. J. Biol. Chem. 275 (2000), 10421-10428). The N-cap (Ser- 154) and N3 (Asp-157) residues were each substituted with an Ala in hGST A1-1 to investigate the role of this motif in the folding of hGST A1-1. Both substitutions resulted in thermal sensitive mutants compared to that of the wild-type. The N3 substitution (D157A) was however too disruptive, where the yields of this mutant were insufficient for any further studies to be carried out. For the N-cap mutant (S154A), the unfolding kinetic studies revealed a significantly destabilised core in domain 2 compared to that of the wild-type. The kinetic folding studies monitored by fluorescence spectroscopy, revealed that the N-cap motif contributes to the efficient folding and dimerisation of the subunits, and to a far lesser extent towards the final tight packing and reorganisation of tertiary interactions in hGST A1-1. Since no changes in the burst-phase of S154A was evident compared to that of the wild-type, it seems unlikely that this motif is a folding nucleation site in hGST A1-1. These results do not exclude the possibility that this motif contributes to the rapid formation secondary structure during the burst-phase of folding. Due to the highly conserved region surrounding helix 6 , the role of this motif contributing to the stability of hGST A1-1 could be a general feature for GSTs and GSTlike proteins. In this study, further insight into the mechanism of folding for hGST A1-1 was gained. The hydrophobic core packing surrounding helix 6 occurred as a late folding event, that is during the final packing and reorganisation of tertiary interactions of the protein. The N-cap motif is an important structural feature for the fast folding of domain 2. This N-cap motif is a unique structural feature important for the efficient folding of the monomers, which is exclusive to its role in stabilising helix 6 in hGST A1-1.

Magnetic Resonance Imaging instruments rely on a contrast agent to provide high-resolution images of tissues in vivo. However, current clinical contrast agents are hindered by low relaxivity and fast correlation time, necessitating high injection dosages. These concerns, among others, have driven the development of a class of protein-based contrast agents (ProCAs), by design of lanthanide binding sites into a scaffold protein. ProCA1 has a higher reported relaxivity and dosage efficiency than current contrast agents. In this study, expression and Glutathione-S-Transferase purification procedures were optimized, and a refolding method for rapid production of ProCA1 has been developed to enable studies of conformation, metal binding, relaxivity, and in vivo applications. Several ProCA1 variants with 4-5 charged ligand residues were shown to have strong gadolinium binding affinity (Kd of 10-12 M) and metal selectivity. Several options to improve ProCA1 have been explored, including addition of a polyethylene chain or a bombesin tag.

Dosage compensation is the key regulatory process employed in <i>Drosophila melanogaster</i> to equalize the level of gene transcripts between the single X chromosome in males (XY) and the two X chromosomes in females (XX). Dimorphic sex chromosomes evolved by the severe degeneration of the Y chromosome, giving rise to an imbalance between the heterogametic sex and the homogametic sex. Vital to the viability of male Drosophila is the dosage compensation complex (DCC), a ribonucleoprotein complex that mediates the precise two-fold transcription of the single male X chromosome. The DCC is comprised of five proteins: male-specific-lethal proteins (MSL) 1, 2, and 3, male absent-on-the-first (MOF), maleless (MLE), and two non-coding RNAs. The complex specifically co-localizes along the male X chromosome in a reproducible manner, resulting in acetylation of lysine 16 of the N-terminal tail of histone H4. The exact mechanism of recruitment and spreading of the DCC along the male X chromosome remains unclear; recent studies propose a multi-step mechanism involving DNA sequence elements, epigenetic marks, and transcription. Understanding how dosage compensation functions provides insight into the interplay between gene regulation and chromatin remodelling. The goal of this project was to better understand how <i>Drosophila</i> MSL1, MSL3, and MOF interact and how their interaction modulates MOFs acetyltransferase activity. Recombinant protein constructs were cloned and over-expressed in a bacterial expression system permitting future structure determination by X-ray crystallography. The dMSL1820-1039 construct consisted of the C-terminal domain, reported to be able to interact with both dMSL3 and dMOF. dMSL3186-512 contained the domain required for the interaction with dMSL1 and dMOF. dMOF371-827 was comprised of the catalytic domain, the CCHC zinc finger, and the chromodomain, as the N-terminal region does not encode any known domains. All three recombinant proteins were successfully cloned, over-expressed, and purified to homogeneity. Recombinant dMOF371-827 was determined to acetylate histones. Interaction studies using GST pull-down assays and size exclusion chromatography determined that dMSL1820-1039 and dMOF371-827 did not interact above background levels. Moreover, size exclusion chromatography revealed dMSL3186-512 and dMOF371-827 did not interact nor did the three recombinant proteins form a stable complex.

