Biological Sciences Student Research Showcase 2010

Master's Abstracts


Cry5B: A crystal protein among a new and powerful class of anthelmintics

Sophia Georghiou
Dr. Raffi Aroian

The soil-dwelling bacterium Bacillus thuringiensis produces crystal proteins that are toxic to insects and nematodes.  These (cry) proteins bind to specific receptors on the midgut epithelial cells of these organisms, but are harmless to vertebrates, which lack any receptors for the ingested protein.  Insecticidal spores and proteins produced by this bacterium are most commonly used as environmentally friendly pesticides.  It has been more recently discovered that a select few of these proteins have anthelmintic activity against parasitic nematodes.  Cry5B, in particular, has been shown to have nematicidal activity against Caenorhabditis elegans in vitro and Ancylostoma ceylanicum in vivo as a triplicate dose treatment.  In this study, purified Cry5B and Cry5B-S407C were tested for their efficacy as a single-dose treatment against chronic, gastrointestinal Heligmosomoides polygyrus infections in mice.  In their purified form, Cry5B and the S407C mutant of the protein were found to have the same single dose efficacy against adult H. polygyrus in vivo.  Therapeutic effects were measured in terms of a decrease in final worm burden as well as a decrease in fecal egg counts, before and following treatment, between control and Cry5B/S407C-treated groups.  Compared to control (4Q7-treated) mice, infected animals treated with either Cry5B or S407C showed statistically significant reduction in worm burden and an even more dramatic drop in fecal egg counts.  The results indicate that Cry5B, showing therapeutic effects against a chronic gastrointestinal parasitic nematode, calls for further clinical development as a therapeutic, and biologically and economically important, treatment for humans and veterinary animals.


Inhibition of muscle phosphofructokinase-1 (PFK-1) and lactate dehydrogenase (LDH) by ascorbate derivatives: a proposal for cancer treatment

Duyen-Anh Pham
Dr. Percy Russell

The role of vitamin C (ascorbate) was re-examined after a controversial history in cancer treatment. We showed that ascorbate specifically inhibited three glycolytic isozymes – phosphofructokinase-1 (RMPFK-1) and lactate dehydrogenase (RMLDH) and adenylate kinase (RMAK). Most cancer cells display an elevation of aerobic glycolysis to provide the high ATP supply needed for cancer cell the proliferation. The energy source upregulation in cancer cells and the dependence of cancer cells on glycolysis to initiate the energy supply suggested that ascorbate derivatives might have some therapeutic value in the treatment of cancer.  The amphipathic character of fatty acid derivatives of ascorbic acid L-ascorbic acid 6-stearate (AAS), L-ascorbic acid 6-palmitate (AAP), L-ascorbic acid 2,6-dipalmitate (AADP), L-ascorbic 2,6 dibutyrate (AADB) should facilitate crossing the membrane process fatty acid bilayer.  These studies show that the effect of ascorbate derivatives on RMPFK-1 and RMLDH are several fold more potent inhibitors than ascorbate itself. Each derivative showed interesting enzyme specificities.  For example, AADP shows very potent inhibitions of RMPFK-1 but not of RMLDH, while AADB shows very potent inhibitions of RMLDH but not of RMPFK-1. Studies with the structural analog of ascorbate, L-gulono-gama-lactone (LGGL), and esters of the fatty acid groups in ascorbate derivatives suggest that the carbonyl double bond of ascorbate is the inhibition site.


Picky eater syndrome: The pesticide imidacloprid on honey bee (Apis mellifera) alters sucrose response threshold and, potentially, colony health.

