R-3 Repository :: Browsing by Author "Gustin, Michael C."

Browsing by Author "Gustin, Michael C."

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A gene therapy approach for tissue engineering applications
(2007) Lau, Ying Ka Ingar; West, Jennifer L.; Cameron, Isabel C.; Grande-Allen, K. Jane; Gustin, Michael C.
In this work, gene therapy was combined with cell therapy to tackle three tissue engineering applications. The goal of the first project was to promote endothelialization of tissue engineering vascular grafts (TEVGs). We developed a system called the collagen-based gene-activated matrix (GAM) which was able to retain plasmid DNA (pDNA) and allowed smooth muscle cells (SMCs) embedded to gradually take up and express the gene of interest, in this case, vascular endothelial growth factor (VEGF). To obtain better transfection efficiency, pDNA was complexed with polyethyleneimine (PEI) which dramatically improved transfection of SMCs in GAMs. Continual production of VEGF for approximately one month was observed. VEGF produced by SMCs in GAMs was bioactive and induced both enhanced migration and proliferation of endothelial cells (ECs) on collagen which is a common biomaterial for TEVGs. The goal of the second project was to potentiate angiogenesis through overexpression of VEGF in 10T1/2 cells for treatment of ischemic diseases and vascularization of tissue engineered constructs. 10T1/2 cells were transfected with the VEGF transgene successfully via retroviral transfection. VEGF-producing 10T1/2 cells were able to induce enhanced migration, proliferation, as well as invasion of underlying matrix in ECs. Potentiation of angiogenesis was further observed in 3D collagen models when ECs were co-cultured with VEGF-producing 10T1/2 cells. ECs formed extensive network of tubular structures and presence of a lumen in the vessels formed was confirmed by confocal microscopy. VEGF-producing 10TI/2 cells also rescued ECs from starvation and induced them to form organized tubular structures. The goal of the third project was to enhance mechanical strength in dermal wound through increased cross-linking of extracellular matrix (ECM) proteins via overexpression of lysyl oxidase (LO). Using the GAM system we developed and embedding transgene encoding LO with fibroblasts, we obtained enhanced mechanical strength in collagen constructs in vitro. We also demonstrated the same efficacy of these LO-producing GAMs in a dermal wound healing model in vivo.
A molecular dynamics approach towards evaluating osmotic and thermal stress in the extracellular environment
(Taylor & Francis, 2018) Fuentes, David; Muñoz, Nina M.; Guo, Chunxiao; Polak, Urzsula; Minhaj, Adeeb A.; Allen, William J.; Gustin, Michael C.; Cressman, Erik N.K.
OBJECTIVE: A molecular dynamics approach to understanding fundamental mechanisms of combined thermal and osmotic stress induced by thermochemical ablation (TCA) is presented. METHODS: Structural models of fibronectin and fibronectin bound to its integrin receptor provide idealized models for the effects of thermal and osmotic stress in the extracellular matrix. Fibronectin binding to integrin is known to facilitate cell survival. The extracellular environment produced by TCA at the lesion boundary was modelled at 37 °C and 43 °C with added sodium chloride (NaCl) concentrations (0, 40, 80, 160, and 320 mM). Atomistic simulations of solvated proteins were performed using the GROMOS96 force field and TIP3P water model. Computational results were compared with the results of viability studies of human hepatocellular carcinoma (HCC) cell lines HepG2 and Hep3B under matching thermal and osmotic experimental conditions. RESULTS: Cell viability was inversely correlated with hyperthermal and hyperosmotic stresses. Added NaCl concentrations were correlated with a root mean square fluctuation increase of the fibronectin arginylglycylaspartic acid (RGD) binding domain. Computed interaction coefficients demonstrate preferential hydration of the protein model and are correlated with salt-induced strengthening of hydrophobic interactions. Under the combined hyperthermal and hyperosmotic stress conditions (43 °C and 320 mM added NaCl), the free energy change required for fibronectin binding to integrin was less favorable than that for binding under control conditions (37 °C and 0 mM added NaCl). CONCLUSION: Results quantify multiple measures of structural changes as a function of temperature increase and addition of NaCl to the solution. Correlations between cell viability and stability measures suggest that protein aggregates, non-functional proteins, and less favorable cell attachment conditions have a role in TCA-induced cell stress.
