Faculty Bibliography

Various biological processes exhibit characteristics that vary dramatically in response to different input conditions or changes in the history of the process itself. One of the examples studied here, the Ras-PKC-mitogen-activated protein kinase (MAPK) bistable pathway, follows two distinct dynamics (modes) depending on duration and strength of EGF stimulus. Similar examples are found in the behavior of the cell cycle and the immune system. A classification methodology, based on time-frequency analysis, was developed and tested on these systems to understand global behavior of biological processes. Contrary to most traditionally used statistical and spectral methods, our approach captures complex functional relations between parts of the systems in a simple way. The resulting algorithms are capable of analyzing and classifying sets of time-series data obtained from in vivo or in vitro experiments, or in silico simulation of biological processes. The method was found to be considerably stable under stochastic noise perturbation and, therefore, suitable for the analysis of real experimental data

Genomes hold within them the record of the evolution of life on Earth. But genome fusions and horizontal gene transfer (HGT) seem to have obscured sufficiently the gene sequence record such that it is difficult to reconstruct the phylogenetic tree of life. HGT among prokaryotes is not random, however. Some genes (informational genes) are more difficult to transfer than others (operational genes). Furthermore, environmental, metabolic, and genetic differences among organisms restrict HGT, so that prokaryotes preferentially share genes with other prokaryotes having properties in common, including genome size, genome G+C composition, carbon utilization, oxygen utilization/sensitivity, and temperature optima, further complicating attempts to reconstruct the tree of life. A new method of phylogenetic reconstruction based on gene presence and absence, called conditioned reconstruction, has improved our prospects for reconstructing prokaryotic evolution. It is also able to detect past genome fusions, such as the fusion that appears to have created the first eukaryote. This genome fusion between a deep branching eubacterium, possibly an ancestor of the cyanobacterium and a proteobacterium, with an archaeal eocyte (crenarchaea), appears to be the result of an early symbiosis. Given new tools and new genes from relevant organisms, it should soon be possible to test current and future fusion theories for the origin of eukaryotes and to discover the general outlines of the prokaryotic tree of life.

Gene signatures that predict aggressive tumor behavior at the earliest stages of disease, ideally before overt tissue abnormalities, are urgently needed. To search for such genes, we generated a transgenic model of survivin, an essential regulator of cell division and apoptosis overexpressed in cancer. Transgenic expression of survivin in the urinary bladder did not cause histologic abnormalities of the urothelium. However, microarray analysis revealed that survivin-expressing bladders exhibited profound changes in gene expression profile affecting extracellular matrix and inflammatory genes. Following exposure to a bladder carcinogen, N-butyl-N-(4-hydroxybutyl) nitrosamine (OH-BBN), survivin transgenic animals exhibited accelerated tumor progression, preferential incidence of tumors as compared with premalignant lesions, and dramatically abbreviated survival. Conversely, transgenic expression of a survivin Thr(34)-->Ala dominant-negative mutant did not cause changes in gene expression or accelerated tumor progression after OH-BBN treatment. Therefore, survivin expression induces global transcriptional changes in the tissue microenvironment that may promote tumorigenesis. Detection of survivin or its associated gene signature may provide an early biomarker of aggressive tumor behavior before the appearance of tissue abnormalities

The precise genetic and molecular defects underlying epithelial ovarian cancer (EOC) remain largely unknown, and treatment options for patients with advanced disease are limited. Cyclooxygenases (COX-1 and COX-2) catalyze the conversion of arachidonic acid to prostaglandins. Whereas overwhelming evidence suggests a role for COX-2 in a variety of cancers, the contribution of COX-1 remains much less explored. The expression status of COX isoforms in ovarian cancers also remains confusing. We have previously shown that human epithelial ovarian tumors have increased levels of COX-1 but not COX-2. To more carefully examine the role of COXs in ovarian cancer, we used a mouse model of EOC in which genetic and oncogenic modifications were experimentally engineered into ovarian surface epithelial cells (OSE) thought to be the cells of origin for human EOC. These OSE cells produce tumors when allografted into host mice. Using multiple approaches, we observed that OSE cells and the tumors comprised of these cells express high levels of COX-1 but not COX-2. Prostacyclin (PGI(2)) is the major prostaglandin generated downstream of COX-1 in these cells, and SC-560, a COX-1-selective inhibitor, dramatically inhibits PGI(2) production. More importantly, SC-560 reduced the growth of tumors when OSE cells were allografted in nude female mice. In contrast, the COX-2-selective inhibitor celecoxib had little effect on tumor growth. The growth inhibitory effects of SC-560 result from reduced cell proliferation and/or accelerated apoptosis. Our results imply COX-1 as a target for the prevention and/or treatment of EOC.

