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Biomedical Sciences Seminar Series

Winter 2005 Poster Presentation

January 28, 2005 at 1 pm

Physical Sciences Lobby

Jose Miranda, Marisa Monreal, Anahid Mirzatoni, Ramon Mercardo, Erika Reynoso, Evelyn Soriano, Iris Rauda, Marco Orozco, Iris Cruz, Rayshonda Williams, Maritza Hernandez, Freddy Toledo, Grace Masangkay, Ruth Avila, Shamaine Bonds

Poster #1

Detection of AMP-Activated Protein Kinase Alpha Subunit Proteins in the Rat Ovary

Jose Miranda

Abstract: The ovary is a crucial component of the female reproductive system, playing key roles in follicular maturation and production of hormones such as estradiol. AMPK is an enzyme involved in protection of cells from metabolic stress, particularly in liver and skeletal muscle. Preliminary studies in our laboratory have shown mRNA for AMP-Activated Protein Kinase (AMPK) is expressed in the rat ovary in a cell-specific manner, and suggest roles of AMPK in regulating hormone production and cell viability. The purpose of this study is to examine the presence of AMPK ±1 and ±2 subunit protein in the ovary. For immunoblot analysis, whole ovary and liver were obtained from rats and proteins extracted. Proteins were run through SDS-gel electrophoresis and blotted onto a nitrocellulose membrane. Blots were incubated with AMPK immunopurified goat anti-human AMPK ±1- and ±2-specific antibodies, and detected with rabbit anti-goat secondary antibodies. Specific immunoreactive signal of the expected molecular weights (60 kD) were observed in both ovary and liver homogenates. To examine the cell-specific location of AMPK ±1 and ±2 subunit proteins in the ovary, immunohistochemical studies are being performed. Twenty-one day old mice were treated with eCG and hCG to induce follicle development and ovulation, respectively, and ovaries collected at different time points and fixed with paraformaldehyde. Ovaries were then processed for immunohistochemical analysis, and 10 ºM paraffin sections were places onto slides. Ongoing immunohistochemical analysis with a confocal microscopy system will reveal the cell-specific localization of AMPK ±1 and ±2 subunit proteins in the ovary, providing new insight into the possible roles of this enzyme in regulating reproductive functions.


Poster #2

Preparation and Characterization of a Silica Gel Supported Zirconium(IV) Complex

Marisa Monreal

Abstract: The preparation and characterization of a heterogeneous chiral Lewis acid catalyst, silica gel supported diethyl-L-tartrate zirconium(IV), is reported. Early results from [4+2] cycloaddition reactions of cyclopentadiene with methyl acrylate catalyzed by II at room temperature demonstrate endo/exo ratios similar to controls. Previous
work on the analogous homogeneous catalyst m-tolyl-tetraphenylcyclopentadienyl-zirconium indicated a selectivity dependence on nature of dienophile and temperature; these variables, in addition to new reactivity methods, are being manipulated in the current system to maximize selectivity. Elemental analysis, IR, and
solid-state NMR methods were used to confirm the structure of II. These preliminary results are guiding our current work on improving the synthesis and maximizing the selectivity of II, as well as our investigations into derivatives of II.


Poster #3

Expression and Regulation of Adenylyl Cyclase Types V and VI in the Rat Ovary

Anahid Mirzatoni, Dr. Philip S. LaPolt

Abstract:Adenylyl cyclases (ACs) are enzymes that convert ATP into the second messenger cAMP. So far, nine isoforms of ACs have been cloned in mammals and their expression has been investigated in various tissues and cells. The regulation and distribution of AC isoforms is very diverse and complex. It has been shown that usually more than one isoform is expressed in a tissue or cell. In ovarian granulosa cells, AC activity is stimulated by follicle-stimulating hormone (FSH), resulting in increased cAMP levels and production of the steroid hormone estradiol (E2). Little is known regarding the regulated expression and localization of AC isoforms in the ovary. This study tested the hypothesis that AC isoforms V and/or VI are expressed in the rat ovary in a cell-specific, regulated manner. RT-PCR confirmed the presence of transcripts encoding AC V and VI isoforms in whole ovary RNA. The identity of these PCR products was confirmed using restriction mapping and DNA sequencing, and expression was also confirmed by Northern blot hybridization analysis. Immunohistochemical analyses of ACV/VI protein during ovarian development, ovulation, and luteinization revealed localized expression of ACV/VI in granulosa cells & oocytes of developing follicles, as well as cumulus cells of preovulatory follicles. After ovulation, ACV/VI was also detected in cells of the corpus luteum. These findings provide new insights into the regulated expression of ACV and VI in the rat ovary, and suggest that ACV/VI may play roles in ovarian functions such as steroidogenesis, oocyte maturation and function of the corpus luteum


