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We have used a novel estrogen receptor-(ER) negative, mixed basal/luminal, aggressive cell line, TMX2-28, as a model to study breast cancer. cDNA microarray comparison of TMX2-28 and its parent non-aggressive, ER-positive cell line, MCF-7, identified 1402 differentially expressed transcripts. Two-hundred upregulated transcripts were sorted by biological function and the expression of selected genes was assessed in TMX2-28 cells, MCF-7 cells, non-tumorigenic human mammary epithelial cells (HMECs), and thirty frozen human breast carcinoma specimens using real time RT-PCR. Four genes were selected for further studies: phospholipase D1 (PLD1), mitogen-inducible gene 2 (MIG2), S-phase kinase-associated protein 2 (SKP2), and paralemmin (PALM). PLD1 mRNA is expressed ten times more in TMX2-28 cells than in MCF-7 cells, and PLD1 is moderately expressed in non-tumorigenic HMEC lines 184, 184A1 and 184AA2. PLD1 mRNA levels were higher in breast tumors that expressed high mRNA levels of basal CKs 5 and/or 17. PLD1 protein was overexpressed in 10 of 42 (24%) breast tumors examined by IHC. Evaluating the expression of PLD1 with other signaling molecules, phospho-Akt and phospho-mTOR, we found that five PLD1-positive tumors were negative for phospho-Akt expression, but positive for phospho-mTOR expression. MIG2 is overexpressed 17-fold in TMX2-28 cells compared to non-aggressive MCF-7 cells and HMECs. MIG2 showed high mRNA levels in 30% of the human breast carcinoma specimens. siRNA-mediated suppression of MIG2 in TMX2-28 cells reduced TMX2-28 cell invasion by 48% compared to cells transfected with siRNAs against GAPDH. We also found that MIG2 protein was expressed in half of the breast tumors tested by IHC and showed heterogeneous expression among 21 tissues from reduction mammoplasty. SKP2 is overexpressed in TMX2-28 cells compared to MCF-7 cells by 13-fold, and SKP2 was not expressed in the HMEC lines. SKP2 mRNA levels were higher in breast tumors that expressed basal cytokeratins (CK) 5 and/or 17. Lastly, PALM is overexpressed in TMX2-28 breast cancer cells compared to MCF-7 and HMECs. We further evaluated the expression of PALM in human breast carcinomas and found that PALM is highly expressed on the plasma membrane of cells in roughly half of the breast tumors.

The import of proteins into chloroplasts is a tightly regulated process that ultimately affects the development both of the plant and the plastid. The Toc159 family of chloroplast outer membrane GTPases has been shown to be a preprotein receptor. The small family of four receptors define distinct entry translocons. In this report, I provide evidence that the large N-terminal A-domain of atToc159 family proteins, working in conjunction with the GTPase domains determines the formation of these translocons.

Workshop cluster 1 (WC1) molecules are exclusively expressed on the surface of γδ T cells. They belong to the scavenger receptor cysteine-rich (SRCR) superfamily and are encoded by a multiple-gene family. WC1 molecules are divided into three major groups, WC1.1, WC1.2 and WC1.3, on the basis of antibody reactivity. The expression of WC1 molecules from these groups correlates with differences in γδ T cell responses. Particularly, the expression of receptors within the serologically-defined WC1.1 group correlates with the capacity to respond to Leptospira antigen. The potential role of WC1 as a co-stimulatory molecule for the γδ TCR is suggested by the presence of several tyrosine-based motifs in their intracellular domains. In this study, we found that WC1 was constitutively phosphorylated in ex vivo bovine γδ T cells and associated with src family tyrosine kinases. Crosslinking of WC1 molecules resulted in an increase in WC1 phosphorylation and co-crosslinking of WC1 and γδ TCR together prolonged WC1 phosphorylation. We identified the second tyrosine residue on the WC1 intracellular tail as the primary phosphorylation target in WC1.1 and WC1.2 intracellular sequences in both in vitro and in vivo assays. And phosphorylation of the second tyrosine was required for the WC1-mediated potentiation of TCR-induced T cell proliferation, suggesting that WC1 acts as a co-stimulatory molecule for γδ TCR. The cytoplasmic tails of WC1.1 and WC1.2 were also phosphorylated on serine and PKC activity was required for phosphorylation-dependent endocytosis of WC1.1 or WC1.2. Finally, we used RNA interference to directly investigate the role of WC1 expression in the response to Leptospira borgpeterseneii . We found that when a subset of WC1 transcripts were down-regulated by RNA interference, the proliferation of cells in response to Leptospira antigen and the production of IFN-γ was significantly reduced. Our data directly demonstrate that the co-receptors in the WC1 family act as an essential component for Leptospira recognition and/or activation of γδ T cells.