Dosage compensation is the key regulatory process employed in <i>Drosophila melanogaster</i> to equalize the level of gene transcripts between the single X chromosome in males (XY) and the two X chromosomes in females (XX). Dimorphic sex chromosomes evolved by the severe degeneration of the Y chromosome, giving rise to an imbalance between the heterogametic sex and the homogametic sex. Vital to the viability of male Drosophila is the dosage compensation complex (DCC), a ribonucleoprotein complex that mediates the precise two-fold transcription of the single male X chromosome. The DCC is comprised of five proteins: male-specific-lethal proteins (MSL) 1, 2, and 3, male absent-on-the-first (MOF), maleless (MLE), and two non-coding RNAs. The complex specifically co-localizes along the male X chromosome in a reproducible manner, resulting in acetylation of lysine 16 of the N-terminal tail of histone H4. The exact mechanism of recruitment and spreading of the DCC along the male X chromosome remains unclear; recent studies propose a multi-step mechanism involving DNA sequence elements, epigenetic marks, and transcription. Understanding how dosage compensation functions provides insight into the interplay between gene regulation and chromatin remodelling. The goal of this project was to better understand how <i>Drosophila</i> MSL1, MSL3, and MOF interact and how their interaction modulates MOFs acetyltransferase activity. Recombinant protein constructs were cloned and over-expressed in a bacterial expression system permitting future structure determination by X-ray crystallography. The dMSL1820-1039 construct consisted of the C-terminal domain, reported to be able to interact with both dMSL3 and dMOF. dMSL3186-512 contained the domain required for the interaction with dMSL1 and dMOF. dMOF371-827 was comprised of the catalytic domain, the CCHC zinc finger, and the chromodomain, as the N-terminal region does not encode any known domains. All three recombinant proteins were successfully cloned, over-expressed, and purified to homogeneity. Recombinant dMOF371-827 was determined to acetylate histones. Interaction studies using GST pull-down assays and size exclusion chromatography determined that dMSL1820-1039 and dMOF371-827 did not interact above background levels. Moreover, size exclusion chromatography revealed dMSL3186-512 and dMOF371-827 did not interact nor did the three recombinant proteins form a stable complex.

Several cell culture and animal studies demonstrated that citrus limonoids have protective effects against certain types of cancer. These chemopreventive properties of citrus limonoids are attributed to the induction of phase II enzyme, glutathione Stransferase (GST). In the current study, six citrus limonoids and two modified limonoids were utilized for the evaluation of NAD(P)H: quinone reductase (QR) activity and glutathione S-tranferase (GST) activity against 1-chloro-2,4-dinitrobenzene (CDNB) and 4-nitroquinoline 1-oxide (4NQO) in A/J female mice. In liver, limonoids that induced phase II enzyme activity were limonin-7- methoxime (32% CBNB), (270% 4NQO), (65% QR); and deacetylnomilin (180% QR). In stomach, limonin-7-methoxime (51% 4NQO); deacetyl nomilinic acid glucoside (55% 4NQO), nomilin (58% CDNB), (75% 4NQO); isoobacunoic acid (25% CDNB); deacetylnomilin (19% CDNB); limonoid mixture (45% 4NQO), (200% QR). Furthermore, in intestine, nomilin (280% 4NQO); deacetylnomilin (73% 4NQO), (22% QR); and the limonoid mixture (93% 4NQO) increase enzymatic activity. Finally in lung, deacetyl nomilinic acid glucoside (67% CDNB); limonin-7-methoxime (32% QR); and defuran limonin (45% QR) diplayed induction properties. Furthermore, D-glucaric acid (GA), a chemoprotective compound found in fruits and vegetables, was quantified using High Performance Liquid Chromatography (HPLC) grapefruit. Nine widely used grapefruit varieties were analyzed for the levels of D-glucaric acid. Seasonal levels of GA in each of the grapefruit varieties tested were found to be Thompson (58.36-126.8 mg/100ml), Henderson (29.6-49.7 mg/100ml), Rio Red (40.0-58.8mg/100ml), Star Ruby (25.5-46.7 mg/100ml), I-48 (26.6-58.3 mg/100ml), Ruby Red (49.3-63.0 mg/100ml), Ray’s Ruby (58.2-72.1 mg/100ml), Marsh (53.7-65.8 mg/100ml) and Duncan (50.17 mg/100ml). The HPLC method developed for the quantification of D-glucaric acid was found to be simple, fast, and reproducible. Additionally, the labor intensity and cost of sample preparation were greatly reduced.

