Faculty Bibliography

Studies of Caenorhabditis elegans vulval development provide a paradigm for pattern formation during animal development. The fates of the six vulval precursor cells are specified by the combined action of an inductive signal that activates the EGF receptor mitogen-activated PK signaling pathway (specifying a primary fate) and a lateral signal mediated by LIN-12/Notch (specifying a secondary fate). Here we use methods devised for the engineering of complex reactive systems to model a biological system. We have chosen the visual formalism of statecharts and use it to formalize Sternberg and Horvitz's 1989 model [Sternberg, P. W. & Horvitz, H. R. (1989) Cell 58, 679-693], which forms the basis for our current understanding of the interaction between these two signaling pathways. The construction and execution of our model suggest that different levels of the inductive signal induce a temporally graded response of the EGF receptor mitogen-activated PK pathway and make explicit the importance of this temporal response. Our model also suggests the existence of an additional mechanism operating during lateral specification that prohibits neighboring vulval precursor cells from assuming the primary fate

Translation is the process by which the genetic information contained in mRNA is used to link amino acids in a predetermined sequential order into a polypeptide chain, which then folds into a protein. Transfer RNAs (tRNAs) are the adapter molecules designed to provide the 'lookup' from codons to amino acids. Cryo-EM has provided evidence that the ribosome, as a molecular machine, undergoes many structural changes during translation. Recent findings show that the tRNA structure itself undergoes large conformational changes as well, and that the decoding process must be seen as a complex dynamic interplay between tRNA and the ribosome

Mutations in the A class of ATP-binding cassette transporters (ABCA) are causally implicated in three human diseases: Tangier disease (ABCA1), Stargadt's macular degeneration (ABCA4), and neonatal respiratory failure (ABCA3). Both ABCA1 and ABCA4 have been shown to transport lipids across cellular membranes, and ABCA3 may play a similar role in transporting pulmonary surfactant. Although the functions of the other 10 ABCA class transporters identified in the human genome remain obscure, ABCA7-transfected cells have been shown to efflux lipids in response to stimulation by apolipoprotein A-I. In an effort to elucidate the physiologic role of ABCA7, we generated mice lacking this transporter (Abca7-/- mice). Homozygous null mice were produced from intercrosses of heterozygous null mice at the expected Mendelian frequency and developed normally without any obvious phenotypic abnormalities. Cholesterol and phospholipid efflux stimulated by apolipoprotein A-I from macrophages isolated from wild type and Abca7-/- mice did not differ, suggesting that these activities may not be central to the physiological role of the transporter in vivo. Abca7-/- females, but not males, had significantly less visceral fat and lower total serum and high density lipoprotein cholesterol levels than wild type, gender-matched littermates. ABCA7 expression was detected in hippocampal and cortical neurons by in situ hybridization and in brain and white adipose tissue by Western blotting. Induction of adipocyte differentiation from 3T3 fibroblasts in culture led to a marked increase in ABCA7 expression. These studies suggest that ABCA7 plays a novel role in lipid and fat metabolism that Abca7-/- mice can be used to elucidate

Ischemia is a known stimulus for vascular growth. Bone marrow (BM)-derived endothelial progenitor cells (EPCs) are believed to contribute to new blood vessel growth, but the mechanism for this contribution is unknown. To elucidate how BM cells are able to form new blood vessels, a novel murine model of soft tissue ischemia was developed in lethally irradiated mice with BM reconstituted from either tie2/lacZ or ROSA/green fluorescent protein (GFP) mice (n = 24). BM-derived EPCs were recruited to ischemic tissue within 72 hours, and the extent of recruitment was directly proportional to the degree of tissue ischemia. At 7 days, there were persistently elevated levels of vascular endothelial growth factor (VEGF) (2.5-fold) and circulating VEGF receptor-2/CD11(-) (flk-1(+)/CD11(-)) cells (18-fold) which correlated with increased numbers of BM-derived EPCs within ischemic tissue. The cells were initially located extravascularly as proliferative clusters. By day 14, these clusters coalesced into vascular cords, which became functional vessels by day 21. In vitro examination of human EPCs from healthy volunteers (n = 10) confirmed that EPC proliferation, adhesion, and chemotaxis were all significantly stimulated in hypoxic conditions. We conclude that BM-derived cells produce new blood vessels via localized recruitment, proliferation, and differentiation of circulating cells in a sequence of events markedly different from existing paradigms of angiogenesis

Detailed descriptions of mammalian sperm morphology across a range of closely related taxa are rare. Most contributions have been generalized descriptions of a few distantly related mammalian species. These studies have emphasized a generalized ungulate sperm morphology, but have not underscored several important morphological differences in ungulate sperm, such as head shape. The present study is the first to document descriptions of sperm head morphology using cold field-emission scanning electron microscopy (FE-SEM) for a large number of closely related mammalian species. In total, the sperm of 36 species in three orders: Artiodactyla (even-toed ungulates), Cetacea (whales, porpoises, and dolphins), and Perissodactyla (odd-toed ungulates) were examined to gather new information relevant to the debate about the phylogenetic placement of cetaceans relative to terrestrial ungulates. In all species examined, the sperm heads were generally flattened and ovate in shape with a distinct apical ridge, although considerable variation in sperm head shape was detected, both within and between orders. In artiodactylans, the sperm head was uniformly flat in lateral view, whereas perissodactylan and cetacean sperm heads showed a distinct posterior thickening. In both artiodactylans and perissodactylans, the mitochondria were elongate and wound in a tight helix around the midpiece, whereas in cetaceans the mitochondria were rounded and appeared to be randomly arranged around the midpiece. Additionally, prominent ridges running along the anterior-posterior axis were observed in the postacrosomal region of the sperm head in four species of cetaceans. These ridges were not observed in any of the terrestrial ungulates examined. Pits or fenestrations were detected in the postacrosomal region in most artiodactylan species examined; these structures were not detected in perissodactylans or cetaceans. The equatorial segment of the acrosome was detected in the artiodactylan species examined, tentatively identified in perissodactylans, but not found in cetaceans. Its shape and location are described for relevant taxa. The presence of a recently reported substructure within the equatorial segment (the equatorial subsegment; Ellis et al. [2002] J Struct Biol 138:187-198) was detected in artiodactylans, and its shape is described for the species examined.

