Faculty Bibliography

BACKGROUND: Trigeminal sensory neurons detect thermal and mechanical stimuli in the skin through their elaborately arborized peripheral axons. We investigated the developmental mechanisms that determine the size and shape of individual trigeminal arbors in zebrafish and analyzed how these interactions affect the functional organization of the peripheral sensory system. RESULTS: Time-lapse imaging indicated that direct repulsion between growing axons restricts arbor territories. Removal of one trigeminal ganglion allowed axons of the contralateral ganglion to cross the midline, and removal of both resulted in the expansion of spinal cord sensory neuron arbors. Generation of embryos with single, isolated sensory neurons resulted in axon arbors that possessed a vast capacity for growth and expanded to encompass the entire head. Embryos in which arbors were allowed to aberrantly cross the midline were unable to respond in a spatially appropriate way to mechanical stimuli. CONCLUSIONS: Direct repulsive interactions between developing trigeminal and spinal cord sensory axon arbors determine sensory neuron organization and control the shapes and sizes of individual arbors. This spatial organization is crucial for sensing the location of objects in the environment. Thus, a combination of undirected growth and mutual repulsion results in the formation of a functionally organized system of peripheral sensory arbors

A dramatic overexpression of IGFBP-1 is responsible for growth inhibition, in response to a low-protein diet feeding. It has been demonstrated that a fall in the amino acid concentration was directly responsible for IGFBP-1 induction. In this report, we sought to determine the mechanism by which amino acid limitation upregulates IGFBP-1 expression. Our results show that both transcriptional activation and mRNA stabilization are involved. We also demonstrate that (i) the mGCN2/ATF4 pathway is not involved in this regulation and (ii) the 3'UTR of IGFBP-1 mRNA is responsible for its destabilization and regulates its stability in response to amino acid starvation.

Genetic deficiency of the plasma phospholipid transfer protein (PLTP) in mice unexpectedly causes a substantial impairment in liver secretion of apolipoprotein-B (apoB), the major protein of atherogenic lipoproteins. To explore the mechanism, we examined the three known pathways for hepatic apoB secretory control, namely endoplasmic reticulum (ER)/proteasome-associated degradation (ERAD), post-ER pre-secretory proteolysis (PERPP), and receptor-mediated degradation, also known as re-uptake. First, we found that ERAD and cell surface re-uptake were not active in PLTP-null hepatocytes. Moreover, ER-to-Golgi blockade by brefeldin A, which enhances ERAD, equalized total apoB recovery from PLTP-null and wild-type cells, indicating that the relevant process occurs post-ER. Second, because PERPP can be stimulated by intracellular reactive oxygen species (ROS), we examined hepatic redox status. Although we found previously that PLTP-null mice exhibit elevated plasma concentrations of vitamin E, a lipid anti-oxidant, we now discovered that their livers contain significantly less vitamin E and significantly more lipid peroxides than do livers of wild-type mice. Third, to establish a causal connection, the addition of vitamin E or treatment with an inhibitor of intracellular iron-dependent peroxidation, desferrioxamine, abolished the elevation in cellular ROS as well as the defect in apoB secretion from PLTP-null hepatocytes. Overall, we conclude that PLTP deficiency decreases liver vitamin E content, increases hepatic oxidant tone, and substantially enhances ROS-dependent destruction of newly synthesized apoB via a post-ER process. These findings are likely to be broadly relevant to hepatic apoB secretory control in vivo.