Translocation is the process by which the ribosome advances the mRNA:tRNA complex by a one-codon step during the elongation cycle of translation. In the pre-translocation state, the ribosome transitions between two metastable states, Global state 1(GS1 or classical state) and Global state 2 (GS2 or hybrid state). EF-G interacts with GS2 and catalyzes translocation. The dynamics of the L1 stalk of the 50S subunit E-site, which is composed of a portion of the 23S rRNA and protein L1, are finely tuned to the GS1 to GS2 transitions. Interaction of the L1 stalk with the elbow of the P/E tRNA both stabilizes the tRNA and facilitates its passage from the P/E to the E/E site. Disruption of these interactions should destabilize the hybrid state, or GS2, and reduce the efficiency of translocation. Mutations were made in the 23S rRNA of the L1 stalk, with the aim of interfering with segments that contribute to its function. The segments targeted for mutagenesis were: (1) The L1 binding fold, which binds protein L1 and also interacts with the elbow of the deacylated tRNA. (2) Helix 76 (H76), the flexibility of which enables the dynamics of the L1 stalk. (3) Helix 79 (H79), which supports the L1 stalk and may coordinate the mobility of the stalk with the functional state of the ribosome. Mutants in which the L1-binding fold was likely to be disrupted, were mostly dominant lethal. Here, the loss of interaction of the L1 stalk with the tRNA may increase the thermodynamic barrier for the mutant ribosomes to attain GS2, such that the efficiency of translocation falls below a growth sustaining threshold. Diminishing the flexibility of H76 did not produce any observable defect. These mutants, which can support growth in a strain lacking all wild-type rRNA, displayed only mild growth defects or mild subunit association defects. Partial deletion of H79 also had no affect on the ability of the ribosome to support growth in the absence of wild-type rRNA, although it lead to defects in subunit association, suggesting a role for H79 in the formation of intersubunit bridge/s. In general, mutants of the 23S rRNA component of the L1 stalk that supported cell growth as the sole source of rRNA did not affect the efficiency or accuracy of translocation, or of reverse translocation, in vitro. Although they may affect the rates of GS1 to GS2 transitions, these mutants do not appear to impair life-supporting translational rates.

Aminoacylphosphatidylglycerol synthases (aaPGSs) are integral membrane proteins that use aminoacyl-tRNAs as substrates to catalyze the addition of amino acids to phosphatidylglycerol (PG) in the cytoplasmic membranes of bacteria. Addition of amino acids to PG decreases the net negative charge of the membrane, conferring resistance to various classes of antibacterial agents (i.e., cationic antimicrobial peptides, beta-lactams, glycopeptides, and lipopeptides) and protecting the cell against osmotic stress and acidic conditions. aaPGS homologs are found in a variety of clinically relevant microorganisms, including Enterococcus faecium, which is increasingly found to be the etiologic agent of antibiotic-resistant nosocomial infections. Although the broad distribution of these virulence factors across bacterial species makes them attractive targets for therapeutic strategies, little is known about the structure of aaPGSs. Two aaPGS paralogs are found in E. faecium, one of which exhibits relaxed substrate specificity and is responsible for the transfer of Arg (R), Ala (A), and Lys (K) to PG (RakPGS). The catalytic site of RakPGS is located in the hydrophilic C-terminal domain, which is localized in the cytoplasm. The N-terminus contains an integral membrane domain that is thought to harbor flippase activity that translocates the neosynthesized aa-PG from the inner to the outer leaflet of the membrane. We are currently developing the substituted cysteine accessibility method (SCAM) and a dual-reporter fusion system, which exploits alkaline phosphatase (Pho) and β-galactosidase (LacZ) activities, for investigating the membrane topology of RakPGS in E. faecium.

