Biochemistry & Molecular Biology.
A maize RNA binding motif protein-48 (RBM48) is a master-gene critical in the development and viability of seeds in maize. Mutations in the RBM48 master-gene cause severe developmental defects and exhibit prolific growth upon tissue culture. In this research project, RNA extracted from the mutant RBM48 and the wild-type RBM48 gene are compared using next-generation RNA sequencing to generate a set of candidate genes that are expressed differentially in the mutant gene. Next, RT-PCR (Reverse Transcriptase-Polymerase Chain Reaction) is used to validate differential gene expression by making cDNA primers from mutant and wild-type RNA, amplifying the results using PCR, and using gel electrophoresis to experimentally validate the identities of differentially expressed genes. Bioinformatics tools make connections between the genes of interest and their biological function. For example, tools such as BLAST are used to find homologous genes in maize and other species to help us understand the functions of the differentially expressed genes in RBM48. Bioinformatics tools can further identify possible gene mechanisms of action that may be responsible for prolific growth with the hope that insights into the development of cancer may become possible. The research is significant from agricultural perspectives regardless since maize is a vital US crop.
Comparing the Effects of Two Isoforms of Vascular Endothelial Growth Factor A 165 on Retinal Endothelial Cells. Megan Moore, Oakland University
The endothelial cells that line blood vessels in the back of the eye form the blood-retinal barrier which allows selective exchanges of fluids and cells between the inside of the vessels and the surrounding tissues. Excessive levels of Vascular Endothelial Growth Factor A in the retina lead to an increase in vascular permeability often related to diabetic retinopathy, wet age-related macular degeneration, and vision loss.
The protein most responsible for this, VEGFA 165, exists in a regular and a bisoform because of differential splicing. The regular isoform causes inflammation, hut some researchers call the h-isoform antiangiogenic. However, our lab has found that the b-isoform can cause inflammation similar to that caused by the regular isoform.
By culturing human retinal microvascular endothelial cells we have been able to compare the cellular transduction of the two isoforms. In-cell Western assays show the different levels of protein phosphorylation between the two isoforms. Despite this, preliminary PCR data based on the expression of genes related to inflammation and the breakdown of the blood-retinal barrier suggest that both isoforms induce similar changes in the retinal endothelial cells. This is a continuing project in our laboratory.
Repair of G-Quadruplex DNA Facilitated by the FANCJ Helicase and the REV1 Polymerase. Mena Jirjees and Colin G. Wu, Oakland University
G-quadruplexes (G4s) are DNA structures formed by guanine rich nucleic acids. Damaged G4s can interfere with essential cellular processes such as DNA replication and RNA transcription. The mechanism by which damaged G4s are recognized and processed is not well-understood. The human FANCJ helicase facilitates DNA replication through G4 forming regions and participates in DNA crosslink repair. In a previous study, we have identified an AKKQ amino acid motif within FANCJ that allows this helicase to target G4 structures. In this work, we used fluorescence spectroscopy and biolayer interferometry (BL1) based binding assays to show that a FANCJ AKKQ peptide binds to G4 DNA with high affinity. Our BLI experiments also indicate that a separate FANCJ motif, known as the "PCNA Interacting Peptide" (PIP) can interact directly with the REV1 DNA repair polymerase via its C-terminus. Taken together, these results are consistent with a model where FANCJ targets a stalled replication fork at a G4containing DNA site, and then recruits REV1 to efficiently replicate DNA across from the unfolded quadruplex.
Assessment of the Influence of Temperature and Pressure on Medicinal Properties of Black Chokeberry Extracts Using Supercritical Carbon Dioxide with an Ethanol Modifier. Allison Seeley, Scott Constine, Cameron Krivitsky, Cheryl Samaniego, Lihua Wang, Michelle Ammerman, and Jonathan Wenzel, Kettering University
Berries of the black chokeberry (Aronia melanocarpa) plant, a shrub native to the Great Lakes Region, are rich in phenolics and other antioxidants. To date, the most efficient methods of chokeberry extraction employ unfavorable, toxic solvents, or are very time-consuming Here, we present the extraction of antioxidants from chokeberry pomace using nontoxic supercritical carbon dioxide with an ethanol modifier in only fifteen minutes. Variables such as pressure, temperature, percent ethanol by weight, and extraction time are investigated to determine the effects on total phenolic content antioxidant values, and the optimal conditions for extractions are reported. Furthermore, these plants appear to have anti-proliferative effects on cancer cell lines which do not directly correlate with antioxidant activity.
