Browsing by Author "Harcombe, Kimberley"
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Item 9-(2-Phosphonyl-methoxyethyl)-adenine promotes erythrocytic differentiation and disrupts cell replication in chronic myelogenous leukemia K562 cells(2021) Wiseman, Brittany; Harcombe, Kimberley; Bernstein, NinaDisruption during cellular differentiation can cause hematopoietic stem cells to proliferate uncontrollably, resulting in the development of cancer. Differentiation therapies are being investigated as a type of cancer treatment which involve inducing agents that promote the differentiation of cancer cells into those with similar properties to normal blood cells. These cells can then undergo apoptosis at an accelerated and controlled rate compared to cancer cells, making this a potential therapeutic technique. In this study, the ability of human chronic myelogenous leukemia K562 cells to undergo cellular differentiation in response to the inducing agent 9-(2-Phosphonyl-methoxy ethyl)-adenine (PMEA) is investigated. PMEA has previously been shown to disrupt cell replication, and promote erythrocytic differentiation in K562 cells. In order to further test the effectiveness of this inducer, cell proliferation was measured with a cell growth curve, hemoglobin presence was measured with benzidine staining, and gamma-globin expression (a protein subunit of fetal hemoglobin) was measured in both induced and uninduced K562 cell cultures via RT-qPCR and western blotting. The results indicate that PMEA slows cell replication, and promotes hemoglobin (and subsequently gamma-globin) expression in treated cells. In summary, the findings support the conclusion that PMEA is able to promote erythrocytic differentiation in K562 cells, and provides information that supports differentiation therapies as a method for cancer treatment.Item Antimicrobial properties of medicinal teas and their interactions with antibiotics(2020) Maziarz, Sydney; Harcombe, KimberleyThe rise of antibiotic-resistant bacteria has greatly increased the need for new drugs to be developed. These drugs may be new antibiotics, or synergists of existing antibiotics. Plants have been identified as a valuable source of new drugs, as plants produce secondary metabolites that have antimicrobial and synergistic activity. In addition, plants are the basis of many natural health products and dietary supplements, and public interest in these products continues to increase. The increasing popularity of natural health products also increases the possibility for antagonistic interactions to occur when these products are used in conjunction with antibiotics. The present study investigated the antimicrobial properties of aqueous extracts of three medicinal teas and their interactions with common antibiotics. The antimicrobial properties of the aqueous extracts of Bronchitis tea, Eyebright tea, and Hyssop tea were assessed via disk diffusion. It was found that both Eyebright tea and Hyssop tea extracts have antimicrobial properties, likely due to the phenolic acids and flavonoids found in extracts of these plants. Interactions between the tea extracts and antibiotics were assessed using a double disk diffusion method, where it was found that all three tea extracts interact antagonistically with sulfadiazine. Additionally, Eyebright tea extract was found to interact antagonistically with chloramphenicol. While further research is required to determine the mechanism of this interaction and the specific compounds involved, this research has identified that these teas should potentially be used with caution in combination with antibiotics. In addition, the antimicrobial properties of both Hyssop and Eyebright tea extracts warrant further research as these extracts may be the basis for new antimicrobial drugs in the future.Item Antimicrobial screening of phytochemicals produced by Albertan invasive weeds(2021) Supina, Brittany; Bott, Tina; Harcombe, KimberleyAntibiotic resistance has rendered many clinically-used antibiotics ineffective, creating an urgent need for new antimicrobial agents. Phytochemicals (secondary metabolites produced by plants) are produced in response to environmental stressors, and can inhibit the growth of bacteria, fungi and surrounding plants. Therefore, these phytochemicals offer an alternative source of antimicrobial compounds. The diversity and abundance of phytochemicals produced by plants can increase during the invasion of new habitats, making invasive weeds strong candidates for antimicrobial discovery. Despite this increase in phytochemical production, invasive plant species are often overlooked in