Faculty Bibliography

Recombinant human prion-protein (PrP23-231) stimulates plasminogen activation by tissue-type plasminogen activator (t-PA). The stimulatory activity is conserved in the N-terminal fragment (PrP23-110). It has further been shown by others that PrP(c) binds to kringle-domains of plasminogen. We compared the stimulatory activity of recombinant PrP23-231 and PrP23-110 on plasminogen activation catalyzed by t-PA, urokinase (u-PA), streptokinase and Desmodus salivary plasminogen activator (DSPAalpha1). As these plasminogen activators are distinct, with respect to their kringle domains we studied their binding to immobilized PrP23-110. Plasminogen activation was measured in a chromogenic assay in vitro and binding studies were carried out using surface plasmon resonance technology. We found that recombinant full-length prion protein, PrP23-231, and PrP23-110 specifically stimulate t-PA mediated plasminogen activation. Two hundred nanomoles per liter of PrP23-110 stimulated 1.8 nmol L(-1) t-PA 48-fold, 180 nmol L(-1) DSPA(alpha1) 2.5-fold, 1.8 nmol L(-1) u-PA 1.1-fold, and 1.8 nmol L(-1) streptokinase 1.8-fold. Our data show no specific binding for streptokinase. In contrast all plasminogen activators carrying a kringle domain bound to PrP23-110. We further studied the effect of lysine on binding to PrP23-110 and on plasminogen activation by DSPA(alpha1) or t-PA. Lysine decreased both the binding of t-PA to PrP23-110 and the stimulation of plasmin generation by t-PA. Both binding and plasminogen activation of DSPA(alpha1) were not influenced by the presence of lysine. All plasminogen activators tested bearing kringle domains bind to PrP23-110. Binding to PrP23-110 is not sufficient for stimulation of plasmin generation. Thus the lysine-binding site of kringle 2 that is unique to t-PA appears to mediate the specific stimulation of plasminogen activation by the cellular prion protein.

Presenilin 1 (PS1) plays a pivotal role in the production of the amyloid-beta protein (Abeta) that is central to the pathogenesis of Alzheimer's disease. PS1 regulates the intramembranous proteolysis of a 99-amino-acid C-terminal fragment of the amyloid precursor protein (APP-C99), a cleavage event that releases Abeta following a reaction catalyzed by an enzyme termed 'gamma-secretase'. The molecular mechanism of PS1-mediated, gamma-secretase cleavage remains largely unresolved. In particular, controversy surrounds whether PS1 includes the catalytic site of the gamma-secretase protease or whether instead PS1 mediates gamma-secretase activity indirectly, perhaps by regulating the trafficking or presentation of substrates to the 'authentic' protease, which may be a molecule distinct from PS1. To address this issue, the baculovirus expression system was used to co-express: (i) APP-C99; (ii) a pathogenic, constitutively active mutant form of PS1 lacking exon 9 (PS1DeltaE9); (iii) nicastrin and (iv) tropomyosin in Spodoptera frugiperda (Sf9) cells. Cells infected with APP-C99 alone produced an Abeta-like species, and levels of this species were enhanced by the addition of baculoviruses bearing the PS1DeltaE9 mutation. The addition to APP-C99-infected cells of baculoviruses bearing nicastrin, also a transmembrane protein, had a neutral or inhibitory effect on the reaction; tropomyosin viruses had the same effect as nicastrin viruses. These results suggest that PS1DeltaE9 molecules expressed in Sf9 cells retain the ability to modulate Abeta levels. Baculoviral-expressed PS1DeltaE9 provides a source of microgram quantities of bioactive molecules for use as starting material for purifying and reconstituting gamma-secretase activity from its individual purified component parts

The utility of HPLC with a size exclusion chromatography (SEC) column for the fractionation of environmental samples was examined to expand the applicability of recombinant yeast assays (RYAs) for the measurement of estrogenic activity in environmental samples. The elution of steroidal hormone standards through SEC was tested, and river water and sediment samples were subjected to SEC fractionation. The retention times of all estrogen standards were longer than those of the major components with absorbance at 230 nm of the environmental sample matrix. The estrogenic activity of SEC fractions of environmental samples from highly polluted sites was measured using RYA and revealed the existence of estrogenic compounds that could not be detected or quantified in extracted samples before fractionation. The fractions from environmental samples that corresponded to the retention time of estrone, 17[small beta]-estradiol, and estriol were analyzed with LC/MS/MS. These three estrogens were separated into three different fractions, and the concentration of estrone coincided with the estrogenic activity of the fraction in which it was detected. The profile of estrogenic activity of SEC fractions indicated the molecular size of the estrogenic compounds and the yeast growth inhibitors in samples.

