Faculty Bibliography

Clinical pregnancy rate (CPR) and implantation rate (IR) in 1,177 patients who had 1,788 fresh, nondonor, nonPGD IVF cycles were highest in cycle 1, significantly declined in cycle 2, and reached a plateau for cycles 3-5 at a rate lower than in cycle 2. In patients >38 years of age CPR and IR in cycles 1 and 2 were significantly lower than in younger patients, but there was no decline in CPR or IR with advancing IVF attempts

We tested the hypothesis that chronically ischemic (IS) myocardium induces autophagy, a cellular degradation process responsible for the turnover of unnecessary or dysfunctional organelles and cytoplasmic proteins, which could protect against the consequences of further ischemia. Chronically instrumented pigs were studied with repetitive myocardial ischemia produced by one, three, or six episodes of 90 min of coronary stenosis (30% reduction in baseline coronary flow followed by reperfusion every 12 h) with the non-IS region as control. In this model, wall thickening in the IS region was chronically depressed by approximately 37%. Using a nonbiased proteomic approach combining 2D gel electrophoresis with in-gel proteolysis, peptide mapping by MS, and sequence database searches for protein identification, we demonstrated increased expression of cathepsin D, a protein known to mediate autophagy. Additional autophagic proteins, cathepsin B, heat shock cognate protein Hsc73 (a key protein marker for chaperone-mediated autophagy), beclin 1 (a mammalian autophagy gene), and the processed form of microtubule-associated protein 1 light chain 3 (a marker for autophagosomes), were also increased. These changes, not evident after one episode, began to appear after two or three episodes and were most marked after six episodes of ischemia, when EM demonstrated autophagic vacuoles in chronically IS myocytes. Conversely, apoptosis, which was most marked after three episodes, decreased strikingly after six episodes, when autophagy had increased. Immunohistochemistry staining for cathepsin B was more intense in areas where apoptosis was absent. Thus, autophagy, triggered by ischemia, could be a homeostatic mechanism, by which apoptosis is inhibited and the deleterious effects of chronic ischemia are limited.

Gastrulation is the process by which the germ layers become positioned in an embryo. C. elegans gastrulation serves as a model for studying the molecular mechanisms of diverse cellular and developmental phenomena, including morphogenesis, cell polarization, cell-cell signaling, actomyosin contraction and cell-cell adhesion. One distinct advantage of studying these phenomena in C. elegans is that genetic tools can be combined with high resolution live cell imaging and direct manipulations of the cells involved. Here we review what is known to date about the cellular and molecular mechanisms that function in C. elegans gastrulation

Over 90% of all human neoplasia is derived from epithelia. Significant progress has been made in the identification of stem cells of many epithelia. In general, epithelial stem cells lack differentiation markers, have superior in vivo and in vitro proliferative potential, form clusters in association with a specialized mesenchymal environment (the 'niche'), are located in well-protected and nourished sites, and are slow-cycling and thus can be experimentally identified as 'label-retaining cells'. Stem cells may divide symmetrically giving rise to two identical stem cell progeny. Any stem cells in the niche, which defines the size of the stem cell pool, may be randomly expelled from the niche due to population pressure (the stochastic model). Alternatively, a stem cell may divide asymmetrically yielding one stem cell and one non-stem cell that is destined to exit from the stem cell niche (asymmetric division model). Stem cells separated from their niche lose their stemness, although such a loss may be reversible, becoming 'transit-amplifying cells' that are rapidly proliferating but have a more limited proliferative potential, and can give rise to terminally differentiated cells. The identification of the stem cell subpopulation in a normal epithelium leads to a better understanding of many previously enigmatic properties of an epithelium including the preferential sites of carcinoma formation, as exemplified by the almost exclusive association of corneal epithelial carcinoma with the limbus, the corneal epithelial stem cell zone. Being long-term residents in an epithelium, stem cells are uniquely susceptible to the accumulation of multiple, oncogenic changes giving rise to tumors. The application of the stem cell concept can explain many important carcinoma features including the clonal origin and heterogeneity of tumors, the occasional formation of tumors from the transit amplifying cells or progenitor cells, the formation of precancerous 'patches' and 'fields', the mesenchymal influence on carcinoma formation and behavior, and the plasticity of tumor cells. While the concept of cancer stem cells is extremely useful and it is generally assumed that such cells are derived from normal stem cells, more work is needed to identify and characterize epithelial cancer stem cells, to address their precise relationship with normal stem cells, to study their markers and their proliferative and differentiation properties and to design new therapies that can overcome their unusual resistance to chemotherapy and other conventional tumor modalities

We have expressed, purified, and characterized one small heat shock protein of the fission yeast Schizosaccharomyces pombe, SpHsp16.0. SpHsp16.0 was able to protect citrate synthase from thermal aggregation at 45 degrees C with high efficiency. It existed as a hexadecameric globular oligomer near the physiological growth temperature. At elevated temperatures, the oligomer dissociated into small species, probably dimers. The dissociation was completely reversible, and the original oligomer reformed immediately after the temperature dropped. Large complexes of SpHsp16.0 and denatured citrate synthase were observed by size exclusion chromatography and electron microscopy following incubation at 45 degrees C and then cooling. However, such large complexes did not elute from the size exclusion column incubated at 45 degrees C. The denatured citrate synthase protected from aggregation was trapped by a GroEL trap mutant at 45 degrees C. These results suggest that the complex of SpHsp16.0 and denatured citrate synthase at elevated temperatures is in the transient state and has a hydrophobic nature. Analyses of the interaction between SpHsp16.0 and denatured citrate synthase by fluorescence cross-correlation spectrometry have also shown that the characteristics of SpHsp16.0-denatured citrate synthase complex at the elevated temperature are different from those of the large complex obtained after the shift to lowered temperatures.

