Faculty Bibliography

Conditional targeted cell ablation in zebrafish would greatly expand the utility of this genetic model system in developmental and regeneration studies, given its extensive regenerative capabilities. Here, we show that, by combining chemical and genetic tools, one can ablate cells in a temporal- and spatial-specific manner in zebrafish larvae. For this purpose, we used the bacterial Nitroreductase (NTR) enzyme to convert the prodrug Metronidazole (Mtz) into a cytotoxic DNA cross-linking agent. To investigate the efficiency of this system, we targeted three different cell lineages in the heart, pancreas, and liver. Expression of the fusion protein Cyan Fluorescent Protein-NTR (CFP-NTR) under control of tissue-specific promoters allowed us to induce the death of cardiomyocytes, pancreatic beta-cells, and hepatocytes at specific times. Moreover, we have observed that Mtz can be efficiently washed away and that, upon Mtz withdrawal, the profoundly affected tissue can quickly recover. These findings show that the NTR/Mtz system is effective for temporally and spatially controlled cell ablation in zebrafish, thereby constituting a most promising genetic tool to analyze tissue interactions as well as the mechanisms underlying regeneration.

During open reduction of an irreducible anterior dislocation of a total hip replacement with an Oxinium femoral head, it was observed that the head had been significantly damaged. Gross and scanning electron microscopic examination revealed cracking, gouging, and delamination of the surface. Because of the risk which this poses for damaging the polyethylene acetabular liner, it is strongly recommended that patients with this type of prosthetic head be carefully monitored after a dislocation

We surveyed native populations of the chestnut blight fungus, Cryphonectria parasitica, in Japan and China, and C. nitschkei, a sympatric species on chestnut trees in Japan, to learn more about the diversity of hypoviruses and other double-stranded (ds) RNA viruses. In a sample of 472 isolates of C. parasitica and 45 isolates of C. nitschkei from six prefectures in Japan, we found 27 containing one or more dsRNAs. Twelve isolates of C. parasitica and two isolates of C. nitschkei were infected with Cryphonectria hypovirus 1 (CHV-1); four of these 12 C. parasitica isolates also contained other dsRNAs that did not hybridize to CHV-1. In China, only one of 85 C. parasitica isolates was CHV-1-infected; no dsRNAs were detected in the other isolates from China. No other known hypoviruses were found in this study. However, we found two previously undescribed dsRNAs in Japan approximately 9kb in size that did not hybridize to each other or to any known dsRNAs from C. parasitica. We also found three additional groups of dsRNAs, one of which represents the genome of a new member of the virus family Chrysoviridae and was found only in C. nitschkei; the other two dsRNAs were found previously in isolates of C. parasitica from Japan or China. The most significant result of this survey is the discovery of novel dsRNAs that can be characterized in future research.

Due to the aging population and the increasing need for total joint replacements, osseointegration is of a great interest for various clinical disciplines. Our objective was to investigate the molecular and cellular foundation that underlies this process. Here, we used an in vivo mouse model to study the cellular and molecular response in three distinct areas of unloaded implants: the periosteum, the gap between implant and cortical bone, and the marrow space. Our analyses began with the early phases of healing, and continued until the implants were completely osseointegrated. We investigated aspects of osseointegration ranging from vascularization, cell proliferation, differentiation, and bone remodeling. In doing so, we gained an understanding of the healing mechanisms of different skeletal tissues during unloaded implant osseointegration. To continue our analysis, we used a micromotion device to apply a defined physical stimulus to the implants, and in doing so, we dramatically enhanced bone formation in the peri-implant tissue. By comparing strain measurements with cellular and molecular analyses, we developed an understanding of the correlation between strain magnitudes and fate decisions of cells shaping the skeletal regenerate.

