1 gb display only

138 UL2

Redesigning Serine Proteases for Resistance to Inhibition

By: Weichao Zhuo

Biochemistry

Faculty Advisor: Dr. Teaster Baird Jr.

Using computational and molecular biology approaches, we are using the model serine protease, trypsin, to identify amino acid residues that are important in the selection and binding of macromolecular substrates and inhibitors. This work will help us engineer trypsin and similar proteases to be more resistant to inhibition so that they may be more effective as therapeutic agents. In several trypsin inhibitor co-crystal structures, the backbone carbonyl oxygen of phenylalanine 41 (F41) accepts a hydrogen bond from the P2’ backbone amide hydrogen of the inhibitor. F41 is conformational restricted by its interaction with the methylene groups of lysine 60, which presumably optimizes the backbone carbonyl oxygen of F41 for hydrogen bonding with the inhibitor. By making substitutions at position 41, we may be able to increase the conformational flexibility of its backbone carbonyl and reduce the affinity of trypsin for inhibitors. To test this hypothesis, we have made two trypsin variants, F41L and F41G. Both F41L and F41G trypsin variants were expressed in Pichia pastorisas zymogens,were purified using hydrophobic chromatography. The mature forms of both variants were obtained by auto-activation at pH 8.00. The mature variants were isolated using affinity chromatography. Lastly, activity assays were performed with and without inhibitors using 1nM enzyme and varying concentrations of Z-GPA-pNA at 25 °C. Variants were kinetically similar to wild-type trypsin in KM (7.55 ± 1.82 µM), kcat (4736 ± 200 min-1), and kcat/KM (627±109 uM-1 min-1) and in the KI value for benzamidine, a small molecule inhibitor (6.1 ± 1.0 µM). These data suggest that the substitutions do not affect the active site structure or the chemistry of the enzyme. We observed differences in the variants when we measured the effect of a macromolecular inhibitor (BPTI) on activity. The association rate for BPTI (kon) with wild-type and F41L were similar (8110.5 ± 460.62 M-1s-1 versus 9262.8 ± 593.6 M-1s-1 respectively. However, the kon value for F41G variant was significantly lower (4991.2 ± 187.67 M-1s-1). The results with BPTI suggest that glycine residue at position 41 decreased the affinity of trypsin for BPTI. Collectively, the data show that the substitutions do not affect the activity of the enzyme and support the hypothesis that increasing the flexibility at position 41 is a plausible approach to decreasing the sensitivity of trypsin (and trypsin-fold proteases) to inhibition by macromolecular inhibitors.

139 UL2

Effects of Caloric Restriction on Neurogenesis in the adult brain of Acheta Domesticus

By: Amanda Carbajal, Mae Calamucha, and Linda Kok

Microbiology

Faculty Advisor: Dr. Christopher Moffatt

Neurogenesis, the birth of new neurons, occurs throughout the lifespan in many species, from development and through adulthood. What is comparatively poorly understood are the factors that regulate this process. The house cricket, Acheta domesticus, is an ideal model to study these processes because neurogenesis occurs throughout their lifespan and occurs in area of the brain crucial for learning and memory in this species, the mushroom bodies. Our studies address the role of acute caloric restriction on the regulation of neurogenesis. Utilizing bromodeoxyuridine (BrdU) to label proliferating cells we examined the effect that four days of starvation had on the rate of neurogenesis in the mushroom bodies. We found that four days of starvation did not affect the rate of cell proliferation: The number of BrdU-immunoreactive cells present in the mushroom bodies two hours after the BrdU injection did not differ between freely feeding crickets and those that had been starved for four days. Continuing experiments are replicating this observation and are exploring whether or not starvation affects the survival of newly generated neurons. Elucidating how neurogenesis is regulated in crickets, and understanding the ways in which it is similar and different from that in mammals has great potential to improve our understanding of a process that plays such an important role in human neuropathology, as well as learning and memory.