The Effects of BPA on ER[alpha] and p53 Expression in MCF-7 Breast Cancer Cells. Betsy Purakal, Victoria Lloyd, and Sumi Dinda, Oakland University
Bisphenol A (BPA) is a chemical found in polycarbonate plastics such as water bottles, food packaging, and storage containers. BPA is classified as an endocrine disrupting chemical (EDC), which mimics endogenous hormones and interferes with the original hormone's signaling. These EDCs may lead to an increased risk of certain cancers. This project will examine the effects of BPA on the expression of estrogen receptor alpha (ER[alpha]) and p53 in ER-positive MCF-7 breast cancer cells. ER[alpha] is a receptor that is activated by estrogen and is associated with breast cancer. Cells will be cultured and treated with varying concentrations of BPA and BPA in combination with hormones and antihormones, and Western blot analyses will be performed to determine BPA's effects on ER[alpha] and p53 protein expression. A cellular viability assay will also be conducted following treatment of the cells with various hormones and antihormones to measure the amount of live and dead cells in a population. The results of this project may reveal how BPA affects breast cancer development through the ER[alpha] and p53 protein signaling pathways, contributing to current research on the topic and adding new information about endocrine cancers.
Using Embryonic Zebrafish to Evaluate the Effects of Exposure to Novel Bisphenol Analogues. Laura Bruton, Xinrui Zhou, Shannon C. Timmons, and Jeffery M. Morrissette, Lawrence Technological University
Bisphenol A (BPA) is a well-established endocrine disruptor that has been shown to induce developmental and behavioral changes in both human and animal models. Gestational exposure of embryonic zebrafish to low nanomolar doses of bisphenol A (or S) altered hypothalamic neurogenesis during development and induced hyperactive swimming behaviors. There are many potential bisphenol analogues, but little is known about their toxicity or ability to act as endocrine disruptors. We have synthesized a small library of novel bisphenol analogues and are testing their toxicity in a zebrafish larval toxicity assay. We are also using an ELISA-based estrogen binding assay to assess the analogues' affinity for estrogen receptors. We will be exposing zebrafish embryos to ranging concentrations of bisphenol analogues during time periods of 10-16 hpf (hours post-fertilization), 16-24 hpf, and 24-36 hpf. At 5 days post-fertilization zebrafish locomotor activity in control and analogue exposed fish will be assessed using video-tracking software quantifying bursts of hyperactivity. Results from these experiments will be discussed.
DNA Recognition by the BRCA1 Tumor Suppressor Protein. Ann J. Fuelle, Francesca Iacovo, and Colin G. Wu, Oakland University
BRCA1 is a human tumor suppressor protein that repairs double-stranded DNA breaks in cells. BRCA1 malfunctions are closely associated with the early onset of breast and ovarian cancers. To date, the molecular mechanism by which BRCA1 assembles onto damaged DNA sites remains unclear. In this work, we examined the DNA binding properties of three BRCA1 fragments in isolation: BRCT, DBD1, and DBD2. These protein domains were produced from E. coli, and they were purified from all other cellular proteins by nickel affinity chromatography. The affinities of BRCT, DBD1, and DBD2 for various DNA repair intermediates were determined using biolayer interferometry (BLI) and other biophysical binding assays. These results were used to rank their preferences for double-stranded DNA, single-stranded DNA, or a G-quadruplex DNA structure. We find that the BRCT domain associates strongly with a G-quadruplex DNA, while DBD1 and DBD2 bound tightest to single-stranded DNA. The modular nature of these BRCA1-DNA interactions may provide a regulatory mechanism to control its DNA repair functions inside the cell. Therefore, we plan to perform DNA repair studies in human cell lines alongside these in vitro binding experiments to further test the link between DNA binding activity and repair of DNA lesions.