Re-epithelialization of wounded skin is necessary for wound closure and restoration of barrier function and requires directional keratinocyte migration towards the center of the wound. The electric field (EF) generated immediately upon wounding could be the earliest signal keratinocytes receive to initiate directional migration and healing. Keratinocytes express many beta2-adrenergic receptors (beta2-ARs), but their role in the epidermis is unknown. We have previously shown that beta-AR agonists decrease keratinocyte migration in a cyclic AMP (cAMP) independent mechanism involving the activation of protein phosphatase 2A (PP2A). Here, we ask whether beta2-ARs play a role in keratinocyte galvanotaxis. We report a bimodal response. When keratinocytes were exposed to higher concentrations of beta-AR agonist (0.1 microM), their tracked migratory speed was inhibited, in both the presence (directional migration) and the absence (random migration) of a 100 mV mm(-1) EF, as expected. At lower agonist concentrations (0.1 pM to 0.1 nM), there was no effect on migratory speed; however, all directionality was lost - essentially, cells were 'blinded' to the directional cue. Preincubating the cells with beta-antagonist restored directional migration, demonstrating that the 'blindness' was beta2-AR mediated. Incubation of keratinocytes with agents known to increase intracellular cAMP levels, such as sp-cAMP, pertussis toxin and forskolin, resulted in similar 'blinding' to the EF, whereas random migration was unaffected. The inactive cAMP analog rp-cAMP had no effect on keratinocyte migration, whether directional or random. However, rp-cAMP pretreatment before beta-agonist addition fully restored galvanotaxis, demonstrating the complete cAMP dependence of the attenuation of keratinocyte directional migration. This is the first report that cAMP is capable of mediating keratinocyte galvanotaxis. beta-AR agonists and antagonists could be valuable tools for modulating re-epithelialization, an essential step in the wound-healing process. Thus, beta-ARs regulate the two distinct components of keratinocyte directional migration differently: migration speed via a cAMP-independent mechanism and galvanotaxis by a cAMP-dependent one

cDNAs encoding a high-affinity sulfate transporter and an adenosine 5'-phosphosulfate reductase from potato (Solanum tuberosum L. cv Desiree) have been cloned and used to examine the hypothesis that sulfate uptake and assimilation is transcriptionally regulated and that this is mediated via intracellular O-acetylserine (OAS) pools. Gas chromotography coupled to mass spectrometry was used to quantify OAS and its derivative, N-acetylserine. Treatment with external OAS increased sulfate transporter and adenosine 5'-phosphosulfate reductase gene expression consistent with a model of transcriptional induction by OAS. To investigate this further, the Escherichia coli gene cysE (serine acetyltransferase EC 2.3.1.30), which synthesizes OAS, has been expressed in potato to modify internal metabolite pools. Transgenic lines, with increased cysteine and glutathione pools, particularly in the leaves, had increased sulfate transporter expression in the roots. However, the small increases in the OAS pools were not supportive of the hypothesis that this molecule is the signal of sulfur (S) nutritional status. In addition, although during S starvation the content of S-containing compounds decreased (consistent with derepression as a mechanism of regulation), OAS pools increased only following extended starvation, probably as a consequence of the S starvation. Taken together, expression of these genes may be induced by a demand-driven model, via a signal from the shoots, which is not OAS. Rather, the signal may be the depletion of intermediates of the sulfate assimilation pathway, such as sulfide, in the roots. Finally, sulfate transporter activity did not increase in parallel with transcript and protein abundance, indicating additional posttranslational regulatory mechanisms.

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

The Hedgehog-Gli (Hh-Gli) signaling pathway controls many aspects of tissue patterning, cell proliferation, differentiation and regeneration and regulates cell number in various organs. In adults, the Hh-Gli pathway remains active in a number of stem cells and regenerating tissues. Inappropriate and uncontrolled HH-GLI pathway activation has been demonstrated in a variety of human cancers. Three recent papers show that components of the pathway are expressed in human prostate tumors and, more importantly, that prostate cancers depend on sustained HH-GLI signaling. These data raise the possibility of a new therapeutic approach to treat this often lethal disease.

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.

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.