Faculty Bibliography

Cardiovascular disease is one of the prime causes of mortality throughout the world and there is a need for targeted and effective contrast agents to allow noninvasive imaging of the cholesterol-rich atherosclerotic plaques in arteries. A new, fully synthetic, high-density lipoprotein (HDL)-mimicking MRI contrast agent is developed, which enhances macrophage-rich areas of plaque in a mouse model of atherosclerosis by 94%. Confirmation of the targeting of this nanoparticulate agent is achieved using confocal microscopy by tracking a fluorescent lipid incorporated into the nanoparticle.

This article is designed for the general cardiologist, endocrinologist, and internist caring for patients with diabetes and coronary artery disease. Despite the burden of coronary disease in diabetics, little is known about the impact of commonly used oral hypoglycemic agents on cardiovascular outcomes. As the untoward effects of insulin resistance (IR) are increasingly recognized, there is interest in targeting this defect. Insulin resistance contributes to dyslipidemia, hypertension, inflammation, hypercoagulability, and endothelial dysfunction. The aggregate impact of this process is progression of systemic atherosclerosis and an increased risk of adverse cardiovascular outcomes. As such, much attention has been paid to the peroxisome-proliferator-activated receptor gamma (PPARg) agonists rosiglitazone and pioglitazone (thiazolidinediones [TZDs]). Many studies have demonstrated a beneficial effect on the atherosclerotic process; specifically, these agents have been shown to reduce markers of inflammation, retard progression of carotid intimal thickness, prevent restenosis after coronary stenting, and prevent cardiovascular death and myocardial infarction in 1 large trial. Such benefits come at the risk of fluid retention and heart failure (HF) exacerbation, and the net effect on plasma lipids is still poorly understood. Thus, the aggregate risk-benefit ratio is poorly defined. A recent meta-analysis has raised significant concerns regarding the overall cardiovascular safety of 1 particular PPARg agonist (rosiglitazone), prompting international debate and regulatory changes. This review scrutinizes the clinical evidence regarding the cardiovascular risks and benefits of PPARg agonists. Future studies of PPARg agonists, and other emerging drugs that treat IR and diabetes, must be designed to look at cardiovascular outcomes

Chromatin remodeling complexes control the availability of DNA binding sites to transcriptional regulators. Two distinct conserved forms of the SWI/SNF class of complexes are characterized by the presence of specific accessory subunits. In Drosophila, the core Brahma complex associates either with Osa to form the BAP complex or with Bap170 and Bap180 to form the PBAP complex. osa mutations reproduce only a subset of the developmental phenotypes caused by mutations in subunits of the core complex. To test whether the PBAP complex performs the remaining functions, we generated mutations in bap170 and bap180. Surprisingly, we found that Bap180 is not essential for viability, although it is required in ovarian follicle cells for normal eggshell development. Bap170 is necessary to stabilize the Bap180 protein, but a mutant form that retains this function is sufficient for both survival and fertility. The two subunits act redundantly to allow metamorphosis; using gene expression profiling of bap170 bap180 double mutants, we found that the PBAP complex regulates genes involved in tissue remodeling and immune system function. Finally, we generated mutants lacking Bap170, Bap180, and Osa in the germ line to demonstrate that the core Brahma complex can function in oogenesis without any of these accessory subunits

Mononuclear phagocytes (MPCs) at the tumor site can be divided into subclasses, including monocyte-lineage myeloid-derived suppressor cells (MDSCs) and the immunosuppressive tumor-infiltrating macrophages (TIMs). Cancer growth coincides with the expansion of MDSCs found in the blood, secondary lymphoid organs, and tumor tissue. These MDSCs are thought to mature into macrophages and to promote tumor development by a combination of growth-enhancing properties and suppression of local antitumor immunoresponses. As little is known about either subset of MPCs, we investigated MPCs infiltrating into murine adenocarcinoma MCA38 tumors. We found that these MPCs displayed immunosuppressive characteristics and a MDSC cell-surface phenotype. Over 70% of the MPCs were mature (F4/80(+)Ly6C(-)) macrophages, and the rest were immature (F480(+) Ly6C(+)) monocytes. MPC maturation was inhibited when the cells infiltrated a tumor variant expressing IL-2 and soluble TNF type II receptor (sTNFRII). In addition, the IL-2/sTNFRII MCA38 tumor microenvironment altered the MPC phenotype; these cells did not survive culturing in vitro as a result of Fas-mediated apoptosis and negligible M-CSFR expression. Furthermore, CD4(+) tumor-infiltrating lymphocytes (TILs) in wild-type tumors robustly expressed IL-13, IFN-gamma, and GM-CSF, and CD4(+) TILs in IL-2/sTNFRII-expressing tumors expressed little IL-13. These data suggest that immunotherapy-altered Th cell balance in the tumor microenvironment can affect the differentiation and maturation of MPCs in vivo. Furthermore, as neither the designation MDSC nor TIM can sufficiently describe the status of monocytes/macrophages in this tumor microenvironment, we believe these cells are best designated as MPCs