It is shown that molecular structure and dynamics of a uniformly labeled membrane protein can be studied under magic-angle-spinning conditions. For this purpose, dipolar recoupling experiments are combined with novel through-bond correlation schemes that probe mobile protein segments. These NMR schemes are demonstrated on a uniformly [13C,15N] variant of the 52-residue polypeptide phospholamban. When reconstituted in lipid bilayers, the NMR data are consistent with an alpha-helical trans-membrane segment and a cytoplasmic domain that exhibits a high degree of structural disorder.

The Hedgehog-Gli (Hh-Gli) signaling pathway is essential for numerous events during the development of many animal cell types and organs. In particular, it controls neural cell precursor proliferation in dorsal brain structures and regulates the number of neural stem cells in distinct embryonic, perinatal, and adult niches, such as the developing neocortex, the subventricular zone of the lateral ventricle of the forebrain, and the hippocampus. We have proposed that Hh-Gli signaling regulates dorsal brain growth during ontogeny and that its differential regulation underlays evolutionary change in the morphology (size and shape) of dorsal brain structures. It is also critically involved in sporadic brain tumorigenesis--as well as several other human cancer--suggesting that tumors derive from stem cells or progenitors maintaining an inappropriate active Hh-Gli pathway. Importantly, we and others have demonstrated that human sporadic tumors from the brain and other organs require sustained HH-GLI signaling for sustained growth and survival. Modulating HH-GLI signaling thus represents a novel rational avenue to treat, on one hand, brain degeneration and injury by inducing controlled HH-GLI-mediated regeneration and growth, and on the other hand, to combat cancer by blocking its abnormal activity in tumor cells.

A fraction derived from presynaptic specializations (presynaptic particle fraction; PPF) can be separated from postsynaptic densities (PSD) by adjusting the pH of Triton X-100 (TX-100) extraction of isolated transsynaptic scaffolds. Solubilization of the PPF corresponds to disruption of the presynaptic specialization. We show that the PPF is insoluble to repeated TX-100 extraction at pH 6.0 but becomes soluble in detergent at pH 8.0. By immunolocalization, we find that the major proteins of the PPF, clathrin and dynamin, are concentrated in the presynaptic compartment. By using multidimensional protein identification technology, we compared the protein compositions of the PPF and the PSD fraction. We identified a total of 341 proteins, 50 of which were uniquely found in the PPF, 231 in the PSD fraction, and 60 in both fractions. Comparison of the two fractions revealed a relatively low proportion of actin and associated proteins and a high proportion of vesicle or intracellular compartment proteins in the PPF. We conclude that the PPF consists of presynaptic proteins not connected to the actin-based synaptic framework; its insolubility in pH 6 and solubility in pH 8 buffered detergent suggests that clathrin might be an anchorage scaffold for many proteins in the PPF.

BACKGROUND: Ischemia is a limiting factor during distraction osteogenesis. The authors sought to determine the extent of ischemia in the distraction zone and whether endothelial progenitor cells home to the distraction zone and participate in local vasculogenesis. METHODS: Laser Doppler imaging was used to assess the extent of blood flow in the distraction zone in gradually distracted, immediately distracted, and osteotomized rat mandibles during activation and consolidation. Animals (n = 50; 25 rats with unilateral gradual distraction and contralateral osteotomy as an internal control, and 25 rats with unilateral immediate distraction) were examined on postoperative days 4, 6, and 8 of activation, and after 1 and 2 weeks of consolidation. Endothelial progenitor cells isolated from human peripheral blood were labeled with fluorescent DiI dye, and 0.5 x 10 cells were injected intra-arterially under direct vision into each carotid artery at the start of activation in nude rats (n = 18) that then underwent the distraction protocol outlined above. RESULTS: Doppler flow analysis demonstrated relative ischemia during the activation period in the distraction osteogenesis group and increased blood flow in the osteotomized control group as compared with flow in a normal hemimandible [normal, 1 (standardized); distraction osteogenesis, 0.58 +/- 0.05; control, 2.58 +/- 0.21; p < 0.05 for both results]. We observed a significantly increased endothelial progenitor cell population at the generate site versus controls at midactivation and at 1 and 2 weeks of consolidation [25 +/- 1.9 versus 1 +/- 0.3 DiI-positive cells per high-power field (p < 0.05), 124 +/- 21 versus 8 +/- 4 DiI-positive cells per high-power field (p < 0.05), and 106 +/- 18 versus 9 +/- 3 DiI-positive cells per high-power field (p < 0.05), respectively]. CONCLUSIONS: These data suggest that the distraction zone becomes relatively ischemic during activation and that endothelial progenitor cells home to the ischemic generate site during the activation phase and remain during the consolidation phase. Selective expansion of these stem cells may be useful in overcoming ischemic limitations of distraction osteogenesis. Moreover, their homing capability may be used to effect site-specific transgene delivery to the generate