Malcolm R. Wood

Src blockade stabilizes a Flk/cadherin complex, reducing edema and tissue injury following myocardial infarction

Ischemia resulting from myocardial infarction (MI) promotes VEGF expression, leading to vascular permeability (VP) and edema, a process that we show here contributes to tissue injury throughout the ventricle. This permeability/edema can be assessed noninvasively by MRI and can be observed at the ultrastructural level as gaps between adjacent endothelial cells. Many of these gaps contain activated platelets adhering to exposed basement membrane, reducing vessel patency. Following MI, genetic or pharmacological blockade of Src preserves endothelial cell barrier function, suppressing VP and infarct volume, providing long-term improvement in cardiac function, fibrosis, and survival. To our surprise, an intravascular injection of VEGF into healthy animals, but not those deficient in Src, induced similar endothelial gaps, VP, platelet plugs, and some myocyte damage. Mechanistically, we show that quiescent blood vessels contain a complex involving Flk, VE-cadherin, and beta-catenin that is transiently disrupted by VEGF injection. Blockade of Src prevents disassociation of this complex with the same kinetics with which it prevents VEGF-mediated VP/edema. These findings define a molecular mechanism to account for the Src requirement in VEGF-mediated permeability and provide a basis for Src inhibition as a therapeutic option for patients with acute MI.

Oxidative Stress Induces a Form of Programmed Cell Death with Characteristics of Both Apoptosis and Necrosis in Neuronal Cells

Abstract: Oxidative stress is implicated in a number of neurological disorders including stroke, Parkinsons disease, and Alzheimers disease. To study the effects of oxidative stress on neuronal cells, we have used an immortalized mouse hippocampal cell line (HT‐22) that is particularly sensitive to glutamate. In these cells, glutamate competes for cystine uptake, leading to a reduction in glutathione and, ultimately, cell death. As it has been reported that protein kinase C activation inhibits glutamate toxicity in these cells and is also associated with the inhibition of apoptosis in other cell types, we asked if glutamate toxicity was via apoptosis. Morphologically, glutamate‐treated cells underwent plasma membrane blebbing and cell shrinkage, but no DNA fragmentation was observed. At the ultrastructural level, there was damage to mitochondria and other organelles although the nuclei remained intact. Protein and RNA synthesis inhibitors as well as certain protease inhibitors protected the cells from glutamate toxicity. Both the macromolecular synthesis inhibitors and the protease inhibitors had to be added relatively soon after the addition of glutamate, suggesting that protein synthesis and protease activation are early and distinct steps in the cell death pathway. Thus, the oxidative stress brought about by treatment with glutamate initiates a series of events that lead to a form of cell death distinct from either necrosis or apoptosis.

GW182 is critical for the stability of GW bodies expressed during the cell cycle and cell proliferation

A novel cytoplasmic compartment referred to as GW bodies was initially identified using human autoantibodies to a 182 kDa protein named GW182. GW bodies are small, generally spherical, cytoplasmic domains that vary in number and size in several mammalian cell types examined to date. Based on our earlier studies, GW bodies were proposed to be cytoplasmic sites for mRNA storage and/or degradation. In the present study, immunogold electron microscopy identified electron dense structures of 100-300 nm diameter devoid of a lipid bilayer membrane. These structures appeared to comprise clusters of electron dense strands of 8-10 nm in diameter. By costaining with CENP-F and PCNA, and employing a double-thymidine block to synchronize HeLa cells, GW bodies were observed to be small in early S phase and larger during late S and G2 phases of the cell cycle. The majority of GW bodies disassembled prior to mitosis and small GW bodies reassembled in early G1. The analysis of GW bodies in two experimental models of cell proliferation using reversal of 3T3/serum-starvation and concanavalin A stimulation of mouse splenocytes and T cells, revealed that proliferating cells contained larger, brighter, and more numerous GW bodies as well as up to a fivefold more total GW182 protein than quiescent cells. In vitro gene knockdown of GW182 led to the disappearance of GW bodies demonstrating that GW182 is a critical component of GW bodies. The incremental expression of the GW182 protein in cells induced to proliferate and the cyclic formation and breakdown of GW bodies during mitosis are intriguing in view of the notion that GW bodies are specialized centers involved in maintaining stability and/or controlling degradation of mRNA.