Prostate cancer is the second leading cause of cancer-related death, and the most commonly diagnosed type of cancer in men. By conventional paradigm, serine proteases have been shown to play a significant role in cancer cell invasion. Prostasin is a serine protease found in abundance in prostate epithelial cells, and also in other tissues and cell types in the body. Prostasin is absent in prostate cancer cell lines. When re-expressed in these prostate cancer cell lines, the invasiveness was decreased. The structural domain responsible for the anti-invasion activity exhibited by prostasin has not yet been characterized. Through site-directed mutagenesis of the serine active residue to an alanine residue, the role that the serine active site plays in anti- invasion can be explored. The cDNA clone of the mutated prostasin was transfected into the 293-EBNA cell line and cultured (293/ProM). The alanine-prostasin mutant, Ala-Pro, was partially purified and confirmed by Western blotting. Additionally, a binding assay was performed using mPBP (mouse prostasin-binding protein), a serpin class serine protease inhibitor. By way of immunoblotting, it was shown that normal wild-type prostasin forms an 82-kDa complex with the mPBP; however, the mutated prostasin does not. In future studies the invasiveness of prostate cancer cells transfected with the mutant prostasin cDNA can be investigated to determine whether prostasin's serine protease activity provides a mechanism for anti-invasion.

Molecular imaging modalities are important tools to evaluate the efficacy of gene delivery systems and cell-based therapies. Development and application of these modalities will advance our understanding of the mechanism of transgene expression and cell fate and functions. Physical gene transfer methods hold many advantages over viral vectors among gene therapeutic strategies. Here, we evaluated the efficacy of biolistic (“gene gun”) gene targeting to tissues with non-invasive bioluminescence imaging (BLI) methods. Plasmids carrying the firefly luciferase reporter gene were transfected into mouse skin and liver using biolistics, and BLI was measured at various time points after transfer. With optimized DNA loading ratio (DLRs), reporter gene expression reached to peak 1day after transfer to mouse skin, and the maximum depth of tissue penetration was between 200-300μm. Similar peak expression of reporter gene was found in mouse liver but the expression was relatively stable 4-8 days postbiolistic gene transfer and remained for up to two weeks afterward. Our results demonstrated BLI was an efficient strategy for evaluation of reporter gene expression in the same animals over a period of up to two weeks in vivo. Different tissues showed different expression kinetics, suggesting that this is an important parameter to consider when developing gene therapy strategies for different target tissues. We also employed BLI to measure differentiation of mouse embryonic stem (ES) cells into beating cardiomyocytes in vitro and in vivo. A subset of these cardiomyocytes appears to be derived from an adrenergic lineage that ultimately contribute to iv substantial numbers of cardiomyocytes primarily on the left side of the heart. At present, it is unclear what the precise role of these cardiac adrenergic cells is with respect to heart development, though it is known that adrenergic hormones (adrenaline and noradrenaline) are essential for embryonic development since mice lacking them die from apparent heart failure during the prenatal period. To identify and characterize cardiac adrenergic cells, we developed a novel mouse genetic model in which the nuclear-localized enhanced green fluorescent protein (nEGFP) reporter gene was targeted to the first exon of the Phenylethanoamine N-transferase (Pnmt) gene, which encodes for the enzyme that converts noradrenaline to adrenaline, and hence serves as a marker for adrenergic cells. Our results demonstrate this knock-in strategy effectively marked adrenergic cells in both fetal and adult mice. Expression of nEGFP was found in Pnmt-positive cells of the adult adrenal medulla, as expected. Pnmt-nEGFP expression also recapitulated endogenous Pnmt expression in the embryonic mouse heart. In addition, nEGFP