An Ig superfamily cell-adhesion molecule, L1, forms an adhesion complex at the cell membrane containing both signaling molecules and cytoskeletal proteins. This complex mediates the transduction of extracellular signals and generates actin-mediated traction forces, both of which support axon outgrowth. The L1 cytoplasmic region binds ezrin, an adapter protein that interacts with the actin cytoskeleton. In this study, we analyzed L1-ezrin interactions in detail, assessed their role in generating traction forces by L1, and identified potential regulatory mechanisms controlling ezrin-L1 interactions. The FERM domain of ezrin binds to the juxtamembrane region of L1, demonstrated by yeast two-hybrid interaction traps and protein binding analyses in vitro. A lysine-to-leucine substitution in this domain of L1 (K1147L) shows reduced binding to the ezrin FERM domain. Additionally, in ND7 cells, the K1147L mutation inhibits retrograde movement of L1 on the cell surface that has been linked to the generation of the traction forces necessary for axon growth. A membrane-permeable peptide consisting of the juxtamembrane region of L1 that can disrupt endogenous L1-ezrin interactions inhibits neurite extension of cerebellar cells on L1 substrates. Moreover, the L1-ezrin interactions can be modulated by tyrosine phosphorylation of the L1 cytoplasmic region, namely, Y1151, possibly through Src-family kinases. Replacement of this tyrosine together with Y1176 with either aspartate or phenylalanine changes ezrin binding and alters colocalization with ezrin in ND7 cells. Collectively, these data suggest that L1-ezrin interactions mediated by the L1 juxtamembrane region are involved in traction-force generation and can be regulated by the phosphorylation of L1.

A common request of proteomics core facilities is protein identification. However, in some instances primary sequence information for the protein in question is not present in public databases. In other cases, the amino acid sequence of a protein may differ in some way from the sequence predicted from the gene sequence in a database as a result of gene mutation, gene splicing, and/or multiple posttranslational modifications. Thus, it may be necessary to determine the sequence of one or more peptides de novo in order to identify and/or adequately characterize the protein of interest. The primary goal of this study was to give participating laboratories an opportunity to evaluate their proficiency in sequencing unknown peptides that are not included in any published database. Samples containing 3-6 pmol each of five synthetic peptides with amino acid sequences that were not present in public databases were sent to 106 laboratories. One nonstandard amino acid was present in one of the peptides. From a comparison of the results obtained by different strategies, participating laboratories will be able to gauge their own capabilities and establish realistic expectations for the approaches that can be used for this determination

Mechanisms of neuronal loss in Alzheimer's disease (AD) are poorly understood. Here we show that apoptosis is a major form of neuronal cell death in PS/APP mice modeling AD-like neurodegeneration. Pyknotic neurons in adult PS/APP mice exhibited apoptotic changes, including DNA fragmentation, caspase-3 activation, and caspase-cleaved alpha-spectrin generation, identical to developmental neuronal apoptosis in wild-type mice. Ultrastructural examination using immunogold cytochemistry confirmed that activated caspase-3-positive neurons also exhibited chromatin margination and condensation, chromatin balls, and nuclear membrane fragmentation. Numbers of apoptotic profiles in both cortex and hippocampus of PS/APP mice compared with age-matched controls were twofold to threefold higher at 6 months of age and eightfold higher at 21 to 26 months of age. Additional neurons undergoing dark cell degeneration exhibited none of these apoptotic features. Activated caspase-3 and caspase-3-cleaved spectrin were abundant in autophagic vacuoles, accumulating in dystrophic neurites of PS/APP mice similar to AD brains. Administration of the cysteine protease inhibitor, leupeptin, promoted accumulation of autophagic vacuoles containing activated caspase-3 in axons of PS/APP mice and, to a lesser extent, in those of wild-type mice, implying that this pro-apoptotic factor is degraded by autophagy. Leupeptin-induced autophagic impairment increased the number of apoptotic neurons in PS/APP mice. Our findings establish apoptosis as a mode of neuronal cell death in aging PS/APP mice and identify the cross talk between autophagy and apoptosis, which influences neuronal survival in AD-related neurodegeneration

Atomic force microscopy (AFM) observation of a crystal surface of the thermostable isocitrate dehydrogenase (ICDH) from a thermophilic eubacterium, Thermus thermophilus HB8, suggested that the crystal consists of huge homo-complexed ellipsoidal bodies of the protein, with averaged long- and short-axis diameters of 18.6 nm and 10.9 nm respectively. Thick diamond-shaped crystals of about 0.4 mm on the longest axis were obtained by the vapor diffusion method from a solution of 100 mM sodium cacodylate, pH 6.6-8.4, containing 1.4 M sodium acetate as the precipitate, and diffracted X-rays at 3.7 A resolution. The crystals belonged to the monoclinic lattice type with space group C2 and had cell dimensions of a=495.5, b=189.2, c=336.2 A, and beta=126.4 degrees , indicating that an asymmetric unit contained more than 33 molecules with a molecular mass of 54.2 kDa. Calculations based on data obtained by the X-ray method showed good agreement with AFM observation. These results suggest the possibility that the residing T. thermophilus HB8 ICDH molecules are piled one on top another as a preformed supramolecular nano-architecture in the crystal lattice. The system appears suitable for further investigation using a bottom-up approach to the self-associated construction of nano-architectures with proteins.