140 UL2

Investigation of Antibiotic Resistance Profiles of Enterobacteria at SFSU

By: Ruth Keoviphone, Mindy Le, Lady Jane Tabalno Pacaldo, and Man Wai Lee

Microbiology

Faculty Advisor: Dr. Lily Chen and Darleen Franklin

Carbapenems, a group of broad-spectrum antibiotics, are used as a last resort treatment for highly resistant Enterobacteriaceae infections such as bacteremia and urinary tract infections (UTIs). Currently, there is a group of carbapenem-resistant Enterobacteriaceae (CRE) that are becoming more problematic to treat worldwide due to its increasing carbapenem resistance. This is especially true for those who are immunocompromised, hospitalized or on long-term antibiotic treatment. / The purpose of our study was to determine the resistance of enterobacteria to carbapenem antibiotics used in BIOL 431 Medical Microbiology laboratory course. / To accomplish this, the Kirby-Bauer susceptibility test was used to measure the antibiotic resistance profiles (i.e. zone of inhibition) on bacterial lawns. Three carbapenem antibiotics (Ertapenem, Meropenem, and Imipenem) along with three bacterial control strains (S. marcescens, E. coli, and S. Saintpaul) were used. A total of 3 independent trials were conducted with two bacterial duplicates per trial. / Our data show that these strains are susceptible to the carbapenems. Overall, ertapenem was shown to be the most effective in bacterial killing, followed by meropenem and then imipenem. This data suggests that these strains have not developed drug resistance against carbapenems. Therefore, the enterobacteria strains currently used in BIOL 431 are safe for students to use. /

141 UL2

Two New Cytotoxic Meroterpenoids Produced by the Marine Sediment-Derived Streptomyces sp. CP26-58

By: Stephanie Gee

Microbiology

Faculty Advisor: Dr. Taro Amagata

In search for new anticancer agents, we focused our attention on discovering novel cytotoxic compounds produced by marine sediment-derived actinomycetes. A chemical library containing 474 organic extracts has been applied to an image-based anticancer screening using HeLa cells. This screening identified 41 strains that showed potent cytotoxic effects against HeLa cells (>95% cytotoxicity at 50 μg/mL). One of the active strains, Streptomyces sp. CP26-58, showed a unique chemical profile of the secondary metabolites based on LCMS analysis. Bioassay-guided HPLC purification using the results obtained from the image-based screening led to isolation of two new meroterpenoids with a naphthoquinone core (1 and 2). The structures of the two new compounds possessing an unprecedented six-fused ring system were firmly assembled by comprehensive 1D and 2D NMR analysis. In addition, their absolute configurations were deduced based on ab intio electronic circular dichroism (ECD) spectral calculations. Isolation, structure elucidation, and cytotoxic effects of the new compounds will be presented.

142 UL2

Determining the Molecular Genetic Mechanism of Extreme Desication-Tolerence in Talbotia Elegans

By: Stephanie Parker, Thuy Tran, and Theresa Iahey

Microbiology

Faculty Advisor:

1 / Determining the Molecular Genetic Mechanism of / Extreme Desiccation-Tolerance in Talbotia elegans / Abstract / California is currently experiencing its worst drought in history. The leading factor of / daily water use from urban households is outdoor watering, especially of grass lawns that require / consistent watering to stay green. Talbotia elegans is a flowering grass plant known for its / desiccation-tolerant (Hallam et al., 1980) and resurrection (Levitt, 1972) characteristics, which / could potentially be genetically incorporated into conventional grass. In order to create / resurrection grass as a possible solution to relieving California’s drought, preliminary studies are / needed to understand the genetic mechanism behind T. elegans ability to resurrect or restore / back to its hydrated state after being dehydrated. By performing protein inhibitor assays, we will / look for proteins that are cell autonomous, and target those proteins to see how they behave over / time within the dehydration of T. elegans. Potential target proteins from this plant could possibly / lead to sequencing the genome of T. elegans, which has yet to be sequenced. With the / mechanistic knowledge of this novel species, we can in the future possibly create desirable grass, / crops, and ornamental plants capable of resurrection. Overall, this study could help Californians / eliminate the majority of outdoor watering and reduce the current drought.

