A comparison of the structure of isolated and in situ chromatin fibers was performed using starfish (Patiria miniata) sperm nuclei. The simple protein content consisting of five major histones was found. A DNA repeat length of 222bp was calculated. Compact chromatin fibers solubilized from detergent isolated nuclei showed diameters of 38 to 44nm by electron microscopy. Chromatin fibers observed in whole cells and in mildly treated nuclear preparations revealed fiber diameters of approximately 24nm when embedded in epoxy resin. Investigation of the chromatin isolation process uncovered a mechanism where fiber integrity was lost during nuclear isolation. Following nuclease digestion and chromatin release, fiber-like structures reformed in solution. The relationship of the isolated "fibers" to the native structures is not known. Nuclear permeability has been found to be a factor in both release of cleaved chromatin from nuclei and inward diffusion of nuclease. Permeability to various sized dextrans was consistent with a mechanism where cut chromatin exits nuclei as nucleosomal chains and aggregates in solution to form the isolated chromatin state. Attempts to isolate detergent free nuclei revealed that completely isolated nuclei were susceptible to the loss of chromatin fiber integrity suggesting that the medium was incapable of maintaining fiber structure. Other buffer compositions did not improve stability of in situ fibers underscoring a general lack of understanding of the nuclear environment.

Control of neurosecretion of neurotransmitters appears to be regulated through second messengers that change the phosphorylation state of critical enzymes and proteins. The effect of protein phosphorylation promoting agents on secretion, desensitization and Na$\sp+$/Ca$\sp{2+}$ exchange activity were investigated and protein phosphatases were identified in bovine chromaffin cells. Cells exposed to 8-Br-cAMP, PDBu or okadaic acid alone show slightly decreased rates of desensitization. Okadaic acid plus 8-Br-cAMP potentiated secretion with repeated stimulations. The protein kinase inhibitor H7 increased the desensitization rate. These phenomena are observed during secretion evoked with elevated K$\sp+$ as well as by a nicotinic agonist. Thus, the effect of phosphorylation is at a post-receptor site. Cells treated with dbcAMP, PDBu, okadaic acid or calyculin A show lowered Na$\sp+$/Ca$\sp{2+}$ exchange activity and prolong cytosolic Ca$\sp{2+}$ transients caused by depolarization. Conversely, H7 enhances Na$\sp+$/Ca$\sp{2+}$ exchange activity. Na$\sp+$/Ca$\sp{2+}$ exchange activity in isolated membrane vesicles is inhibited by PKA and PKC. The results indicate that reversible protein phosphorylation modulates Na$\sp+$/Ca$\sp{2+}$ exchange activity and suggest that modulation of the exchanger may play a role in the regulation of secretion. Four distinct peaks of phosphatase activity were observed in homogenized bovine adrenal medulla cells when fractionated using an HPLC ion exchange DEAE column. Of these phosphatases, peaks II, III and IV show preferential dephosphorylation of the $\alpha$ subunit of phosphorylase kinase relative to the $\beta$ subunit and therefore are classified as protein phosphatases type 2. These phosphatases have broad substrate specificity and distinctly different relative specific activities toward the different substrates. Phosphatase 2A is likely to be the major enzyme in bovine adrenal medulla cells. ATP induced a greater secretory response than did acetylcholine without causing preferential secretion of norepinephrine or epinephrine. These data show that the response to ATP found in cultured cells is not an artifact of cell culture, and that ATP corelease with catecholamines from the storage vesicles has a significant physiological role. Freshly isolated cells were separated on a density gradient; the lower density cells develop a much stronger response to ATP than do the higher density cells.

No description available.

<p> Throughout development, canonical Wnt signaling contributes to the formation and maintenance of a wide array of cells, tissues, and organs. Dys-regulated Wnt signaling during embryonic development is implicated in developmental defects known as neurochristopathies, including craniofacial and heart defects, as well as defects in neural development. Due to its roles in stem cell maintenance and self-renewal, tissue homeostasis, and regeneration, aberrant Wnt signaling in adult tissues can result in various forms of cancer, including colorectal cancer, breast cancer, lung cancer, and gastro-intestinal cancer, among others. Dys-regulated Wnt signaling is also implicated in other pathologies including bone disease, and metabolic diseases, such as Type II diabetes. Our lab has previously identified a novel crosstalk between canonical Wnt signaling and ephrin signaling. Ephrin signaling occurs through the interaction of ephrin ligands and Eph receptor tyrosine kinases, and is bidirectional. Due to the roles of ephrin signaling in tissue development and maintenance, aberrant ephrin signaling is implicated in many diseases including bone remodeling diseases, diabetes, and cancer. The molecular mechanism of the crosstalk between canonical Wnt signaling and ephrin-B signaling remains unknown. &beta;-catenin is a key intracellular effector of canonical Wnt signaling that transduces the signal to the nucleus, where &beta;-catenin interacts with the TCF/LEF transcription factors and activates transcription of target genes. Due to its central role in transducing the canonical Wnt signal to the nucleus, we predict that ephrin-B signaling antagonizes canonical Wnt signaling by affecting the stability and/or sub-cellular localization of &beta;-catenin, or the interaction between &beta;-catenin and TCF/LEF transcription factors. By employing mouse ephrin-B constructs in human cell lines, we show that the canonical Wnt - ephrin-B crosstalk is conserved between frogs and mammals. We also found that ephrin-B antagonism of canonical Wnt signaling is likely independent of ubiquitin proteasome system (UPS)-mediated degradation of &beta;-catenin. Furthermore, confocal immunofluorescence microscopy revealed that overexpression of ephrin-B in HEK293T cells treated with lithium chloride (LiCl) seems to promote membrane localization of &beta;-catenin, particularly at the apical Z sections. These results suggests that re-localization of &beta;-catenin to the cell membrane may contribute to the ephrin-B antagonism of canonical Wnt signaling.</p>