A Multifaceted Approach to Enhance the Current Understanding and Treatment of Calcific Aortic Valve Disease
(2013-11-27) Wiltz, Dena; Grande-Allen, K. Jane; Gustin, Michael C.; Mikos, Antonios G.
Calcific aortic valve disease (CAVD) is a serious condition with unclear mechanisms driving this disease. This research focused on investigating the role of lysophosphatidylcholine (LPC) in CAVD and evaluating the efficacy of Raman spectroscopy (RS) to aid in current tissue engineering methods of heart valve replacements used to treat CAVD. Appropriate culture conditions for in vitro studies of CAVD were established. Specifically, the application of gentamicin in valvular interstitial cell (VIC) cultures was determined to significantly decrease mineralization of VICs in vitro in both normal and pre-calcified VIC culture conditions. Next, in vitro studies were conducted examining the role of LPC in a comparison of aortic and mitral VIC mineralization. Results indicated a higher percentage of LPC in calcified regions of tissue compared to non-calcified regions. In addition, 10000 nM LPC led to an increase in VIC mineralization, and aortic VICs displayed greater mineralization compared to mitral VICs. The role of the ryanodine receptor (RyR) in LPC-induced mineralization was evaluated. The presence of RyR isoforms 2 and 3 were confirmed in VICs. Next, in the presence of 10 µM LPC, the RyR was blocked and mineralization in VIC cultures significantly decreased compared to LPC treated cultures in which the RyR was not blocked. Several strategies exist for utilizing mesenchymal stem cells (MSCs) for tissue engineering of heart valves (TEHV) for valve replacement therapies. In this research, RS was able to detect distinct molecular characteristics of MSCs from different sources. This research has a significant impact on the study and understanding of CAVD. It suggests that gentamicin be used cautiously with in vitro studies of calcification, and suggest that mechanisms by which gentamicin acts in VICs may reverse calcification. In addition, these results showed that LPC has the capacity to promote VIC calcification, by interacting with the RyR, and that aortic VICs have a greater propensity for mineralization compared to mitral VICs. Also, RS may be used in future research to characterize MSCs prior to their use in TEHV. This research has highlighted the need for future investigations of LPC and the use of RS in understanding and treating CAVD.
Broad-Complex, a mediator of ecdysone induced stress response in Drosophila melanogaster
(2004) Hall-O'Connell, Veronica; Gustin, Michael C.
The better an animal can react to stressful conditions the longer it can live and possibly reproduce. Stress response and lifespan are under the control of two pathways of the endocrine system: the insulin/IGF-1 signaling pathway and a steroid hormone-signaling pathway. Mutations that alter the expression or function of the components of these pathways increase stress tolerance and lifespan. In D. melanogaster, mutants that reduce signaling the steroid hormone ecdysone show an increase in stress tolerance and lifespan. Although ecdysone signaling is important for stress resistance and lifespan, the molecular mechanism responsible remains to be elucidated. My thesis research focused on the ecdysone-induced family of zinc finger transcription factors known as the Broad-Complex (BR-C) as a possible downstream target of ecdysone mediated stress resistance and lifespan. I examined the role of BR-C in stress resistance and longevity by measuring the survival under varying conditions of flies that have altered BR-C expression. BR-C expression was altered by one of three methods: a P-element insertion into the BR-C loci that causes the misexpression of BR-C transcripts, heatshock overexpression of BR-C transgenes, and reducing BR-C transcript levels by RNAi. The results from these experiments suggest a complex correlation between BR-C expression and stress response. Global overexpression or underexpression of BR-C suggests a positive correlation between BR-C expression levels and stress resistance. Misexpression of BR-C transcripts by P-element insertion into the BR-C loci indicates an inverse correlation between BR-C expression levels and stress resistance. My thesis research allows us to conclude that BR-C mediates stress resistance and may be the downstream target of ecdysone mediated stress resistance at the molecular level.
Cloning and characterization of genes required for osmoregulation in the yeast Saccharomyces cerevisiae
(1994) Brewster, Jay Lynn; Gustin, Michael C.