Flavonoids, present in fruits, vegetables and beverages derived from plants, have been described as health-promoting, disease-preventing dietary supplements, and have activity as cancer preventive agents. The cancer protective effects of flavonoids have been attributed to a wide variety of mechanisms, including modulating enzyme activities resulting in the decreased carcinogenicity of xenobiotics. Cytochrome P4501A1 (CYP1A1) is a Phase I enzyme which is known to be involved in the activation of procarcinogens and Glutathione S-Transferase (GST) is a Phase II enzyme which is largely responsible for the detoxification of carcinogens. In this study, it was aimed to investigate the mechanisms of inhibition of CYP1A1 and GST activities of fish by phenolic compounds/flavonoids. Leaping mullet (Liza saliens), captured from highly polluted sites of izmir Bay, expressing high levels of CYP1A, were used in order to investigate these effects. It was demonstrated that all of the phenolic compounds/flavonoids used, exert an inhibitory effect on both CYP1A1 associated 7-Ethoxyresorufin-O-deethylase (EROD) activity and GST activities of fish, although the degree of inhibition was varied with the flavonoid used. Of the flavonoids tested, the most potent inhibitor of CYP1A1 associated EROD activity was found to be quercetin. The potency of the phenolic compounds/flavonoids to inhibit CYP1A1 associated EROD activity follow the sequence of quercetin &gt / resveratrol &gt / naringenin &gt / hesperidin &gt / rutin with IC50 values of 1.32 &micro / M, 3.59 &micro / M, 9.78 &micro / M, 98.5 &micro / M and 0.64 mM respectively. Quercetin, resveratrol, hesperidin and rutin were found to inhibit EROD activity in a competitive manner, on the other hand, naringenin was found to inhibit EROD activity in a non-competitive manner. Inhibition constant (Ki) values of quercetin, resveratrol, naringenin, hesperidin and rutin were calculated from Dixon plots as 0.12 &micro / M, 0.67 &micro / M, 2.63 &micro / M, 18 &micro / M and 0.1 mM, respectively. In the case of GST enzyme, it was demonstrated that all of the phenolic compounds/flavonoids used, exert an inhibitory effect on both total GST and GST-Mu activities of fish. Of the flavonoids tested, the most effective inhibitor of total GST activity was found to be resveratrol. The potency of the phenolic compounds/flavonoids to inhibit total GST activity follow the sequence of resveratrol &gt / quercetin &gt / rutin &gt / naringenin &gt / hesperidin with IC50 values of 7.1 &micro / M, 24.5 &micro / M, 89 &micro / M, 116 &micro / M and 118 &micro / M respectively. Resveratrol, quercetin and hesperidin were found to inhibit total GST activity in a competitive manner, on the other hand, rutin and naringenin were found to inhibit GST activity in a mixed type manner. Ki values of resveratrol, quercetin, hesperidin, naringenin and rutin were calculated from Dixon plots as 3.2 &micro / M, 12.5 &micro / M, 45 &micro / M, 128 &micro / M and 150 &micro / M respectively. In the case of GST-Mu activity, the most potent inhibitor was found to be rutin. The potency of the phenolic compounds/flavonoids to inhibit GST-Mu activity follow the sequence of rutin &gt / resveratrol &gt / quercetin &gt / naringenin &gt / hesperidin with IC50 values of 66.5 &micro / M, 72.3 &micro / M, 113.5 &micro / M, 135.5 &micro / M and 196 &micro / M, respectively. In conclusion, this study indicated that flavonoids were the strong inhibitors of CYP1A1 associated EROD activity and GST activities of mullet liver. The modulation of drug-metabolizing enzymes by flavonoids is important in terms of human health, since these enzymes can activate or inactivate carcinogens. The potential role of xenobiotic metabolizers CYP1 family in the activation of carcinogens and inactivation of chemotherapeutics suggests a potential therapeutic benefits in inhibiting these enzymes. The results of the present study support the hypothesis that flavonoids may be involved in the prevention of malignant transformation, by reducing the formation of carcinogens through inhibition of enzymes such as CYP1A1 which is known to be involved in carcinogen activation.