<p> Past sightings of red-tailed (<i>Cercopithecus ascanius</i>) x blue (<i>Cercopithecus mitis</i>) hybrid monkeys in Uganda indicates the potential for hybridization between <i>C. ascanius</i> and <i> C. mitis</i> individuals. Apart from Gombe Stream National Park, there is no of evidence suggestive of <i>C. ascanius &times; C. mitis</i> monkey hybridization at investigated East African locations. Phylogenetic analysis was examined using Mitochondrial DNA (mtDNA) sequence data of twelve <i>C. mitis stuhlmanni</i> samples (from two populations) in Kibale National Park (KNP), Uganda to test for any evidence of hybridization. </p><p> Strict mono-phylogeny among two new <i>C. mitis</i> haplotypes were detected. Genetic diversity measurements support neither interspecific or intraspecific hybridization among <i>C. mitis</i> individuals from populations within Kibale National Park. To intensify the implications of this study further examination should include an increase in sample size(s), mtDNA comparison of <i>C. mitis</i> subspecies from additional populations at East African locations, and assessment of nuclear and genomic DNA.</p><p>

<p> The genus <i>Ophiocordyceps</i> contains the most diverse assemblage of fungi attacking ants worldwide and are remarkably well adapted to the specific ecologies of their hosts. <i>Desmidiospora myrmecophila</i> is closely related to ant-pathogenic species within <i>Ophiocordyceps</i>, possibly specific to queens, but the sheer infrequency of encounters and previously unsuccessful attempts to culture this fungus has precluded any meaningful assessment until now. A new record of <i>Desmidiospora myrmecophila</i> from Louisiana was found infecting a foundress <i>Camponotus pennsylvanicus </i> queen, the same host species favored by the more common and ubiquitous ant-pathogenic <i>Ophiocordyceps</i> unilateralis found in the same geographic locality. To evaluate a long-held assumption that these fungi represent synanamorphs of a single species, we sampled our Desmidiospora specimen along with the local <i>O. unilateralis</i> population for molecular comparison. We are able to present for the first time the <i>in vitro </i> characteristics and morphology of <i>Desmidiospora myrmecophila</i> as well as a phylogenetic context for this fungus based on combined molecular analysis of representative members of the Ophiocordycipitaceae. Our results place the <i>Desmidiospora myrmecophila</i> lineage within the genus <i>Ophiocordyceps</i> but with a basal affiliation to the ant-pathogen clade. These results further implicate <i>Desmidiospora myrmecophila</i> as an important and quintessential example of cryptic diversity among an already taxonomically diverse and ecologically important group of fungi.</p><p>

Thesis (Ph.D.)--Brown University, 2008. / Vita. Advisor : Kristi A. Wharton. Includes bibliographical references.

Thesis (Ph.D.)--Brown University, 2008. / Vita. Adviser: Wen-Ming Chu. Includes bibliographical references.

<p> Adult echinoderms form a mineralized skeleton, but only sea urchins and brittle stars form larval skeletons. In the sea urchin <i>Strongylocentrotus purpuratus</i>, the gene regulatory network (GRN) leading to skeleton formation has been characterized. This <i>S. purpuratus</i> GRN includes several members of the <i>ets</i> family, including <i> Erg, Ets1/2</i> and <i>Gabp</i>. The brittle star <i> Ophiocoma wendtii</i> forms an embryonic skeleton similar to <i> S. purpuratus</i>. The goal of this proposal is to see if expression of the <i>ets</i> family members is conserved as part of the skeletogenic GRN in <i>O. wendtii</i>. Four genes were identified in <i> O. wendtii</i>; homologous to <i>S. purpuratus Erg, Ets1/2, Ets4 </i> and <i>Gabp</i> based on phylogenetic analysis. The coding sequences of these <i>O. wendtii</i> genes were obtained and their temporal expression was determined. These results suggest that sea urchins and brittle stars share a GRN leading to skeleton formation that has been activated in the embryos of both.</p>

<p> The identification of beige fat within the last decade, its ability to burn energy in adult humans, and its great potential for therapeutic applications has motivated many to work towards understanding how beige fat is made. Although several proteins have been identified as important for beige fat differentiation, it is clear that the differences that distinguish beige fat from other types of fat cannot be explained by the presence of these proteins alone. Additional regulatory proteins, including transcription factors and their co-factor proteins, must be involved. I took advantage of two important developments in the field of fat differentiation to develop two high throughput approaches that identified new transcription factors involved in beige fat: (1) our ability to culture beige fat cells by differentiating white fat pre-adipocytes in the presence of Rosiglitazone and (2) the established role for PRDM16 as required for beige fat differentiation. In brief, I combined RNA-seq data from beige fat cells and proteomics data from Rosiglitazone-dependent PRDM16 protein complexes to identify a set of candidate transcription factors involved in beige fat differentiation. The most promising candidate among this pool of putative beige fat regulatory transcription factors was GTF2IRD1. I determined that GTF2IRD1 is a PRDM16-interacting transcription factor that is enriched in beige and brown fat cells. In vivo, GTF2IRD1 is enriched in brown adipose tissue and is increased in beige and brown fat in response to beta-3-adrenergic stimulus. In the presence of the potent PPARgamma agonist Rosiglitazone, GTF2IRD1 overexpression enhances and shRNA-mediated knockdown reduces beige fat differentiation. GTF2IRD1 represses TGF-beta-mediated inhibition of beige fat differentiation. In summary, my data strongly supports that GTF2IRD1 is an essential regulator of beige fat differentiation through interaction with PRDM16 and inhibition of TGF-beta-mediated repression of differentiation.</p>