Salt stress induces multiple physiological responses in unicellular organisms. Responses include both immediate effects upon membrane permeability to select cytoplasmic solutes and changes in gene expression. This study has sought to isolate genes required for osmoregulation in the yeast, Saccharomyces cerevisiae. Eighteen mutants, with an inability to grow under osmotically stressed conditions ($\rm Osm\sp{s}$), and deficient in salt induced glycerol accumulation were isolated. Each of the mutants fell into one of four complementation groups hog1 through hog4 (High Osmolarity Glycerol response). Three genes, HOG1, HOG2, and HOG4, that rescued growth of representative mutants under osmotically stressed conditions were cloned and disruption alleles generated. Disruption mutants of each gene displayed $\rm Osm\sp{S}$ growth and depressed glycerol accumulation following salt stress. Sequence analysis of HOG1 revealed striking sequence homology with mitogen-activated protein (MAP) kinases, including 50% identity with FUS3, a MAP kinase in the yeast mating response pathway. Immunoblots of normal and osmotically stressed cells detected an inducible tyrosine phosphorylation of HOG1, an event shown to activate homologous kinases. Site-directed mutagenesis of HOG1 on residues required for activation in related MAP kinases resulted in a loss of complementing activity on high salt plates and a lack of tyrosine phosphorylation of Hog1p when $\rm Tyr\sp{176}$ was mutated to Phe. HOG4 was identified to be a previously sequenced gene, PBS2, with strong sequence homology to MAPK kinases, kinases upstream of MAP kinases and thought to be responsible for the activating phosphorylation events. Hog1p was not tyrosine phosphorylated in a pbs2 mutant consistent with PBS2 being the kinase responsible for Hog1p phosphorylation. Osmotic stress was shown to result in aberrant terminal morphology (elongate, multi-budded, multi-nucleate cells) in both hog1 and pbs2 null mutants. Further characterization of the mutants using time lapse microscopy revealed that salt stress induces bud abandonment and aberrant bud site selection. HOG1 and PBS2 therefore are two genes involved in carrying an osmostress induced signal to stimulate adaptive cellular responses and are required for normal bud site selection following salt stress.
Effects of mechanical loading on osteoblast function using a three dimensional celijpolymer model
(1998) Jen, Anna Hsiao-Chieh; McIntire, Larry V.; Mikos, Antonios G.; Rudolph, Frederick B.; Gustin, Michael C.; Farach-Carson, Cindy
Mechanisms which trigger bone modeling/remodeling in response to changes in the mechanical environment are still unclear. In a three part study, effects of loading on osteoblast function were investigated using a three dimensional (3-D) ceWpolymer model. The 3-D model has advantages of cell culture while maintaining the natural matrix architecture of bone. Such celVpolymer constructs have been shown to form bone in vitro. Osteoblasts in 3-D ceWpolymer constructs were cyclically loaded (5% ). After five days, compressed constructs decreased in alkaline phosphatase activity, a marker of osteoblast maturation. After three weeks, loaded constructs showed lower alkaline phosphatase activity but higher RNA level of L-type calcium channels, involved in calcium signaling cascades. No difference was detected after twelve weeks. Results suggest osteoblasts sensed loading and altered functional activities in response. Use of the 3-D model to study other osteoblast functions under mechanical loading may increase understanding of regulated functional adaptation by bone.
Evolution of the Perlecan/HSPG2 gene and Regulation of its Expression by Inflammatory Cytokines in Normal Tissue Models and Cancer
(2014-04-21) Warren, Curtis Robert; Gustin, Michael C.; Wagner, Daniel S.; Carson, Daniel D.; Grande-Allen, K. Jane; Farach-Carson, Cindy
Perlecan is the large heparan sulfate proteoglycan common to all basement membranes. It has numerous functions in maintenance of BM integrity, cell signaling and scaffolding protein interactions. Perlecan accumulation is elevated in wound healing and is essential to organismal development. In this work the evolution of perlecan and its role in the simplest and most ancient animals are explored. Transcriptional regulation of the HSPG2 gene also is examined in human prostate cancer and associated stromal cells. The protein was elevated in the reactive stroma of primary prostate cancer and TNF-α was identified as the primary driver of HSPG2 expression induction in various prostate cancer, prostate stromal and bone marrow stromal cell lines. Various aspects of this response echo the fibroblastic response to wounding and tumor progression. HSPG2 homologues were found in the genomes of the cnidarian, Nematostella vectensis, and the placozoan, Trichoplax adhaerens. Thus the last common ancestor to encode a perlecan homologue is the placozoan Trichoplax adhaerens. N. vectensis perl elevation was identified as part of the gene expression profile of complex regenerating structures in the oral region of the animal following wounding. This is a conserved expression pattern of the gene which is still found in wound healing of modern mammals. These studies both demonstrate a role for perlecan in wound healing and pathological states, corroborating the hypothesis that the perlecan gene’s primary evolutionary role is to support tissues in times of remodeling.