Daren Eiri
Dr. James Nieh

In recent years, beekeepers have experienced high honey bee (Apis mellifera) loss, some reporting a loss of 30-90 percent of their hives. Unlike some other honey bee diseases, beekeepers report a sudden disappearance of adult bees and few dead bees inside or around the colony.  Where have the bees gone? This phenomenon, known as colony collapse disorder (CCD), has been attributed to parasites carrying diseases, pathogens, management techniques, and pesticides. Lately, much more attention has been focused on a combination of these stresses.  Using the proboscis extension reflex (PER) assay and assessing an individual’s sucrose response threshold, this study examines the effects of minute, sublethal doses of the pesticide, imidacloprid, on honey bee foraging behavior.  Imidacloprid is a commonly used pesticide (Gaucho®) and has been banned in some countries because of its effects on honey bees. Our results indicate that foragers treated with imidacloprid have a higher sucrose response threshold (will only feed on the highest sucrose concentrations) than those not treated with the pesticide. This suggests that bee foragers that are exposed to imidacloprid respond only to high concentrations of sucrose. Such “picky feeding” could reduce the number of nectar calories collected by a colony, thereby leading to reduced health, because most natural nectar sources are fairly dilute. In addition, some honey bees specialize on pollen foraging and this is associated with low sucrose response thresholds. If imidacloprid causes a general increase in sucrose response threshold, the number of pollen foragers should decrease and this will reduce the number of brood that can be reared, leading to declining numbers of foragers.


Seasonal priority effects: Implications for invasion and restoration in California coastal sage scrub

Claire Wainwright
Dr. Elsa Cleland

Exotic annual grasses (EAGs) threaten native plant communities in California, and pose a challenge for habitat restoration. Several mechanisms have been hypothesized to explain the success of EAGs, including suppression of native seedlings by high litter accumulation, exhaustion of soil moisture by EAGs, and low seed rain of natives relative to exotics. EAGs may also exert priority effects by being active earlier in the growing season; EAGs sometimes germinate with a smaller threshold rain event, whereas native species germinate with cooler temperatures and larger winter rains. We  hypothesized that 1) EAGs (Avena fatua, Bromus hordeaceus) would germinate with an experimentally applied late-summer rain, but native species would not germinate until cool autumn rains, and 2) EAGs which germinated in late summer would not survive until the winter rains begin, due to either desiccation or herbivory. EAGs and native species were seeded into known areas of experimental plots, and a pulse of water was applied in late summer to a disturbed site within a coastal sage scrub community. Four levels of summer watering were included (none=control, addition of 10, 20 or 30 mm). Germination, survival, and community level patterns of abundance were monitored for both native and exotic species.


The Importance of Matrix Metalloprotainase 9 in Hypoxia-Induced Lung Remodeling

Mary Nguyen
Dr. Gabriel Haddad

Rationale: Hypoxia is a condition associated with many pulmonary diseases.  Hypoxia can induce tissue and vascular remodeling, which can result in many pathological conditions; pulmonary hypertension is one such disease. Hypoxia-induced pulmonary hypertension is increasingly prevalent in children since hypoxia is common in many diseases. Pulmonary hypertension is associated with high mortality and the pathology is marked by vascular remodeling. This process includes increased smooth muscle proliferation and matrix deposition, such as fibronectin and collagen. Since Matrix Metalloproteinases (MMP) family can alter the components of the matrix and thus is an important facilitator of the remodeling process; we  chose to study the impact of MMP-9, a member of the MMP family, in hypoxia-induced lung remodeling in immature mice because of its association in many hypoxia-induced diseases.   Methods: MMP-9 knockout (KO) mice and FVB controls (background for MMP-9 KO) were both exposed to either 11%O2 or room air for 2 weeks, from postnatal days 2 to 17. At postnatal day 17, mice were sacrificed and lungs were processed for protein and histology.  Results: The survival rate of MMP-9 KO mice exposed to chronic hypoxia was significantly lower than FVB mice exposed to the same condition. MMP-9 KO mice that survived hypoxia exposure were significantly smaller than FVB mice. Histology revealed that MMP-9 KO mice that survived hypoxia had approximately a four-fold increase in muscularized vessels than FVB mice. Protein analysis demonstrated that a four-fold increase in fibronectin levels in MMP-9 KO mice compared to FVB exposed to chronic hypoxia.  Conclusions: Our study shows that MMP-9 plays a critical role in hypoxia-induced vascular remodeling. Deletion of this protein results in increased mortality that may be associated with increased smooth muscle proliferation. These results may be due directly to MMP-9 deletion or indirectly to an increase in expression of fibronectin, a substrate of MMP-9, which is also associated with pulmonary hypertension and smooth muscle proliferation.


