Biochemistry and Molecular Biology
Presenter(s): Haley Reinhard, Farida Mossaad, Yen Nguyen
Advisor(s): Dr. Aftab Ahmed
The World Health Organization (WHO) is responsible for the international public health published a book entitles "WHO traditional medicine strategy 2014-2023". One of the major issues on complementary medicine is the lack of research data. In the developing world, over 80% of the population use traditional and complementary medicine to treat various ailments. Our laboratory focus is in the evaluation and characterization of bioactive proteins and peptides from various plant components use as the complementary medicine for various disorders. In this poster we are presenting our preliminary data on the Mulberry fruit found mainly in Asia and North America. It has health benefits against multiple disease, lower high cholesterol, diabetes, and reduce cancer risk. The presence of plant compounds such as anthocyanins, rutin, chlorogenic acid and myricetin, but lacks data on proteins and peptides from Mulberry. We are reporting here our preliminary findings on the extraction and purification of proteins and peptides from Mulberry fruit using column chromatography and gel electrophoresis. The SDS-PAGE profile revealed that Mulberry fruit extract has several proteins and peptides ranging from 5-75 kDa. The partial purification of proteins achieved using gel filtration and reversed phase HPLC. Further study is in progress to evaluate their homogeneity and primary structure using Edman sequencing and LC/MS/MS technologies.
Type I Diabetes Mellitus Adversely Impacts Ocular Surface Glycocalyx and Mucins
Presenter(s): Jacob Baker, Saleh Alfuraih, Kiumars Shamloo
Advisor(s): Dr. Ajay Sharma
Purpose: It is well known that poor glycemic control in patients of diabetes mellitus causes retinopathy and cataract. Recent evidence suggests that diabetes mellitus can also have a detrimental impact on the anterior segment of the eye. The present study was designed to examine the effect of type I diabetes mellitus on corneal glycocalyx, tear film and corneal epithelial defects. Methods: Type I diabetes was induced in C57 mice by a single intraperitoneal injection of streptozotocin (150 mg/kg). The blood glucose was monitored using a glucometer. Tear film volume was quantified using phenol red thread test. The corneas were stained using fluorescein for epithelial defects and imaged using cobalt filter slit lamp for corneal keratopathy scoring. The corneas were harvested at week 1 and week 2 and the glycocalyx was stained using wheat germ agglutinin. The stained corneas were imaged using a confocal microscope to obtain Z stack images. Results: Induction of type I diabetes after streptozotocin injection in the mice was confirmed by high blood glucose levels (>500 mg/dl). The diabetic mice showed a significant decrease in the tear volume as early as week 1 after the induction of diabetes. Slit-lamp imaging revealed a high corneal keratopathy score of 5-9 in these diabetic mice. Digital quantification of confocal images showed a 12- 20% decrease in the area of stained corneal glycocalyx in these diabetic mice. Conclusions: Our data demonstrates that type I diabetes mellitus negatively impacts tear film, causes epithelial surface defects as revealed by corneal keratopathy and a reduction in the corneal glycocalyx.
A Comparative Analysis of Cytokine Gene Expression in Human Conjunctival and Corneal Epithelial Cells in Response to Hyperosmotic Stress
Presenter(s): Priya Mistry, Kiumars Shamloo
Advisor(s): Dr. Ajay Sharma
Increase in tear osmolarity and chronic inflammation are cardinal feature of dry eye disease. The ocular surface is covered by conjunctival and corneal epithelial cells. Exposure of ocular surface epithelial cells to hyperosmolar stress may initiate inflammatory response. Therefore, the present study was designed to investigate the differential changes in the gene expression of proinflammatory cytokines in human conjunctival and corneal epithelial cells upon exposure to hyperosmolar stress. The cultured human corneal and conjunctival epithelial cells were exposed to hyperosmolar stress for 12 and 24 hours. The mRNA was isolated from these cells and was reverse transcribed into cDNA. The cDNA was used for quantification of IL1, IL6 and TNF-alpha gene expression by real-time PCR. Hyperosmotic stress caused a 1.5- and 2-fold increase in the gene expression of IL1 in the human conjunctival and corneal epithelial cells respectively. Hyperosmotic stress also caused a 3- 4 fold increase in IL6 expression in conjunctival cells, whereas the increase in IL6 gene expression was much more robust in human corneal epithelial cells. Similarly, hyperosmotic stress caused a 2-3 fold increase in TNF-alpha in human conjunctival cells, whereas a >10-fold increase in TNF-alpha expression was observed in the human corneal epithelial cells. Our data suggests that hyperosmolar stress causes an increase in the gene expression of proinflammatory cytokines in cultured human corneal and conjunctival epithelial cells. Corneal epithelial cells seem to be more sensitive to the detrimental effect of hyperosmotic stress. The hyperosmolar stress-mediated increase in proinflammatory cytokine milieu may be partly responsible for dry eye associated chronic low-grade inflammation.