favour of medicinal and edible plants, and few studies have characterized their antimicrobial activity. In this research, we used successive Soxhlet extractions with hexane, ethyl acetate, and methanol to extract the phytochemicals from Albertan invasive weed species collected from the Edmonton area. Using Kirby-Bauer disk diffusion assays, extracts were assessed for their ability to inhibit the growth of tester bacterial species including Escherichia coli, Bacillus subtilis, Pseudomonas aeruginosa, and Staphylococcus aureus, which represent a range of common pathogens and bacterial types. Preliminary characterizations of extracts from multiple plant species, including common tansy (Tanacetum vulgare) and woolly burdock (Arctium tomentosum), showed promising inhibitory activity against several bacterial species, warranting further investigation. This study provides a starting point for further bioactivity and chemical characterizations of Albertan invasive weeds and highlights these invasive plant species as potential leads for the development of new antimicrobial treatments.Item BldG and SCO3548 interact antagonistically to control key developmental processes in streptomyces coelicolor(2009) Parashar, Archana; Bignell, Dawn R. D.; Leskiw, Brenda K.; Harcombe, KimberleyThe similarity of BldG and the downstream coexpressed protein SCO3548 to anti-anti-sigma and anti-sigma factors, respectively, together with the phenotype of a bldG mutant, suggests that BldG and SCO3548 interact as part of a regulatory system to control both antibiotic production and morphological differentiation in Streptomyces coelicolor. A combination of bacterial two-hybrid, affinity purification, and far-Western analyses demonstrated that there was self-interaction of both BldG and SCO3548, as well as a direct interaction between the two proteins. Furthermore, a genetic complementation experiment demonstrated that SCO3548 antagonizes the function of BldG, similar to other anti-anti-sigma/anti-sigma factor pairs. It is therefore proposed that BldG and SCO3548 form a partner-switching pair that regulates the function of one or more sigma factors in S. coelicolor. The conservation of bldG and sco3548 in other streptomycetes demonstrates that this system is likely a key regulatory switch controlling developmental processes throughout the genus Streptomyces.Item Expression of ccaR, encoding the positive activator of cephamycin C and clavulanic acid production in Streptomyces clavuligerus, is dependent on bldG(2005) Leskiw, Brenda K.; Tahlan, Kapil; Harcombe, Kimberley; Bignell, Dawn R. D.; Jensen, Susan E.In Streptomyces coelicolor, bldG encodes a putative anti-anti-sigma factor that regulates both aerial hypha formation and antibiotic production, and a downstream transcriptionally linked open reading frame (orf3) encodes a putative anti-sigma factor protein. A cloned DNA fragment from Streptomyces clavuligerus contained an open reading frame that encoded a protein showing 92% identity to the S. coelicolor BldG protein and 91% identity to the BldG ortholog in Streptomyces avermitilis. Sequencing of the region downstream of bldG in S. clavuligerus revealed the presence of an open reading frame encoding a protein showing 72 and 69% identity to the ORF3 proteins in S. coelicolor and S. avermitilis, respectively. Northern analysis indicated that, as in S. coelicolor, the S. clavuligerus bldG gene is expressed as both a monocistronic and a polycistronic transcript, the latter including the downstream orf3 gene. High-resolution S1 nuclease mapping of S. clavuligerus bldG transcripts revealed the presence of three bldG-specific promoters, and analysis of expression of a bldGp-egfp reporter indicated that the bldG promoter is active at various stages of development and in both substrate and aerial hyphae. A bldG null mutant was defective in both morphological differentiation and in the production of secondary metabolites, such as cephamycin C, clavulanic acid, and the 5S clavams. This inability to produce cephamycin C and clavulanic acid was due to the absence of the CcaR transcriptional regulator, which controls the expression of biosynthetic genes for both secondary metabolites as well as the expression of a second regulator of clavulanic acid biosynthesis, ClaR. This makes bldG the first regulatory protein identified in S. clavuligerus that functions upstream of CcaR and ClaR in a regulatory cascade to control secondary metabolite production.Item A LexA-related protein regulates redox-sensitive expression of the cyanobacterial RNA helicase, crhR(2006) Owttrim, George W.; Harcombe, Kimberley; Patterson-Fortin, Laura