A method for the flexible docking of high-resolution atomic structures into lower resolution densities derived from electron microscopy is presented. The atomic structure is deformed by an iterative process using combinations of normal modes to obtain the best fit of the electron microscopical density. The quality of the computed structures has been evaluated by several techniques borrowed from crystallography. Two atomic structures of the SERCA1 Ca-ATPase corresponding to different conformations were used as a starting point to fit the electron density corresponding to a different conformation. The fitted models have been compared to published models obtained by rigid domain docking, and their relation to the known crystallographic structures are explored by normal mode analysis. We find that only a few number of modes contribute significantly to the transition. The associated motions involve almost exclusively rotation and translation of the cytoplasmic domains as well as displacement of cytoplasmic loops. We suggest that the movements of the cytoplasmic domains are driven by the conformational change that occurs between nonphosphorylated and phosphorylated intermediate, the latter being mimicked by the presence of vanadate at the phosphorylation site in the electron microscopy structure

The identification of bone marrow-derived endothelial progenitor cells has altered our understanding of new blood vessel growth and tissue regeneration. Previously, new blood vessel growth in the adult was thought to only occur through angiogenesis, the sprouting of new vessels from existing structures. However, it has become clear that circulating bone marrow-derived cells can form new blood vessels through a process of postnatal vasculogenesis, with endothelial progenitor cells selectively recruited to injured or ischemic tissue. How this process occurs has remained unclear. One common element in the different environments where vasculogenesis is believed to occur is the presence of a hypoxic stimulus. We have identified the chemokine stromal cell-derived factor-1 (SDF-1) and its receptor CXCR4 as critical mediators for the ischemia-specific recruitment of circulating progenitor cells. We have found that the endothelial expression of SDF-1 acts as a signal indicating the presence of tissue ischemia, and that its expression is directly regulated by hypoxia-inducible factor-1. Stromal cell-derived factor 1 is the only chemokine family member known to be regulated in this manner. Later events, including proliferation, patterning, and assembly of recruited progenitors into functional blood vessels, are also influenced by tissue oxygen tension and hypoxia. Interestingly, both SDF-1 and hypoxia are present in the bone marrow niche, suggesting that hypoxia may be a fundamental requirement for progenitor cell trafficking and function. As such, ischemic tissue may represent a conditional stem cell niche, with recruitment and retention of circulating progenitors regulated by hypoxia through differential expression of SDF-1

The present study was undertaken to clarify the relationship between the p34cdc2 kinase activity of in vitro-aged or enucleated rat oocytes and the premature chromosome condensation (PCC) of microinjected cumulus cell nuclei. Wistar rat oocytes were placed in vitro up to 120 min after the animal was killed. The p34cdc2 kinase activity of the oocytes decreased in a time-dependent manner. The incidence of PCC was higher when nuclear injection into intact oocytes was completed in 15-45 min rather than 46-120 min. When rat oocytes were enucleated for subsequent nuclear injection, the p34cdc2 kinase activity transiently increased soon after enucleation but drastically decreased after 30 min. Removal of the cytoplasm instead of the meta-phase-plate did not affect the p34cdc2 kinase activity even after 60 min. PCC occurred in intact and cytoplasm-removed oocytes but not in enucleated oocytes. In contrast, oocytes from BDF1 mice exhibited a p34cdc2 kinase level twice that of rat oocytes and supported PCC despite enucleation. The p34cdc2 kinase level of intact rat oocytes was reduced to the equivalent level of aged (120 min) or enucleated (+60 min) oocytes by a 45 min treatment with roscovitine, an inhibitor of p34cdc2 kinase. None of the roscovitine-treated oocytes supported PCC while half of the control oocytes did. When rat oocytes were treated with MG132, a proteasome inhibitor, delayed inactivation of the p34cdc2 kinase was observed in the MG132-treated oocytes. A significantly higher proportion of the MG132-treated oocytes supported PCC when compared with the control oocytes. Moreover, a higher proportion of MG132-treated and enucleated oocytes carried two pseudo-pronuclei after cumulus cell injection and developed to the two-cell stage when compared with the enucleated oocytes at the telophase-II stage. These results suggest that the decreased level of p34cdc2 kinase activity in aged or enucleated rat oocytes is responsible for their inability to support PCC of microinjected donor cell nuclei and that inhibition of p34cdc2 kinase inactivation by chemicals such as MG132 is in part effective for rat oocytes to promote PCC and further development