Faculty Bibliography

Metastasis comprises several subsequent steps including local invasion and intravasation at the primary site, then their adhesion/arrest within the vessels of host organs followed by their extravasation and infiltration into the target organ stroma. In contrast to previous studies which have used aspartate-glycine-arginine (RGD) peptides and antibodies against integrins, we used rare collagen- and laminin-antagonizing integrin inhibitors from snake venoms to analyze the colonization of the liver by tumor cells both by intravital microscopy and in vitro. Adhesion of liver-targeting tumor cells to the sinusoid wall components, laminin-1 and fibronectin, is essential for liver metastasis. This step is inhibited by lebein-1, but not by lebein-2 or rhodocetin. Both lebeins from the Vipera lebetina venom block integrin interactions with laminins in an RGD-independent manner. Rhodocetin is an antagonist of alpha2beta1 integrin, a collagen receptor on many tumor cells. Subsequent to tumor cell arrest, extravasation into the liver stroma and micrometastasis are efficiently delayed by rhodocetin. This underlines the importance of alpha2beta1 integrin interaction with the reticular collagen I-rich fibers in liver stroma. Antagonists of laminin- and collagen-binding integrins could be valuable tools to individually block the direct interactions of tumor cells with distinct matrix components of the Disse space, thereby reducing liver metastasis.

The Hedgehog (Hh) signaling pathway plays critical instructional roles during embryonic development. Misregulation of Hh/Gli signaling is a major causative factor in human congenital disorders and in a variety of cancers. The zebrafish is a powerful genetic model for the study of Hh signaling during embryogenesis, as a large number of mutants that affect different components of the Hh/Gli signaling system have been identified. By performing global profiling of gene expression in different Hh/Gli gain- and loss-of-function scenarios we identified known (e.g., ptc1 and nkx2.2a) and novel Hh-regulated genes that are differentially expressed in embryos with altered Hh/Gli signaling function. By uncovering changes in tissue-specific gene expression, we revealed new embryological processes that are influenced by Hh signaling. We thus provide a comprehensive survey of Hh/Gli-regulated genes during embryogenesis and we identify new Hh-regulated genes that may be targets of misregulation during tumorigenesis.

We take advantage of an array of hybrid baboons (Papio anubis x Papio hamadryas) living in the same social group to explore the causes and consequences of different male mating strategies. Male hamadryas hold one-male units and exhibit a sustained, intense interest in adult females, regardless of the latter's reproductive state. Anubis baboons, by contrast, live in multi-male, multi-female groups where males compete for females only when the latter are estrous. These two taxa interbreed to form a hybrid zone in the Awash National Park, Ethiopia, where previous work has suggested that hybrid males have intermediate and ineffective behavior. Here, we first examine male mating strategies with respect to morphological and genetic measures of ancestry. We found significant relationships between behavioral measures and morphology; males with more hamadryas-like morphology had more hamadryas-like behavior. However, genetic ancestry was not related to behavior, and in both cases intermediates displayed a previously unreported level of behavioral variation. Furthermore, male behavior was unrelated to natal group. Second, we evaluated reproductive success by microsatellite-based paternity testing. The highest reproductive success was found for individuals exhibiting intermediate behaviors. Moreover, over nine years, some genetically and morphologically intermediate males had high reproductive success. We conclude that the behavior of hybrid males is therefore unlikely to be an absolute barrier to admixture in the region.

A retrospective study of 132 patients (63 spinal anesthesia and 69 general anesthesia) undergoing total hip arthroplasty was performed by 4 fellowship-trained adult reconstructive surgeons to determine the influence of anesthesia type on postoperative limb length and medial offset. Limb length discrepancy occurred in 87.0% of patients who received regional anesthesia as opposed to 47.6% patients who had general anesthesia (P<.001). Differences in postoperative medial offset measurements between the 2 groups were not statistically significant. It was concluded that surgeons operating on patients who receive regional anesthesia should supplement intraoperative tests for assessing hip stability with meticulous preoperative templating to avoid overlengthening the operative limb

The activation of the blastocyst, a process by which it gains competency to attach with the receptive uterus, is a prerequisite for successful implantation. However, the molecular basis of blastocyst activation remains largely unexplored. Combining molecular, pharmacological and physiological approaches, we show here that silencing of Wnt-beta-catenin signaling in mice does not adversely affect the development of preimplantation embryos to blastocysts and uterine preparation for receptivity, but, remarkably, blocks blastocyst competency to implantation. Using the physiologically relevant delayed implantation model and trophoblast stem cells in culture, we further demonstrate that a coordinated activation of canonical Wnt-beta-catenin signaling with attenuation of the non-canonical Wnt-RhoA signaling pathway ensures blastocyst competency to implantation. These findings constitute novel evidence that Wnt signaling is at least one pathway that determines blastocyst competency for implantation.

