Digital Commons Network^™

Imaging Activity Of Neuronal Populations With New Long-Wavelength Voltage-Sensitive Dyes, Joseph P. Wuskell, Leslie M. Loew

UCHC Articles - Research

We have assessed the utility of five new long-wavelength fluorescent voltage-sensitive dyes (VSD) for imaging the activity of populations of neurons in mouse brain slices. Although all the five were capable of detecting activity resulting from activation of the Schaffer collateral-CA1 pyramidal cell synapse, they differed significantly in their properties, most notably in the signal-to-noise ratio of the changes in dye fluorescence associated with neuronal activity. Two of these dyes, Di-2-ANBDQPQ and Di-1-APEFEQPQ, should prove particularly useful for imaging activity in brain tissue and for combining VSD imaging with the control of neuronal activity via light-activated proteins such as channelrhodopsin-2 …

Use Of An Alpha-Smooth Muscle Actin (Smaa) Gfp Reporter To Identify An Osteoprogenitor Population, Zana Kalajzic, Haitao Li, Li-Ping Wang, Xi Jiang, Katie B. Lamothe, Douglas J. Adams, Hector L. Aguila, David W. Rowe, Ivo Kalajzic

UCHC Articles - Research

Identification of a reliable marker of skeletal precursor cells within calcified and soft tissues remains a major challenge for the field. To address this, we used a transgenic model in which osteoblasts can be eliminated by pharmacological treatment. Following osteoblast ablation a dramatic increase in a population of α-smooth muscle actin (α-SMA) positive cells was observed. During early recovery phase from ablation we have detected cells with the simultaneous expression of SMAA and a preosteoblastic 3.6GFP marker, indicating the potential for transition of α-SMA⁺ cells towards osteoprogenitor lineage. Utilizing α-SMAGFP transgene, α-SMAGFP⁺ positive cells were detected in the …

A Crucial Role In Cell Spreading For The Interaction Of Abl Pxxp Motifs With Crk And Nck Adaptors, Susumu Antoku, Gonzalo M. Rivera, Bruce J. Mayer

UCHC Articles - Research

The dynamic reorganization of actin structures helps to mediate the interaction of cells with their environment. The Abl non-receptor tyrosine kinase can modulate actin rearrangement during cell attachment. Here we report that the Abl PxxP motifs, which bind Src homology 3 (SH3) domains, are indispensable for the coordinated regulation of filopodium and focal adhesion formation and cell-spreading dynamics during attachment. Candidate Abl PxxP-motif-binding partners were identified by screening a comprehensive SH3-domain phage-display library. A combination of protein overexpression, silencing, pharmacological manipulation and mutational analysis demonstrated that the PxxP motifs of Abl exert their effects on actin organization by two distinct …

Detection Of Biomarkers By Mrs Requires Error Analysis, Jeffrey C. Hoch, Mark W. Maciejewski, Michael R. Gryk

UCHC Articles - Research

Using MRS imaging and singular value decomposition (SVD), Manganas et al. (Science 318, 980,2007) reported a nuclear magnetic resonance diagnostic for neural progenitor cells. Characteristics of MRS experiments are known to be problematic for SVD-based methods. Absent detection using alternate methods of spectrum analysis or controls to quantify the false discovery rate, little significance can be attached to their result.

Microsome Biocolloids For Rapid Drug Metabolism And Inhibition Assessment By Lc-Ms, Besnik Bajrami, Sadagopan Krishnan, James F. Rusling

UCHC Articles - Research

Rat liver microsomes attached to nanoparticles were used for LC-MS studies of CYP3A and 2E1 enzymes in metabolism of N-nitroso compounds. Using these biocolloids, turnover rates were measured within 2 min. Inhibitor IC₅₀ values for ketoconazole (KET) and 4-methylpyrazole (4-MEP) were estimated.