143 UL2

An Enhancer / Suppressor Screen for the Endoplasmic Reticulum Membrane Protein / Jagunal in the Drosophila compound eye

By: Emmanuel Valenciano, Ronnie Marania, and Catherine Lugar

Physiology

Faculty Advisor: Dr. Blake Riggs

An Enhancer / Suppressor Screen for the Endoplasmic Reticulum Membrane Protein / Jagunal in the Drosophila compound eye / / Emmanuel Valenciano, Ronnie Marania, Catherine Lugar, and Blake Riggs / Department of Biology, San Francisco State University / / The endoplasmic reticulum (ER) is a continuous network of membrane tubules and / flattened cisternae. It is a multifunctional organelle, which involves synthesis of membrane / lipids, membrane and secretory proteins, and other functions. During the cell cycle, the ER / experiences a change in its structure and localization with the most dramatic changes / occurring during mitosis. However, the molecular mechanism driving these changes is poorly / understood. Recently, the ER membrane protein Jagunal (Jag) was shown to be essential for / ER organization during Drosophila oogenesis, by facilitating vesicular traffic to the cortex / necessary for oocyte growth. Here we hypothesize that Jag is necessary for the ER cortical / interactions involved in mitotic ER reorganization. In order to identify regulatory components / of Jag, we will perform an enhancer / suppressor screen of Jag in the Drosophila eye. We / ectopically expressed Jag double stranded RNA (dsRNA) to inhibit Jag expression in the / compound eye using tissue specific Gal4 / UAS driver. We found that knockdown of Jagunal / produced a rough eye phenotype in 71% of the progeny. This Jag dsRNA phenotype will be / placed in combination with individual lines from a collection of deficiencies covering the entire / right third chromosome and examine for enhancement or suppression of the rough eye / phenotype. Results from this screen will be examined further for their role in ER organization / during mitosis and identify Jagunal pathway components.

144 UL2

Modifier Screen for Atlastin, the ER associated GTPase, in the D. Melanogaster Compound Eye

By: Heidi Hoffman, Molly Starkovich, and Zainab Abbas

Physiology

Faculty Advisor: Dr. Blake Riggs

The Endoplasmic Reticulum (ER) is the largest organelle in the cell and recently has been shown to change its shape and localization throughout the cell cycle, experiencing the most dramatic changes during mitosis. Recently several studies have identified the GTPase Atlastin as being responsible for the homotypic fusion of ER tubules and thus helps maintain the structural shape of the ER. Disruption of Atlastin in the ER of D. melanogaster compound eye expresses a rough phenotype. However, the mechanisms driving the dynamic rearrangement of the change is poorly understood.The goal of this study is to characterize a molecular pathway for ER dynamics by identifying regulatory proteins that interact with Atlastin. In order to identify these regulatory proteins we are using a genetic screen, where we cross Atlastin knockdowns with specific genetic deficiencies. So far we have screened through 54 of known deficiencies in the 3rd right arm of the D. melanogaster chromosome. We have identified 21 deficiencies that have acted as strong dominant enhancers. We have also identified 6 deficiencies that suppressed the Atlastin phenotype. These deficiencies can be used to identify the genetic components that interact with ER organization.

145 UL2

Andreas Vesalius and the Birth of Modern Anatomy

By: Charles Barbieri and Catherine Lugar

Physiology

Faculty Advisor: Gloria Nusse and Ryan Marder

Andreas Vesailius is considered by many to be the father of modern anatomy because his publication of De Humani Corporis Fabrica shifted human anatomy education away from a 1200 year tradition of hands-off human analog dissections to a humanistic; cadaver center approach. This publication was revolutionary for artists and anatomists alike because it provided the first accurate, detailed images of human anatomical systems, at a time when most medical texts contained minimal graphical representations. This fundamental shift in anatomical study has carried over 500 years, into the modern anatomical laboratory where instructor-led student dissections and graphical models and diagrams play essential roles in anatomical education. / / The aim of this study was to create a bridge between modern anatomy and its origins within Vesailius’ prints in De Humani Corporis Fabrica. Further study of Vesailius’ research methods and his work on the intalio “Muscle Men” plates with the artist Titian, reveals Vesailius’ dedication to a meticulously accurate and evolving anatomical study. The “Muscle Men” are a series of anatomical depictions that when placed side-by-side show a progressive dissection of a man in the Euganean Hills surrounding the Padua University in Italy. This method of showing a progressive dissections is still used in modern anatomy education. We digitally colorized the Muscle Men series by referencing human cadavers donated to San Francisco State University from University of California: San Francisco. We then stitched the individual “Muscle Men” into a panoramic view to illustrate how powerful Vesailius’ work can be as a teaching tool.

