Faculty Bibliography

The RNA-dependent RNA polymerase L protein of vesicular stomatitis virus, a prototype of nonsegmented negative-strand (NNS) RNA viruses, forms a covalent complex with a 5'-phosphorylated viral mRNA-start sequence (L-pRNA), a putative intermediate in the unconventional mRNA capping reaction catalyzed by the RNA:GDP polyribonucleotidyltransferase (PRNTase) activity. Here, we directly demonstrate that the purified L-pRNA complex transfers pRNA to GDP to produce the capped RNA (Gpp-pRNA), indicating that the complex is a bona fide intermediate in the RNA transfer reaction. To locate the active site of the PRNTase domain in the L protein, the covalent RNA attachment site was mapped. We found that the 5'-monophosphate end of the RNA is linked to the histidine residue at position 1,227 (H1227) of the L protein through a phosphoamide bond. Interestingly, H1227 is part of the histidine-arginine (HR) motif, which is conserved within the L proteins of the NNS RNA viruses including rabies, measles, Ebola, and Borna disease viruses. Mutagenesis analyses revealed that the HR motif is required for the PRNTase activity at the step of the enzyme-pRNA intermediate formation. Thus, our findings suggest that an ancient NNS RNA viral polymerase has acquired the PRNTase domain independently of the eukaryotic mRNA capping enzyme during evolution and PRNTase becomes a rational target for designing antiviral agents.

ABSTRACT: BACKGROUND: Increased levels of cryptic collagen epitope HU177 in the sera of melanoma patients have been shown to be associated with thicker primary melanomas and with the nodular histologic subtype. In this study, we investigate the association between HU177 shedding in the sera and clinical outcome in terms of disease-free survival (DFS) and overall survival (OS). METHODS: Serum samples from 209 patients with primary melanoma prospectively enrolled in the Interdisciplinary Melanoma Cooperative Group at the New York University Langone Medical Center (mean age=58, mean thickness=2.09 mm, stage I=136, stage II=41, stage III=32, median follow-up=54.9 months) were analyzed for HU177 concentration using a validated ELISA assay. HU177 serum levels at the time of diagnosis were used to divide the study cohort into two groups: low and high HU177. DFS and OS were estimated by Kaplan-Meier survival analysis, and the log-rank test was used to compare DFS and OS between the two HU177 groups. Multivariate Cox proportional hazards regression models were employed to examine the independent effect of HU177 category on DFS and OS. RESULTS: HU177 sera concentrations ranged from 0-139.8 ng/ml (mean and median of 6.2 ng/ml and 3.7 ng/ml, respectively). Thirty-eight of the 209 (18%) patients developed recurrences, and 34 of the 209 (16%) patients died during follow-up. Higher HU177 serum level was associated with an increased rate of melanoma recurrence (p=0.04) and with increasing mortality (p=0.01). The association with overall survival remained statistically significant after controlling for thickness and histologic subtype in a multivariate model (p=0.035). CONCLUSIONS: Increased shedding of HU177 in the serum of primary melanoma patients is associated with poor prognosis. Further studies are warranted to determine the clinical utility of HU177 in risk stratification compared to the current standard of care

Rational: Vascular smooth muscle cells (VSMCs) switching from a contractile/differentiated to a synthetic/dedifferentiated phenotype has an essential role in atherosclerosis, postangioplastic restenosis and hypertension. However, how normal VSMCs maintain the differentiated state is less understood. Objective: We aimed to indentify the effect of cartilage oligomeric matrix protein (COMP), a normal vascular extracellular matrix, on modulation of VSMCs phenotype. Methods and Results: We demonstrated that COMP was associated positively with the expression of VSMC differentiation marker genes during phenotype transition. Knockdown of COMP by small interfering (si)RNA favored dedifferentiation. Conversely, adenoviral overexpression of COMP markedly suppressed platelet-derived growth factor-BB-elicited VSMC dedifferentiation, characterized by altered VSMC morphology, actin fiber organization, focal adhesion assembly, and the expression of phenotype-dependent markers. Whereas alpha7 integrin coimmunoprecipitated with COMP in normal rat VSMCs and vessels, neutralizing antibody or siRNA against alpha7 integrin inhibited VSMC adhesion to COMP, which indicated that alpha7beta1 integrin is a potential receptor for COMP. As well, blocking or interference by siRNA of alpha7 integrin completely abolished the effect of COMP on conserving the contractile phenotype. In accordance, ectopic adenoviral overexpression of COMP greatly retarded VSMC phenotype switching, rescued contractility of carotid artery ring, and inhibited neointima formation in balloon-injured rats. Conclusions: Our data suggest that COMP is essential for maintaining a VSMC contractile phenotype and the protective effects of COMP are mainly mediated through interaction with alpha7beta1 integrin. Investigations to identify the factors affecting the expression and integrity of COMP may provide a novel therapeutic target for vascular disorders

