College of Science

2013 Presentations

Martha Zamora: ABRCMS 2013: Nov 13-16 Nashville, TN

Investigating the Role of Aqueous Garlic Extract in Mediating LPS-induced TNF-α Secretion Through Modulation of Mitogen Activated Protein Kinase Pathway”

Julio Sanchez: ABRCMS 2013: Nov 13-16 Nashville, TN

Viral Induced Inhibition of Interferon by Modification of Innate Immune Signaling Proteins

Maribel Marquez: ABRCMS 2013: Nov 13-16 Nashville, TN

In vitro model of mesenchymal stem cells for the treatment of myocardial hibernation

Maribel P. Marqueza,c,e, Cayla M. Duffya–c, Rachel E. Leed, Tammy A. Buttericka,c Jesús A. Cabreraa,b, Joshua P. Nixona,c, Yuanxiang Zhaoe, Rosemary F. Kellya,b, Edward O. McFallsa,b

a Veterans Affairs Medical Center, Minneapolis, MN
b Department of Surgery, University of Minnesota, Minneapolis, MN
c Department of Food Science and Nutrition, University of Minnesota, St. Paul, MN
d Department of Integrative Biology, University of Minnesota, Minneapolis, MN
e Department of Biological Sciences, California State Polytechnic University, Pomona, CA

Background: The use of bone marrow-derived mesenchymal stem cells (MSCs) for cell-based therapies in chronic cardiovascular diseases has been under intense investigation. Although the mechanisms by which MSCs protect the ischemic heart are poorly defined, evidence suggests they may contribute to cardiovascular tissue repair by favorably altering mitochondrial function. Our lab group has previously demonstrated that expression of key mitochondrial proteins within the electron transport chain proteins (ETCs) from a porcine model of hibernating myocardium (HIB) is reduced. In the present study, we hypothesized that addition of MSCs to an in vitro model of chronic myocardial ischemia that simulates HIB improves myocyte function by promoting improved mitochondrial function and reducing indicators of oxidant stress.

Methods: A model of hibernation was established using H9c2 rat cardiomyoblasts (ATCC ,CRL-1446) in a modular incubator chamber with a gas mixture of 5% O2 + 5% CO2 (balanced with N2). H9c2 cells were exposed to hypoxic conditions (0, 120 and 150 min) and then re-oxygenated in the presence or absence of primary rat MSCs (conditioned media or co-cultured conditions). Temporal changes in mitochondrial function, oxidant stress markers and ETC proteins are measured by ATP assays, qRT-PCR and Western blot analysis and compared between groups.

Results: The induction of hypoxia was validated using both decreases in O2 saturation in cell culture media and changes in cellular respiration (via resazurin based assay). Changes in hypoxia-related gene expression, such as HIF-1 alpha, were measured by qRT-PCR. Caspase 3/7 activity and ETC proteins (ATPase V) in H9c2 will be determined by enzymatic assay and/or Western blots following the exposure of rMSCs (co-cultured or conditioned media). These studies are ongoing and data will be evaluated to determine whether rMSCs can reverse hypoxia-induced HIB stress in H9c2 cells.

Conclusions: Although the results of these preliminary studies are incomplete, preliminary data supports the validity of the hypoxia induction methodology, as a means of testing paracrine effects of MSCs on the viability of myocytes exposed to brief stress. Data from current and planned studies will be evaluated to determine if rMSCs can reverse hypoxia-induced HIB stress in H9c2 cells, potentially by enhancing mitochondrial function. The long-term goal of our in vitro model of HIB with the addition of rMSC treatment is to assist in elucidating mitochondrial targeted mechanisms for cell based therapies that might translate into new therapies for patients with chronically ischemic hearts and the hibernating phenotype.

Rachel Sanchez: CSUPERB 2013: Jan 3-5th,Anaheim, CA

Fast Events Revealed by Concentration Jumps in ϒ-Aminobutyric Acid Transporters

Rachel V. Sanchez, Cynthia M. Anderson, Sepehr Eskandari
Biological Sciences Department, California State Polytechnic University, Pomona

ϒ-Aminobutyric acid (GABA) transporters (GAT) are electrogenic transport proteins that couple the cotranslocation of Na+, Cl–, and GABA across the plasma membrane of neurons and glia. We used rapid extracellular Na+ or Cl– concentration jumps over intact, voltage-clamped Xenopus laevis oocytes expressing GAT1 to gain insight into the nature of partial reactions responsible for ion binding to the transporter. In the presence or absence of external Cl–, Na+ concentration jumps yielded capacitive charge movements that were characteristic of positive charge entering/leaving the membrane electric field. Sodium removal led to an outward capacitive current, and subsequent reintroduction of Na+ led to an inward capacitive current of equal magnitude. The charge movements were characterized by a rapid transient rising phase followed by two-component exponential decay to a steady-state. The steady-state component was also seen in control cells and was subtracted from the total current to yield the transporter-mediated charge movement. The charge movements evoked by Na+ concentration jumps were voltage-dependent, saturating at membrane potentials more negative than –90 mV, but decreased sharply at more positive potentials. In the nominal absence of external Cl–, there was a ~70% reduction in the total charge moved in response to Na+ concentration jumps. The time-to-peak for the rising phase, characteristics of the two components, and the time constants of the relaxation of the charge movements were not altered significantly in the absence of external Cl–. Chloride concentration jumps in the presence of Na+ exhibited charge movements that were qualitatively similar to those observed with Na+ jumps. Rapid Cl– removal led to an outward capacitive current and Cl– reintroduction led to an inward capacitive current. At any given voltage, the total charge moved in response to Cl– jumps was ~30% of the charge moved with Na+ jumps in the presence of Cl–. In the absence of Na+, Cl– did not evoke any charge movements. A specific inhibitor of GAT1, SKF-89976A, also led to charge movements. Altogether, the results suggest that external Cl– does not play a significant role in Na+ binding to the transporter, but rather it stabilizes the fully Na+-loaded transporter state. We propose Na+ binding constitutes the rate-limiting step in the GAT transport cycle, whereas GABA binding and translocation across the plasma membrane are rapid events. Supported by NIH grant SC1GM086344.

