Faculty Bibliography

Staphylococcus aureus is the single most common cause of osteomyelitis in humans. Incidences of osteomyelitis caused by S. aureus have increased dramatically in recent years, in part due to the appearance of community-acquired antibiotic resistant strains. Therefore, understanding the pathogenesis of this organism has become imperative. Recently, we have described the surprising ability of bone-forming osteoblasts to secrete a number of important immune mediators when exposed to S. aureus in vitro. In the present study, we provide the first evidence for the in vivo production of such molecules by osteoblasts during bacterial infection of bone. These studies demonstrate the expression of the key inflammatory cytokine interleukin-6 by osteoblasts in organ cultures of neonatal mouse calvaria, and in vivo using a mouse model that closely resembles the pathology of trauma-induced staphylococcal osteomyelitis, as determined by confocal microscopic analysis. Importantly, we have established the clinical relevancy of these findings in infected human bone tissue from patients with S. aureus-associated osteomyelitis. As such, these studies demonstrate that bacterial challenge of osteoblasts during bone diseases, such as osteomyelitis, induces cells to produce inflammatory molecules that can direct appropriate host responses or contribute to progressive inflammatory damage.

8-Prenylnaringenin is a recently discovered phytoestrogen. Using an in vitro model of angiogenesis in which endothelial cells can be induced to invade a three-dimensional collagen gel within which they form capillary-like tubes, we demonstrate that 8-prenylnaringenin inhibits angiogenesis induced by basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), or the synergistic effect of the two cytokines in combination, with an IC(50) of between 3 and 10 microM. This effect was seen with bovine microvascular endothelial cells derived from the adrenal cortex (BME cells) and with endothelial cells from the bovine thoracic aorta (BAE cells). The inhibitory effects of 8-prenylnaringenin were found to be roughly equipotent to those of genistein that has previously been shown to inhibit angiogenesis in vitro. Early chorioallantoic membrane (CAM) assay results showed reductions in both vessel lengths and vein diameters, with similar potency in the 8-prenylnaringenin and genistein groups. Similar effects on the CAM vessels were seen when the two substances were co-added. These findings suggest that 8-prenylnaringenin has potential therapeutic applications for diseases in which angiogenesis is an important component.

Abstract The enzyme gamma-secretase catalyzes the intramembrane proteolytic cleavage that generates the amyloid beta-peptide from the beta-amyloid precursor protein. The presenilin (PS) protein is one of the four integral membrane protein components of the mature gamma-secretase complex. The PS protein is itself subjected to endoproteolytic processing, generating stable N- and C-terminal fragment (NTF and CTF, respectively) heterodimers. Here we demonstrate that coexpression of PS1 NTF and CTF functionally mimics expression of the full-length PS1 protein and restores gamma-secretase activity in PS-deficient mammalian cells. The coexpressed fragments re-associate with each other inside the cell, where they also interact with nicastrin, another gamma-secretase complex component. Analysis of gamma-secretase activity following the expression of mutant forms of NTF and CTF, under conditions bypassing endoproteolysis, indicated that the putatively catalytic Asp257 and Asp385 residues have a direct effect on gamma-secretase activity. Moreover, we demonstrate that expression of the wild-type CTF rescues endoproteolytic cleavage of C-terminally truncated PS1 molecules that are otherwise uncleaved and inactive. Recovery of cleavage is critically dependent on the integrity of Asp385. Taken together, our findings indicate that ectopically expressed NTF and CTF restore functional gamma-secretase complexes and that the presence of full-length PS1 is not a requirement for proper complex assembly

The epidermal growth factor receptor (EGFR) is overexpressed and/or constitutively activated in a variety of human malignancies. Detection of increased expression levels of EGFR in cancer and the association between overexpression and decreased patient survival has led to the development of several therapeutic strategies to target this receptor. The results of early-phase clinical trials to date suggest that targeting EGFR alone may not be sufficient to eradicate established tumors. This limited antitumor efficacy as monotherapy has led to combining EGFR inhibitors with chemotherapy or radiation therapy for advanced disease, or incorporating EGFR inhibition to cancer prevention approaches. This review will discuss the role of EGFR signaling in carcinogenesis and the rationale for EGFR inhibition as a clinical prevention and treatment strategy.

Binding of fibroblast growth factor (FGF) to the high affinity receptor-1 (FGFR-1) leads to activation of its endogenous tyrosine kinase activity. A number of substrates for the FGFR-1 kinase have been identified. Among those, FGF receptor-substrate-2 (FRS-2) was identified by virtue of its interaction with p13suc, a yeast protein involved in cell cycle regulation. We have used immobilized p13suc to identify a new substrate for FGRF-1, which is identical to 'translocated in liposarcoma' (TLS). TLS is a RNA/DNA-binding protein which occurs in fusion products with different transcription factors in a variety of solid tumours. We show that TLS is tyrosine phosphorylated in intact cells by a number of different growth factors, indicating a role in growth regulation