Biological Signatures Of Vaccine Responses, Lorena Nunez

Honors Scholar Theses

The set of host- and pathogen-specific molecular features of a disease comprise its “signature”. We hypothesize that biological signatures enables distinctions between vaccinated vs. infected individuals. In our research, using porcine samples, protocols were developed that could also be used to identify biological signatures of human disease. Different classes of molecular features will be tested during this project, including indicators of basic immune capacity, which are being studied at this instance.

These indicators of basic immune response such as porcine cytokines and antibodies were validated using Enzyme-linked immunosorbent assay (ELISA). This is an established method that detects antigens by their …

Synergistic Metabolic Toxicity Screening Using Microsome/Dna Electrochemiluminescent Arrays And Nanoreactors, Sadagopan Krishnan, Eli G. Hvastkovs, Besnik Bajrami, Dharamainder Choudhary, John B. Schenkman, James F. Rusling

UCHC Articles - Research

Platforms based on thin enzyme/DNA films were used in two-tier screening of chemicals for reactive metabolites capable of producing toxicity. Microsomes were used for the first time as sources of cytochrome (cyt) P450 enzymes in these devices. Initial rapid screening involved electrochemiluminescent (ECL) arrays featuring spots containing ruthenium poly(vinylpyridine), DNA, and rat liver microsomes or bicistronically expressed human cyt P450 2E1 (h2E1). Cyt P450 enzymes were activated via the NADPH/reductase cycle. When bioactivation of substrates in the films gives reactive metabolites, they are trapped by covalent attachment to DNA bases. The rate of increase in ECL with enzyme reaction time …

Subcutaneous Late Phase Responses Are Augmented During Local Inhalational Tolerance In A Murine Asthma Model, Anurag Singh, Roger S. Thrall, Linda A. Guernsey, William F. Carson Iv, Eric R. Secor Jr, Robert E. Cone, Thiruchandurai V. Rajan, Craig M. Schramm

UCHC Articles - Research

Acute exposure of sensitized mice to antigen elicits allergic airway disease (AAD) characterized by Th2 cytokine-dependent pulmonary eosinophilia, methacholine hyperresponsiveness and antigen-specific IgE elevation. However, chronic exposure induces a local inhalational tolerance (LIT), with resolution of the airway responses but persistent systemic IgE production. To further determine if systemic immunologic responses were maintained during LIT, we assessed subcutaneous late phase responses to ovalbumin in this model. Sensitized and AAD mice developed small subcutaneous responses to ovalbumin, with footpad thickness increasing to 113.7 and 113.6% of baseline, respectively. In comparison, LIT mice developed marked foot swelling (141.6%). Histologic examination confirmed increased …

Molecular Biology: Power Sequencing, Brenton R. Graveley

UCHC Articles - Research

Advances in DNA-sequencing technology provide unprecedented insight into the entire collection of four genomes' transcribed sequences; they herald a new era in the study of gene regulation and genome function.

Ontogenetic Effects Of Hatching Plasticity In The Spotted Salamander (Ambystoma Maculatum) Due To Egg And Larval Predators, Leah Brown-Wilusz

Honors Scholar Theses

The ability to respond plastically to the environment has allowed amphibians to evolve a response to spatial and temporal variation in predation threat (Benard 2004). Embroys exposed to egg predation are expected to hatch out earlier than their conspecifics. Larval predation can induce a suite of phenotypic changes including growing a larger tail area. When presented with cues from both egg and larval predators, embryos are expected to respond to the egg predator by hatching out earlier because the egg predator presents an immediate threat. However, hatching early may be costly in the larval environment in terms of development, morphology, …

Investigating The Affects Of Cucurbitacin-I On Cellular Motility, Rebecca Lafleur

Honors Scholar Theses

Cellular migration is an integral component of many biological processes including immune function, wound healing and cancer cell metastasis. A complete model illustrating the mechanism by which cells accomplish movement is still lacking. Exploring the affects of various drugs on cell motility may be instrumental in discovering new proteins which mediate cell movement. This project aims ultimately to characterize the molecular target of the drug Cucurbitacin-I, a natural plant product. This drug has been shown to inhibit migration of epithelial sheets and may have anti-tumor activity.