The axonal signals that regulate oligodendrocyte myelination during development of the central nervous system (CNS) have not been established. In this study, we have examined the regulation of oligodendrocyte myelination by the type III isoform of neuregulin-1 (NRG1), a neuronal signal essential for Schwann cell differentiation and myelination. In contrast to Schwann cells, primary oligodendrocytes differentiate normally when cocultured with dorsal root ganglia (DRG) neurons deficient in type III NRG1. However, they myelinate type III NRG1-deficient neurites poorly in comparison to wild type cultures. Type III NRG1 is not sufficient to drive oligodendrocyte myelination as sympathetic neurons are not myelinated even with lentiviral-mediated expression of NRG1. Mice haploinsufficient for type III NRG1 are hypomyelinated in the brain, as evidenced by reduced amounts of myelin proteins and lipids and thinner myelin sheaths. In contrast, the optic nerve and spinal cord of heterozygotes are myelinated normally. Together, these results implicate type III NRG1 as a significant determinant of the extent of myelination in the brain and demonstrate important regional differences in the control of CNS myelination. They also indicate that oligodendrocyte myelination, but not differentiation, is promoted by axonal NRG1, underscoring important differences in the control of myelination in the CNS and peripheral nervous system (PNS)

Osteoblasts are derived from two distinct embryonic lineages: cranial neural crest, and mesoderm. Both populations of cells are capable of forming bone and cartilage during fetal development and during adult bone repair, but whether they use equivalent molecular pathways to achieve osteoblast differentiation is unknown. We addressed this question in the context of cranial repair and focused on the role of Wnt signaling in mandibular skeletal healing. Transgenic Wnt reporter mice were used to pinpoint Wnt-responsive cells in the injury callus, and in situ hybridization was used to identify some of the Wnt ligands expressed by cells during the repair process. A gene transfer technique was employed to abrogate Wnt signaling during mandibular healing, and we found that reparative intramembranous ossification requires a functional Wnt pathway. Finally, we evaluated how constitutive activation of the Wnt pathway, caused by mutation of the LRP5 receptor, affected bone repair in the mandible. Taken together, these data underscore the functional requirement for Wnt signaling in cranial skeletal healing.

We describe a new approach for the identification and characterization by mass spectrometry of proteins that have been electroblotted onto nitrocellulose. Using this method (Blotting and Removal of Nitrocellulose (BARN)), proteins can be analyzed either as intact proteins for molecular weight determination or as peptides generated by on-membrane proteolysis. Acetone is used to dissolve the nitrocellulose and to precipitate the adsorbed proteins/peptides, thus removing the nitrocellulose which can interfere with MS analysis. This method offers improved protein coverage, especially for membrane proteins, such as uroplakins, because the extraction step after in-gel digestion is avoided. Moreover, removal of nitrocellulose from the sample solution allows sample analysis by both MALDI- and (LC) ESI-based mass spectrometers. Finally, we demonstrate the utility of BARN for the direct identification of soluble and membrane proteins after Western blotting, obtaining comparable or better results than with in-gel digestion

Solar ultraviolet radiation (UVR) is a major environmental hazard for the skin, and UVB (280-320 nm) has been proposed to be a main factor for melanoma development. In response to sunlight exposure, the skin has adapted a number of innate resistance mechanisms. Among them is the small heat shock protein of 27 kDa (HSP27) known to play a role in the protection of cells from variety of environmental insults including UV irradiation. In this study, we demonstrated that UVB irradiation of cultured normal epidermal melanocytes initiates changes in HSP27 phosphorylation and localization. In unstressed melanocytes, HSP27 was present as the non-phosphorylated isoform. UVB irradiation with a physiological dose (7-25 mJ/cm(2)) resulted in the formation of a mono-phosphorylated isoform and sometimes a bi-phosphorylated isoform. The UVB-induced HSP27 phosphorylation was inhibited when melanocytes were treated with the antioxidant N-acetyl cysteine or inhibitor of p38 MAP kinase prior to UVB exposure, suggesting that UVB induced HSP27 phosphorylation through reactive oxygen species/p38 MAP kinase pathway. In response to UBV irradiation, HSP27 in melanocytes translocated from the cytoplasm to the nucleus. The HSP27 responses may provide some protective role against UVB-induced cell damage in the skin

The N-ribosyldihydronicotinamide (NRH):quinone oxidoreductase 2 (NQO2) gene encodes an enzyme that catalyzes activation of quinones. Blood DNA from 80 control individuals and 118 age-matched Parkinson's disease patients were analyzed for NQO2 gene promoter polymorphisms. The results revealed three allelic variants, designated I-29, I-16, and D. These results were confirmed in fibroblast cell lines. In patients with Parkinson's disease, there was a significant increase in the frequency of the D allele, but there was no difference in the frequency of the alleles in familial compared to sporadic Parkinson's disease. The D and I-16 promoters direct higher NQO2 gene expression that results in higher enzyme activity. Overexpression of NQO2 in the catecholaminergic neuroblastoma SH-SY5Y cells resulted in increased production of reactive oxygen species when exposed to exogenous dopamine. The results suggest that the association of the D promoter with Parkinson's disease may be due to an increase in expression of the NQO2 gene.