Faculty Bibliography

Besides its well-established role in wound healing and fibrinolysis, tissue-type plasminogen activator (t-PA) has been shown to contribute to cognitive processes and memory formation within the central nervous system, and to promote glutamate receptor-mediated excitotoxicity. The t-PA gene is expressed and regulated in neuronal cells but the regulatory transcriptional processes directing this expression are still poorly characterized. We have used DNase I-hypersensitivity mapping and in vivo foot printing to identify putative regulatory elements and transcription factor binding sites in two human neuroblastomal (KELLY and SK-N-SH) and one human glioblastomal (SNB-19) cell lines. Hypersensitive sites were found in the proximal promoter region of all cell lines, and within the first exon for KELLY and SNB-19 cells. Mapping of methylation-protected residues in vivo detected a cluster of protected residues corresponding to a cAMP response element (CRE) and Sp1 sites in the proximal promoter previously shown to be essential for basal expression in other cell types. Protected residues were also found at other sites, notably a kappaB element at position bp -3081 to -3072 that was partly protected in KELLY and SNB-19 cells. Analysis of transfected reporter constructs in KELLY and SNB-19 cells confirmed that this particular element is functionally significant in the transactivation of the t-PA promoter in both cell types. This study defines, by in vivo and in vitro methods, a previously undescribed kappaB site in the t-PA gene promoter that influences t-PA expression in neuronal cells.

Mutations in T-box genes are the cause of several congenital diseases and are implicated in cancer. Tbx20-null mice exhibit severely hypoplastic hearts and express Tbx2, which is normally restricted to outflow tract and atrioventricular canal, throughout the heart. Tbx20 mutant hearts closely resemble those seen in mice overexpressing Tbx2 in myocardium, suggesting that upregulation of Tbx2 can largely account for the cardiac phenotype in Tbx20-null mice. We provide evidence that Tbx2 is a direct target for repression by Tbx20 in developing heart. We have also found that Tbx2 directly binds to the Nmyc1 promoter in developing heart, and can repress expression of the Nmyc1 promoter in transient transfection studies. Repression of Nmyc1 (N-myc) by aberrantly regulated Tbx2 can account in part for the observed cardiac hypoplasia in Tbx20 mutants. Nmyc1 is required for growth and development of multiple organs, including the heart, and overexpression of Nmyc1 is associated with childhood tumors. Despite its clinical relevance, the factors that regulate Nmyc1 expression during development are unknown. Our data present a paradigm by which T-box proteins regulate regional differences in Nmyc1 expression and proliferation to effect organ morphogenesis. We present a model whereby Tbx2 directly represses Nmyc1 in outflow tract and atrioventricular canal of the developing heart, resulting in relatively low proliferation. In chamber myocardium, Tbx20 represses Tbx2, preventing repression of Nmyc1 and resulting in relatively high proliferation. In addition to its role in regulating regional proliferation, we have found that Tbx20 regulates expression of a number of genes that specify regional identity within the heart, thereby coordinating these two important aspects of organ development.

The ribosomal protein L30e is an indispensable component of the eukaryotic 80S ribosome, where it is part of the large (60S) ribosomal subunit. Here, we determined the localization of L30e in the cryo-EM map of the 80S wheat germ (wg) ribosome at a resolution of 9.5 A. L30e is part of the interface between large and small subunits, where it dynamically participates in the formation of the two intersubunit bridges eB9 and B4

Caspofungin acetate (CAS) is a member of a new class of clinically-approved echinocandin drugs to treat invasive aspergillosis. CAS inhibits the activity of beta-1,3-D-glucan synthase (GS), thus damaging the fungal cell wall. Although no clinical resistance of Aspergillus to CAS has been reported as yet, the development of in vitro reduced susceptibility is presumed to be inevitable. By contrast, echinocandin resistance in laboratory strains of Candida albicans and Saccharomyces cerevisiae has been well documented. To study the potential for clinical resistance in Aspergillus, two classes of Aspergillus fumigatus mutant strains were isolated that exhibited reduced susceptibility to CAS. In the first class, a site-directed mutation within the target gene (AfFKS1, encoding the putative catalytic subunit of GS) was introduced and shown to confer low-level (16-fold) reduced susceptibility. A second class of spontaneous mutants were sensitive to low levels of drug but displayed nearly normal growth above 0.5 microg/ml, suggesting induction of an unknown resistance mechanism. At higher levels of drug (> or = 16 microg/ml), the mutants displayed partially restored sensitivity. Preliminary studies indicate that neither target site mutations, nor changes in target gene expression are present in these strains, as has been documented for several yeasts. Instead, preliminary results indicate that the molecular mechanism(s) underlying reduced susceptibility of CAS in the A. fumigatus strains is novel, possibly due to remodeling of the cell wall components.