Analyzing Restored Nitrogen Fixation Through CowN in G. diazotrophicus
Presenter(s): Chloe Nicole Garcia, Kiersten Chong, Terrence Lee, Max Strul, Ruchita Kharwa, Emily Wong, Kevin Bretzing
Advisor(s): Dr. Cedric Owens
Nitrogen fixation occurs when atmospheric dinitrogen gas is reduced to ammonia. In certain bacteria, such as Gluconacetobacter diazotrophicus, nitrogen fixation is catalyzed by the enzyme nitrogenase, a multisubunit protein composed of an iron protein subunit (FeP) and a molybdenum-iron protein subunit (MoFeP). Nitrogenase is inhibited by carbon monoxide (CO). In certain soil conditions, CO levels are sufficiently high to inhibit nitrogenase activity and prevent nitrogen fixation. However, G. diazotrophicus expresses a protein called CowN that prevents the inhibition of nitrogenase by CO. This current research attempts to understand the mechanism of how CowN prevents inhibition of nitrogenase to restore nitrogen fixation. We have expressed G. diazotrophicus CowN heterologously in E. coli and purified the protein to homogeneity. In vitro studies with FeP, MoFeP, and CowN in the presence of CO showed that CowN effectively restores nitrogenase activity for CO concentrations up to 0.1 atm. The activity of CowN exhibits Michaelis-Menten-like kinetics with a Km of approximately 8 µM. Our experiments further show that CowN, which exists in both a monomeric and oligomeric state, is only active as a monomer. Further studies have partially elucidated how CowN binds to nitrogenase and if the prevention of inhibition is due to CowN directly preventing CO access to the active site of nitrogenase.
Protection of Nitrogen Fixation by CowN in G. diazotrophicus
Presenter(s): Kiersten Chong, Chloe Garcia, Terrence Lee, Max Strul, Ruchita Kharwa, Emily Wong, Kevin Bretzing
Advisor(s): Dr. Cedric Owens
Nitrogen fixation is the process in which atmospheric dinitrogen is reduced to ammonia. Nitrogen fixation occurs naturally in certain bacteria, such as Gluconacetobacter diazotrophicus, via the enzyme nitrogenase, a multisubunit protein with an iron protein subunit (FeP) and a molybdenum-iron protein subunit (MoFeP). Nitrogenase is inhibited by carbon monoxide (CO). In some soil conditions, CO levels are sufficient to inhibit nitrogen fixation. However, nitrogenase inhibition is prevented by CowN, a protein expressed by G. diazotrophicus. This research attempts to understand the mechanism behind CowN-mediated protection of nitrogenase from CO inhibition. We expressed G. diazotrophicus CowN heterologously in E. coli and purified the protein to homogeneity. In vitro, in presence of CowN, nitrogenase is able to tolerate CO concentrations up to 0.1 atm, a concentration that completely inhibits the enzyme in the absence of CowN. The activity of CowN exhibits Michaelis-Menten-like kinetics with a Km of approximately 8 µM. Our experiments further show that CowN, which exists in a monomeric and oligomeric state, is only active as a monomer. Future work will aim to elucidate if/how CowN binds to nitrogenase and if CowN protects nitrogenase by directly preventing CO access to the active site or if it engenders CO reduction abilities to nitrogenase.