Factors affecting bone cell growth and differentiation under differing culture conditions
(2000) Helmke, Christopher D.; Gustin, Michael C.
Marrow stromal cell (MSC) differentiation into osteoblasts is an important part of the bone growth and remodeling process. This process can be exploited to help solve the problem of bone wound healing. Because of problems with bone grafts, implantation of biodegradable 3D scaffolds seeded with MSCs has been suggested. However, differences in osteoblast differentiation in 2D versus 3D cultures remain unclear. In this study, rat marrow stromal cells (MSCs) were grown both on plastic and in 3D polymer scaffolds and their differentiation into osteoblasts studied. MSCs cultured in a synthetic 3D matrix differentiate faster into osteoblasts than those grown on plastic; the osteoblast differentiation markers alkaline phosphatase and osteocalcin peak in mRNA expression first in 3D in vitro cultures. The culture conditions of MSCs grown in 3D scaffolds were studied to determine the optimal conditions for osteoblastic differentiation. Factors such as cell density, scaffold seeding method, scaffold thickness and secreted soluble factors were investigated. Soluble factors secreted by the differentiated cells into the culture medium were found to be critical for timely differentiation. Lack of such factors promoted cellular proliferation over differentiation. Constant perfusion of cell culture medium through the scaffolds enhanced osteoblastic differentiation. Mature osteoblasts have been shown to undergo chemotaxis, and it is possible that their progenitor cells (MSCs) could as well. Very little is known about MSC chemotaxis. It is possible that they, like osteoblasts, can be recruited to a site where bone formation is needed. Under agarose chemotaxis assays were performed to investigate MSC chemotaxis toward osteoblast matrix factors or other cell types. MSCs did not appear to move under any of the conditions studied.
From START to FINISH: The Influence of Osmotic Stress on the Cell Cycle
(Public Library of Science, 2013) Radmaneshfar, Elahe; Kaloriti, Despoina; Gustin, Michael C.; Gow, Neil A.R.; Brown, Alistair J.P.; Grebogi, Celso; Romano, M.Carmen; Thiel, Marco
The cell cycle is a sequence of biochemical events that are controlled by complex but robust molecular machinery. This enables cells to achieve accurate self-reproduction under a broad range of different conditions. Environmental changes are transmitted by molecular signalling networks, which coordinate their action with the cell cycle. The cell cycle process and its responses to environmental stresses arise from intertwined nonlinear interactions among large numbers of simpler components. Yet, understanding of how these pieces fit together into a coherent whole requires a systems biology approach. Here, we present a novel mathematical model that describes the influence of osmotic stress on the entire cell cycle ofﾠS. cerevisiaefor the first time. Our model incorporates all recently known and several proposed interactions between the osmotic stress response pathway and the cell cycle. This model unveils the mechanisms that emerge as a consequence of the interaction between the cell cycle and stress response networks. Furthermore, it characterises the role of individual components. Moreover, it predicts different phenotypical responses for cells depending on the phase of cells at the onset of the stress. The key predictions of the model are: (i) exposure of cells to osmotic stress during the late S and the early G2/M phase can induce DNA re-replication before cell division occurs, (ii) cells stressed at the late G2/M phase display accelerated exit from mitosis and arrest in the next cell cycle, (iii) osmotic stress delays the G1-to-S and G2-to-M transitions in a dose dependent manner, whereas it accelerates the M-to-G1 transition independently of the stress dose and (iv) the Hog MAPK network compensates the role of the MEN network during cell division of MEN mutant cells. These model predictions are supported by independent experiments inﾠS. cerevisiaeﾠand, moreover, have recently been observed in other eukaryotes.
Genetic modification of smooth muscle cells to enhance the performance of tissue engineered vascular grafts
(2003) Elbjeirami, Wafa Mohammad; Gustin, Michael C.
