Browsing by Author "Blacklock, Brenda"

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    Aerobic Uptake of Cholesterol by Ergosterol Auxotrophic Strains in Candida glabrata & Random and Site-Directed Mutagenesis of ERG25 in Saccharomyces cerevisiae
    (2012-09-27) Whybrew, Jennafer Marie; Bard, Martin; Lees, N. Douglas; Blacklock, Brenda
    Candida albicans and Candida glabrata are opportunistic human pathogens that are the leading cause of fungal infections, which are increasingly becoming the leading cause of sepsis in immunosuppressed individuals. C. glabrata in particular has become a significant concern due to the increase in clinical isolates that demonstrate resistance to triazole antifungal drugs, the most prevalent treatment for such infections. Triazole drugs target the ERG11 gene product and prevent C-14 demethylation of the first sterol intermediate, lanosterol, preventing the production of the pathways end product ergosterol. Ergosterol is required by yeast for cell membrane fluidity and cell signaling. Furthermore, C. glabrata, and not C. albicans, has been reported to utilize cholesterol as a supplement for growth. Although drug resistance is known to be caused by an increase in expression of drug efflux pumps, we hypothesize a second mechanism: that the overuse of triazole drugs has lead to the increase of resistance by C. glabrata through a 2-step process: 1) the accumulation of ergosterol auxotrophic mutations and 2) mutants able to take up exogenous cholesterol anaerobically in the body acquire a second mutation allowing uptake of cholesterol aerobically. Two groups of sterol auxotrophic C. glabrata clinical isolates have been reported to take up sterol aerobically but do not produce a sterol precursor. Sterol auxotrophs have been created in C. glabrata by disrupting different essential genes (ERG1, ERG7, ERG11, ERG25, and ERG27) in the ergosterol pathway to assess which ergosterol mutants will take up sterols aerobically. Random and site-directed mutagenesis was also completed in ERG25 of Saccharmoyces cerevisiae. The ERG25 gene encodes a sterol C-4 methyloxidase essential for sterol biosynthesis in plants, animals, and yeast. This gene functions in turn with ERG26, a sterol C-3 dehydrogenase, and ERG27, a sterol C-3 keto reductase, to remove two methyl groups at the C-4 position on the sterol A ring. In S. cerevisiae, ERG25 has four putative histidine clusters, which bind non-heme iron and a C-terminal KKXX motif, which is a Golgi to ER retrieval motif. We have conducted site-directed and random mutagenesis in the S. cerevisiae wild-type strain SCY876. Site-Directed mutagenesis focused on the four histidine clusters, the KKXX C-terminal motif and other conserved amino acids among various plant, animal, and fungal species. Random mutagenesis was completed with a procedure known as gap repair and was used in an effort to find novel changes in enzyme function outside of the parameters utilized for site-directed mutagenesis. The four putative histidine clusters are expected to be essential for gene function by acting as non-heme iron binding ligands bringing in the oxygen required for the oxidation-reduction in the C-4 demethylation reaction.
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    Glycine max and Glycine soja are capable of cold acclimation
    (Wiley, 2017-12) Robison, Jennifer; Arora, Nigam; Yamasaki, Yuji; Saito, M.; Boone, J.; Blacklock, Brenda; Randall, Stephen; Biology, School of Science
    Soybean has been considered a cold intolerant species; based largely upon seed germination and soil emergent evaluations. This study reports a distinct acquisition of cold tolerance, in seedlings, following short acclimation periods. Diversity in cold responses was assessed in eight cultivars of Glycine max and six accessions of G. soja. All varieties of soybean significantly increased in freezing tolerance following acclimation. This study indicates soybean seedlings are indeed capable of sensing cold and acquiring cold tolerance. Germination rates after cold imbibition were negatively correlated with maturity group, but positively correlated with cold acclimation potential in G. soja. Seed fatty acid composition was varied between the species, with Glycine soja accessions containing about 2-times more linolenic acid (18:3) than G. max. Furthermore, high levels of linoleic acid (18:2) in seeds were positively correlated with germination rates following cold imbibition in G. soja only. We suggest that domestication has not impacted the overall ability of soybean to cold acclimate at the seedling stage and that there is little variation within the domesticated species for ability to cold acclimate. Thus, this brief comparative study reduces the enthusiasm for the “wild” species as an additional source of genetic diversity for cold tolerance.