Markers of cardiomyocyte injury in acute Kawasaki disease

Yuichiro Sato
Dr. Jane Burns

Endomycocardial biopsies have demonstrated that subclinical myocarditis is a universal feature of acute Kawasaki disease (KD). We investigated biochemical evidence of myocardial strain and cardiomyocyte injury in acute and convalescent KD, and febrile (FC) and healthy control (HC) subjects by measuring blood levels of N-terminal propeptide B-type natriuretic peptide (NT-proBNP), midregional pro-atrial natriuretic peptide (MR-proANP), interleukin-1 receptor family member ST2, cardiac troponin I (cTnI), C-terminal pro-vasopressin (copeptin), C-terminal pro-endothelin-1 (CT-proET-1), midregional adrenomedullin (MR-proADM), and procalcitonin (PCT). Biomarker concentrations were determined for the following subjects: 55 acute KD (30 with paired convalescent samples), 55 age-similar FC, and 20 HC children. Levels of NT-proBNP and MR-proANP were elevated in paired acute vs. convalescent KD (p<0.0001; p=0.002, respectively), vs. FC (p<0.0001; p=0.001), and vs. HC (both p<0.0001). Both markers were positively correlated with ALT and GGT, and negatively with age and illness days.  Levels of ST2 and PCT were elevated in acute KD vs. convalescent KD (p=0.001; p=0.002), FC (p<0.0001; p=0.0008), and HC (both p<0.0001).  CT-proET and MR-proADM were elevated in acute KD vs. HC (p=0.0009; p=0.005), but only the latter was significantly different vs. convalescent KD (p=0.003). The natriuretic peptides negatively correlated with measures of diastolic function (MV E:A ratio and deceleration time) and positively correlated with LAD Z score, MV A, and LAD/RCA Z worst.  In conclusion, the natriuretic peptides were markers of impaired myocardial relaxation and coronary artery dilatation in acute KD subjects. Elevated levels of natriuretic peptides and PCT with normal levels of cTnI suggest that potentially reversible cardiomyocyte injury rather than cell death is a feature of myocardial inflammation in acute KD.


Toll-like Receptor 7 Tolerance in Anti-Neuroinflammation in Murine Experimental Autoimmune Encephalomyelitis

Linda Vuong
Dr. Dennis Carson

Multiple sclerosis (MS) is an autoimmune disease that results in demyelination and neurodegeneration of the central nervous system (CNS).  This disease has a chronic progressive or a relapsing course that is partially recapitulated in murine models such as experimental autoimmune encephalomyelitis (EAE).  Toll-like receptors (TLRs) are a family of pattern-recognition receptors that mediates the innate and adaptive immune responses.  TLR tolerance is a phenomenon in which repeated stimulation of a TLR will lead to hyporesponsiveness.  To test the potential for TLR7 hyporesponsiveness to limit CNS inflammation, SJL/J mice immunized with proteolipid protein (PLP) 139-151 were treated with the synthetic TLR7 agonist 9-benzyl-8-hydroxy-2-(2-methoxyethoxy) adenine (SM360320, designated here as 1V136).  Daily low-dose 1V136 treatment significantly decreased disease severity.  Concordantly, the number of CNS-infiltrating immune cells was significantly reduced in 1V136-treated mice.  A microglia-enriched cell population tested for response to TLR agonists confirmed that 1V136 treatment induces hyporeponsiveness to subsequent TLR7 stimulation.  Splenocytes from 1V136-treated mice exhibited a specific decrease in interferon (IFN)-γ and interleukin (IL)-17 secretion.  Serum samples from 1V136-treated mice showed no difference in the humoral response.  In summary, chronic treatment with 1V136 can induce innate immune system hyporesponsiveness and inhibit an abnormal adaptive immune response.  The direct effects of 1V136 on the CNS may contribute to a reduction in the clinical severity of a murine model of MS.