Ca(2+)-ATPase belongs to the family of P-type ATPases and maintains low concentrations of intracellular Ca(2+). Its reaction cycle consists of four main intermediates that alternate ion binding in the transmembrane domain with phosphorylation of an aspartate residue in a cytoplasmic domain. Previous work characterized an ultrastable phosphoenzyme produced first by labeling with fluorescein isothiocyanate, then by allowing this labeled enzyme to establish a maximal Ca(2+) gradient, and finally by removing Ca(2+) from the solution. This phosphoenzyme is characterized by very low fluorescence and has specific enzymatic properties suggesting the existence of a high energy phosphoryl bond. To study the structural properties of this phosphoenzyme, we used cryoelectron microscopy of two-dimensional crystals formed in the presence of decavanadate and determined the structure at 8-A resolution. To our surprise we found that at this resolution the low fluorescence phosphoenzyme had a structure similar to that of the native enzyme crystallized under equivalent conditions. We went on to use glutaraldehyde cross-linking and proteolysis for independent structural assessment and concluded that, like the unphosphorylated native enzyme, Ca(2+) and vanadate exert a strong influence over the global structure of this low fluorescence phosphoenzyme. Based on a structural model with fluorescein isothiocyanate bound at the ATP site, we suggest that the stability as well as the low fluorescence of this phosphoenzyme is due to a fluorescein-mediated cross-link between two cytoplasmic domains that prevents hydrolysis of the aspartyl phosphate. Finally, we consider the alternative possibility that phosphate transfer to fluorescein itself could explain the properties of this low fluorescence species

MicroRNAs (miRNAs) are small RNAs that regulate gene expression posttranscriptionally. To block all miRNA formation in zebrafish, we generated maternal-zygotic dicer (MZdicer) mutants that disrupt the Dicer ribonuclease III and double-stranded RNA-binding domains. Mutant embryos do not process precursor miRNAs into mature miRNAs, but injection of preprocessed miRNAs restores gene silencing, indicating that the disrupted domains are dispensable for later steps in silencing. MZdicer mutants undergo axis formation and differentiate multiple cell types but display abnormal morphogenesis during gastrulation, brain formation, somitogenesis, and heart development. Injection of miR-430 miRNAs rescues the brain defects in MZdicer mutants, revealing essential roles for miRNAs during morphogenesis

Various biological processes exhibit characteristics that vary dramatically in response to different input conditions or changes in the history of the process itself. One of the examples studied here, the Ras-PKC-mitogen-activated protein kinase (MAPK) bistable pathway, follows two distinct dynamics (modes) depending on duration and strength of EGF stimulus. Similar examples are found in the behavior of the cell cycle and the immune system. A classification methodology, based on time-frequency analysis, was developed and tested on these systems to understand global behavior of biological processes. Contrary to most traditionally used statistical and spectral methods, our approach captures complex functional relations between parts of the systems in a simple way. The resulting algorithms are capable of analyzing and classifying sets of time-series data obtained from in vivo or in vitro experiments, or in silico simulation of biological processes. The method was found to be considerably stable under stochastic noise perturbation and, therefore, suitable for the analysis of real experimental data

Genomes hold within them the record of the evolution of life on Earth. But genome fusions and horizontal gene transfer (HGT) seem to have obscured sufficiently the gene sequence record such that it is difficult to reconstruct the phylogenetic tree of life. HGT among prokaryotes is not random, however. Some genes (informational genes) are more difficult to transfer than others (operational genes). Furthermore, environmental, metabolic, and genetic differences among organisms restrict HGT, so that prokaryotes preferentially share genes with other prokaryotes having properties in common, including genome size, genome G+C composition, carbon utilization, oxygen utilization/sensitivity, and temperature optima, further complicating attempts to reconstruct the tree of life. A new method of phylogenetic reconstruction based on gene presence and absence, called conditioned reconstruction, has improved our prospects for reconstructing prokaryotic evolution. It is also able to detect past genome fusions, such as the fusion that appears to have created the first eukaryote. This genome fusion between a deep branching eubacterium, possibly an ancestor of the cyanobacterium and a proteobacterium, with an archaeal eocyte (crenarchaea), appears to be the result of an early symbiosis. Given new tools and new genes from relevant organisms, it should soon be possible to test current and future fusion theories for the origin of eukaryotes and to discover the general outlines of the prokaryotic tree of life.

