Faculty Bibliography

Processing of the Alzheimer amyloid precursor protein (APP) into the amyloid beta-protein and the APP intracellular domain is a proteolysis event mediated by the gamma-secretase complex where presenilin (PS) proteins are key constituents. PS is subjected to an endoproteolytic cleavage, generating a stable heterodimer composed of an N-terminal and a C-terminal fragment. Here we aimed at further understanding the role of PS in endoproteolysis, in proteolytic processing of APP and Notch, and in assembly of the gamma-secretase complex. By using a truncation protocol and alanine scanning, we identified Tyr-288 in the PS1 N-terminal fragment as critical for PS-dependent intramembrane proteolysis. Further mutagenesis of the 288 site identified mutants differentially affecting endoproteolysis and gamma-secretase activity. The Y288F mutant was endoproteolyzed to the same extent as wild type PS but increased the amyloid beta-protein 42/40 ratio by approximately 75%. In contrast, the Y288N mutant was also endoproteolytically processed but was inactive in reconstituting gamma-secretase in PS null cells. The Y288D mutant was deficient in both endoproteolysis and gamma-secretase activity. All three mutant PS1 molecules were incorporated into gamma-secretase complexes and stabilized Pen-2 in PS null cells. Thus, mutations at Tyr-288 do not affect gamma-secretase complex assembly but can differentially control endoproteolysis and gamma-secretase activity

An amphiphilic hexa-peri-hexabenzocoronene self-assembles to form a pi-electronic, discrete nanotubular object. The object is characterized by an aspect ratio greater than 1000 and has a uniform, 14-nanometer-wide, open-ended hollow space, which is an order of magnitude larger than those of carbon nanotubes. The wall is 3 nanometers thick and consists of helical arrays of the pi-stacked graphene molecule, whose exterior and interior surfaces are covered by hydrophilic triethylene glycol chains. The graphitic nanotube is redox active, and a single piece of the nanotube across 180-nanometer-gap electrodes shows, upon oxidation, an electrical resistance of 2.5 megohms at 285 kelvin [corrected]. This family of molecularly engineered graphite with a one-dimensional tubular shape and a chemically accessible surface constitutes an important step toward molecular electronics.

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.

Acetylcholine receptor (AChR) genes are transcribed selectively in synaptic nuclei of skeletal muscle fibers, leading to accumulation of the mRNAs encoding AChR subunits at synaptic sites. The signals that regulate synapse-specific transcription remain elusive, though Neuregulin-1 is considered a favored candidate. Here, we show that motor neurons and terminal Schwann cells express neuregulin-2, a neuregulin-1-related gene. In skeletal muscle, Neuregulin-2 protein is concentrated at synaptic sites, where it accumulates adjacent to terminal Schwann cells. Neuregulin-2 stimulates AChR transcription in cultured myotubes expressing ErbB4, as well as ErbB3 and ErbB2, but not in myotubes expressing only ErbB3 and ErbB2. Thus, Neuregulin-2 is a candidate for a signal that regulates synaptic differentiation

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

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

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