Faculty Bibliography

The phosphoprotein (P protein) of vesicular stomatitis virus (VSV) is an essential subunit of the viral RNA-dependent RNA polymerase complex and plays a central role in viral transcription and replication. Using both the yeast two-hybrid system and coimmunoprecipitation assays, we confirmed the self-association of the P protein of Indiana serotype (Pind) and heterotypic interaction between Pind and the P protein of New Jersey serotype (Pnj). Furthermore, by using various truncation and deletion mutants of Pind, the self-association domain of the Pind protein was mapped to amino acids 161 to 210 within the hinge region. The self-association domain of Pind protein is not required for its binding to nucleocapsid and large proteins. We further demonstrated that the self-association domain of Pind protein is essential for VSV transcription in a minireplicon system and that a synthetic peptide spanning amino acids 191 to 210 in the self-association domain of Pind protein strongly inhibited the transcription of the VSV genome in vitro in a dose-dependent manner. These results indicated that the self-association domain of Pind protein plays a critical role in VSV transcription.

Macroautophagy, a lysosomal pathway responsible for the turnover of organelles and long-lived proteins, has been regarded mainly as an inducible process in neurons, which is mobilized in states of stress and injury. New studies show, however, that macroautophagy is also constitutively active in healthy neurons and is vital to cell survival. Neurons in the brain, unlike cells in the periphery, are protected from large-scale autophagy induction because they can use several different energy sources optimally, receive additional nutrients and neurotrophin support from glial cells, and benefit from hypothalamic regulation of peripheral nutrient supplies. Due to its exceptional efficiency, constitutive autophagy in healthy neurons proceeds in the absence of easily detectable autophagic vacuole intermediates. These intermediates can accumulate rapidly, however, when late steps in the autophagic process are blocked. Autophagic vacuoles also accumulate abnormally in affected neurons of several major neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease, where they have been linked to various aspects of disease pathogenesis including neuronal cell death. The build-up of autophagic vacuoles in these neurological disorders and others may reflect either heightened autophagy induction, impairment in later digestive steps in the autophagy pathway, or both. Determining the basis for AV accumulation is critical for understanding the pathogenic significance of autophagy in a given pathologic state and for designing possible therapies based on modulating autophagy. In this review, we discuss the special features of autophagy regulation in the brain, its suspected roles in neurodevelopment and plasticity, and recent progress toward understanding how dysfunctional autophagy contributes to neurodegenerative disease.

The confluence of recent advances in microscopy instrumentation and image analysis, coupled with the widespread use of GFP-like proteins as reporters of gene expression, has opened the door to high-throughput in vivo studies that can provide the morphological and temporal context to the biochemical pathways regulating cell function. We are now able to quantify the concentration and three-dimensional distribution of multiple spectrally resolved GFP-tagged proteins. Using automatic segmentation and tracking we can then measure the dynamics of the processes in which these elements are involved. In this way, parallel studies are feasible where multiple cell colonies treated with drugs or gene expression repressors can be monitored and analyzed to study the dynamics of relevant biological processes

PURPOSE OF REVIEW: Amyloid deposits are a defining feature of several age-related and debilitating diseases. Their widespread presence in atherosclerotic plaques suggests a potential role in lesion development. This review discusses the proteins known to accumulate in atheroma and examines the evidence that amyloid-like structures activate macrophage signaling pathways linked to inflammation and prothrombotic potential. RECENT FINDINGS: Numerous proteins that accumulate in atherosclerotic plaques form amyloid fibrils in vivo, including apolipoproteins, beta-amyloid, and alpha1-antitrypsin. In addition, oxidation or enzymatic modification of low-density lipoproteins induces a structural reorganization of the particle, including the acquisition of amyloid-like properties. Similarly, glycation of serum albumin, as observed in diabetes, is accompanied by the formation of aggregates with all the hallmarks of amyloid. Several receptors implicated in atherogenesis modulate the fate of amyloid fibrils by mediating their clearance (scavenger receptors A and B-I), activating inflammatory signaling cascades (receptor for advanced glycation endproducts), or both (CD36). Finally, recent studies indicate that amyloid deposition accelerates diet-induced atherosclerosis in mice. SUMMARY: Given the substantial evidence that amyloid fibrils or preamyloidogenic species are cytotoxic, the aberrant deposition of amyloid in the intima may be pathologically important in vascular inflammation and the promotion of atherosclerosis

Use of a dished polyethylene insert in 114 total knee arthroplasties, all with the posterior cruciate ligament resected or recessed, was retrospectively studied. Patients were evaluated at a mean follow-up of 8.3 years. Mean range of motion increased from 92 degrees to 111 degrees . Mean Knee Society pain and function scores increased from 35.2 and 39.7 to 91.3 and 74.7, respectively. WOMAC scores improved significantly in each category evaluated, including pain, stiffness, and physical function. Kaplan-Meier survivorship was 95% at 10 years (95% confidence interval, 82%-99%). The use of a dished polyethylene insert in primary total knee arthroplasty provides good to excellent midterm results regardless of whether the posterior cruciate ligament is recessed or sacrificed

The need for more specific and selective contrast agents for magnetic resonance imaging motivated us to prepare a new nanoparticle agent based on high-density lipoproteins (HDL). This second generation contrast agent can be prepared in three different ways. The HDL nanoparticles (rHDL) were fully characterized by FPLC and gel electrophoresis. The flexibility of the platform also allows us to incorporate optical probes into rHDL for localization ex vivo by confocal fluorescence microscopy. The contrast-agent-containing nanoparticles were injected into mice that develop atherosclerotic lesions. Magnetic resonance imaging of the animals showed clear enhancement of the atherosclerotic plaques.