Cytokines Induce Proliferation via an NCX-1 Dependent Mechanism

Edwin Yoo
Dr. Tomothy Bigby

RATIONALE: Airway smooth muscle hyperplasia is a characteristic of airway remodeling in asthma and this is thought to be, at least in part, cytokine mediated.  Because cytosolic free Ca2+ ([Ca2+]cyt) plays an important role in smooth muscle proliferation, we chose to examine the role of [Ca2+]cyt , focusing on the expression of the Na+/Ca2+ exchanger 1 (NCX1) protein and its link to human airway smooth muscle proliferation.  METHODS: Human bronchial smooth muscle cells (HBSMC) were purchased and grown in a defined HBSMC medium (Science Cell, SD, CA) on polylysine coated tissue culture plates.  For immunofluorescent microscopy, HBSMC cells were grown on coated glass slides (Labtek).  Cells were also grown in the presence of 20 ng/ml of TNFa, IL-13, or IL-33.  Immunoreactive NCX1 was detected using the R3F1, a murine monoclonal antibody in immunoblots and in immunohistochemistry.  mRNA for NCX1 was detected by quantitative RT-PCR.  Cell growth was assessed using the Cell Titer-96 cell proliferation assay.   RESULTS: mRNA encoding for NCX1 was detected in HBSMC and increased in response to TNFa 3-fold at 6 h. Immunoreactive NCX1 protein increased ~2-fold in response to conditioning with TNFa and IL-33 at 24 h.  Immunofluorescent staining of NCX1 in cultured cells increased in 24 h.  Cell culture of HBSMC in the presence or absence of TNFa, IL-13, or IL-33 increased cell number ~2-fold at 24 h compared to control and further growth occurred at 48 and 72h.  When cells were treated with the specific NCX1 inhibitor SN-6 (10 M), increases in cell number were blocked at 24 h, while cell viability was unchanged.  CONCLUSIONS:  We conclude that NCX1 is expressed in HBSMC and that this expression is increased by cytokines associated with asthma.  Moreover, cells proliferate with these cytokines, which is blocked by NCX1 inhibition.  These data suggest that NCX1 may play an important role in airway remodeling associated with asthma.


Up-regulation of Chemokine receptor-like 2 in an in vitro model of cerebral ischemia

Alice Chen
Dr. Gabriel Haddad

Ischemic penumbral cell death accounts for the progressive infarct expansion which exacerbates neurological outcome in both stroke patients and animal models. The underlying mechanism of penumbral cell death has not been well understood. In this study, we investigated the signaling mechanism of penumbral cell death using an in vitro model (ischemic solution, IS model) that simulates the ischemic penumbral cell death (Yao, et al, J. Neurochem, 2007, 100, 1224-33) together with qRT-PCR and Western blot analysis. Our study showed a 9-, 9- and 35-fold increase in chemokine receptor-like 2 (CCRL2) RNA expression following a 2, 6 and 12 h of IS treatment (n=3, p<0.05 for all groups), respectively. Western blot analysis showed a 7-fold increase in CCRL2 protein expression after 12.5 h of IS treatment, suggesting a potential role of CCRL2 in ischemic penumbral cell death. In a separate study, we examined the activity of caspase 3 in IS-treated slices. Our data exhibited a 2- and a 17-fold increase in cleaved caspase-3 after a 4 and 14 hrs of IS treatment, respectively, suggesting apoptotic cell death in IS-treated slices. In addition, the involvement of c-JUN N-terminal kinase (JNK) in CCRL2-mediated ischemic cell death was studied in brain slices derived from both CCRL2 KO and WT mice. Our preliminary data show that the expression level of phosphorylated JNK was higher in WT than that in CCRL2 KO slices. Finally, since ChemR23 shares the same ligand (chemerin) with CCRL2, we have examined the expression profile of ChemR23. Our data showed a 2-fold increase of ChemR23 expression following IS treatment in WT slices and a further 1.5 increase in CCRL2 KO slices. Taken together, our data suggests that a) IS induces apoptotic cell death in brain slices; b) CCRL2 and ChemR23 expression are enhanced in penumbral cells and ChemR23 increases its expression level when CCRL2 is absent c) JNK may be involved in CCRL2 mediated penumbral cell death. (Acknowledgement: We thank Dr. Brian A. Zabel from Department of Pathology, Stanford University for providing CCRL2 antibody).