Poster #4

Optimal Concentration of Dietary Jojoba Oil that Alters High Density Lipoprotein
Cholesterol Concentration in New Zealand White Rabbits

Ramon Mercado

Abstract: Jojoba oil is a wax ester that consists of eicosenoic acid and eicosenol. Previous studies in our laboratory have shown that female New Zealand White rabbits fed a 2% jojoba oil diet for seven days have a significantly increased HDL-C concentration. Our objective is to determine the optimal dietary concentration of jojoba oil that increases HDL-C concentration and the optimal time for maximizing the amount of eicosenoate in the blood. Female New Zealand White rabbits were fed a rabbit chow supplemented with 3 or 9% (w/w) jojoba oil for 39 days. Blood was collected from an ear vein at 0, 14, 28, and 39 days. Serum was obtained by centrifugation. Lipids were extracted from serum, feces, and feed by the method of Bligh and Dyer, followed by trans-esterification with sulfuric acid in methanol and quantification of the fatty acid methyl esters with gas-liquid chromatography. The HDL fraction was separated from the VLDL+LDL fraction by polyanion precipitation of the serum, and total cholesterol concentration was measured enzymatically. The highest concentration of HDL-C in the blood occurs with the 3% jojoba oil diet, and the highest concentration of eicosenoate in the blood occurs at 28 days. Therefore, the 3% jojoba oil diet is better than the 9% diet in altering HDL-C metabolism, and the optimal time for feeding the diet is 28 days. (Supported by MBRS-RISE grant R25 GM61331.)


Poster #5

A New Tool to Study Processing of Human Defensin 5, An Important Mediator of Mucosal Immunity

Erika Reynoso 

Abstract:A New Tool to Study Processing of Human Defensin 5, An Important Mediator of Mucosal Immunity
Paneth cells (PC) in the small intestine contribute to innate mucosal immunity by secreting defensins, natural peptide antibiotics that are small cationic amphipathic molecules with a membrane disrupting activity. Many defensins are produced as prepropeptides that undergo posttranslational sequential processing. Human defensin 5 (HD5) is found in PC granules as a propeptide (proHD5) and is further processed by PC derived trypsin. As biological activities of the different HD5 forms vary and processing may be altered in diseases of the gastrointestinal tract, it is important to have a tool to identify the various HD5 forms. We describe here epitope mapping of monoclonal antibodies raised against proHD5 employing fluorimetric ELISA, dot blot and Western immunoblot analysis. As antigens, recombinant and natural HD5 forms with varying N-terminus and synthetic peptide fragments were used. Polyclonal rabbit antisera raised against fully processed rHD5 reacted with all HD5 forms, except the synthetic fragments. Most monoclonal forms were reactive only with incompletely processed HD5 forms and non-reactive with fully processed HD5. These antibodies will be useful in the study of HD5 processing and intestinal diseases involving PC.



Poster #6


Expression and function of 5'-AMP-activated protein kinase alpha subunit
mRNAs in rat ovarian granulosa cells

Evelyn S. Soriano and Dr. Philip S. LaPolt

Abstract:Adenosine monophosphate-activated protein kinase (AMPK) is a signaling enzyme activated by increased AMP levels in liver and skeletal muscle, protecting cells against metabolic stress associated with low ATP levels. AMPK is a heterotrimeric protein kinase with multiple isoforms for each subunit (alpha, beta, and gamma). Whereas only the alpha-1 and alpha-2 subunits are catalytic, beta and gamma subunits are regulatory and necessary for full enzymatic activity. Previous studies indicate that AMPK is expressed in ovarian oocytes, and may play a role in oocyte maturation. Preliminary data in our laboratory suggests a role of AMPK in hormone production and viability of granulosa cells. To better understand the role of AMPK in the ovary, we studied the expression and potential function of AMPK alpha subunits in cultured rat granulosa cells. Using reverse transcription-polymerase chain reaction (RT-PCR) and specific primers for AMPK alpha-1 and alpha-2 subunits, PCR products corresponding to the expected sizes were obtained, indicating the presence of AMPK alpha-1 and alpha-2 subunits in rat granulosa cells. To determine possible functions of AMPK in granulosa cells, we are utilizing an antisense oligonucleotide approach to inhibit alpha-1 and alpha-2 expression in cultured cells. Culture of granulosa cells with a fluorescently labeled version of this antisense oligonucleotide DNA revealed uptake into cells, as determined by confocal microscopy. The effectiveness of the antisense oligonucleotide in inhibiting AMPK expression, and possible effects on hormone production, are being determined. These studies demonstrate the expression of AMPK alpha-1 and alpha-2 subunit mRNAs in rat granulosa cells, and will provide new information regarding possible functions of this enzyme in the ovary.