Inflammation resultant from tumor growth, infection with certain pathogens, or in some cases, trauma, can result in systemic release of cytokines, especially GM-CSF, that in turn stimulate the abundant production and activation of a population of immature myeloid cells, termed myeloid suppressor cells (MSCs), that have potent immunosuppressive functions. In this issue of the JCI, Gallina and colleagues have illuminated some complex issues concerning the development, activation, and function of MSCs (see the related article beginning on page 2777). They show that activation of MSCs is initiated in response to IFN-gamma, presumably produced in situ by antitumor T cells in the tumor microenvironment. After this triggering event, MSCs express 2 enzymes involved in l-arginine metabolism, Arginase I and iNOS, whose metabolic products include diffusible and highly reactive peroxynitrites, the ultimate biochemical mediators of T cell immune suppression. The multifaceted regulation of this complex suppressive effector system provides several potential therapeutic targets

OBJECTIVE: Clinical scales evaluating arm function after stroke are weak at detecting quality of movement. Therefore a new scale, the Motor Evaluation Scale for Upper Extremity in Stroke Patients (MESUPES), was developed, comprising 22 items pertaining to arm and hand performance. The scale was investigated for validity and unidimensionality using the Rasch measurement model, and for inter-rater reliability. SETTING: Twelve hospitals and rehabilitation centres in Belgium, Germany and Switzerland. PATIENTS: There were 396 patients (average age 63.38+/-12.89 years) in the Rasch study and 56 patients (average age 65.68+/-12.75 years) in the reliability study. MAIN MEASURES: The scale was examined on its fit to the Rasch model, thereby evaluating the scale's unidimensionality and validity. Differential item functioning was performed to test the stability of item hierarchy on several variables. Inter-rater reliability was examined with kappa values, weighted percentage agreement and intraclass correlation coefficients (ICC). RESULTS: Based on Rasch analysis, five items were removed. The MESUPES was divided in two tests: the MESUPES-arm test (8 items) and MESUPES-hand test (9 items). Both scales fitted the Rasch model. All items were stable among the subgroups of the sample. ICCs were 0.95 (95% confidence interval (CI) 0.91 -0.97) and 0.97 (95% CI 0.95-0.98) for the total score on arm and hand test respectively. The scale was also reliable at item level (weighted kappa 0.62 -0.79, weighted percentage agreement 85.71 -98.21). CONCLUSION: The MESUPES-arm and MESUPES-hand meet the statistical properties of reliability, validity and unidimensionality. Both tests provide a useful clinical and research tool to qualitatively evaluate arm and hand function during recovery after stroke.

BACKGROUND AND PURPOSE: No treatments have been identified to lower the risk of intracerebral hemorrhage due to cerebral amyloid angiopathy (CAA). A potential approach to prevention is the use of agents that interfere with the pathogenic cascade initiated by the beta-amyloid peptide (Abeta). Tramiprosate (3-amino-1-propanesulfonic acid) is a candidate molecule shown in preclinical studies to reduce CAA in a transgenic mouse model. METHODS: We performed a 5-center phase 2 double-blinded trial to evaluate the safety, tolerability, and pharmacokinetics of tramiprosate in subjects with lobar intracerebral hemorrhage. Twenty-four subjects age > or =55 years with possible or probable CAA were randomized to receive 12 weeks of tramiprosate at 1 of 3 oral doses (50, 100, or 150 mg twice daily). Subjects were followed for clinical adverse effects, laboratory, vital sign, electrocardiogram, cognitive, or functional changes, appearance of new symptomatic or asymptomatic hemorrhages, and pharmacokinetic parameters. RESULTS: Enrolled subjects were younger (mean age 70.8+/-5.4, range 61 to 78) and had more advanced baseline disease (measured by number of previous hemorrhages) than consecutive subjects in a CAA natural history cohort. No concerning safety issues were encountered with treatment. Nausea and vomiting were the most common adverse events and were more frequent at high doses. Nine subjects had new symptomatic or asymptomatic hemorrhages during treatment; all occurred in subjects with advanced baseline disease, with no apparent effect of drug dosing assignment. CONCLUSIONS: These data suggest that tramiprosate can be given safely to subjects with suspected CAA and support future efficacy trials.

The extracellular space is an environment hostile to unmodified polypeptides. For this reason, many eukaryotic proteins destined for exposure to this environment through secretion or display at the cell surface require maturation steps within a specialized organelle, the endoplasmic reticulum (ER). A complex homeostatic mechanism, known as the unfolded protein response (UPR), has evolved to link the load of newly synthesized proteins with the capacity of the ER to mature them. It has become apparent that dysfunction of the UPR plays an important role in some human diseases, especially those involving tissues dedicated to extracellular protein synthesis. Diabetes mellitus is an example of such a disease, since the demands for constantly varying levels of insulin synthesis make pancreatic beta-cells dependent on efficient UPR signaling. Furthermore, recent discoveries in this field indicate that the importance of the UPR in diabetes is not restricted to the beta-cell but is also involved in peripheral insulin resistance. This review addresses aspects of the UPR currently understood to be involved in human disease, including their role in diabetes mellitus, atherosclerosis, and neoplasia