M.Expression of the cyanobacterial DEAD-box RNA helicase, crhR, is regulated in response to conditions, which elicit reduction of the photosynthetic electron transport chain. A combination of electrophoretic mobility shift assay (EMSA), DNA affinity chromatography and mass spectrometry identified that a LexA-related protein binds specifically to the crhR gene. Transcript analysis indicates that lexA and crhR are divergently expressed, with lexA and crhR transcripts accumulating differentially under conditions, which respectively oxidize and reduce the electron transport chain. In addition, expression of the Synechocystis lexA gene is not DNA damage inducible and its amino acid sequence lacks two of three residues required for activity of prototypical LexA proteins, which repress expression of DNA repair genes in a range of prokaryotes. A direct effect of recombinant LexA protein on crhR expression was confirmed from the observation that LexA reduces crhR expression in a linear manner in an in vitro transcription/translation assay. The results indicate that the Synechocystis LexA-related protein functions as a regulator of redox-responsive crhR gene expression, and not DNA damage repair genes.Item Method optimization for the antimicrobial screening of pigmented invasive weed extracts(2020) Supina, Brittany; Giebelhaus, J. Duncan; Bott, Tina; Harcombe, KimberleyNew antimicrobial sources are required to address the growing prevalence of antibiotic-resistant pathogens. Secondary metabolites produced by plants (phytochemicals) are widely studied for their bacteria-inhibiting activity. Although the antimicrobial activity of medicinal and edible plants is well-known, few studies have examined compounds derived from invasive weeds. Invasive weeds produce large amounts of phytochemicals and can affect the bacterial composition of soils, making them a potential source of new bacteria-inhibiting compounds. Previously, the antimicrobial activity of Albertan invasive weeds was analyzed using Kirby-Bauer disk diffusion, broth microdilution, and drop check assays. Although the results showed promising bioactivity, plant pigment molecule interference at 600 nm prevented the accurate quantification of this antimicrobial activity during spectrophotometric analysis of broth microdilution assays. These issues highlighted a need for improved antimicrobial screening methods in the presence of pigmented plant extracts. Using ultraviolet-visible spectrophotometry, this study identified 750 nm as a wavelength that is minimally absorbed by phytochemicals in Albertan invasive weed extracts. As verified by bacterial growth curve analysis, this wavelength detects bacterial growth and may be used in broth microdilution assays to quantify antimicrobial activity. These findings offer a method for resolving pigment interference, improving the accuracy of antimicrobial screening of Albertan invasive weed extracts. These findings may also be applicable to the antimicrobial screening of other pigmented plant extracts and compounds. Overall, this method optimization may assist in the identification of new antimicrobial compounds derived from pigmented plant sources.Item Proteins and clades: a lab exercise using molecular methods to illustrate phylogenetic relationships among fish(2014) Harcombe, Kimberley; Das, MrinalExtended abstract a description of a student lab experiment using molecular methods to illustrate phylogenetic relationships among fish.Item The putative anti-anti sigma factor BldG is post-translationally modified by phosphorylation in Streptomyces coelicolor(2003) Harcombe, Kimberley; Bignell, Dawn R. D.; Lau, Leon H.; Leskiw, Brenda K.The Streptomyces coelicolor bldG gene encodes a protein showing similarity to the SpoIIAA and RsbV anti-anti-sigma factors of Bacillus subtilis. Purified maltose binding protein-BldG could be phosphorylated in vitro by wild-type S. coelicolor crude extract, and both the phosphorylated and unphosphorylated forms of BldG could be detected in vivo using isoelectric focusing. ATP was shown to serve as the phosphoryl group donor, and phosphorylation of BldG was abolished when the putative phosphorylation site was changed from a serine to an alanine residue. A bldG mutant strain expressing the non-phosphorylatable BldG protein was unable to undergo morphological differentiation or produce antibiotics even after prolonged incubation, suggesting that phosphorylation of BldG is necessary for proper development in S. coelicolor.Item Research techniques in molecular biology(2014) Harcombe, Kimberley; Hills, MelissaExtended abstract describing a new laboratory course