Inorganic arsenic, an environmental contaminant, produces a variety of stress responses in mammalian cells, including metabolic abnormalities accompanied by growth inhibition and carcinogenesis. Much of the toxicity of arsenic is known to stem from its uncoupling effects on mitochondria. Because previously we had shown that mitochondrial dysfunction can disrupt oocyte and embryo development, we investigated effects of arsenite on meiotic progression and early embryo development in mice. Six-week-old CD-1 mice were treated with 0 (solvent as control), 8 mg/kg (a dose previously established in mice as the maternal no-observed-adverse-effect level), and 16 mg/kg doses of sodium arsenite every 2 days for a total of seven i.p. injections ver a period of 14 days. The incidence of meiotic anomalies, characterized by spindle disruption and/or chromosomal misalignment, was significantly increased in arsenite-treated groups (25% after 8 mg/kg and 62.5% after 16 mg/kg), compared to normal metaphase II in control oocytes. Further, arsenite treatment significantly decreased cleavage rates of zygotes at 24 h, morula formation at 72 h, and development to blastocysts at 96 h in a dose-dependent manner. The total cell number in developed blastocysts did not differ significantly between the 8 mg/kg arsenite treatment and control groups, but was significantly reduced in the 16 mg/kg arsenite treatment group. Moreover, the percentage of apoptotic nuclei was significantly increased in blastocysts following 16 mg/kg arsenite treatment. These data suggest that arsenite causes meiotic aberrations, which may contribute to decreased cleavage and preimplantation development, as well as increased apoptosis

BACKGROUND: Five patients with DiGeorge syndrome presented with infections, skin rashes, and lymphadenopathy after the newborn period. T-cell counts and function varied greatly in each patient. Initial laboratory testing did not suggest athymia in these patients. OBJECTIVE: The purpose of this study was to determine whether the patients had significant immunodeficiency. METHODS: Research testing of peripheral blood included immunoscope evaluation of T-cell receptor beta variable gene segment repertoire diversity, quantification of T-cell receptor rearrangement excision circles, and detection of naive T cells (expressing CD45RA and CD62L). RESULTS: The patients were classified as having DiGeorge syndrome on the basis of syndromic associations and heart, parathyroid, and immune abnormalities. Immunoscope evaluation revealed that the T-cell repertoires were strikingly oligoclonal in all patients. There were few recent thymic emigrants, as indicated by the very low numbers of naive T cells (<50/mm(3)) and the absence of T-cell receptor rearrangement excision circles. These studies showed that all 5 patients were athymic. Two patients died, one from infection. No thymus was found during the complete autopsy performed on one patient. CONCLUSION: Patients with DiGeorge syndrome, skin rash, and lymphadenopathy should undergo analysis of naive T-cell numbers and of T-cell receptor beta variability segment repertoire to determine whether they are athymic, even if they have T cells with mitogen responsiveness. It is important for physicians to realize that patients with complete DiGeorge syndrome remain profoundly immunodeficient after development of these atypical features (rash, lymphadenopathy, and oligoclonal T cells). Prompt diagnosis is necessary for appropriate management.

The horizontal gene transfer (HGT) being inferred within prokaryotic genomes appears to be sufficiently massive that many scientists think it may have effectively obscured much of the history of life recorded in DNA. Here, we demonstrate that the tree of life can be reconstructed even in the presence of extensive HGT, provided the processes of genome evolution are properly modeled. We show that the dynamic deletions and insertions of genes that occur during genome evolution, including those introduced by HGT, may be modeled using techniques similar to those used to model nucleotide substitutions that occur during sequence evolution. In particular, we show that appropriately designed general Markov models are reasonable tools for reconstructing genome evolution. These studies indicate that, provided genomes contain sufficiently many genes and that the Markov assumptions are met, it is possible to reconstruct the tree of life. We also consider the fusion of genomes, a process not encountered in gene sequence evolution, and derive a method for the identification and reconstruction of genome fusion events. Genomic reconstructions of a well-defined classical four-genome problem, the root of the multicellular animals, show that the method, when used in conjunction with paralinear/logdet distances, performs remarkably well and is relatively unaffected by the recently discovered big genome artifact.

Schwann cell factor 1 (SC1), a p75 neurotrophin receptor-interacting protein, is a member of the positive regulatory/suppressor of variegation, enhancer of zeste, trithorax (PR/SET) domain-containing zinc finger protein family, and it has been shown to be regulated by serum and neurotrophins. SC1 shows a differential cytoplasmic and nuclear distribution, and its presence in the nucleus correlates strongly with the absence of bromodeoxyuridine (BrdU) in these nuclei. Here, we investigated potential transcriptional activities of SC1 and analyzed the function of its various domains. We show that SC1 acts as a transcriptional repressor when it is tethered to Gal4 DNA-binding domain. The repressive activity requires a trichostatin A-sensitive histone deacetylase (HDAC) activity, and SC1 is found in a complex with HDACs 1, 2, and 3. Transcriptional repression exerted by SC1 requires the presence of its zinc finger domains and the PR domain. Additionally, these two domains are involved in the efficient block of BrdU incorporation by SC1. The zinc finger domains are also necessary to direct SC1's nuclear localization. Lastly, SC1 represses the promoter of a promitotic gene, cyclin E, suggesting a mechanism for how growth arrest is regulated by SC1