It is well known that tumor necrosis factor (TNF) can have both contrary and pleiotropic effects in anti-tumor immune response. In the present study, we prepared two different tumor cell-based immunotherapy models: MCA38 adenocarcinoma and GL261 glioma intracranial interleukin-2 (IL-2)-based. Each tumor was transfected to express IL-2 with or without expression of the soluble form of tumor necrosis factor receptor type II (sTNFRII). Although mice in which TNF is blocked survive longer than IL-2 alone (35.2 versus 26 days), the reverse was observed for GL261 glioma. The differential effect on tumor growth implies enhanced TNF sensitivity of GL261 compared to MCA38. This notion is supported by the observation that TNF induces apoptosis in GL261 but not MCA38 tumors. We further examined tumor infiltrating CD11b+F4/80+ macrophages (or tumor-associated macrophages: TAM) for TNF production in vivo and found that TAM express cell surface TNF implying a role in eliminating glioma cells mediated by the cell surface form of TNF

The biogenesis and accumulation of the beta amyloid protein (Abeta) is a key event in the cascade of oxidative and inflammatory processes that characterises Alzheimer's disease. The presenilins and its interacting proteins play a pivotal role in the generation of Abeta from the amyloid precursor protein (APP). In particular, three proteins (nicastrin, aph-1 and pen-2) interact with presenilins to form a large multi-subunit enzymatic complex (gamma-secretase) that cleaves APP to generate Abeta. Reconstitution studies in yeast and insect cells have provided strong evidence that these four proteins are the major components of the gamma-secretase enzyme. Current research is directed at elucidating the roles that each of these protein play in the function of this enzyme. In addition, a number of presenilin interacting proteins that are not components of gamma-secretase play important roles in modulating Abeta production. This review will discuss the components of the gamma-secretase complex and the role of presenilin interacting proteins on gamma-secretase activity.

Wound healing is a complex and well-orchestrated biological process. Corneal epithelial cells (CECs) must respond quickly to trauma to rapidly restore barrier function and protect the eye from noxious agents. They express a high level of beta2-adrenergic receptors but their function is unknown. Here, we report the novel finding that they form part of a regulatory network in the corneal epithelium, capable of modulating corneal epithelial wound repair. Beta-adrenergic receptor agonists delay CEC migration via a protein phosphatase 2A-mediated mechanism and decrease both electric field-directed migration and corneal wound healing. Conversely, beta-adrenergic receptor antagonists accelerate CEC migration, enhance electric field-mediated directional migration, and promote corneal wound repair. We demonstrate that CECs express key enzymes required for epinephrine (beta-adrenergic receptor agonist) synthesis in the cytoplasm and can detect epinephrine in cell extracts. We propose that the mechanism for the pro-motogenic effect of the beta-adrenergic antagonist is blockade of the beta2-adrenergic receptor preventing autocrine catecholamine binding. Further investigation of this network will improve our understanding of one of the most frequently prescribed class of drugs

OBJECTIVES: Because the trigone is a unique region in the caudal bladder with a higher risk of neoplasia, we hypothesized that this area would have a high proportion of progenitor cells. As yet there is no marker nor methodology to specifically isolate urothelial stem cells, and thus demonstrate multi-potential differentiation and self-renewal. Here, our goal was to evaluate the distribution of progenitor cells that carry two general major attributes of stem cells: clonogenicity and proliferative capacity. MATERIALS AND METHODS: The bladders of Fisher rats were divided into caudal and cephalic segments and primary cultures were established from the harvested urothelial cells. RESULTS: We found that colony-forming efficiency was almost 2-fold higher for cells from the caudal bladder compared to the cephalic bladder. Doubling time was significantly faster for cells harvested from the caudal bladder at initial plating. This suggested that the caudal bladder harbours a higher density of urothelial progenitor cells. With passage to p4, the differences between the upper and lower bladder were lost, suggesting selection of proliferative cells with serial passage. Based on Ki-67 staining, there was no geographical difference in cell proliferation under normal homeostatic in vivo conditions. CONCLUSIONS: These results demonstrate geographical sequestration of urothelial progenitor cells to the area of the bladder that encompasses the bladder neck and trigone, which may be a factor in pathological disparities between the trigone and remaining bladder

During infection, human immunodeficiency virus type 1 integrase engages a number of molecules and mechanisms, both of viral and cellular origin. In one of such instances, integrase is thought to be degraded by the N-end rule proteasome pathway a process that targets the N-terminal residue of its substrates. Here we describe the properties of HIV-1 viruses in which the first amino acid residue of integrase has been substituted to render it resistant to the N-end rule pathway. As result of this exchange, we observe a set of class I and class II defects that result in a large decrease of viral replication efficiency. Specifically, reverse transcription and integration are the steps that appear to be affected. We propose that the severe deficiency of these mutants exert a strong selective pressure that leads to the near total conservation of the N-terminal residue of integrase in HIV-1, HIV-2 and SIV.