Development of small-diameter blood vessels using tissue engineering has been promising as a novel therapy for patients who require coronary artery bypass grafting but lack suitable donor tissue. However, the performance of tissue engineered vascular grafts (TEVGs) has been challenged by the lack of a complete endothelium and by poor mechanical properties which contribute to blood incompatibility and burst failure in vivo respectively. In this work, these challenges have been addressed by genetically modifying vascular smooth muscle cells (VSMCs) to produce factors that will promote endothelialization and improve mechanical properties. Genetic engineering of VSMCs was explored using different methods to obtain the optimal transfection system. Viral transduction of cells resulted in the highest efficiency and stability of expression. Neomycin selection of virally transduced SMCs resulted in a homogenous population with high expression levels. To promote endothelialization of TEVGs, VSMCs were virally-transduced to produce vascular endothelial growth factor (VEGF) which acts as a chemoattractant and mitogen of endothelial cells (ECs). The proliferation of ECs significantly increased after exposure to VEGF-transfected SMCs or their conditioned media. The chemotactic response of ECs to the VEGF-producing cells was explored by different in vitro models which demonstrated increased migration of ECs in response to VEGF-transfected cells on both tissue culture treated surfaces as well as collagen-coated surfaces. To improve mechanical properties, lysyl oxidase (LO) was utilized to enzymatically crosslink extracellular matrix (ECM) proteins, particularly collagen and elastin, to enhance the mechanical integrity of the ECM and thereby impart mechanical strength to the engineered tissue. Viral transduction of VSMCs resulted in increased LO expression using Northern and Western analysis. Increased LO activity was demonstrated using a fluorescent enzyme substrate assay, and as increased levels of desmosine, a product of LO crosslinking, in the ECM. The mechanical effects of altered crosslink densities within tissue engineered constructs were demonstrated in a VSMC-populated collagen gel model. VSMCs transfected with lysyl oxidase were seeded in collagen gels; the tensile strength and elastic modulus in these constructs increased by approximately three-fold compared to constructs seeded with mock transfected VSMCs. Compositional analysis of the ECM deposited by the transformed cells showed similar collagen and elastin levels, and cell proliferation rates were similar as well, thus attributing increased mechanical properties to ECM crosslinking.
Hypertonic shock and the cell cycle: Identification of a stress-induced G2 delay in Saccharomyces cerevisiae
(1999) Alexander, Matthew Robert; Gustin, Michael C.
Exposure of S. cerevisiae cells to an increase in external osmolarity induces a temporary growth arrest. Recovery from this stress is mediated by the accumulation of intracellular glycerol and the transcription of several stress response genes. This study demonstrates that an additional effect is the triggering of a cell cycle delay during G2/M. Increasing the extracellular osmolarity results in an accumulation of 1N and 2N cells, and a depletion of S phase cells from an asynchronous culture. Additionally, hypertonic stress causes a decrease in mRNA from the B-type cyclin CLB2, phosphorylation of the cyclin dependent kinase Cdc28p, and inhibition of Clb2p-Cdc28p kinase activity, while Clb2 protein levels are unaffected. The osmotic stress induced G2 delay is dependent upon the kinase Swe1p. Surprisingly, this delay is not correlated with inhibition of Clb2p-Cdc28p kinase activity. Deletion of SWE1 prevents the phosphorylation of Cdc28p in response to hypertonic shock, and removes the block to cell cycle progression. Deletion of SWE1 also causes synchronized cultures stressed in G2 to accumulate cells with mislocalized nuclei. However, deletion of SWE1 does not prevent the hypertonic stress induced inhibition of Clb2p-Cdc28p kinase activity. Conversely, deletion of HOG1 does prevent Clb2p-Cdc28p inhibition, but does not block Cdc28p phosphorylation or significantly remove the block to cell cycle progression. Deletion of HOG1 does however further disrupt post-stress nuclear localization in combination with a swe1Delta mutation. Finally, preventing Cdc28p phosphorylation by a Y19F substitution does not disrupt the cell cycle delay to the same degree as deletion of SWE1. Together, these results suggest that osmotic stress-induced cell cycle delay is mediated in a novel way by Swe1p, with a contribution from the MAP kinase Hog1p.
Identification and characterization of yeast genes involved in defense against oxidative stress induced by macrophages
(2003) Ullmann, Breanna Diane; Gustin, Michael C.