Structural and Biophysical Characterization of CowN from Gluconacetobacter diazotrophicus
Presenter(s): Christine Lo Verde, Alejandro Espinoza, Kevin Bretzing
Advisor(s): Dr. Cedric Owens
The bacterium Gluconacetobacter diazotrophicus expresses the enzyme nitrogenase, which converts atmospheric dinitrogen to ammonia- a critical source of nitrogen for plants. In the presence of the environmental gas-carbon monoxide (CO)-nitrogenase is inhibited. However, CowN, a protein found within many diazotrophs, can prevent CO from exerting its inhibitory effects on nitrogenase. CowN not only protects nitrogenase against CO, but is shown to have elevated expression in cold temperatures, suggesting that CowN may function as a cold response protein. Given the limited research conducted on CowN, its structure and function remain mostly unknown. Therefore, we aim to gain an understanding of the structural and biophysical properties of CowN and determine how CowN shields nitrogenase from CO. Similar to the amyloidogenic protein Aβ, CowN aggregates as a result of changes in temperature, concentration of salt and concentration of protein. Aiming to explore how these various factors change the oligomeric state of CowN, we hypothesize that a) CowN aggregation is caused by protein secondary structural changes that are thermally induced, b) aggregation is driven by increased salt and protein concentration, and c) monomeric and oligomeric states have different activities. To test these hypotheses, CowN was purified following expression within E. coli and then functionally examined using dynamic light scattering, circular dichroism spectroscopy, and Fourier transform infrared spectroscopy. Results indicate that there are two different aggregation mechanisms occurring. At pH 7.5 and above, CowN aggregates when heated. Under these conditions, aggregation and thermal denaturation both occur around 45⁰C. Aggregation is likely caused by an amyloid-like secondary structure change from α-helices to β-sheets. At pH 7 and below, CowN aggregates at room temperature prior to unfolding. This mechanism is likely driven by electrostatics, as the pH approaches the protein’s pI. Together, data suggests that CowN is active within a narrow pH, temperature and concentration window.
Investigating the Interactions Between Individual Calmodulin and HIV-1 Matrix Protein Domains
Presenter(s): Riley Kendall
Advisor(s): Dr. Jerry LaRue
The World Health Organization found that 37.9 million people were living with HIV by the end of 2018. HIV is a virus that weakens the immune system through viral replication and the destruction of CD4+ T-cells, which are white blood cells that detect infection and make antibodies. A cure for HIV has not yet been discovered. HIV-1 contains a Gag polyprotein which regulates the stages of viral replication. Previous studies suggest that the myrisoyl group of a matrix protein peptide found on the Gag polyprotein, MA, forms a complex with a calcium-binding, multifunctional regulatory protein called Calmodulin (CaM). CaM has also been found to be upregulated upon HIV infection. The MA/CaM complex induces extended conformation and causes a decrease in the compact structure of MA, which is predicted to impact the accessibility of interaction sites within MA and lead to rapid HIV viral production. Through hindering the myristoyl group on MA, it is possible that production of HIV can be greatly decreased. Before exploring this possibility, it is first necessary that the site and mechanism of the protein-protein interaction are identified and understood. For this reason, our lab is investigating the interactions of the independent N-terminal and C-terminal domains of MA and CaM. The MA protein has tryptophan-containing helices on its domains which allows for quantification using fluorescence spectroscopy and anisotropy. By investigating each protein domain and tryptophan signal separately, the location where binding occurs can be isolated and it can be determined if the interaction of one CaM or MA domain is required, or a prerequisite, for the interaction of the other. Identifying how each protein domain is involved enhances current understanding of HIV production and is a significant step in determining a possible solution for inhibiting HIV-1 replication.