Poster #7

Studies of Amyloidogenic Protein Interactions at the Surface/Solution Interface
via Atomic Force Microscopy

Iris E. Rauda

Abstract:The aggregation of _-synuclein, an amyloidogenic protein, has been implicated as a critical step in the development of Parkinson's disease (PD). Recently, it has been demonstrated that amyloid proteins aggregate via partially folded intermediates to form ordered aggregates, such as protofibrils and fibrils as well as disordered amorphous aggregates. These _-synuclein fibrils are found in the form of deposits in the cell. The molecular mechanisms leading to protein deposition of _-synuclein are not well understood. We hypothesize that interactions between _-synuclein and certain surfaces may catalyze the aggregation process, such as hydrophobic versus hydrophilic surfaces and positive versus negative surfaces. We also believe that interactions between _-synuclein and other molecules can inhibit or promote aggregation. We plan to study these biomolecular interactions at the surface/solution interface using atomic force microscopy (AFM), which is a powerful technique for studying the morphology and structure of amyloid fibrils, with high resolution and imaging ability in air and fluid conditions. In addition, AFM is extremely powerful for studying the amyloidogenic protein aggregation and fibrillation at surfaces under physiologically relevant conditions. We would like to obtain useful information about the factors leading to _-synuclein protein aggregation so that mechanisms involving the pathogenesis of Parkinson's disease can be formulated.


Poster #8

Determination of Reduced Phosphorus in Geothermal Waters

Marco Orozco

Abstract:Phosphorus is one of the most abundant nutrients in the environment. However, even though it is abundant, little is known about how it exists in nature. Past methods have not specifically measured the different forms of inorganic phosphorus (phosphate, phosphite, and hypophosphite) in natural waters. Using ion chromatography, a new method has been developed that is selective and can measure low concentrations of reduced phosphorus. Water samples were collected from Hot Creek near Mammoth Lake, CA during the summer of 2004. Preliminary analyses suggest these samples may contain phosphite. The implication of this study will be discussed.


Poster #9

Synthesis of Unsymmetrical Adamanate

Iris Cruz

Abstract:Siderophores are iron binding ligands, which serve to gather iron for microbial cells. Under an iron deficient environment bacteria produce and excrete siderophores. After sequestering environmental ferric ion, these siderophores are recognized and transported into the cell by receptors located in the outer membrane. Escherichia coli (E.coli) expresses on its surface the FepA receptor, which discriminately transports iron through the siderophore enterobactin(1). Studies have found that FepA also transports analogs of enterobactin containing catecholate type binding units attached to simpler backbones. Much is known about the fate and role of siderophores, however little is known about the mechanistic interaction of a siderophore during its transportation of iron. In order to elucidate some of this we have proposed the use of bifunctional analogs.
More specifically the utilization of adamantane derivative consisting of three 2,3-dihydroxybenzoyl- arms in the 3,5,7-positions for iron binding and a fourth position occupied by group R, which potentially could be conjugated to fluorescent probe or an antibiotic through a linker. Proton is used as a protecting group for unsymmetrical amidation of tetra(aminomethyl)adamantane. Here we present synthesis of tricatecholateadamantane(4) protected intermediate starting from inexpensive adamantane.


Poster #10

Synthesis and Characterization of Pentaphenylcyclopentadienyltris(dimethylamido)
Zirconium and Chloride Derivatives

Rayshonda Williams

Abstract: We have prepared pentaphenylcyclopentadienyltris (dimethylamido) zirconium (C5Ph5)Zr(NMe2)3 (I) and two chloride derivatives: (C5Ph5)Zr(NMe2)2Cl (II) and (C5Ph5)Zr(NMe2)Cl2 (III) . I was synthesized by reacting pentapheylcyclopentadiene (HC5Ph5) with tetrakisdimethylamidozirconium Zr(NMe2)4. Next, II was produced by reacting I with one equivalent of Me2NHoHCl which was added using a slow addition method at room temperature. III was prepared by reacting I with two equivalents of Me2NHo HCl which was added using the same slow addition method but at -78oC. The compounds were characterized by 1H and 13C NMR spectroscopy. They will be used in the synthesis of chiral Lewis acids, which will be catalysts in C-C bond formation reactions.


Poster #11

The Effect of Dietary Jojoba Oil on HDL Metabolism in New Zealand White Rabbits

Maritza Hernandez, Monica Hernandez & Raymond E. Garcia

Abstract: We have prepared pentaphenylcyclopentadienyltris (dimethylamido) zirconium (C5Ph5)Zr(NMe2)3 (I) and two chloride derivatives: (C5Ph5)Zr(NMe2)2Cl (II) and (C5Ph5)Zr(NMe2)Cl2 (III) . I was synthesized by reacting pentapheylcyclopentadiene (HC5Ph5) with tetrakisdimethylamidozirconium Zr(NMe2)4. Next, II was produced by reacting I with one equivalent of Me2NHoHCl which was added using a slow addition method at room temperature. III was prepared by reacting I with two equivalents of Me2NHo HCl which was added using the same slow addition method but at -78oC. The compounds were characterized by 1H and 13C NMR spectroscopy. They will be used in the synthesis of chiral Lewis acids, which will be catalysts in C-C bond formation reactions.