BACKGROUND CONTEXT: In patients with spinal osteoporosis, the early achievement and maintenance of a biological bond between the pedicle screw and bone is important to avoid screw loosening complications. There are few reports of in vivo investigations involving biomechanical and histological evaluations in the osteoporotic spine. PURPOSE: To evaluate the effect of hydroxyapatite (HA)-coating on the pedicle screw in the osteoporotic lumbar spine and to investigate the relationship between resistance against the screw pull-out force and bone mineral density (BMD) of the vertebral body. STUDY DESIGN/SETTING: Mechanical and pathological investigations in the lumbar spine. METHODS: Two 24-month-old female beagle dogs were fed a calcium-free dog chow for 6 months after ovariectomy (OVX). BMD (in g/cm2) was measured by dual energy X-ray absorptiometry at pre-OVX and 6 months after OVX. Pedicle screws were placed from L1 to L6 at 6 months after OVX. Twenty-four pure titanium cortical screws (Synthes, #401-114) were used as pedicle screws (Ti-PS). Of these, 12 screws had HA-coating (HA-PS). The HA-PS screws were inserted into the right pedicles and the Ti-PS were inserted into the left pedicles. Ten days after this procedure, the lumbar spines were removed en bloc for screw pull-out testing and histological evaluation. RESULTS: The mean BMD value of the lumbar vertebrae 6 months after the OVX was 0.549+/-0.087 g/cm2, which was significantly less than the pre-OVX mean BMD of 0.603+/-0.092 g/cm2 (p < 0.001). The mean resistance against the pull-out force for the HA-PS was significantly greater at 165.6+/-26.5N than in the Ti-PS (103.1+/-30.2N, p < .001). The histological sections in the HA-PS clearly revealed new bone bonding with the apatite coating but only fibrous tissue bonding in the Ti-PS. CONCLUSIONS: The results of this study showed that the resistance to the pull-out force of HA-PS is 1.6 times that of Ti-PS. Furthermore, HA-PS has superior biological bonding to the surrounding bone, as early as 10 days after surgery in this osteoporotic spine model. Thus, in patients with osteoporosis, coating of the pedicle screw with HA may provide better stability and bonding between the pedicle screw and bone in the early postoperative period.

Osteoblasts produce an array of immune molecules following bacterial challenge that could recruit leukocytes to sites of infection and promote inflammation during bone diseases, such as osteomyelitis. Recent studies from our laboratory have shed light on the mechanisms by which this cell type can perceive and respond to bacteria by demonstrating the functional expression of members of the Toll-like family of cell surface pattern recognition receptors by osteoblasts. However, we have shown that bacterial components fail to elicit immune responses comparable with those seen following challenge with the intracellular pathogens salmonellae and Staphylococcus aureus. In the present study, we show that UV-killed bacteria and invasion-defective bacterial strains elicit significantly less inflammatory cytokine production than their viable wild-type counterparts. Importantly, we demonstrate that murine osteoblasts express the novel intracellular pattern recognition receptors Nod1 and Nod2. Levels of mRNA encoding Nod molecules and protein expression are significantly and differentially increased from low basal levels following exposure to these disparate bacterial pathogens. In addition, we have shown that osteoblasts express Rip2 kinase, a critical downstream effector molecule for Nod signaling. Furthermore, to begin to establish the functional nature of Nod expression, we show that a specific ligand for Nod proteins can significantly augment immune molecule production by osteoblasts exposed to either UV-inactivated bacteria or bacterial lipopolysaccharide. As such, the presence of Nod proteins in osteoblasts could represent an important mechanism by which this cell type responds to intracellular bacterial pathogens of bone.