Genotype and Phenotype Testing to Predict Azathioprine-Induced Toxicity in Pediatric Inflammatory Bowel Disease (IBD)
Presenter(s): Natalie Paterson
Advisor(s): Dr. John Miklavcic
Inflammatory bowel disease (IBD) is a collective of chronic conditions characterized by a dysregulated immune response to tissue injury that causes inflammation, swelling, ulcers, and intense pain of the intestines. The immunomodulator Azathioprine serves to suppress patient’s overactive immune response, decreasing their disease symptoms, and helping improve their quality of life. Of children with IBD, 20% have a polymorphism in the gene that codes for the enzyme thiopurine methyltransferase (TPMT). Polymorphisms that result in low TPMT activity are correlated to decreased efficacy of Azathioprine metabolism which increases drug toxicity and can result in life-threatening myelosuppression (reduction of bone marrow). We hypothesize that genotype and phenotype tests for TPMT can be used to predict patient response to Azathioprine. To explore this, we utilized data from ImproveCareNow (ICN) which is a vast network containing anonymized data from over 40,000 pediatric IBD patients. We found 17% of patients in ICN are currently prescribed Azathioprine, 29.9% of those patients have been genotyped and 66.8% have been phenotyped. Treatment success was measured by remission status. We found that of the patients who failed treatment, 25.8% had never completed genotype testing and 31.5% had never completed phenotype testing. Overall, these findings are informative and suggest a potential benefit of such tests but to draw statistically viable conclusions a clinical study is necessary.
Exploring the Effect of the Diarylpentanoid Curcumin Analog 27 in Androgen Receptor-Positive Breast Cancer Cells
Presenter(s): Avrita Brar
Advisor(s): Dr. Marco Bisoffi
We have previously synthesized diarylpentanoid analogs of the natural product curcumin (diferuloylmethane), one of which, ca27, has been shown to markedly down-regulate the androgen receptor (AR) in prostate cancer (PCa) cells. The AR in PCa is a major driver of uncontrolled cell proliferation and thus, a major target for therapeutic intervention in the clinical management of PCa. Therefore, ca27 has potential to foster the development of novel therapeutic organic molecules. In this work, we have begun to test ca27 in two breast cancer (BCa) cell models. MCF-7 cells express the estrogen receptor (ER), the progesterone receptor (PR), and the epidermal growth factor receptor 2 (EGFR2/Her2), while MDA-MB-231 cells do not express these receptors, which makes them of the triple-negative character. This makes them resistant against several therapies targeted to ER, PR, and EGFR2/Her2, which represents an aggressive BCa phenotype. Both cell models express the AR, which also drives cancer progression, and may be an effective target in triple-negative BCa. The effect of ca27, compared to curcumin and vehicle control, was tested using the 96-well plate based colorimetric WST assay in which 1,3,5-triphenyltetrazolium formazan is converted by mitochondrial dehydrogenases and reductases as a measure of metabolic activity and cell viability. The cells were treated with 0.1-50 micromolar ca27, curcumin, vehicle control, or nothing for 24 hours Reference absorbances at 660 nanometer were determined to account for differences in cell number between individual wells, and metabolic activity was measured over the period of four hours by determining absorbances at 440 nanometer. Reference-controlled results indicate a dose-responsive effect of ca27 and curcumin and a stronger effect of ca27 compared to curcumin. Future experimentation includes Western blot analyses to determine whether AR expression is differentially affected in the two cell models. Ca27 may be developed into an effective therapeutic modality against triple-negative BCa.
Downregulating the Androgen Receptor in Pancreatic Cancer Cells
Presenter(s): Alex Graden
Advisor(s): Dr. Marco Bisoffi
Pancreatic cancer affects hundreds of thousands of people globally every year, and with a current survival rate of 7%, there is a high demand for new and effective treatments. Pancreatic cancer has been shown to express the androgen receptor (AR), a member of the steroid receptor protein family that mediates cell differentiation, growth, and survival. While in prostate cancer, AR overexpression and hyperactivation is an oncogenic factor, and thus a therapeutic target, the role of the AR in pancreatic cancer remains unknown and inconclusive. The goal of the present project is to determine the role of the AR in the pancreatic cancer cell model COLO 357 with respect to cell viability using the small interference RNA (siRNA) method. Three different plasmids with a pLKO.1 backbone, pLKO.1 empty vector, as well as two vectors coding for two different siRNAs targeting two different segments of the 3’ region of the AR mRNA, pLKO.1/462 siRNA and pLKO.1/730 siRNA, were amplified in Escherichia coli by heat shock transformation and liquid culture followed by plasmid isolation using silica-based column chromatography. An additional plasmid coding for an enhanced green fluorescence protein (pEGFP) under the cytomegalo virus (CMV) promoter was also amplified. COLO 357 cells were transfected with the plasmids using the lipofectamine 2000 reagent and were incubated for 72 hours. Fluorescence microscopic analysis of pEGFP transfected cells indicated a transfection efficiency of ~30-50%. RNA was isolated from the cells by silica-based column chromatography and subjected to AR-specific quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) to determine the expression level of the AR. Preliminary data showed AR repression by pLKO.1/730 siRNA and AR induction by pLKO.1/462 siRNA. Future studies include a repetition of these inconclusive results, as well as cell viability and cell death assays to determine the importance of the AR in pancreatic cancer cells.