Chinese Medicinal Herbs Scutellaria barbata and Oldenlandia diffusa Modulate the Mutagenicity of PhIP (2-Amino-l-methyl-6-phenylimidazo[4,5-b]pyridine). Rayford Alva, Brian Y. Y. Wong, Ryan Hayes, John Tanner, and Snow Y. L. X. Wan, Andrews University
Scutellaria barbata (SB) and Oldenlandia diffusa (OD) have been used in Chinese medicine for treating liver, lung and rectal tumors. PhIP (2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine) is produced from the high cooking temperature of creatine, sugars, and amino acids and is one of the most abundant heterocyclic amines (HCAs) in cooked meat. Its high intake and exposure are linked to prostate, breast, lung, esophagus, stomach, and colorectum cancer. In this study, the effects of aqueous extracts of SB and OD on PhIP-induced mutagenesis using Salmonella typhimurium TA98 as the bacterial tester strain and Aroclor 1254-induced rat liver 9000 x g supernatant (S9) as the metabolic activation system were tested. Our results showed that SB and OD significantly inhibited the mutagenicity of PhIP (0.1[micro]g/plate) by S9 (800 mg/plate) in a dose-response (1.5, 3, and 6 mg/plate) fashion (SB > OD, p < 0.05). The percent inhibition of revertants formation of these two herbs against PhIP were 77%, 91%, and 95% for SB and 43%, 80%, and 93% for OD. There was a slight additive SB + OD inhibition effect. Futther study of their modulation effects on metabolism and DNA binding of PhIP is warranted to reveal their anti-mutagenicity mechanism and potential chemopreventive property against PhIP and other HCAs.
Stress Conditions Promote Increased MMP9 Activity in Breast Cancer Cells. Holly Attebury, Larry Kuipers, Brandi Kaaykati, and Joseph F. Sucic, University of Michigan-Flint
Cancer remains one of humanity's most dreaded diseases. All cancers have two important characteristics: uncontrolled cell division and metastasis. Metastasis poses the gravest threat to cancer patients, as 90% of cancer mortality is the result of metastatic tumors. While uncontrolled cell proliferation has been researched extensively and the mechanisms associated with it are fairly well understood, the underlying causes of metastasis remain largely mysterious. We have hypothesized that metastasis is triggered by stressful conditions within the tumor environment. To test this hypothesis, we placed breast cancer cells under various stress conditions, including hypoxia, heat shock, and a combination of both. In earlier experimentation, we showed that these stressors caused increased expression of both Matrix-degrading metalloproteinase 9 (MMP9), which is a key mediator of metastasis, and the proprotein convertase, furin, which is involved in activating MMP9. In this project, we examined for MMP9 activity in the conditioned medium derived from cells exposed to these stress conditions. Results suggest that the stressed cells secreted more active MMP9 than control cells. These results are consistent with our earlier analysis of MMP9 expression and also support the hypothesis that metastasis could be triggered by stress conditions within the tumor.
Reduction of Cytochrome C by Diphenolic Compounds. Evan Kindl, Montserrat Rabago Smith, and Veronica R. Moorman, Kettering University
The redox protein cytochrome c is involved in cellular respiration within mitochondria, has an apoptotic role in the cytosol, and is known to interact with numerous other proteins. Specifically, cytochrome c can be reduced by a variety of compounds, including diphenolic ones. These diphenols, including certain flavonoids and hormones, have been shown to exhibit anti-oxidant activities, anti-cancer properties, and an ability to protect against cardiovascular diseases. To better understand the chemical underpinnings behind the reaction between cytochrome c and biologically relevant diphenols, we spectroscopically quantified the redox reaction of equine cytochrome c and numerous diphenolic compounds. Reduction rates and rate constants were subsequently compared to various physical properties.
Methods of Studying Chemotaxis in C. Elegans for Research and Educational Purposes. Ahron Wayne, Lawrence Technological University and Hebrew University of Jerusalem; Donovan Dennis, Lawrence Technological University
The nematode C. elegans possesses a number of unique traits which make it an invaluable model organism for neuroscience. In addition to being easy to care for, having a short life cycle of just 72 hours, and heing naturally vulnerable to genetic manipulation, the adult hermaphrodite possesses just 302 neurons, with a nearly invariable, fully-mapped connectome. Despite having such a small nervous system, individuals display a variety of stereotyped and complex behaviors. Here, we compare two methods for studying chemotaxis behavior in C. elegans. The first, a classic assay invented by Cori Bargmann, is simple, end-point based, and produces an easily interpreted, quantifiable result in the form of a chemotactic index. This assay is compared to a method utilizing machine vision, wherein a camera and multi-animal tracker are used to follow the movement of many worms over an extended period of time. The former assay is ideal for an educational setting, and is used to demonstrate basic experimental methodology in an undergraduate biology course. The latter method requires more preparation and analysis, but produces richer data that can shed light on a broader range of topics, such as food leaving behavior.