Poster #12

Metallothionein (MT) & Lys-Cys-Thr-Cys-Cys-Ala (FT) as Biosensors for the Detection of Trace Metals by Surface Plasmon Resonance (SPR)

Freddy Toledo

Abstract:MT and FT immobilized onto a modified gold surface were used as biosensors to detect metal ions such as Cd, Zn by surface plasmon resonance. Injecting solutions of Cd and Zn onto MT or FT we find that there is a conformational change of the metalloproteins upon metal sequestration. Using 0.01 M HCl for acidic conditions, we found that MT and FT can release any metals bound to their cysteine groups, thereby, regenerating the biosensor surface. Therefore, MT and FT as biosensor chips have proved to be effective sensors for trace metals by SPR.


Poster #13

Preliminary Steps For The Recombination And Expression of

Grace Aldana Masangkay

Abstract: Reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) can produce hydroxyl radicals (OHo) that damage DNA. Oxidative damage affects the function or reproduction of cells leading to diseases such as cancer and neurodegenerative disorders. Living organisms have developed defense mechanisms such as antioxidants that can prevent or repair oxidation. Peroxiredoxins (prx), ubiquitous proteins that are found in organisms ranging from bacteria to humans, can act as antioxidants reducing H2O2 to H2O. In eukaryotic cells, prx are also regulators of H2O2-mediating signaling. In the presence of high levels of H2O2, peroxiredoxin becomes overoxidized, forming sulfinic acid. The overoxidized prx can no longer reduce H2O2 to H2O, allowing H2O2 to participate in signaling. The formation of sulfinic acid was believed to be irreversible; however, recent studies have shown that a yeast protein called sulfiredoxin (srx) can reduce the cysteine-sulfinic acid of yeast peroxiredoxin TsaI back to its thiol form, (Biteau, B., Labarre, J., Toledano, M.B. (2003) Nature 425, 980-984). . Our goal is to express srx cDNA (from cancerous human lung tissue) in prokaryotic cells so that other lab members can show that it can repair the oxidative damage in human peroxiredoxin I. We plan to make a donor vector and fuse it with an acceptor vector via Cre recombinase to form a recombinant plasmid that will express the srx. Our project began by finding the sulfiredoxin sequence on the NCBI website (AAH47707) and obtaining the srx cDNA as an expressed sequence tagged DNA from Invitrogen. This plasmid was transformed into E. coli cells and colonies were picked and analyzed by restriction digestion. The srx coding sequence was subcloned by Polymerase Chain Reaction (PCR) technique, analyzed by agarose gel, and ligated into a plasmid to form a donor vector. This vector was purified by column chromatography and sequenced at City of Hope. In the future, we plan to form a recombinant plasmid that will express the srx and use it to repair overoxidized prx.


Poster #14

Synthesis and Characterization of a Photoreversible Calcium Chelator Based on Azobenzene

Ruth Avila

Abstract:Calcium plays an integral role in cell signaling. The release and uptake of calcium in an oscillatory manner triggers various important physiological processes. In order to study the effects of calcium oscillations in proteins and cells, calcium signals will be mimicked artificially using a light-responsive chelator. The aim of this project is to synthesize a molecule based on azotoluene that can bind and release calcium reversibly in response to irradiation. It is expected that different wavelengths of light will isomerize the chelator, interconverting it between high affinity (cis) and low affinity (trans) forms. This interconversion should mimic biochemical calcium signals. The synthesis of this molecule requires three steps. The first step is the synthesis of 4,4'-dimethylazobenzene, which yielded 84.56% of expected product. The second step is the synthesis of 4,4'-Bis(bromomethyl) azobenzene, resulting in 73.40% yield of product, followed by the third step: substitution of iminodiacetic acid for bromine, which yielded 90.85% of pure product. Irradiation of the chelator both in the presence and absence of metal ions such as calcium resulted in the successful interconversion between cis and trans forms. These findings form the basis for further studies involving calcium binding and photochemistry in aqueous media, which will be presented.

Poster #15

The Effect of Low-Pass Filtering, Exposure, and Delay on Voice Quality and Recognition

Shamaine Bonds

Abstract: Accuracy of voice recognition and perceptual voice quality were tested for twelve digitally recorded female voices.  The task was to recognize whether the voices were previously heard or not.  Overall performance was 74% correct, and significant effects were evident in voice quality ratings, but not for delay, filtering, and exposure repetition.


Poster #16


Poster #17

Abstract: .


Poster #18



Poster #19



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