Exploring the Effect of the Diarylpentanoid ca27 on the Translation of the Androgen Receptor in Prostate Cancer Cells
Presenter(s): Sarah Hester
Advisor(s): Dr. Marco Bisoffi
The androgen receptor (AR) is a steroid receptor that plays a key role in male sexual differentiation. Under normal physiological conditions the AR acts as a ligand-mediated nuclear transcription factor that binds with androgens and is able to interact with DNA to induce the transcription of genes that can lead to proliferation or apoptosis. In prostate cancer, the AR is often over-expressed in order to increase the proliferation and survival of cancer cells. ca27, an analog of the natural product curcumin, has been shown to down-regulate AR expression in prostate cancer cells. While it has potential as a novel prostate cancer therapeutic, the exact mechanism of action of ca27 is still unknown. In this experiment we treated human LNCaP prostate cancer cells with ca27 as well as additional inhibitors of transcription, translation, and protein degradation. This allowed us to monitor the downregulation of the AR in LNCaP cells as well give an indication of at which point in protein production ca27 works to decrease AR expression (transcription, translation, or protein degradation). In conducting a Western Blot, we found the greatest downregulation of AR expression to be in cells treated with a combination of cycloheximide (a translational inhibitor) and ca27. In addition, qRT-PCR supported these findings by revealing low levels of AR mRNA in cells treated with both cycloheximide and ca27. Knowledge of the mechanism of action of ca27 will help develop further analogs of ca27 with increased AR-downregulatory capacity.
Hybridization and Gene Flow Patterns in a Novel Contact Zone Between Two Species of Native California Sunflowers
Presenter(s): Olivia Durant
Advisor(s): Dr. Jennifer Funk, Shana Welles
When closely related species have overlapping ranges hybridization may occur, and successful survival and reproduction of the resulting hybrid propagules can result in the creation of a hybrid lineage or introgression. Encelia californica and Encelia farinosa are two species of sunflowers native to southern California. E. californica’s range is restricted to coastal regions of Southern California and Baja Mexico while E. farinosa occurs in more arid regions including the Southwest United States and Northern Mexico. Recent conservation efforts have led to individuals of E. farinosa being planted west of its natural range such that they co-occur with populations of E. californica, resulting in a novel contact zone. Individuals with morphological traits from both species (putative hybrids) have been observed within this contact zone. Previous studies have shown that Encelia hybrids are likely to be restricted to first-generation except for in areas of human disturbance, where the formation of hybrid lineages and backcrossing with parental species is thought to occur more frequently. In this study, we used a reduced-representation next generation sequencing approach to determine whether hybridization has occurred between E. californica and E. farinosa in this novel contact zone and whether there is evidence of gene flow between species. To address these questions, leaves of E. californica, E. farinosa, and of presumptive hybrids based on morphological traits were collected from five different locations in southern California. DNA was extracted from each sample using a modified CTAB protocol, quantified, and prepared for sequencing using restriction digests (PST1 and MSE) and sequenced on IIllumina’s Hi-Seq platform. Following sequencing data was aligned and filtered. Genetic data confirms the presence of hybrids and suggests that gene flow has occurred between E. farinosa and E. californica. This demonstrates that in conservation efforts it is important to consider the potential for hybridization before moving species to areas that are outside of their current range.