The Effect of Octopamine and Chelrythrine Chloride on Phonotaxis in Unexposed Acheta domesticus. Beatrice Cho and Cassie Kim, Andrews University
Female crickets Acheta domesticus exhibit phonotaxis in response to male calls, signifying a prospective mate. In the lab, females may respond to calls with syllable periods (SP) ranging from 30 ms to 90 ms. Young (5-10 days), virgin females are most likely to respond phonotactically to calls with SPs of 50-70 ms. In older, virgin females (greater than 20 days), there is more variability in the range and number of SPs responded to. Neuromodulators like juvenile hormone III (JHIII) have been shown to affect selectivity in phonotactic response. The effects of many neuromodulators on cricket phonotaxis remain unknown. One such neuromodulator, octopamine, is a monohydroxylic correspondent of norepinephrine found in nervous systems of invertebrates and vertebrates. Preliminary nanoinjections of octopamine in the prothoracic ganglion show decreased phonotactic responsiveness in young females. A second neuromodulator, chelerythrine chloride (CC) is a protein kinase C (PKC) blocker. PKC is believed to be part of the signaling mechanism in crickets, particularly regarding JHIII. When older females are injected with JHIII, selectivity increases. When CC is injected into young females, selectivity decreases. Older unexposed female crickets are already unselective, therefore no change in selectivity following CC injection is expected. The current study seeks to identify the effect of injecting the selected compounds on phonotactic selectivity.
The Role of Vitamin D in Non-Melanoma Skin Cancer. Rawia Khasawneh, Central Michigan University; Archana Unnikrishnan, University of Oklahoma; Bassel Mahmoud, University of Massachusetts; Iltefat Hamzavi, Henry Ford Hospital; Ahmad R. Heydari, Wayne State University and Barbara Ann Karmanos Cancer Institute
The relationship between vitamin D and non-melanoma skin cancer is not clear. Epidemiological studies are inconsistent and inconclusive, and mechanistic studies are lacking. It has been suggested that vitamin D possesses antiproliferative and pro-differentiation effects by negatively regulating key signaling pathways through its nuclear receptor (VRD), that is a nuclear transcription factor. In particular, VDR target gene REDD1 (regulated in development and DNA damage response 1) inhibits mTOR pathway, thus impacting cellular growth.
Our study aimed to explore the role of vitamin D on the onset, progression and possibly treatment of basal cell carcinoma (BCC). Three tissue samples were collected from 20 BCC patients (Cancer, Proximal, and Distal), and one sample from 6 cancer-free individuals. BCC and its feeding cells seemed to up-regulate vitamin D activation enzymes, VDR, and its co-activators, thus proposing higher local activity of vitamin D. Despite the significantly increased protein levels of REDD1 in the cancer tissue, our data showed that VDR's ability to down-regulate mTOR pathway through REDD1 was diminished. Interestingly, vitamin D negative regulation of Hedgehog-Gli pathway was also lost in BCC tissue, and Kras mutation was detected. In conclusion, we propose that the anticipated role of vitamin D is not conserved in BCC.
Bacterial Production of Biologically Active Nome's Disease Protein. Jennifer Felisky, Wendy Dailey, Mei Cheng, and Kenneth Mitton, Oakland University
Human proteins, like Norrin (Nome's Disease Protein) fold into their functional native state with the assistance of chaperone proteins within an environment that is more oxidizing than the bacterial cytoplasm. Disulfide bonds are difficult to make in bacteria for these reasons. A Maltose Binding Protein (MBP)-Norrin construct was made for expression in SHuffle strain of E. coli modified that has a more oxidizing cytoplasm and a disulfide shuffling chaperone, DsbC. The expression construct included an HRV3C protease cleavage site between the MBP and Norrin domains. Version 1 was uncleavable. Models of the domains suggested that the cut site could be inaccessible. Version 2 incorporated several flexible and rigid linker domains, to improve access for HRV3C. This was successful. Norrin had poor affinity for the Frizzled-4 receptor, and failed to stimulate human retinal endothelial cells (HRECs). A method was developed to refold Norrin into its native conformation. Refolding was performed in vitro with disulfide reshuffling using Glutathione. Refolded Norrin bound Frizzled-4, and stimulated AXIN-2 expression. We developed a strategy for improving protease accessibility that can be used in the bacterial production of human proteins using the MBP system. This can be combined with disulfide reshuffling to obtain biologically active proteins.