Humans exist in a world fraught with exposure to microbial threats. Even baker's yeast has begun displaying virulence in an increasing number of cases. Our bodies employ a vast arsenal of antimicrobial defenses to ward off infection. Indeed, there is an ever expanding body of knowledge concerning the defenses mounted against pathogens. One potent antimicrobial defense involves the generation of radical species. These highly reactive molecules are capable of damaging nearly every cellular component of the microbe. Despite the significant threat posed by pathogenic organisms, correspondingly little is known about how microbes evade and resist elimination by the host. Even the contribution of antioxidant responses to virulence has only been studied minimally. Furthermore, nonpathogenic organisms also often possess defenses against oxidants that are similar to defenses found in pathogens. How is it, then, that the antioxidant defenses of virulent organisms help them more successfully evade oxidant-producing immune cells? To address this topic, a physiologically relevant macrophage co-culture system has been developed and applied to study oxidative stress responses in the typically avirulent baker's yeast, Saccharomyces cerevisiae, and in its pathogenic relative Candida albicans . The production of reactive nitrogen intermediates by macrophages appears to factor heavily in the killing of each of these yeast. Comparative studies of the nitric oxide (NO) defenses employed by S. cerevisiae and C. albicans reveal that both yeast possess flavohemoglobin-dependent mechanisms for NO consumption. However, C. albicans NO defenses are highly inducible and appear to be NO specific, setting them apart from similar defenses in Saccharomyces and various bacteria. Furthermore, C. albicans flavohemoglobin expression is induced during exposure to NO-producing macrophages. There is no flavohemoglobin homologue in the human genome, which may point to the Candida albicans flavohemoglobin-dependent NO defense system as a potential chemotherapeutic target for the development of new antifungal technologies. Although much remains to be learned about flavohemoglobin-based defenses, it seems likely that the unique NO consumptive characteristics of C. albicans contributes to its virulent status.
MAPK pathways of the yeast Saccharomyces cerevisiae: Cell integrity and filamentation/invasion pathway interaction with the hog pathway
(1999) Davenport, Kenneth Ray; Gustin, Michael C.
Mitogen Activated Protein Kinase (MAPK) cascades are frequently used signal transduction mechanisms in eukaryotes. Of the five MAPK cascade containing signaling pathways in the budding yeast Saccharomyces cerevisiae , the High Osmolarity Glycerol response (HOG) pathway functions to sense and respond to hypertonic stress. Mutants in the members of a second MAPK pathway, the cell integrity pathway, are sensitive to conditions of low osmolarity raising the possibility that this pathway may respond to hypotonic stress. We demonstrate that the cell integrity pathway MAPK Slt2p is phosphorylated in response to hypotonic stress in a rapid (<15 seconds), transient, and solute independent manner. The phosphorylation of Slt2p following hypotonic stress requires the upstream components of the cell integrity pathway as well as calcium, though a calcium influx into the cell is not sufficient to produce Slt2p phosphorylation. Interestingly, the HOG pathway MAPK Hog1p is rapidly de-phosphorylated in response to hypotonic stress in a cell integrity pathway dependent manner. This may indicate a cell integrity mediated regulation of the HOG pathway. It is possible that other pathways and proteins influence HOG pathway signaling or growth in various osmotic conditions. To investigate this possibility we utilized a mutant in the HOG pathway, pbs2-3, in a high copy suppressor screen to identify proteins that modulate growth on high osmolarity media. Three high copy suppressors of pbs2-3 osmosensitivity were identified: MSG5, CAK1, and TRX1. Msg5p is a dual specificity phosphatase that was previously demonstrated to dephosphorylate MAPKs in yeast. Deletions of the putative MAPK targets of Msg5p revealed that kss1Delta could suppress the osmosensitivity of pbs2-3. The filamentation/invasion pathway MAPK Kss1p is phosphorylated in response to hyperosmotic shock in a pbs2-3 strain, but not in a wild type strain or in a pbs2-3 strain overexpressing MSG5. Both TEC1 and FRE::lacZ expression is activated in strains lacking a functional HOG pathway during osmotic stress in a filamentation/invasion pathway dependent manner. Finally, the cellular projections formed by a pbs2-3 mutant on high osmolarity are absent in strains lacking KSS11 or STE7. These data suggest that the loss of filamentation/invasion pathway repression contributes to the HOG mutant phenotype.
Microorganisms and methods for their use
(1996-08-13) Gustin, Michael C.; Rice University; United States Patent and Trademark Office
A novel group of mutant yeasts and novel methods for selecting and using the mutant microorganisms are set out. A novel process for using the mutant yeasts to produce fermentation products with a lower than usual glycerol content is also disclosed. Novel beverages, including beers, wines, ales, and sake, and novel low glycerol baked goods are also possible using the novel mutants of the invention. The mutant yeasts are of the genus Saccharomyces or genus Torulaspora. The mutants are also used to isolate DNA which can be inserted into yeasts to produce transformed yeasts having either greater or smaller glycerol production. The transformed yeasts may also be used to produce novel beverages, baked goods, or glycerol.
