Faculty Bibliography

Undirected mismatch repair initiated by the incorporation of the base analog 2-aminopurine kills DNA-methylation-deficient Escherichia coli dam cells by DNA double-strand breakage. Subsequently, the chromosomal DNA is totally degraded, resulting in DNA-free cells.

Cryo-electron tomography of frozen-hydrated specimens holds considerable promise for high-resolution three-dimensional imaging of organelles and macromolecular complexes in their native cellular environment. While the technique has been successfully used with small, plunge-frozen cells and organelles, application to bulk mammalian tissue has proven to be difficult. We report progress with cryo-electron tomography of frozen-hydrated sections of rat liver prepared by high-pressure freezing and cryo-ultramicrotomy. Improvements include identification of suitable grids for mounting sections for tomography, reduction of surface artifacts on the sections, improved image quality by the use of energy filtering, and more rapid tissue excision using a biopsy needle. Tomographic reconstructions of frozen-hydrated liver sections reveal the native structure of such cellular components as mitochondria, endoplasmic reticulum, and ribosomes, without the selective attenuation or enhancement of ultrastructural details associated with the osmication and post-staining used with freeze-substitution

Using suppressive subtractive hybridization, we have identified a novel gene, which we named early epithelial differentiation associated (EEDA), which is uniquely associated with an early stage of stratified epithelial differentiation. In epidermis, esophageal epithelium, and tongue epithelium, EEDA mRNA, and antigen was abundant in suprabasal cells, but was barely detectable in more differentiated cells. Consistent with the limbal location of corneal epithelial stem cells, EEDA was expressed in basal corneal epithelial cells that are out of the stem cell compartment, as well as the suprabasal corneal epithelial cells. The strongest EEDA expression occurred in suprabasal precortical cells of mouse, bovine, and human anagen follicles. Developmental studies showed that the appearance of EEDA in embryonic mouse epidermis (E 15.5) coincided with morphological keratinization. Interestingly, EEDA expression is turned off when epithelia were perturbed by wounding and by cultivation under both low and high Ca(2+) conditions. Our results indicate that EEDA is involved in the early stages of normal epithelial differentiation, and that EEDA is important for the 'normal' differentiation pathway in a wide range of stratified epithelia. (c) 2005 Wiley-Liss, Inc

Keratinocytes migrate directionally into the wound bed to initiate re-epithelialization, necessary for wound closure and restoration of barrier function. They solely express the beta2-adrenergic receptor (beta2-AR) subtype of beta-ARs and can also synthesize beta-AR agonists generating a hormonal mediator network in the skin. Emerging studies from our laboratory demonstrate that beta-AR agonists decrease keratinocyte migration via a protein phosphatase (PP) 2A-dependent mechanism. Here we have extended our investigations to observe the effects of beta2-AR activation on keratinocyte polarization, migration, and ERK phosphorylation at the wound edge, cytoskeletal organization, phospho-ERK intracellular localization, proliferation, human skin wound re-epithelialization, wound-induced ERK phosphorylation, and murine skin wound healing. We demonstrate that in keratinocytes, beta2-AR activation is anti-motogenic and anti-mitogenic with both mechanisms being PP2A dependent. beta2-AR activation dramatically alters the organization of the actin cytoskeleton and prevents localization of phospho-ERK to the lamellipodial edge and its colocalization with vinculin. Finally, we demonstrate a beta2-AR-mediated delay in re-epithelialization and decrease in wound-induced epidermal ERK phosphorylation in human skin wounds and a delay in re-epithelialization in murine tail-clip wounds. Our work uncovers novel keratinocyte biology and a previously unrecognized role for the adrenergic hormonal mediator network in the wound repair process

EHD1 is a member of the EHD family that contains four mammalian homologs. Among the invertebrate orthologs are a single Drosophila and Caenorhabditis elegans proteins and two plant members. They all contain three modules, a N-terminal domain that contains nucleotide-binding motifs, a central coiled-coil domain involved in oligomerization and a C-terminal region that harbors the EH domain. Studies in C. elegans and EHD1 depletion by RNA interference in human cells have demonstrated that it regulates recycling of membrane proteins. We addressed the physiological role of EHD1 through its inactivation in the mouse. Ehd1 knockout mice were indistinguishable from normal mice, had a normal life span and showed no histological abnormalities. Analysis of transferrin uptake in Ehd1(-/-) embryonic fibroblasts demonstrated delayed recycling to the plasma membrane with accumulation of transferrin in the endocytic recycling compartment. Our results corroborate the established role of EHD1 in the exit of membrane proteins from recycling endosomes in vivo in a mouse model