In this paper, we show that Cucurbitacin-I inhibits the migration of MDCK and B16F1 …

Colocalization And Distribution Of Hcn4 Channels And A Maguk Protein In Mammalian Sinoatrial Myocytes, Padmastuti Akella

Honors Scholar Theses

The sinoatrial node is known as the pacemaker of the heart. It controls the rate at which action potentials are generated and propagate through the sinoatrial myocytes. This thesis is an attempt to identify the colocalization of a specific ion channel (HCN4 channel) with a class of scaffolding protein (MAGUK proteins) within the sinoatrial myocyte. The colocalization of these two variables was performed using immunocytochemistry on murine sinoatrial myocytes and experimental results revealed the existence of a strong colocalization between the two variables. Images collected from the confocal microscope and analyzed using ImageJ software revealed a strong dependent correlation between …

In Vitro Exploration Of Macrophage Migration Inhibitory Factor (Mif) As A Possible Agent Of Lymphatic Dilation In The Pathogenesis Of Lymphatic Filiariasis, Lauren Rosen

Honors Scholar Theses

Abstract:Macrophage Migration Inhibitory Factor (MIF) is a human cytokine that is well known for its role in modulating macrophage function. It has also been shown to cause smooth muscle cell death. A homolog of the MIF protein (BmMIF) has been found in Brugia malayi, one of the etiological agents of lymphatic filariasis. BmMIF shares a significant portion of its sequence homology with human MIF. Infection by Brugia malayi leads to a consistent finding of lymphatic dilation with surrounding smooth muscle cell apoptosis. We hypothesize that Brugia malayi releases BmMIF as a mediator of host smooth muscle cell death in the …

Marine Science Assessment Of Capture-Based Tuna (Thunnus Orientalis) Aquaculture In The Ensenada Region Of Northern Baja California, Mexico, José A. Zertuche-González, Oscar Sosa-Nishizaki, Juan G. Vaca Rodriguez, Raul Del Moral Simanek, Charles Yarish, Barry A. Costa-Pierce

Publications

Final Report of the Binational Scientific Team to the Packard Foundation.

Expression And Function Of Dlx Genes In The Osteoblast Lineage, Haitao Li, Inga Marijanovic, Mark S. Kronenberg, Ivana Erceg, Mary Louise Stover, Dimitrios Velonis, Mina Mina, William B. Upholt, Ivo Kalajzic, Alexander C. Lichtler

UCHC Articles - Research

Our laboratory and others have shown that overexpression of Dlx5 stimulates osteoblast differentiation. Dlx5^−/−/Dlx6^−/− mice have more severe craniofacial and limb defects than Dlx5^−/−, some of which are potentially due to defects in osteoblast maturation. We wished to investigate the degree to which other Dlx genes compensate for the lack of Dlx5, thus allowing normal development of the majority of skeletal elements in Dlx5^−/− mice. Dlx gene expression in cells from different stages of the osteoblast lineage isolated by FACS sorting showed that Dlx2, Dlx5 and Dlx6 are expressed most strongly in less mature …

Roles Of Fgfr3 During Morphogenesis Of Meckel's Cartilage And Mandibular Bones, Bruce A. Havens, Dimitris Velonis, Mark S. Kronenberg, Alex C. Lichtler, Bonnie Oliver, Mina Mina

UCHC Articles - Research

To address the functions of FGFR2 and FGFR3 signaling during mandibular skeletogenesis, we over-expressed in the developing chick mandible, replication-competent retroviruses carrying truncated FGFR2c or FGFR3c that function as dominant negative receptors (RCAS-dnFGFR2 and RCAS-dnFGFR3). Injection of RCAS-dnFGFR3 between HH15−20 led to reduced proliferation, increased apoptosis, and decreased differentiation of chondroblasts in Meckel's cartilage. These changes resulted in the formation of a hypoplastic mandibular process and truncated Meckel's cartilage. This treatment also affected the proliferation and survival of osteoprogenitor cells in osteogenic condensations, leading to the absence of five mandibular bones on the injected side. Injection of RCAS-dnFGFR2 between HH15−20 …