146 UL2

The Lymphatic System: an In-depth Study of an Underrepresented System

By: Charles Barbieri and Eunice Manuel

Physiology

Faculty Advisor: Gloria Nusse and Ryan Marder

The lymphatic system is often overlooked, seen only as a secondary part of the circulatory system. However it is extremely diverse and plays major roles in the circulatory, digestive, and immunological systems. Historically, the lymphatic system was described in the 16th century by Gaspare Aselli. He used the mesentery from a canine to describe the flow of lipids through small vessels called lacteals found in intestinal villi. Since Aselli’s work, the lymphatic system has been clearly described, however, its physiology is still a diverse topic of research.

The goal of our project was to create educational models of lymphatic pathophysiology. To show the connection between Aselli’s work, and modern understanding we compared historical prints of canine mesentery with photographs taken from modern surgery. In addition we removed key structures from a cadaver to present them in an accessible way for study. As well we have created a physical model of the cisterna chyli in order to understand the lymphatic system's role in the circulatory mechanism.

Many diseases are associated with the lymphatic system. One such is chronic lymphocytic leukemia. We are presenting how this intricate system plays a role in metastatic cancer by presenting a case study from one of our cadavers

147 UL2

The Design, Synthesis and Preliminary Evaluation of Hyperpolarized Sodium [1-13C] Glycerate as a Probe for Monitoring Glycolysis via 13C Magnetic Resonance Spectroscopy

By: Andrew Castillo

Physiology

Faculty Advisor: Dr. Kelvin L. Billingsley

The Design, Synthesis and Preliminary Evaluation of Hyperpolarized Sodium [1-13C] Glycerate as a Probe for Monitoring Glycolysis via 13C Magnetic Resonance Spectroscopy / / Andrew Castillo, Heather Lough, and Kelvin L. Billingsley* / / *Department of Chemistry and Biochemistry, San Francisco State University, San Francisco, CA 94132 / / Glycolysis performs an essential role in the regulation of energy and metabolism. Deficiencies in this pathway are also commonly correlated with various diseases. However, the development of non-invasive techniques for the assessment of the cycle in vivo has remained challenging. In this work, we have synthesized a novel imaging agent, sodium [1-13C] glycerate, for hyperpolarized 13C metabolic imaging of glycolytic pathway. The probe was found to have suitable T1 for magnetic resonance (MR) spectroscopic studies. The agent was also formulated with 10 mM trityl-based radical (OX063) in a glassing mixture of glycerol/water, and samples of sodium [1-13C] glycerate were successfully hyperpolarized via dynamic nuclear polarization (DNP) using a Hypersense system at Stanford University. Future studies are focused on the assessment of glycolytic metabolism in cellular models of lung cancer via hyperpolarized 13C MR spectroscopy.

148 UL2

The Formal Synthesis of the Protein Kinase C Modulator (-)-Indolactam V

By: Italia Diaz and Jeremy Haynes-Smith

Physiology

Faculty Advisor: Dr. Kelvin L. Billingsley

Protein kinase C (PKC) modulators are currently of great importance in preclinical and clinical studies directed at cancer, immunotherapy, HIV eradication, and Alzheimer’s disease. In recent decades, several natural products families have been identified that activate PKC. Despite the therapeutic potential, several factors have limited their clinical application: (1) poor potency (diacylglycerols), (2) lack of synthetic accessibility (bryostatins) and (3) capability to promote rather than suppress tumor growth (phorbol esters, aplysiatoxins, and teleocidins). We seek to address these issues through the development of potent, synthetically accessible analogs of teleocidin-based PKC modulators. To this end, we designed a concise synthetic route to prepare the natural product (-)-indolactam V (ILV), the core scaffold of the teleocidin family. The synthesis relied upon a copper-catalyzed amino acid arylation that efficiently provided the C4 carbon-nitrogen bond. Further elaboration of the amino acid moiety allowed for the preparation of an advanced intermediate, which can be subjected to known procedures to access ILV. Future investigations will target strategies for employing ILV in the preparation of more structurally complex teleocidin-based analogs followed by exploring the biological activity of these novel PKC modulators. /

149 UL2

The short- and long-term effects of capsaicin on the “pain” response in an insect