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Farmworkers in Apopka, FL, have been subjected to overhead pesticide exposure since the 1940s. Pesticides including Paraquat (PQ), Metribuzin and Aldicarb were sprayed onto the field while farmworkers worked. In "Fed Up: The High Cost of Cheap Food," farmworkers recalled the physical toll these conditions took on their bodies, blaming pesticides for their diseases, such as chronic kidney disease (CKD). While established that pesticides, specifically PQ, may be involved in some forms of Parkinson's disease, no explicit connection has been identified for SLE, CKD, and other diseases experienced by farm workers. This study evaluated whether pesticides could contribute to kidney disease. We quantified the fluorescence of reactive oxygen species (ROS) following varying PQ exposure in human embryonic kidney 293 (HEK293) cells using a microplate reader. Dosages of 75 and 150 µM were chosen based on previous literature. We also measured expression of KD biomarkers KIM-1 and NGAL upon PQ exposure with RT-qPCR. Glutathione-S-transferase pi 1 (GSTP1) served as an indicator of ROS. We predicted that ROS would increase with increasing PQ concentration, as would the fold change in the expression of the mRNA biomarker levels. The results showed a trend of increased expression of NGAL, KIM-1 and GSTP1 as PQ concentration increased. This study suggests metabolic panels may be an option when assessing patient health, especially patients susceptible to kidney disease. Future in vitro and in vivo examinations of these biomarkers are needed to clinically correlate physiological concentrations of these pesticides, and progression of kidney disease.

This research sought to determine which induction condition resulted in the greatest GST-GFP fusion protein expression. It will hopefully serve as a guide for future researchers trying to produce their own recombinant protein containing GST and GFP-tags. The CDNB Enzyme Assay was used to determine the quantity of GST-GFP fusion protein present and tested three variables: IPTG concentration, duration, and temperature of induction. The findings showed that IPTG concentration, temperature, and induction duration all had a significant impact on protein expression. Induction temperatures of 20 °C and 25 °C showed better protein expression at IPTG concentrations of 1.0 mM IPTG over 0.1 mM IPTG. Induction durations of 20 hours and 5 hours were better than 3 hours. The 25 °C condition had the greatest protein expression, followed by 20 °C condition. In a follow-up experiment, using 1.0 mM IPTG and 20- hour induction duration, the 20 °C condition showed higher GST activity. Analysis of the 20 °C Western blots revealed the presence of possibly truncated/degraded fusion protein (not seen in the 25 °C Western blots). Future experimenters should use C-terminal GST-tags, as opposed to N-terminal GST-tags, to prevent accidental purification of truncated proteins in addition to the target protein. If this is not possible, then the recommendation is to use the 25 °C temperature for induction, as this temperature had similar GST-GFP fusion protein expression and resulted in much cleaner Western blots.

Yes / The governments, nowadays, throughout the world are increasingly becoming dependent on public opinion regarding the framing and implementation of certain policies for the welfare of the general public. The role of social media is vital to this emerging trend. Traditionally, lack of public participation in various policy making decision used to be a major cause of concern particularly when formulating and evaluating such policies. However, the exponential rise in usage of social media platforms by general public has given the government a wider insight to overcome this long pending dilemma. Cloud-based e-governance is currently being realized due to IT infrastructure availability along with mindset changes of government advisors towards realizing the various policies in a best possible manner. This paper presents a pragmatic approach that combines the capabilities of both cloud computing and social media analytics towards efficient monitoring and controlling of governmental policies through public involvement. The proposed system has provided us some encouraging results, when tested for Goods and Services Tax (GST) implementation by Indian government and established that it can be successfully implemented for efficient policy making and implementation.