and Pnmt expression were induced in parallel during differentiation of pluripotent mouse ES cells into beating cardiomyocytes. This new mouse genetic model provides a useful new tool for studying the properties of adrenergic cells in different tissues. We also identified two limitations of the Pnmt-nEGFP model. One is that the amount of nEGFP expressed within individual adrenergic cells was highly variable. Secondly, expression of nEGFP in the embryonic heart was of low abundance and difficult to distinguish from background autofluorescence. To overcome these limitations, we developed two alternative genetic models to investigate adrenergic cells: v (1) Mouse embryonic stem cells, which have been previously targeted with Pnmt-Cre recombinase gene, were additionally targeted with a dual reporter plasmid which covered both a loxP-flanked cDNA of red fluorescence protein (HcRed) and also EGFP. Under the undifferentiated status, cells emit red fluorescence as transcription stops before EGFP coding sequence. After differentiation into beating cardiomyoctyes, some cells switch fluorescence from red to green, indicating that excision of loxP-flanked sequences by Cre since Pnmt had been activated. (2) A surface marker, truncated lowaffinity nerve growth factor receptor (ΔLNGFR) was used as the reporter gene as cells expressing this marker can be enriched by magnetic-activated cell sorting (MACS), a potentially efficient way to yield highly purified positive cells at low input abundance in a population. Through a series of subcloning steps, the targeting construct, Pnmt- ΔLNGFR-Neo-DTA was created and electroporated into 7AC5EYFP embryonic stem cells. Correctly targeted cells were selected by positive and negative screening. These cells provide a new tool with which to identify, isolate, and characterize the function of adrenergic cells in the developing heart, adrenal gland, and other tissues where adrenergic cells make important contributions.

Parkinson’s disease (PD) is a debilitating neurodegenerative disorder affecting one million Americans. Despite its social and economic impact, the pathological cascades that lead to neuron dysfunction and degeneration in PD are poorly understood. Endoplasmic reticulum (ER) stress has been implicated as an initiator or contributing factor in neurodegenerative diseases including PD. The ER is an organelle central to protein folding and intracellular Ca2+ homeostasis. Perturbations of these functions result in ER stress and upregulation of ER stress proteins, of which some have been implicated in counteracting ER stress-induced cell death. The mechanisms that lead to ER stress and how ER stress proteins contribute to the degenerative cascades remain unclear but their understanding is critical to devising effective therapies for PD. Both the accumulation of mutant -synuclein (Syn), which causes an inherited form of PD, and the inhibition of mitochondrial complex I function by PDinducing neurotoxin lead to ER stress. The critical involvement of ER stress in experimental models of PD supports its potential relevance to PD pathogenesis and led us to test the hypothesis whether the homocysteine-inducible ER protein (Herp), an ubiquitin-like domain (UBD) containing ER-resident protein, can counteract mutant Syn- and neurotoxin- induced pathological cascades. In the first part of my study I showed that knockdown of Herp aggravates ER stress-mediated cell death induced by PD-linked mutant Syn. Functionally, Herp plays iv a role in maintaining ER homeostasis by facilitating proteasome-mediated degradation of ER-resident Ca2+ release channels in a neuronal-like cell line expressing the mutant A53T-Syn. Deletion of UBD or pharmacological inhibition of the proteasomes abolishes the Herp-mediated stabilization of ER Ca2+ homeostasis. Furthermore, knockdown or pharmacological inhibition of ER Ca2+ release channels ameliorates ER stress suggesting that impaired homeostatic regulation of Ca2+ channels promotes a protracted ER stress with the consequent activation of ER stress-associated cell death pathways. Interestingly, sustained upregulation of ER stress markers and aberrant accumulation of ER Ca2+ release channels were detected in transgenic mutant A53T- Syn mice. These