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    Molecular dynamics simulations of spore photoproduct containing DNA systems
    (2023-05) Hege, Mellisa; Pu, Jingzhi; Blacklock, Brenda; Georgiadis, Millie; Deng, Yongming
    Bacterial endospores have been a topic of research interest over the last several decades given their high resistance to ultraviolet (UV) damage. Unlike vegetative bacterial cells, which form cyclobutane pyrimidine dimers (CPD) and pyrimidine 6-4 pyrimidone photoproducts (6-4PPs) as the major product upon UV irradiation, endospore bacteria form a spore photoproduct (5-(R-thyminyl)-5,6-dihydrothymine or SP) as the major product. Vegetative bacteria cells are subject to regular cell activities and processes such as division and deoxyribonucleic acid (DNA) replication, which are prone to damage from UV exposure. However, in endospores, which have a largely anhydrous inner environment, the DNA remains dormant when bound to spore-specific small acid-soluble proteins (SASP) and dipicolinic acid, making spores highly resistant to radiation, heat, desiccation, and chemical harm. During spore germination, SP lesions in DNA are repaired by a distinctive repair enzyme, spore photoproduct lyase (SPL). In this thesis, molecular dynamics (MD) simulations were carried out to (i) examine how the formation of the SP lesion in DNA affects the global and local structural properties of duplex DNA and (ii) study how this lesion is recognized and repaired in endospore. The first part of this work was focused on designing and developing a structurally and dynamically stable model for dinucleotide SP molecule (TpT), which was subsequently used as an SP patch incorporated into duplex DNA. Computationally, this requires modifications of the bond and nonbonded force field parameters. The stability of the patch and developed parameters was tested via solution-phase MD simulations for the SP lesion incorporated within the B-DNA dodecamer duplex (PDB 463B). The second part involved applying the new SP patch to simulate the crystallographic structure of the DNA oligomer containing SP lesions. Solution-phase MD simulations were performed for the SP-containing DNA oligomers (modeled based on PDB 4M94) and compared to the simulations of the native structure (PDB 4M95). Our analysis of the MD trajectories revealed a range of SP-induced structural and dynamical changes, including the weakened hydrogen bonds at the SP sites, increased DNA bending, and distinct conformational stability and distribution. In the third part of this thesis project, we carried out MD simulations of SP-containing DNA bound with SASPs to examine how the DNA interacts differently with SASP in the presence and absence of the SP lesion. The simulation results suggested decreased electrostatic and hydrogen bonding interactions between SASP and the damaged DNA at the SP site compared to the undamaged DNA-protein complex. In addition, decreased helicity percentage was observed in the SASPs that directly interact with the SP lesion. The last part of this this thesis work focused on the SP-dimer flipping mechanism, as the lesion is likely flipped out to its extrahelical state to be recognized and repaired by SPL. Using steered molecular dynamic (SMD) simulations and a pseudo-dihedral angle reaction coordinate, we obtained possible SP flipping pathways both in the presence and absence of SASP. Collectively, these simulation results lend new perspectives toward understanding the unique behavior of the SP lesion within the DNA duplex and the nucleoprotein complex. They also provide new insights into how the SP lesion is efficiently recognized and repaired during spore germination.
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    Recent Activities in the Center for Membrane Biosciences
    (Office of the Vice Chancellor for Research, 2011-04-08) Blazer-Yost, Bonnie L.; Randall, Stephen; Minto, Robert; Birch, Garrison; Haydon, Julie; Bacallao, Robert; Gattone, Vincent; Blacklock, Brenda
    The Center for Membrane Biosciences (CMB) is active in facilitating collaborative research among center members and other IUPUI community members. A number of seed grants have been made and the results from two will be presented. Recent major funding from the NSF supports a CMB-centered program that promotes intensive undergraduate research opportunities. Project 1: Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the slow growth of fluid-filled cysts in the kidney tubules and liver bile ducts. We identified LPA (lysophosphatic acid) as a component of cyst fluid that stimulates secretory Cl- and compensatory water flux into cysts through binding of receptors on the basolateral membrane of renal cells. LPA concentrations measured in ADPKD cyst fluid and in normal serum are sufficient to maximally stimulate ion transport. Thus, cyst fluid seepage and/or leakage of vascular LPA into the interstitial space are capable of stimulating secretion from epithelial cells resulting in cyst enlargement. Project 2: Upon the recent acquisition of Center-supported high-resolution mass spectrometers at IUPUI, methods for the analysis of lipid and protein samples to support nascent research endeavors within the CMB are being developed. Identification and quantification of sphingolipids in biological samples as well as other lipidomic experiments will be presented. Project 3: The IUPUI URM Immersion in Interdisciplinary Research in Biological Signaling program targets underrepresented minorities in the biological sciences, and through early and sustained undergraduate research experiences that are intensely mentored at multiple levels, aims to increase the number of underrepresented minorities achieving graduate degrees in the Biological Sciences. The first cohort will begin research in the program during the summer of 2011 and are currently in the selection process.
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    Renal Cyst Fluid From Human Polycystic Kidney Disease Patients Stimulates Cl- Transport: Active Factor and Cl- Channels
    (Office of the Vice Chancellor for Research, 2011-04-08) Blazer-Yost, Bonnie L.; Blacklock, Brenda; Bacallao, Robert L.; Gattone, Vincent H.
    Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the slow growth of fluid-filled cysts predominately in the kidney and in liver bile ducts. The factors involved in modifying the rate of cyst growth through epithelial proliferation or secretion are critical to understanding the progression of the disease. In addition, elucidation of mechanisms that potentiate the normal progression to renal failure will provide the basis for therapeutic intervention. Of note are the observations that the decline in renal function in middle age is precipitous and that renal injury results in an exacerbation of cyst growth. Using electrophysiological and biochemical techniques, we identified LPA (lysophosphatic acid) as a component of cyst fluid that stimulates secretory Cl- transport via two anion channels, CFTR and TMEM16a, in the mpkCCDcl4 model of renal principal cells. The LPA effect is manifested through receptors located on the basolateral membrane of polarized renal cells resulting in stimulation of channel activity in the apical membrane. Concentrations of LPA measured in ADPKD cyst fluid and in normal serum are sufficient to maximally stimulate ion transport. Thus, cyst fluid seepage into the interstitial space and/or leakage of vascular LPA are capable of stimulating epithelial cell secretion resulting in cyst enlargement. Research Support: IUPUI Membrane Biosciences Signature Center Grant