Keywords: neurotransmitter transporter, concentration jump, electrophysiology

CSUPERB 2013

Long term protection in Swiss Webster(SW) mice using a liposomal M2e Influenza A vaccine

J. E. Henriquez, N. Nguyen, E. Chavez, Jill Adler-Moore
Cal Poly Pomona

Long term protection in Swiss Webster(SW) mice using a liposomal M2e Influenza A vaccine J. E. Henriquez, N. Nguyen, E. Chavez, Jill Adler-Moore Cal Poly Pomona

Introduction: Liposomal M2e (L-M2e) Influenza A vaccine was shown in our laboratory to provide significant protection against Influenza A challenge one week post-vaccination in SW mice. The present study focused on how long the protection generated by the vaccine would last, with or without an additional boost.

Methods: Four gps (A-D, n=21/gp) of SW mice were vaccinated with L-M2e (100ug M2e/dose) or non-M2e liposome control (Con) using a Subcutaneous (SC) prime on day 0 and intranasal (IN) boosts on d28 and d56. Mice in gps A (M2e) and B(Con) were given an additional IN boost 4 weeks after completing the standard vaccination regimen, while gps C (M2e) and D(Con) had no extra IN boost. Four days after the gp A boost, serum and spleens from 6 mice/gp were collected and remaining mice in each gp (n=15) challenged with 10X LD50 of influenza A PR8 H1N1. Day 5 post-infection, lungs from 5 mice/gp were collected for viral burden analysis using a foci assay, and the rest of the mice (n=10) were followed for morbidity. Spleens were used for BioPlex cytokine secretion and EliSpot assays; serum was used for determining viral precipitating antibody titers and anti-M2e IgG isotypes by ELISA. 

Results: Vaccinated mice given the extra boost of L-M2e (gp A) showed significantly less weight loss (P<0.0001), and reduced disease signs (P<0.01) as well as prolonged survival (90%) versus non-boosted L-M2e mice (50% survival) (gp C). Viral lung burden was also reduced in boosted mice compared to their Con (P<0.02). However, both boosted and non-boosted mice had significantly prolonged survival versus their respective Con (0% survival) (P<0.0004 and P<0.019, respectively) and IFN-y levels were increased for both gps versus their Con (P<0.02). Boosted mice had significantly higher precipitating antiviral antibody titers compared to their Con(P<0.009) while higher concentrations of anti-M2e IgG1 and IgG2A were detected in boosted and non-boosted mice versus their respective Con(P<0.001).

Conclusion: Long term protection in Swiss Webster mice against lethal influenza virus infection could be achieved by L-M2e vaccination, with or without an extra IN boost. However, the additional IN boost provided an enhanced level of protection since the mice given the extra boost had a higher survival rate with reduced infection severity compared to non-boosted mice. Acknowledgements: NIH MBRS RISE 2 R25 GM061190-05A2, Molecular Express Inc. Key Words: influenza, vaccination, liposomes

Fast Events Revealed by Concentration Jumps in ℽ-Aminobutyric Acid Transporters

Rachel V. Sanchez, Cynthia M. Anderson, Sepehr Eskandari
Biological Sciences Department, California State Polytechnic University, Pomona

ℽ-Aminobutyric acid (GABA) transporters (GAT) are electrogenic transport proteins that couple the cotranslocation of Na+, Cl–, and GABA across the plasma membrane of neurons and glia. We used rapid extracellular Na+ or Cl– concentration jumps over intact, voltage-clamped Xenopus laevis oocytes expressing GAT1 to gain insight into the nature of partial reactions responsible for ion binding to the transporter. In the presence or absence of external Cl–, Na+ concentration jumps yielded capacitive charge movements that were characteristic of positive charge entering/leaving the membrane electric field. Sodium removal led to an outward capacitive current, and subsequent reintroduction of Na+ led to an inward capacitive current of equal magnitude. The charge movements were characterized by a rapid transient rising phase followed by two-component exponential decay to a steady-state. The steady-state component was also seen in control cells and was subtracted from the total current to yield the transporter-mediated charge movement. The charge movements evoked by Na+ concentration jumps were voltage-dependent, saturating at membrane potentials more negative than –90 mV, but decreased sharply at more positive potentials. In the nominal absence of external Cl–, there was a ~70% reduction in the total charge moved in response to Na+ concentration jumps. The time-to-peak for the rising phase, characteristics of the two components, and the time constants of the relaxation of the charge movements were not altered significantly in the absence of external Cl–. Chloride concentration jumps in the presence of Na+ exhibited charge movements that were qualitatively similar to those observed with Na+ jumps. Rapid Cl– removal led to an outward capacitive current and Cl– reintroduction led to an inward capacitive current. At any given voltage, the total charge moved in response to Cl– jumps was ~30% of the charge moved with Na+ jumps in the presence of Cl–. In the absence of Na+, Cl– did not evoke any charge movements. A specific inhibitor of GAT1, SKF-89976A, also led to charge movements. Altogether, the results suggest that external Cl– does not play a significant role in Na+ binding to the transporter, but rather it stabilizes the fully Na+-loaded transporter state. We propose Na+ binding constitutes the rate-limiting step in the GAT transport cycle, whereas GABA binding and translocation across the plasma membrane are rapid events. Supported by NIH grant SC1GM086344
Keywords: neurotransmitter transporter, concentration jump, electrophysiology