Faculty Bibliography

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.

Indoleamine 2,3 dioxygenase (IDO) catabolizes the amino acid tryptophan. IDO-expressing immunoregulatory dendritic cells (DCs) have been implicated in settings including tumors, autoimmunity, and transplant tolerance. However, the downstream molecular mechanisms by which IDO functions to regulate T cell responses remain unknown. We now show that IDO-expressing plasmacytoid DCs activate the GCN2 kinase pathway in responding T cells. GCN2 is a stress-response kinase that is activated by elevations in uncharged tRNA. T cells with a targeted disruption of GCN2 were not susceptible to IDO-mediated suppression of proliferation in vitro. In vivo, proliferation of GCN2-knockout T cells was not inhibited by IDO-expressing DCs from tumor-draining lymph nodes. IDO induced profound anergy in responding wild-type T cells, but GCN2-knockout cells were refractory to IDO-induced anergy. We hypothesize that GCN2 acts as a molecular sensor in T cells, allowing them to detect and respond to conditions created by IDO.

Cholesterol is a major lipid component of the plasma membrane in animal cells. In addition to its structural requirement, cholesterol is essential in cell proliferation and other cell processes. The aim of the present study was to elucidate the stringency of the requirement for cholesterol as a regulator of proliferation and cell cycle progression, compared with other sterols of the cholesterol biosynthesis pathway. Human promyelocytic HL-60 cells were cultured in cholesterol-free medium and treated with different distal inhibitors of cholesterol biosynthesis (zaragozic acid, SKF 104976, SR 31747, BM 15766, and AY 9944), which allow the synthesis of isoprenoid derivatives and different sets of sterol intermediates, but not cholesterol. The results showed that only the inhibition of sterol Delta7-reductase was compatible with cell proliferation. Blocking cholesterol biosynthesis upstream of this enzyme resulted in the inhibition of cell proliferation and cell cycle arrest selectively in G2/M phase

A triazine-based combinatorial library of small molecules was screened in zebrafish to identify compounds that produced interesting phenotypes. One compound (of 1536 screened) induced a dramatic increase in the pigmentation of early stage zebrafish embryos. This compound, PPA, was also found to increase pigmentation in cultured mammalian melanocytes. The cellular target was identified as the mitochondrial F1F0-ATP synthase (ATPase) by affinity chromatography. Oligomycin, a small molecule known to inhibit the mitochondrial ATPase, competed with PPA for its cellular target in melanocytes. In addition, PPA was shown to alter the membrane potential of mitochondria, consistent with inhibition of the mitochondrial ATPase. Thus, PPA has been successfully used as a chemical probe in a forward chemical genetic approach to establish a link between the phenotype and the protein. The results attest to the power of screening small molecule libraries in zebrafish as a means of identifying mammalian targets and suggest the mitochondrial ATPase as a target for modulating pigmentation in both melanocytes and melanoma cells

For approximately 80 years following Alzheimer's description of the disease that bears his name, a gulf divided researchers who believed that extracellular deposits of the amyloid beta (Abeta) peptide were pathogenic from those who believed that the deposits were secondary detritus. Since 1990, the discoveries of missense mutations in the Abeta peptide precursor (APP) and the APP-cleaving enzyme presenilin 1 (PS1) have enabled much progress in understanding the molecular, cellular, and tissue pathology of the aggregates that accumulate in the interstices of the brains of patients with autosomal dominant familial Alzheimer disease (AD). Clarification of the molecular basis of common forms of AD has been more elusive. The central questions in common AD focus on whether cerebral and cerebrovascular Abeta accumulation is (a) a final neurotoxic pathway, common to all forms of AD; (b) a toxic by-product of an independent primary metabolic lesion that, by itself, is also neurotoxic; or (c) an inert by-product of an independent primary neurotoxic reaction. Antiamyloid medications are entering clinical trials so that researchers can evaluate whether abolition of cerebral amyloidosis can mitigate, treat, or prevent the dementia associated with common forms of AD. Successful development of antiamyloid medications is critical for elucidating the role of Abeta in common AD

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.

The aldose reductase pathway has been demonstrated to be a key component of myocardial ischemia reperfusion injury. Previously, we demonstrated that increased lactate/pyruvate ratio, a measure of cytosolic NADH/NAD+, is an important change that drives the metabolic cascade mediating ischemic injury. This study investigated signaling mechanisms by which the aldose reductase pathway mediates myocardial ischemic injury. Specifically, the influence of the aldose reductase pathway flux on JAK-STAT signaling was examined in perfused hearts. Induction of global ischemia in rats resulted in JAK2 activation followed by STAT5 activation. Pharmacological inhibition of aldose reductase or sorbitol dehydrogenase blocked JAK2 and STAT5 activation and was associated with lower lactate/pyruvate ratio and lower protein kinase C activity. Niacin, known to lower cytosolic NADH/NAD+ ratio independent of the aldose reductase pathway inhibition, also blocked JAK2 and STAT5 activation. Inhibition of protein kinase C also blocked JAK2 and STAT5 activation. Transgenic mice overexpressing human aldose reductase exhibited increased JAK2 and STAT5 activation. Pharmacological inhibition of JAK2 reduced ischemic injury and improved functional recovery similar to that observed in aldose reductase pathway inhibited mice hearts. These data, for the first time, demonstrate JAK-STAT signaling by the aldose reductase pathway in ischemic hearts and is, in part, due to changes in cytosolic redox state