Osmotic stress and the yeast actin-based cytoskeleton
(1994) Chowdhury, Sumita; Gustin, Michael C.
In order for cells to grow and survive, they must be able to overcome changes in their external environment. For example, stresses such as high or low temperatures and nutrient deprivation have been shown to elicit a response pathway which eventually results in the transcriptional or translational regulation of various genes. This study focuses on the ability of yeast cells to respond to and recover from the environmental stress of a change in external osmolarity. Because of its unicellular nature, it is vital that the sudden changes in internal turgor pressure caused by osmotic stress are compensated for, usually through the accumulation of glycerol inside the cell. Recently, it was found that strains with mutations in the single essential yeast actin gene, ACT1, were not able to grow at high temperatures or high osmolarity. This phenotype suggested that an underlying physiological process affecting cytoskeletal actin was involved in the response to osmotic stress. In order to understand this process, wild type yeast cells were analyzed at the morphological, genetic and molecular level. Results from this characterization led to the hypothesis that the response of actin to osmotic stress is regulated by interactions with other actin binding proteins. Potential candidates for these interactions were discovered by generating second site suppressors of an actin mutant and analysing them genetically. A dominant suppressor, RAH3, was found with four alleles, each having different phenotypes. Cloning of the recessive single mutant revealed that the gene product was the yeast homolog to fimbrin, a protein thought to regulate stress responses. Sequence analysis of the different alleles indicates that several sites dispersed throughout the fimbrin gene are important in regulating the cytoskeletal response to osmotic stress.
p38a/Mapkapk2a signaling regulates tristetraprolin in the yolk syncytial layer: A role for mRNA degradation in the morphogenesis of a novel embryonic structure in vertebrate development
(2014-04-02) Gomez de la Torre Canny, Sol; Wagner, Daniel S.; Bartel, Bonnie; Diehl, Michael R.; Gustin, Michael C.; Silberg, Jonathan J.
The yolk syncytial layer (YSL) is a novel embryonic structure that is unique to teleost fishes like the zebrafish. How existing genetic mechanisms can change to contribute to the generation of morphological novelties such as the YSL is a fundamental question of evolutionary biology. To address this question we examined the function of mapkapk2a (mk2a). Mk2a is required for YSL morphogenesis. To study the requirement of Mk2a signaling during embryogenesis, we analyzed the betty boop mutant (bbp). Bbp encodes Mk2a, the zebrafish homolog of mammalian MK2, a protein kinase activated by the p38 MAPK signaling pathway. bbp mutants display a striking lysis phenotype. bbp mutant embryos lose the expression of multiple YSL-specific genes. Thus, we examined the role of tristetraprolin (Ttp), a MK2 regulated mRNA-binding protein that promotes degradation of specific mRNA targets. Manipulation of the endogenous activity of Ttp showed that Ttp regulates the stability of YSL-specific mRNA molecules, most notably of mxtx2, which encodes for a zebrafish-specific transcription factor that activates a large proportion of YSL-specific genes. Specific activation of the Mk2a in the YSL inhibits Ttp activity in this cell layer, and prevents expression of Mxtx2 in other cells of the embryo. Expression of Mxtx2 or activation of the p38a /Mk2a pathway outside of the YSL results in dramatic defects in development. MK2 is not required for embryogenesis in mammals. Mutation of MK2 results in impaired inflammatory response and resistance to inflammatory diseases. The ability to manipulate the activity of the members of this conserved pathway in this novel context suggests that epiboly may be a useful platform to probe the molecular mechanism of TTP-dependent mRNA degradation that plays a crucial role in the regulation of the inflammatory response in mammals.