Omega-3 Fatty Acids: Potential Anti-Bipolar Agents. Taejun Ok, Andrews University
Bipolar disorder is a debilitating mood disorder characterized by recurring episodes of mania and depression. It affects 2.6% of adults and has a lifetime prevalence among adults of 3.9%. Current mood stabilizers such as valproate are not always effective for and/or are not well tolerated by a large number of patients. Therefore there is a need to develop or identify more effective and less harmful treatment. Omega-3 fatty acids have been shown to alleviate bipolar disorder symptoms without the harmful side effects. Since depletion of myo-intracellular inositol is one of the mechanisms hypothesized by which mood stabilizers exert their therapeutic effect, the goal of this study was to determine the effects of the omega-3 fatty acids decosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) on intracellular myo-inositol levels and the expression of myo-inositol synthase genes INO1. To do so, cells were grown in the presence and absence of DHA and EPA and myo-inositol was assayed in the cell extracts and gene expression determined by RT-PCR.
Our results showed unlike valproate, the omega-3 fatty acids increased intracellular myo-inositol and decreased INO1 expression. We therefore conclude DHA and EPA may exert their therapeutic effect on bipolar disorder by mediating myo-inositol levels.
Sorting Out Multiple Soybean Orthologs of SLEEPY 1. Sam Henson, Derek Janssens, and Pei-Lan Tsou, Grand Valley State University
Gibberellic acid is an important plant hormone that regulates growth, cell division, and seed germination. SLEEPY1 (SLY1) functions as a positive regulator of gibberellic acid (GA) signaling in Arabidopsis. Components in the GA response pathway in Arabidopsis have been identified and studied. SLY1 is an F-box protein that is up-regulated in response to increased bioactive GA and functions by degrading DELLA proteins. These DELLA proteins, RGA and GAI, inhibit GA signaling. We have identified GmSLY1a and GmSLY1b, two orthologs of SLY1 in soybean. Both of these putative orthologs contain an F-box motif as well as several other domains found in the Arabidopsis SLY1 protein. However, it has not yet been determined whether these orthologs maintain the same function as SLY1. We measured the relative expression of GmSLY1a and GmSLYlb in different soybean tissues and compare them to the expression of SLY1 in Arabidopsis in order to gain insight into the functionality of the two genes. The sly1 mutants in Arabidopsis result in a dwarf phenotype and show variable seed dormancy. We also transformed both genes into Arabidopsis sly1 mutants to further analyze the soybean orthologs and assess their ability to rescue the sly1 mutant phenotype in Arabidopsis.
Detection of Bacterial Cell-Surface Capacity for Lectin Binding Induced by Acquisition of Antibiotic Resistance Using Biosensor Methods. Julia Poirier, Archan Patel, Jacqueline Paul, Arie Dvir, and Xiangqun Zeng, Oakland University
Cell surface carbohydrates (glycans) and adhesin molecules are major components of the outer surface of cells and are often characteristic of the cell types. Glycans and adhesins are the first interfaces to the cell's biotic and abiotic environment. In the recent past, we demonstrated various molecular design and fabrication of glycosurfaces for carbohydrate biosensor development and for their applications in bacterial detection and bacterial antibiotic resistance study. The approach is based on the detection of the phenotypic biomarkers of the bacterial cell surface. In this project, we correlate the fundamental biology of phenotypic changes of bacterial cell surface markers (i.e., LPS) under various antibiotic exposures with the binding signals obtained from the biosensor measurements using both biological method and biosensor methods. We have produced several modified bacterial lines that bear a single antibiotic resistance trait. Cells are analyzed for differences in lipopolysaccharide/sugar profiles for certain antibiotics are of particular interest. Our initial observations provide the basis for differential detection by sensors coated with lectins, which will further lead to future development of rapid analytical tools for determining antibiotic resistance in infected fluids.
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|Date:||Sep 22, 2018|
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