BACKGROUND: Pre-antral and early antral follicles secrete Mullerian inhibiting substance (MIS), suggesting that MIS may directly reflect ovarian reserve. Since little is known about how ovarian reserve affects oocyte quality, we attempt here to assess the predictive value of MIS on embryo morphology and IVF outcome. To do so, we measured MIS at the time of HCG administration 36 h prior to oocyte retrieval. METHODS: A total of 257 patients undergoing IVF were prospectively recruited. We measured MIS levels by enzyme-linked immunosorbent assay at the time of HCG, and compared the MIS values to day 3 FSH levels in the prediction of embryo morphology and IVF outcome. RESULTS: The distribution of MIS levels was skewed, with a median of 2.7 ng/ml (range 0 to 28.5 ng/ml). MIS values at the time of HCG administration inversely correlated with basal FSH levels (P = 0.002), and both correlated significantly with patient age, number of mature follicles, number of oocytes retrieved and serum estradiol levels. MIS levels correlated significantly with a greater number of 6-cell embryos and better embryo morphology score, while basal FSH levels did not correlate with these outcome variables. MIS levels > or =2.7 ng/ml portended improved oocyte quality as reflected in a higher implantation rate (P = 0.001) and a trend toward a better clinical pregnancy rate (P = 0.084). CONCLUSIONS: MIS levels seem to predict not only ovarian reserve, but also embryo morphology. Measurement of MIS at the time of HCG administration may, therefore, in the future improve management of patients undergoing treatments with assisted reproductive technology

Light intensity and atmospheric CO2 partial pressure are two environmental signals known to regulate stomatal numbers. It has previously been shown that if a mature Arabidopsis leaf is supplied with either elevated CO2 (750 ppm instead of ambient at 370 ppm) or reduced light levels (50 micromol m-2 s-1 instead of 250 micromol m-2 s-1), the young, developing leaves that are not receiving the treatment grow with a stomatal density as if they were exposed to the treatment. But the signal(s) that it is believed is generated in the mature leaves and transmitted to developing leaves are largely unknown. Photosynthetic rates of treated, mature Arabidopsis leaves increased in elevated CO2 and decreased when shaded, as would be expected. Similarly, the levels of sugars (glucose, fructose, and sucrose) in the treated mature leaves increased in elevated CO2 and decreased with shade treatment. The levels of sugar in developing leaves were also measured and it was found that they mirrored this result even though they were not receiving the shade or elevated CO2 treatment. To investigate the effect of these treatments on global gene expression patterns, transcriptomics analysis was carried out using Affymetrix, 22K, and ATH1 arrays. Total RNA was extracted from the developing leaves after the mature leaves had received either the ambient control treatment, the elevated CO2 treatment, or the shade treatment, or both elevated CO2 and shade treatments for 2, 4, 12, 24, 48, or 96 h. The experiment was replicated four times. Two other experiments were also conducted, one to compare and contrast gene expression in response to plants grown at elevated CO2 and the other to look at the effect of these treatments on the mature leaf. The data were analysed and 915 genes from the untreated, signalled leaves were identified as having expression levels affected by the shade treatment. These genes were then compared with those whose transcript abundance was affected by the shade treatment in the mature treated leaves (1181 genes) and with 220 putative 'stomatal signalling' genes previously identified from studies of the yoda mutant. The results of these experiments and how they relate to environmental signalling are discussed, as well as possible mechanisms for systemic signalling.

The formation of neuromuscular synapses requires a complex exchange of signals between motor neurons and skeletal muscle fibers. Essential for the formation of neuromuscular junction (NMJ) is the activation of MuSK, a muscle-specific receptor tyrosine kinase (DeChiara et al., 1996). In mice lacking MuSK, motor axons fail to stop and differentiate, acetylcholine receptors (AChRs) fail to cluster, and AChR genes are expressed uniformly in muscle (DeChiara et al., 1996; Gautam et al., 1996). The retrograde signals for presynaptic differentiation are not known. Because synapse-specific transcription, like presynaptic differentiation, is MuSK-dependent, it is possible that retrograde signals for presynaptic differentiation might be encoded by genes that are expressed preferentially by synaptic nuclei. To identify such synapse-specific genes we screened Affymetrix microarrays with RNA from the dissected, synapse-enriched, and extrasynaptic regions of skeletal muscle and further studied those genes that encode for the secreted or cell-surface proteins

Single-particle cryo-electron microscopy (cryo-EM) is the method of choice for studying the dynamics of macromolecular machines both at a phenomenological and, increasingly, at the molecular level, with the advent of high-resolution component X-ray structures and of progressively improving fitting algorithms. Cryo-EM has shed light on the structure of the ribosome during the four steps of translation: initiation, elongation, termination, and recycling. Interpretation of cryo-EM reconstructions of the ribosome in quasi-atomic detail reveals a picture in which the ribosome uses RNA not only to catalyze chemical reactions, but also as a means for signal transduction over large distances