Faculty Bibliography

Macrophage foam cells are critical mediators in atherosclerosis plaque development. A better understanding of the in vivo transcript profile of foam cells during the formation and progression of lesions may lead to novel therapeutic interventions. Toward this goal, we demonstrate for the first time that foam cell-specific RNA can be purified from atherosclerotic arteries, a tissue of mixed cellular composition. Foam cells from apolipoprotein (apo) E-/- mice were isolated by laser capture microdissection (LCM); RNA was extracted and used for molecular analysis by real-time quantitative polymerase chain reaction. Compared to whole tissue, a significant enrichment of foam cell-specific RNA transcripts was achieved. Furthermore, to test the ability to quantify differences in gene expression in response to an inflammatory stimulus, apoE-/- mice were injected with lipopolysaccharide, after which the transcriptional induction of the inflammatory mediators, VCAM, ICAM, and MCP-1, was observed in lesional macrophage foam cell RNA. These approaches will facilitate the study of macrophage gene expression under various conditions of plaque formation, regression, and response to genetic and environmental perturbations.

Surface-enhanced Raman scattering (SERS) spectroscopy is a plasmonics-based spectroscopic technique that combines modern laser spectroscopy with unique optical properties of metallic nanostructures, resulting in strongly increased Raman signals when molecules are adsorbed on or near nanometer-size structures of special metals such as gold, silver, and transition metals. This chapter provides a synopsis of the development and application of SERS-active metallic nanostructures, especially for the analysis of biologically relevant compounds. Some highlights of this chapter include reports of SERS as an immunoassay readout method, SERS gene nanoprobes, near-field scanning optical microscopy SERS probes, SERS as a tool for single-molecule detection, and SERS nanoprobes for cellular studies

In this review I describe the several stages of my research career, all of which were driven by a desire to understand the basic mechanisms responsible for the complex and beautiful organization of the eukaryotic cell. I was originally trained as an electron microscopist in Argentina, and my first major contribution was the introduction of glutaraldehyde as a fixative that preserved the fine structure of cells, which opened the way for cytochemical studies at the EM level. My subsequent work on membrane-bound ribosomes illuminated the process of cotranslational translocation of polypeptides across the ER membrane and led to the formulation, with Gunter Blobel, of the signal hypothesis. My later studies with many talented colleagues contributed to an understanding of ER structure and function and aspects of the mechanisms that generate and maintain the polarity of epithelial cells. For this work my laboratory introduced the now widely adopted Madin-Darby canine kidney (MDCK) cell line, and demonstrated the polarized budding of envelope viruses from those cells, providing a powerful new system that further advanced the field of protein traffic

The major facilitator superfamily represents the largest group of secondary active membrane transporters in prokaryotic and eukaryotic cells. They transport a vast variety of substrates, presumably via similar mechanisms, yet the details of these mechanisms remain unclear. Here we report the 3.3 A resolution structure of a member of this superfamily--GlpT, the glycerol-3-phosphate transporter from the E. coli inner membrane, in the absence of a substrate. The antiporter mediates the exchange of glycerol-3-phosphate for inorganic phosphate across the membrane. Its N- and C-terminal domains exhibit a pseudo 2-fold symmetry along an axis perpendicular to the membrane. Eight of the twelve transmembrane alpha-helices are arranged around a centrally located substrate translocation pore that is closed off at the periplasmic surface. Present at the beginning of the pore are two arginine residues that presumably comprise the substrate-binding site which is accessible only from the cytosol, suggesting an inward-facing conformation for the transporter. The central loop connecting the N- and C-terminal domains is partially disordered and exhibits reduced susceptibility to trypsin in the presence of substrate, indicating conformational changes. We propose that GlpT operates via a single binding-site, alternating-access mechanism

Recent findings regarding pathways of stem/progenitor cell involvement in adult blood vessel growth (postnatal vasculogenesis) suggest new theories for the pathogenesis of vascular anomalies. The somatic growth of vascular malformations and the mysterious pattern of proliferation and involution in infantile hemangioma can no longer be purely understood through the paradigm of angiogenesis. Molecular signals for postnatal vasculogenesis are being discovered in numerous animal models of cancer and ischemia, yet little research has addressed the importance of vasculogenesis in the growth of vascular anomalies. In this review, we discuss early studies that have investigated stem/progenitor cell involvement in the pathophysiology of infantile hemangioma and other congenital vascular anomalies