at MacEwan University in the biological sciences discipline.Item RNA structural rearrangement via unwinding and annealing by the cyanobacterial RNA helicase, CrhR*(2005) Owttrim, George W.; Harcombe, Kimberley; Chamot, Danuta; Kujat-Choy, SonyaRearrangement of RNA secondary structure is crucial for numerous biological processes. RNA helicases participate in these rearrangements through the unwinding of duplex RNA. We report here that the redox-regulated cyanobacterial RNA helicase, CrhR, is a bona fide RNA helicase possessing both RNA-stimulated ATPase and bidirectional ATP-stimulated RNA helicase activity. The processivity of the unwinding reaction appears to be low, because RNA substrates containing duplex regions of 41 bp are not unwound. CrhR also catalyzes the annealing of complementary RNA into intermolecular duplexes. Uniquely and in contrast to other proteins that perform annealing, the CrhR-catalyzed reactions require ATP hydrolysis. Through a combination of the unwinding and annealing activities, CrhR also catalyzes RNA strand exchange resulting in the formation of RNA secondary structures that are too stable to be resolved by helicase activity. RNA strand exchange most probably occurs through the CrhR-dependent formation and resolution of an RNA branch migration structure. Demonstration that another cyanobacterial RNA helicase, CrhC, does not catalyze annealing indicates that this activity is not a general biochemical characteristic of RNA helicases. Biochemically, CrhR resembles RecA and related proteins that catalyze strand exchange and branch migration on DNA substrates, a characteristic that is reflected in the recently reported structural similarities between these proteins. The data indicate the potential for CrhR to catalyze dynamic RNA secondary structure rearrangements through a combination of RNA helicase and annealing activities.Item Site-directed mutagenesis of position 204 threonine to isoleucine failed to generate acid-tolerant EGFP(2023) Vignjevic, Mimi; Harcombe, KimberleyProtonation of green fluorescent protein (GFP) in acidic conditions prevents the emission of fluorescent light and limits the ability to visualize, localize, and study acidic organelles. Therefore, it is critical to introduce mutations into enhanced GFP (EGFP) to generate acid- tolerant fluorescent proteins. This experiment aimed to replicate a threonine to isoleucine mutation at position 204 in EGFP and identify if acid-stable fluorescent proteins would be produced in the BL21(DE3) Escherichia coli system. Site-directed mutagenesis was utilized to generate T204I mutant EGFP. SDS-PAGE and fluorescence microscopy were employed to analyze induction success and fluorescence. Spectrofluorophotometry was used to determine the excitation and emission spectra of T204I mutant EGFP and whether acid-tolerant proteins were generated. Results illustrated that the mutation of threonine to isoleucine at position 204 produced fluorescent proteins at pH 7. However, at pH 6 and 5, proteins failed to fluoresce. The replication of the T204I mutation failed to generate acid-stable EGFP proteins in the BL21(DE3) E. coli system. There is significance in generating acid-stable cellular markers, as currently, no cellular markers thrive in acidic conditions. This limits the ability to study acidic organelles and acidic cellular processes. Creating acid-tolerant markers will permit a greater range of biological research.Item Using anticipated learning outcomes for backward design of a molecular cell biology course-based undergraduate research experience(2020) Hills, Melissa; Harcombe, Kimberley; Bernstein, NinaAnticipated learning outcomes (LOs) were defined and used for the backward design of a Course-based Undergraduate Research Experience (CURE). These LOs reflect the inquiry-based nature of CUREs and capture key knowledge and skills inherent to scientific practice and essential in research. The LOs were used to plan a formative and summative assessment strategy to support and evaluate student achievement. A research question was identified that aligned with the learning goals of the course, provided an opportunity for discovery and iteration, and introduced a variety of molecular, cellular, and biochemical techniques. The course is offered to students in the final year of their degree and delivered over a 12-week period with two 3-hr labs each week. These LOs, and the rigorous assessment strategy used to support them, could be adapted to different projects. Likewise, the laboratory exercises are presented as a series of modules highlighting opportunities for adaptation to a variety of schedules.