Gene therapy is a promising modality for the treatment of soft tissue malignancies. Our laboratory has developed a novel technique of gene transfer using microvascular free flaps that addresses many of the current barriers preventing gene therapy from achieving widespread clinical use. Our previous work has demonstrated our ability to transduce free flaps with an adenovirus encoding the reporter gene lacZ. In this current study, we show that microvascular free flaps can be transduced with an adenovirus encoding the angiogenesis inhibitor endostatin with high levels of local flap expression. These transduced free flaps were able to serve as 'biologic pumps' and were able to secrete endostatin into the serum as demonstrated by enzyme-linked immunosorbent assay. This form of 'biologic brachytherapy' could provide a novel approach for the continuous delivery of therapeutic genes to a localized area while avoiding many of the practical obstacles currently limiting gene therapy

Distraction osteogenesis (DO) requires a long consolidation period and has a low but real failure rate. Bone morphogenic proteins (BMPs) accelerate bone deposition in fractures and critical-sized bone defects, but their effects on mandibular DO are unknown. We investigated the effect of local delivery of adenovirus containing the gene for BMP-2 (Adbmp-2) on mandibular DO in a rat model. Rats (n = 54) were distracted to 3 mm over 6 days. At the start of consolidation (POD 10), Adbmp-2 or adenovirus containing the lacZgene (AdlacZ) was injected directly into the distraction zone. After 1, 2, and 4 weeks of consolidation, mandibles were evaluated for amount of bone deposition. Adbmp-2-treated specimens demonstrated an increased amount of new bone formation by radiographic, histologic, and histomorphometric analysis. This study demonstrates that local, adenovirally-mediated delivery of BMP-2 can increase bone deposition during DO, potentially shortening consolidation and enhancing DO in poorly healing mandibles, such as occurs postirradiation

Diminished production of vascular endothelial growth factor (VEGF) and decreased angiogenesis are thought to contribute to impaired tissue repair in diabetic patients. We examined whether recombinant human VEGF(165) protein would reverse the impaired wound healing phenotype in genetically diabetic mice. Paired full-thickness skin wounds on the dorsum of db/db mice received 20 microg of VEGF every other day for five doses to one wound and vehicle (phosphate-buffered saline) to the other. We demonstrate significantly accelerated repair in VEGF-treated wounds with an average time to resurfacing of 12 days versus 25 days in untreated mice. VEGF-treated wounds were characterized by an early leaky, malformed vasculature followed by abundant granulation tissue deposition. The VEGF-treated wounds demonstrated increased epithelialization, increased matrix deposition, and enhanced cellular proliferation, as assessed by uptake of 5-bromodeoxyuridine. Analysis of gene expression by real-time reverse transcriptase-polymerase chain reaction demonstrates a significant up-regulation of platelet-derived growth factor-B and fibroblast growth factor-2 in VEGF-treated wounds, which corresponds with the increased granulation tissue in these wounds. These experiments also demonstrated an increase in the rate of repair of the contralateral phosphate-buffered saline-treated wound when compared to wounds in diabetic mice never exposed to VEGF (18 days versus 25 days), suggesting that topical VEGF had a systemic effect. We observed increased numbers of circulating VEGFR2(+)/CD11b(-) cells in the VEGF-treated mice by fluorescence-activated cell sorting analysis, which likely represent an endothelial precursor population. In diabetic mice with bone marrow replaced by that of tie2/lacZ mice we demonstrate that the local recruitment of bone marrow-derived endothelial lineage lacZ+ cells was augmented by topical VEGF. We conclude that topical VEGF is able to improve wound healing by locally up-regulating growth factors important for tissue repair and by systemically mobilizing bone marrow-derived cells, including a population that contributes to blood vessel formation, and recruiting these cells to the local wound environment where they are able to accelerate repair. Thus, VEGF therapy may be useful in the treatment of diabetic complications characterized by impaired neovascularization

The goal of animal wound healing models is to replicate human physiology and predict therapeutic outcomes. There is currently no model of wound healing in rodents that closely parallels human wound healing. Rodents are attractive candidates for wound healing studies because of their availability, low cost, and ease of handling. However, rodent models have been criticized because the major mechanism of wound closure is contraction, whereas in humans reepithelialization and granulation tissue formation are the major mechanisms involved. This article describes a novel model of wound healing in mice utilizing wound splinting that is accurate, reproducible, minimizes wound contraction, and allows wound healing to occur through the processes of granulation and reepithelialization. Our results show that splinted wounds have an increased amount of granulation tissue deposition as compared to controls, but the rate of reepithelialization is not affected. Thus, this model eliminates wound contraction and allows rodents' wounds to heal by epithelialization and granulation tissue formation. Given these analogies to human wound healing, we believe that this technique is a useful model for the study of wound healing mechanisms and for the evaluation of new therapeutic modalities

In patients undergoing in vitro fertilization, the presence of higher E(2) levels at the time of hCG administration predict a greater likelihood of ongoing pregnancy