Faculty Bibliography

We have searched for sequence motifs that contribute to the recognition of human pre-mRNA splice sites by comparing the frequency of 8-mers in internal noncoding exons versus unspliced pseudo exons and 5' untranslated regions (5' untranslated regions [UTRs]) of transcripts of intronless genes. This type of comparison avoids the isolation of sequences that are distinguished by their protein-coding information. We classified sequence families comprising 2069 putative exonic enhancers and 974 putative exonic silencers. Representatives of each class functioned as enhancers or silencers when inserted into a test exon and assayed in transfected mammalian cells. As a class, the enhancer sequencers were more prevalent and the silencer elements less prevalent in all exons compared with introns. A survey of 58 reported exonic splicing mutations showed good agreement between the splicing phenotype and the effect of the mutation on the motifs defined here. The large number of effective sequences implied by these results suggests that sequences that influence splicing may be very abundant in pre-mRNA.

We applied a robust combinatorial (multi-test) approach to microarray data to identify genes consistently up- or down-regulated in head and neck squamous cell carcinoma (HNSCC). RNA was extracted from 22 paired samples of HNSCC and normal tissue from the same donors and hybridized to the Affymetrix U95A chip. Forty-two differentially expressed probe sets (representing 38 genes and one expressed sequence tag) satisfied all statistical tests of significance and were selected for further validation. Selected probe sets were validated by hierarchical clustering, multiple probe set concordance, and target-subunit agreement. In addition, real-time PCR analysis of 8 representative (randomly selected from 38) genes performed on both microarray-tested and independently obtained samples correlated well with the microarray data. The genes identified and validated by this method were in comparatively good agreement with other rigorous HNSCC microarray studies. From this study, we conclude that combinatorial analysis of microarray data is a promising technique for identifying differentially expressed genes with few false positives

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

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

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

Vitiligo is an acquired depigmentary disorder of the skin that results from the selective destruction of melanocytes, generally during the second decade of life and affecting approximately 1% of the population worldwide. Loss of cutaneous pigment appears to render the skin susceptible to premature aging and cancer. In addition this disease can be socially devastating for afflicted individuals. The etiology of vitiligo is poorly understood. The present dogma suggests that genetic factors render the melanocyte fragile thus predisposing individuals to developing vitiligo. When subjected to instigating factors, these susceptible, fragile melanocytes undergo apoptosis. Autoimmune factors then perpetuate the removal of the melanocyte component from the skin. In the majority of cases the instigating factors are not known (idiopathic vitiligo), however a small sub-set of individuals develop contact/occupational vitiligo following exposure to particular chemicals. Many of these chemicals have been implicated in both contact/occupational vitiligo and chemical leukoderma. Both conditions present with well-defined, depigmented skin lesions that develop following exposure. Only in the case of vitiligo does the depigmentation spread beyond the areas of contact, probably via an immune-mediated mechanism. The largest class of chemicals known to trigger contact/occupational vitiligo is the phenolic/catecholic derivatives. Many have been demonstrated to be preferentially cytotoxic to melanocytes, with high-dose exposure resulting in the initiation of apoptosis. Phenolic/catecholic derivatives are structurally similar to the melanin precursor tyrosine, and therefore tyrosinase was originally implicated as a mediator of cytotoxicity. However, our data suggests that tyrosinase-related protein-1, rather than tyrosinase, facilitates toxicity, possibly by catalytic conversion of the compounds, which results in the generation of radical oxygen species. The ensuing oxidative stress then triggers activation of cellular free radical scavenging pathways to prevent cell death. Genetic inability of melanocytes to tolerate and/or respond to the oxidative stress may underlie the etiology of contact/occupational vitiligo

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