data establish a causative link between impaired ER Ca2+ homeostasis and chronic ER stress in the degenerative cascades induced by mutant A53T-Syn and suggest that Herp is essential for the resolution of ER stress through maintenance of ER Ca2+ homeostasis. Because oxidants and mitochondria-derived free radicals can target ER-based Ca2+ regulatory proteins and cause uncontrolled Ca2+ release that may contribute to protracted ER stress resulting in cell death, I next determined the impact of the PD causing neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), the precursor of 1-methyl-4-phenylpyridinium (MPP+ ) on ER functions. I demonstrated that knockdown of Herp renders dopaminergic cells vulnerable to MPP+ -induced toxicity by a mechanism involving upregulation of CCAAT/enhancer binding protein homologous protein (CHOP) and depletion of the ER Ca2+ store. Conversely, ectopic expression of Herp confers protection by blocking MPP+ -induced CHOP upregulation, ER Ca2+ store depletion and mitochondrial Ca2+ accumulation in a manner dependent on a functional v ubiquitin-proteasomal protein degradation pathway. Deletion of the UBD or treatment with a proteasomal inhibitor abolished the central function of Herp in ER Ca2+ homeostasis. Collectively, our findings suggest that approaches that aim to increase Herp levels or its ER Ca2+ -stabilizing action may prevent or ameliorate neuronal loss in PD. Though abnormal protein aggregates are characteristic features of the slowly progressive neurodegenerative disorders, they are also found in acute pathological states such as cerebral ischemia. The role of protein aggregation in neuronal pathology after brain ischemia is not clear. In the last part of my work, I show that transient focal ischemia induces the continuous accumulation of insoluble Syn and DJ-1, two proteins linked to early-onset PD, in vulnerable neurons from the onset of reperfusion until delayed neuronal death. Double immunocytochemical analysis reveals that Syn and DJ-1 are co-localized in inclusion-like structures in the vulnerable neurons of the lesioned cortices suggesting that DJ-1 is recruited into the Syn-containing inclusions and thereby precludes this neuroprotective protein from exercising its anti-oxidant and chaperone-like activities. Supporting this notion, knockdown of DJ-1 promotes Syn insolubility and renders neurons vulnerable to an ischemic insult whereas ectopic expression of DJ-1 ameliorates Syn -induced degenerative cascades and reverses ischemic neuronal injury. Furthermore, mice deficient in Syn exhibit significantly smaller infarcts and improved behavioral recovery after ischemia compared to nontransgenic mice. Ablation of Syn ameliorates the accumulation of insoluble DJ-1 and the ensuing oxidative damage following an ischemic insult. Taken together, our data show that aberrant accumulation of Syn plays a precipitating role in ischemic neuronal vi injury and suggest that PD-causing mutations in Syn and DJ-1 can worsen ischemic brain damage. In conclusion, these studies provide insights into the molecular cascade of Syninduced degeneration and may uncover novel therapeutic strategies for PD and stroke.

We report a mechanism based screening technique to rapidly identify eukaryotic topoisomerase I targeting agents. The method is based on genetic tagging of topoisomerase I to immobilize the enzyme on a solid surface in a microtiter well format. DNA is added to the wells and retained DNA is detected by Picogreen fluorescence. Compounds that result in an increase in Picogreen staining represent potential topoisomerase interfacial poisons while those that reduce fluorescence report catalytic inhibitors; therefore, the solid phase assay represents a „bimodal‟ readout that reveals mechanisms of action. The method has been demonstrated to work with known interfacial poisons and catalytic inhibitors. In addition to specific topoisomerase targeting drugs, the method also weakly detects other relevant anticancer agents, such as potent DNA alkylating and intercalating compounds; therefore, topoisomerase I HTS represents an excellent tool for searching and identifying novel genotoxic agents. This method is rapid, robust, economical and scalable for large library screens.