Accurate Dna Fragment Sizing By Capillary Electrophoresis With Laser-Induced Fluorescence Array For Detection Of Sequence Specificity Of Dna Damage, Erwin V. Fundador, Dharamainder Choudhary, John B. Schenkman, James F. Rusling

UCHC Articles - Research

Cancer has been linked to mutations within specific codons in genes that code for critical biomolecules such as tumor suppressor proteins (e.g., p53). Activated metabolites like benzo[a]pyrenediol epoxide act on preferred nucleotide sequences of DNA, and such mutations have been identified in cancers. DNA reaction site identification depends on accurate analysis of oligonucleotide fragment sizes produced by strand breakage at the damaged sites. Herein, we report a new method for DNA fragment sizing using capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). Absolute sizing accuracy and speed are achieved by utilizing a CE-LIF array with two-color fluorescence detection. Accuracy …

A Sex Difference In Seasonal Timing Of Birth In A Livebearing Fish, Eric T. Schultz

EEB Articles

Sex differences in seasonal timing include differences in hatch- or birth-date distribution and differences in the timing of migration or maturation such as protandrous arrival timing (PAT), which is early male arrival at breeding sites. I describe a novel form of protandrous arrival timing, as a sex difference in birth-date distribution in a live-bearing fish (Dwarf Perch, Micrometrus minimus). In this species, birth coincides with arrival at breeding sites because newborn males are sexually active. A series of samples of pregnant females and young of year was collected in Tomales Bay, CA. I analyzed the daily age record in otoliths …

Mating Systems, Copulatory Organ Size, And Scaling Relationship In Mollies (Poecilia Spp.), Martha Divver, Eric T. Schultz

EEB Articles

Copulatory organs rapidly evolve and are subject to complex selective pressures affecting mating success. One feature of copulatory organs that is subject to such selective pressures is size. Benefits of longer organs may include greater signal effectiveness in courtship and longer ‘reach’ when attempting copulations with evasive females. Costs of longer organs may include impaired locomotion, increased energetic cost or reduced mechanical compatibility with female genitalia. The optimal size for a copulatory organ may vary with mating behavior. The objective of this study is to examine among-species variability in copulatory organ size, body size and the relationship between copulatory organ …

A Dynamic Lagrangian, Field-Scale Model Of Dust Dispersion From Agriculture Tilling Operations, Thomas H. Meyer

Department of Natural Resources and the Environment Articles

Dust exposure in and near farm fields is of increasing concern for human health and may soon be facing new emissions regulations. Dust plumes of this nature have rarely been documented due to the unpredictable nature of the dust plumes and the difficulties of accurately sampling the plumes. This article presents a dynamic random‐walk model that simulates the field‐scale PM10 (particle diameter <10 >um) dust dispersion from an agriculture disking operation. The major improvements over traditional plume models are that it can simulate moving sources and plume meander. The major inputs are the friction velocity (u*), wind direction in the simulation …

Biochemical Applications Of Ultrathin Films Of Enzymes, Polyions And Dna, James F. Rusling, Dominic O. Hull, Eli G. Hvastkovs, John B. Schenkman

UCHC Articles - Research

This feature article summarizes recent applications of ultrathin films of enzymes and DNA assembled layer-by-layer (LbL). Using examples mainly from our own research, we focus on systems developed for biocatalysis and biosensors for toxicity screening. Enzyme–poly(l-lysine) (PLL) films, especially when stabilized by crosslinking, can be used for biocatalysis at unprecedented high temperatures or in acidic or basic solutions on electrodes or sub-micron sized beads. Such films have bright prospects for chiral synthesis and biofuel cells. Excellent bioactivity and retention of enzyme structure in these films facilitates their use in detailed kinetic studies. Biosensors and arrays employing DNA–enzyme films show great …