The neuritic plaque in the brain of Alzheimer's disease (AD) patients consists of an amyloid composed primarily of Abeta, an approx 4-kDa peptide derived from the amyloid precursor protein. Multiple lines of evidence suggest that Abeta plays a key role in the pathogenesis of the disease, and potential treatments that target Abeta production and/or Abeta accumulation in the brain as beta-amyloid are being aggressively pursued. Methods to quantitate the Abeta peptide are, therefore, invaluable to most studies aimed at a better understanding of the molecular etiology of the disease and in assessing potential therapeutics. Although other techniques have been used to measure Abeta in the brains of AD patients and beta-amyloid-depositing transgenic mice, the enzyme-linked immunosorbent assay (ELISA) is one of the most commonly used, reliable, and sensitive methods for quantitating the Abeta peptide. Here we describe methods for the recovery of both soluble and deposited Abeta from brain tissue and the subsequent quantitation of the peptide by sandwich ELISA

The brain is a remarkably complex anatomical structure that contains a diverse array of subdivisions, cell types, and synaptic connections. It is equally extraordinary in its physiological properties, as it constantly evaluates and integrates external stimuli as well as controls a complicated internal environment. The brain can be divided into three primary broad regions: the forebrain, midbrain (Mb), and hindbrain (Hb), each of which contain further subdivisions. The regions considered in this chapter are the Mb and most-anterior Hb (Mb/aHb), which are derived from the mesencephalon (mes) and rhombomere 1 (r1), respectively. The dorsal Mb consists of the laminated superior colliculus and the globular inferior colliculus (Fig. 1A and B), which modulate visual and auditory stimuli, respectively. The dorsal component of the aHb is the highly foliated cerebellum (Cb), which is primarily attributed to controlling motor skills (Fig. 1A and B). In contrast, the ventral Mb/aHb (Fig. 1B) consists of distinct clusters of neurons that together comprise a network of nuclei and projections-notably, the Mb dopaminergic and Hb serotonergic and Mb/aHb cholinergic neurons (Fig. 1G and H), which modulate a collection of behaviors, including movement, arousal, feeding, wakefulness, and emotion. Historically, the dorsal Mb and Cb have been studied using the chick as a model system because of the ease of performing both cell labeling and tissue transplants in the embryo in ovo; currently DNA electroporation techniques are also used. More recently the mouse has emerged as a powerful genetic system with numerous advantages to study events underpinning Mb/aHb development. There is a diverse array of spontaneous mutants with both Mb- and Cb-related phenotypes. In addition, numerous gene functions have been enumerated in mouse, gene expression is similar across vertebrates, and powerful genetic tools have been developed. Finally, additional insight into Mb/aHb function has been gained from studies of genetic diseases, such as Parkinson's disease, schizophrenia, cancer, and Dandy Walker syndrome, that afflict the Mb/aHb in humans and have genetic counterparts in mouse. Accordingly, this chapter discusses a spectrum of experiments, including classic embryology, in vitro assays, sophisticated genetic methods, and human diseases. We begin with an overview of Mb and aHb anatomy and physiology and mes/r1 gene expression patterns. We then provide a summary of fate-mapping studies that collectively demonstrate the complex cell behaviors that occur while the Mb and aHb primordia are established during embryogenesis and discuss the integration of both anterior-posterior (A-P) and dorsal-ventral (D-V) patterning. Finally, we describe some aspects of postnatal development and some of the insights gained from human diseases

It is experimentally demonstrated that quantum coherences can be efficiently transferred using adiabatic energy-level crossing. In a cluster of six dipolar-coupled proton spins of benzene, oriented by a liquid-crystalline matrix, a single-quantum coherence between one pair of states has been adiabatically transferred to another pair of states, and the superposition survived even after ten successive energy-level crossings.