Re-epithelialization of wounded skin is necessary for wound closure and restoration of barrier function and requires directional keratinocyte migration towards the center of the wound. The electric field (EF) generated immediately upon wounding could be the earliest signal keratinocytes receive to initiate directional migration and healing. Keratinocytes express many beta2-adrenergic receptors (beta2-ARs), but their role in the epidermis is unknown. We have previously shown that beta-AR agonists decrease keratinocyte migration in a cyclic AMP (cAMP) independent mechanism involving the activation of protein phosphatase 2A (PP2A). Here, we ask whether beta2-ARs play a role in keratinocyte galvanotaxis. We report a bimodal response. When keratinocytes were exposed to higher concentrations of beta-AR agonist (0.1 microM), their tracked migratory speed was inhibited, in both the presence (directional migration) and the absence (random migration) of a 100 mV mm(-1) EF, as expected. At lower agonist concentrations (0.1 pM to 0.1 nM), there was no effect on migratory speed; however, all directionality was lost - essentially, cells were 'blinded' to the directional cue. Preincubating the cells with beta-antagonist restored directional migration, demonstrating that the 'blindness' was beta2-AR mediated. Incubation of keratinocytes with agents known to increase intracellular cAMP levels, such as sp-cAMP, pertussis toxin and forskolin, resulted in similar 'blinding' to the EF, whereas random migration was unaffected. The inactive cAMP analog rp-cAMP had no effect on keratinocyte migration, whether directional or random. However, rp-cAMP pretreatment before beta-agonist addition fully restored galvanotaxis, demonstrating the complete cAMP dependence of the attenuation of keratinocyte directional migration. This is the first report that cAMP is capable of mediating keratinocyte galvanotaxis. beta-AR agonists and antagonists could be valuable tools for modulating re-epithelialization, an essential step in the wound-healing process. Thus, beta-ARs regulate the two distinct components of keratinocyte directional migration differently: migration speed via a cAMP-independent mechanism and galvanotaxis by a cAMP-dependent one

Gene signatures that predict aggressive tumor behavior at the earliest stages of disease, ideally before overt tissue abnormalities, are urgently needed. To search for such genes, we generated a transgenic model of survivin, an essential regulator of cell division and apoptosis overexpressed in cancer. Transgenic expression of survivin in the urinary bladder did not cause histologic abnormalities of the urothelium. However, microarray analysis revealed that survivin-expressing bladders exhibited profound changes in gene expression profile affecting extracellular matrix and inflammatory genes. Following exposure to a bladder carcinogen, N-butyl-N-(4-hydroxybutyl) nitrosamine (OH-BBN), survivin transgenic animals exhibited accelerated tumor progression, preferential incidence of tumors as compared with premalignant lesions, and dramatically abbreviated survival. Conversely, transgenic expression of a survivin Thr(34)-->Ala dominant-negative mutant did not cause changes in gene expression or accelerated tumor progression after OH-BBN treatment. Therefore, survivin expression induces global transcriptional changes in the tissue microenvironment that may promote tumorigenesis. Detection of survivin or its associated gene signature may provide an early biomarker of aggressive tumor behavior before the appearance of tissue abnormalities

The precise genetic and molecular defects underlying epithelial ovarian cancer (EOC) remain largely unknown, and treatment options for patients with advanced disease are limited. Cyclooxygenases (COX-1 and COX-2) catalyze the conversion of arachidonic acid to prostaglandins. Whereas overwhelming evidence suggests a role for COX-2 in a variety of cancers, the contribution of COX-1 remains much less explored. The expression status of COX isoforms in ovarian cancers also remains confusing. We have previously shown that human epithelial ovarian tumors have increased levels of COX-1 but not COX-2. To more carefully examine the role of COXs in ovarian cancer, we used a mouse model of EOC in which genetic and oncogenic modifications were experimentally engineered into ovarian surface epithelial cells (OSE) thought to be the cells of origin for human EOC. These OSE cells produce tumors when allografted into host mice. Using multiple approaches, we observed that OSE cells and the tumors comprised of these cells express high levels of COX-1 but not COX-2. Prostacyclin (PGI(2)) is the major prostaglandin generated downstream of COX-1 in these cells, and SC-560, a COX-1-selective inhibitor, dramatically inhibits PGI(2) production. More importantly, SC-560 reduced the growth of tumors when OSE cells were allografted in nude female mice. In contrast, the COX-2-selective inhibitor celecoxib had little effect on tumor growth. The growth inhibitory effects of SC-560 result from reduced cell proliferation and/or accelerated apoptosis. Our results imply COX-1 as a target for the prevention and/or treatment of EOC.