Mapping the Mouse Brain Microvessel Proteome

Hyun Chun
Dr. Brian Eliceiri

Previous studies have reported on the transcriptome of rodent brain microvessels, containing endothelial cells, astrocytic endfeet and associated basal lamina, termed the neurovascular unit (NVU). To begin to identify membrane proteins composing the NVU, highly enriched, mouse brain microvessels were used as starting material for membrane isolation. To identify proteins present in the membranes, the isolated and tryptically digested proteins were analyzed utilizing two-dimensional liquid chromatography separation of peptides on a microcapillary column with detection via a tandem mass spectrometer.  The novel combination of microvessel isolation and membrane fractionation (n=5 expts) allowed multidimensional protein identification of up to 2634 proteins, of which 53% were determined bioinformatically to be membrane associated proteins.  Additional groups include extracellular matrix components, mitochondrial membrane proteins, as well as other protein candidates found in endothelial cells and astrocytes. To be discussed will be the identity of proteins common to and unique in the NVU as compared to non-microvessels. The findings gleaned from the studies will began to provide a NVU protein database allowing identification of protein constituents of the blood brain barrier in normal and disease states.


Intrathecal botulinum neurotoxin B: effects on spinal primary afferent sensory C-fibers and nociception in the mouse.

Polly Huang
Dr. Tony Yaksh

Botulinum neurotoxin B (BoNT-B) proteolytically cleaves VAMP I and II (synaptobrevins) in the family of soluble N-methylaleimide-sensitive attachment protein receptor (SNARE) proteins. Proteolytic cleavage by BoNT-B inhibits vesicle-membrane fusion and the release of neurotransmitters, such as acetylcholine.To investigate the effects of intrathecally delivered BoNT-B on neurotransmitter release from spinal primary afferent C-fibers, C57/Bl6 mice were treated with single intrathecal (IT) injection of BoNT-B (Myobloc® Solstice Neurosciences) 2 days prior to intraplantar (IPLT) formalin injection in the hind paw. IPLT formalin-evoked paw flinching behavior was assessed using an automated motion detection system (Yaksh TL, J Appl Physiol, 2001) for 40 minutes following IPLT injection. Animals that received IT BoNTB (0.1U-0.5U,5μL) pretreatment showed significant decrease in formalin-evoked flinching behavior in phase 2 (11-40mins) compared to vehicle animals (IT 0.9% NaCl, 5μL). IPLT formalin induces substance P (subP) release from primary afferent C-fibers. SubP specific binding to neurokinin-1 receptors (NK1-R) and the consequent internalization of NK1-R on spinal superficial dorsal horn (laminae I and II) neurons was visualized using immunohistochemistry (IHC) and confocal microscopy. In IT NaCl pre-treated mice, IPLT formalin resulted in a significant increase in the fraction of NK1-R expressing cells in the superficial dorsal horn showing internalization. In contrast, consistent with the effects on flinching behavior, IT BoNT-B (0.5U, 5μL) pre-treated mice showed a reduction in the fraction of dorsal horn NK1-R expressing cells showing receptor internalization. In order to investigate the mechanism of BoNT-B effects on neurotransmitter release, spinal cord tissue lysate was incubated with BoNT-B. Western blot shows decrease in VAMP I/II protein, which suggests cleavage of VAMP I/II by active BoNT-B. We then became interested in the effect of IT BoNT-B on mechanical allodynia following spinal nerve ligation (SNL) using von Frey microfilaments. At 14 days after SNL at the L5 spinal nerve, all animals showed decreased paw withdrawal threshold ipsilateral to the site of ligation when stimulated by von Frey microfilaments. In mice that received a single intrathecal injection of BoNT-B (0.1U-0.5U, 5µL) at 14 days post-SNL, a modest increase in paw withdrawal threshold was observed and maintained up to 15 days after IT BoNT-B, compared to vehicle animals. Overall, the data suggests that intrathecal BoNT-B alleviates formalin-induced nociceptive behavior by blockade of neurotransmitter release from spinal primary afferent C-fibers, and reduces spinal nerve injury-induced mechanical hyperalgesia. These effects are due to cleavage of VAMP I/II by botulinum neurotoxin B.