The p75 neurotrophin receptor, a member of the tumor necrosis factor superfamily of receptors, undergoes an alpha-secretase-mediated release of its extracellular domain, followed by a gamma-secretase-mediated intramembrane cleavage. Like amyloid precursor protein and Notch, gamma-secretase cleavage of the p75 receptor releases an intracellular domain (ICD). However, it has been experimentally challenging to determine the precise subcellular localization and functional consequences of the p75 ICD. Here, we utilized a nuclear translocation assay and biochemical fractionation approaches to follow the fate of the ICD. We found that the p75 ICD can translocate to the nucleus to activate a green fluorescent protein reporter gene. Furthermore, the p75 ICD was localized in nuclear fractions. Chromatin immunoprecipitation experiments indicated that nerve growth factor induced the association of endogenous p75 with the cyclin E(1) promoter. Expression of the p75 ICD resulted in modulation of gene expression from this locus. These results suggest that the p75 ICD generated by gamma-secretase cleavage is capable of modulating transcriptional events in the nucleus

The formation of functional synapses on artificial substrates is a very important step in the development of engineered in vitro neural networks. Spherical supported bilayer lipid membranes (SS-BLMs) are used here as a novel substrate to demonstrate presynaptic vesicle accumulation at an in vitro synaptic junction. Confocal fluorescence microscopy, cryo-transmission electron microscopy (cryo-TEM), and fluorescence recovery after photobleaching (FRAP) experiments have been used to characterize the SS-BLMs. Conventional immunocytochemistry combined with confocal fluorescence microscopy was used to observe the formation of presynaptic vesicles at the neuron-SS-BLM contacts. These results indicate that lipid phases may play a role in the observed phenomenon, in addition to the chemical and electrostatic interactions between the neurons and SS-BLMs. The biocompatibility of lipid bilayers along with their membrane tunability makes the suggested approach a useful "toolkit" for many neuroengineering applications including artificial synapse formation and synaptogenesis in vivo.

Within the catecholaminergic systems, there are contradictory findings regarding ability of estradiol to regulate expression of genes related to catecholamine biosynthesis. Several parameters important for effects of estradiol on the catecholamine (CA) related enzyme gene expression were examined in two CA regions. Ovariectomized (OVX) female rats were given prolonged estradiol treatments, either in a pulsatile fashion by injections or continuously by pellets. The mode affected the response of tyrosine hydroxylase (TH) and GTP cyclohydrolase I (GTPCH) mRNAs differentially in the nucleus of solitary tract (NTS) and the locus coeruleus (LC). In rostral-medial NTS, TH mRNA levels were increased with injections, but declined in rats administered estradiol by pellets. In LC, a significant change was only observed in GTPCH with injections. These differences may reflect activation of different estrogen receptors (ER). The response to estradiol in the presence of ERalpha and ER beta was examined in PC12 cell culture. Estradiol directly regulated promoter activity of TH, GTPCH and dopamine beta-hydroxylase (DBH) genes. With ERalpha, 17 beta-estradiol elevated TH promoter activity, while there was a decline with ERbeta. In contrast, both DBH and GTPCH promoters were enhanced by 17 beta-estradiol over a wide range of concentrations with either ER subtype. Thus, mode of administration, location examined and ER subtype expressed are important considerations in the overall response of catecholamine related enzymes to estradiol.

The complexity of proteomic instrumentation for LC-MS/MS introduces many possible sources of variability. Data-dependent sampling of peptides constitutes a stochastic element at the heart of discovery proteomics. Although this variation impacts the identification of peptides, proteomic identifications are far from completely random. In this study, we analyzed interlaboratory data sets from the NCI Clinical Proteomic Technology Assessment for Cancer to examine repeatability and reproducibility in peptide and protein identifications. Included data spanned 144 LC-MS/MS experiments on four Thermo LTQ and four Orbitrap instruments. Samples included yeast lysate, the NCI-20 defined dynamic range protein mix, and the Sigma UPS 1 defined equimolar protein mix. Some of our findings reinforced conventional wisdom, such as repeatability and reproducibility being higher for proteins than for peptides. Most lessons from the data, however, were more subtle. Orbitraps proved capable of higher repeatability and reproducibility, but aberrant performance occasionally erased these gains. Even the simplest protein digestions yielded more peptide ions than LC-MS/MS could identify during a single experiment. We observed that peptide lists from pairs of technical replicates overlapped by 35-60%, giving a range for peptide-level repeatability in these experiments. Sample complexity did not appear to affect peptide identification repeatability, even as numbers of identified spectra changed by an order of magnitude. Statistical analysis of protein spectral counts revealed greater stability across technical replicates for Orbitraps, making them superior to LTQ instruments for biomarker candidate discovery. The most repeatable peptides were those corresponding to conventional tryptic cleavage sites, those that produced intense MS signals, and those that resulted from proteins generating many distinct peptides. Reproducibility among different instruments of the same type lagged behind repeatability of technical replicates on a single instrument by several percent. These findings reinforce the importance of evaluating repeatability as a fundamental characteristic of analytical technologies

The wiring of the nervous system arises from extensive directional migration of neuronal cell bodies and growth of processes that, somehow, end up forming functional circuits. Thus far, this feat of biological engineering appears to rely on sequences of pathfinding decisions upon local cues, each with little relationship to the anatomical and physiological outcome. Here, we uncover a straightforward cellular mechanism for circuit building whereby a neuronal type directs the development of its future partners. We show that visceral afferents of the head (that innervate taste buds) provide a scaffold for the establishment of visceral efferents (that innervate salivatory glands and blood vessels). In embryological terms, sensory neurons derived from an epibranchial placode--that we show to develop largely independently from the neural crest--guide the directional outgrowth of hindbrain visceral motoneurons and control the formation of neural crest-derived parasympathetic ganglia.