Rbt1 is required for Nitric Oxide Stress Resistance in the Human Commensal Fungus Candida albicans
(2013-12-03) Dou, Yanru; Gustin, Michael C.; Olson, John S.; Zhong, Weiwei
Candida albicans is a human‐resident opportunistic fungal pathogen. Persistence of C. albicans is largely affected by resistance to host‐generate nitric oxide (NO) mediated nitrosative stress. The transcription factor Cta4 was first identified by our group as a regulator of the nitrosative stress resistance gene YHB1. However, little else is known about the molecular mechanism of C. albicans resistance to NO. Here I propose that there are proteins besides Cta4 involved in the regulation of C. albicans nitrosative stress resistance and Rbt1 is one of the positive regulators. Supporting this hypothesis is the observation that Δrbt1 showed significantly inhibited growth when challenged with nitrosative stress producing chemicals nitrite and DPTA Nonoate. Quantitative PCR showed that YHB1 was no longer transcriptionally induced by nitric oxide compared to the wild type strain. Complementation with the plasmid expressing RBT1 gene is needed to confirm the function of Rbt1. But the fact that Δrbt1 mutants generated by other labs showed the same phenotype supported our observation. The screening assay indicated in this thesis yielded several mutants with significant sodium nitrite sensitivity and this suggests that multiple Candida albicans proteins might function synergistically to detoxify in vivo nitric oxide.
The CTA4 transcription factor mediates induction of nitrosative stress response in the fungal pathogen Candida albicans
(2007) Chiranand, Wiriya; Gustin, Michael C.
I have identified regulatory elements in the pathogen Candida albicans that enable response to nitrosative stress. This dimorphic fungus typically resides in the digestive and genitourinary tracts as an innocuous constituent of the normal microflora but can opportunistically cause superficial mucosal infection. In immunocompromised patients, such infections may also progress to potentially lethal systemic disease. One adaptation that facilitates survival of C. albicans against the hostile environment inside the mammalian body is the ability to resist toxic reactive nitrogen species (RNS) generated by macrophages of the host immune system. Recent studies have shown that exposing C. albicans to nitric oxide, one type of RNS, induces upregulation of the flavohemoglobin Yhb1p. This protein confers protection by enzymatically converting nitric oxide to harmless nitrate, but it is unknown how C. albicans is able to detect nitric oxide in its environment and thus initiate this defense only as needed. I therefore analyzed this problem by incrementally mutating the YHB1 regulatory region to identify a nitric oxide-responsive element (NORE) that is required for NO sensitivity. Five transcription factor candidates of the Zn(II)2-Cys6 family were then isolated by using magnetic beads coated with this DNA element in crude whole cell extracts. Of the five, only deletion of the CTA4 gene prevented induction of YHB1 transcription during nitrosative stress and caused growth sensitivity to the nitric oxide donor DPTA NONOate. The virulence of the cta4Delta deletion mutant was also mildly impaired, slightly more so than that of a yhb1Delta deletion mutant. Cta4p is the first protein found to be necessary for nitric oxide response in C. albicans .
The HOG MAPK pathway and yeast stress responses: Roles in oxidative stress and heat shock
(2002) Zhao, Qiang; Gustin, Michael C.
The HOG MAP kinase pathway in the budding yeast Saccharomyces cerevisiae senses and responds to high osmolarity. Here we demonstrated that HOG pathway mutants are hypersensitive to K1 killer toxin, which implies certain defects in their cell wall. Overexpression of the PBS2 gene leads to enhanced resistance to K1 killer toxin. Treating yeast cells with a pore-forming antifungal agent, amphotericin B, lowers the cellular turgor pressure. More importantly, amphotericin B treatment leads to activation of the HOG pathway, supporting the hypothesis that loss of turgor pressure activates the HOG pathway. Deficiencies in the HOG pathway also cause hypersensitivity to hydrogen peroxide and the superoxide-generating drug plumbagin. Hydrogen peroxide, menadione and plumbagin all activate the HOG pathway. The HOG pathway acts parallel to Skn7p and Yap1p in oxidative stress response, evidenced by the additive effect of hog1Delta, skn7Delta and yap1Delta on hydrogen peroxide sensitivity. Both ssn6Delta and sko1Delta suppress hog1Delta mutant sensitivity to oxidants. Oxidative stress induces transcription of HSP12 and HSP26. The HOG pathway regulates HSP12 transcription in this response. Msn2p and Msn4p are important for the oxidative stress-induced transcription of HSP12 and HSP26. The HOG pathway is also involved in heat shock response. Cells lacking the HOG1 gene are hypersensitive to heat stress. A temperature shift from 25°C to 37°C activates the HOG pathway. Such an increase in temperature also induces transcription of HSP12 and HSP26. The HOG pathway regulates HSP12 transcription in the heat shock response. Msn2p and Msn4p are important for the heat shock-induced transcription of HSP12 and HSP26.

Browsing by Author "Gustin, Michael C."

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