By: Cody Burns, Eric Arreola, and Dennis Tabuena

Physiology

Faculty Advisor: Dr. Megumi Fuse

Cody Burns / Megumi Fuse / / The short- and long-term effects of capsaicin on the “pain” response in an insect / / Abstract: / / Nociception is the ability of an organism to sense and process noxious stimuli or irritants, via nociceptors in afferent nerve endings, which results in an appropriate defensive response. Some forms of severe tissue damage and inflammation, however, result in chronic pain such as hyperalgesia, long after the damage is gone, where patients remain hypersensitive to otherwise mild stimuli. In the hornworm, Manduca sexta, a response to irritants has been shown to manifest itself in a defensive strike characterized by the sweeping of the head and biting at the site of the stimulus. Moreover, this response becomes sensitized with severe stimuli, such that animals remain hypersensitized to irritants for at least 20 hours after the stimulus. We suggest that this strike response is regulated similarly to that seen in humans, and thus should show conservation in its responses to irritants known to humans. Capsaicin, an active component of chili peppers, is a common irritant in humans as well as many other animals, which produces a burning sensation via the vanilloid family of TRPV1 nociceptors. It acts as an agonist on afferent nerves where it binds and lowers the threshold for which these ion channels are opened. The opening of these heat-sensing receptors can cause an initial excitatory response in the form of hyperalgesia, however prolonged activation has also been shown to have analgesic effects through desensitization of the nerves. The goal of this experiment was to determine the short- and long-term responses to capsaicin in M. sexta, both injected into the animal and applied topically to the site of a future mechanical or thermal stimulus. For this experiment, capsaicin was diluted to various concentrations and applied subcutaneously and topically to a proleg directly before a noxious stimulus. A baseline strike response was elicited before the noxious stimulus – either a pinch or excessive heat to a proleg – was introduced. The strike response was again tested 1 and 19 hours post stimulus. Preliminary results suggest that only topical application of capsaicin showed early sensitization 1 hour post stimulus, as noted by an increased strike response. In contrast, both topical application and injection of capsaicin decreased sensitization, although responses to control injections were somewhat variable. Thus, M. sexta may be a useful model organism for studying chronic pain, and future directions will explore the exact mechanism by which the nerves respond to capsaicin.

150 UL2

Examination of the possible function of a Pseudomonas aeruginosa Gcn5-related N-acetyltransferase (PA2578) using bioinformatics and enzyme kinetics

By: Brian Amsler and Layton Joe

Physiology

Faculty Advisor: Dr. Misty Kuhn

Gcn5-related N-acetyltransferases (GNATs) belong to a superfamily of enzymes that acetylate a wide variety of molecules, including antibiotics. The GNAT superfamily is seen throughout all domains of life and its members are identified by a characteristic GNAT protein fold. The model bacterium Pseudomonas aeruginosa has more than 35 GNATs in its genome but only a few GNAT functions are known, and most genes are annotated as hypothetical proteins or proteins of unknown function. To increase the number of functionally annotated GNATs from Pseudomonas, we selected the GNAT encoded by the PA2578 gene. Previous screening efforts showed that the enzyme was capable of acetylating chloramphenicol, but the enzyme does not share the same characteristics as chloramphenicol acetyltransferases and we hypothesize that it is not the native substrate for this enzyme. In this study, we used enzyme kinetic assays and bioinformatics tools to investigate possible substrates for this enzyme. We constructed a genome neighborhood network for this gene to explore the functions of neighboring genes that could provide insight into the possible function of PA2578. Examination of the genome neighborhood network suggests that PA2578 is potentially involved in environmental stress responses; however, further assays are necessary to elucidate the native substrate for the enzyme.

151 UL2

Structure elucidation of compounds extracted from actinomycete Streptomyces CP54-7

By: Scott Campit

Physiology

Faculty Advisor: Dr. Taro Amagata

Actinomyces are bacteria well-known for the natural products they produce, such as napthablin and streptomycin. Metabolites produced by terrestrial actinobacteria are well-studied and many pathogens are now resistant to these compounds. However, compounds produced by marine-derived Actinomyces have been isolated and give promise to interesting and novel future pharmaceuticals. A series of organic extraction methods were used to obtain crude extract of Streptomyces CP-54-7 grown in liquid media. Flash chromatography and RP-HPLC were used to purify the crude extract obtained from CP54-7, while H-NMR and 2D NMR were used to elucidate the structure of the compounds which appears to be part of the napthaquinone family of natural products. HRMS performed on one of the compounds showed that the MF was C28H28O5Cl2 with MW = 528.13.