Ultrastructural and Biophysical Characterization of Hepatitis C Virus Particles Produced in Cell Culture

ABSTRACT We analyzed the biochemical and ultrastructural properties of hepatitis C virus (HCV) particles produced in cell culture. Negative-stain electron microscopy revealed that the particles were spherical (∼40- to 75-nm diameter) and pleomorphic and that some of them contain HCV E2 protein and apolipoprotein E on their surfaces. Electron cryomicroscopy revealed two major particle populations of ∼60 and ∼45 nm in diameter. The ∼60-nm particles were characterized by a membrane bilayer (presumably an envelope) that is spatially separated from an internal structure (presumably a capsid), and they were enriched in fractions that displayed a high infectivity-to-HCV RNA ratio. The ∼45-nm particles lacked a membrane bilayer and displayed a higher buoyant density and a lower infectivity-to-HCV RNA ratio. We also observed a minor population of very-low-density, >100-nm-diameter vesicular particles that resemble exosomes. This study provides low-resolution ultrastructural information of particle populations displaying differential biophysical properties and specific infectivity. Correlative analysis of the abundance of the different particle populations with infectivity, HCV RNA, and viral antigens suggests that infectious particles are likely to be present in the large ∼60-nm HCV particle populations displaying a visible bilayer. Our study constitutes an initial approach toward understanding the structural characteristics of infectious HCV particles.

Structural Rearrangement of Ebola Virus VP40 Begets Multiple Functions in the Virus Life Cycle.

Proteins, particularly viral proteins, can be multifunctional, but the mechanisms behind multifunctionality are not fully understood. Here, we illustrate through multiple crystal structures, biochemistry, and cellular microscopy that VP40 rearranges into different structures, each with a distinct function required for the ebolavirus life cycle. A butterfly-shaped VP40 dimer traffics to the cellular membrane. Once there, electrostatic interactions trigger rearrangement of the polypeptide into a linear hexamer. These hexamers construct a multilayered, filamentous matrix structure that is critical for budding and resembles tomograms of authentic virions. A third structure of VP40, formed by a different rearrangement, is not involved in virus assembly but instead uniquely binds RNA to regulate viral transcription inside infected cells. These results provide a functional model for ebolavirus matrix assembly and the other roles of VP40 in the virus life cycle and demonstrate how a single wild-type, unmodified polypeptide can assemble into different structures for different functions.

Short-term fasting induces profound neuronal autophagy

Disruption of autophagy—a key homeostatic process in which cytosolic components are degraded and recycled through lysosomes—can cause neurodegeneration in tissue culture and in vivo. Up-regulation of this pathway may be neuroprotective, and much effort is being invested in developing drugs that cross the blood brain barrier and increase neuronal autophagy. One well-recognized way of inducing autophagy is by food restriction, which up-regulates autophagy in many organs including the liver; but current dogma holds that the brain escapes this effect, perhaps because it is a metabolically-privileged site. Here, we have re-evaluated this tenet using a novel approach that allows us to detect, enumerate, and characterize autophagosomes in vivo. We first validate the approach by showing that it allows the identification and characterization of autophagosomes in the livers of food-restricted mice. We use the method to identify constitutive autophagosomes in cortical neurons and Purkinje cells, and we show that short-term fasting leads to a dramatic up-regulation in neuronal autophagy. The increased neuronal autophagy is revealed by changes in autophagosome abundance and characteristics, and by diminished neuronal mTOR activity in vivo, demonstrated by a reduction in levels of phosphorylated S6 ribosomal protein in Purkinje cells. The increased abundance of autophagosomes in Purkinje cells was confirmed using transmission electron microscopy. Our data lead us to speculate that sporadic fasting might represent a simple, safe and inexpensive means to promote this potentially-therapeutic neuronal response.

Coxsackievirus Infection Induces Autophagy-Like Vesicles and Megaphagosomes in Pancreatic Acinar Cells In Vivo

ABSTRACT Autophagy can play an important part in protecting host cells during virus infection, and several viruses have developed strategies by which to evade or even exploit this homeostatic pathway. Tissue culture studies have shown that poliovirus, an enterovirus, modulates autophagy. Herein, we report on in vivo studies that evaluate the effects on autophagy of coxsackievirus B3 (CVB3). We show that in pancreatic acinar cells, CVB3 induces the formation of abundant small autophagy-like vesicles and permits amphisome formation. However, the virus markedly, albeit incompletely, limits the fusion of autophagosomes (and/or amphisomes) with lysosomes, and, perhaps as a result, very large autophagy-related structures are formed within infected cells; we term these structures megaphagosomes. Ultrastructural analyses confirmed that double-membraned autophagy-like vesicles were present in infected pancreatic tissue and that the megaphagosomes were related to the autophagy pathway; they also revealed a highly organized lattice, the individual components of which are of a size consistent with CVB RNA polymerase; we suggest that this may represent a coxsackievirus replication complex. Thus, these in vivo studies demonstrate that CVB3 infection dramatically modifies autophagy in infected pancreatic acinar cells.

N-(3-oxo-acyl)homoserine lactones signal cell activation through a mechanism distinct from the canonical pathogen-associated molecular pattern recognition receptor pathways.

Innate immune system receptors function as sensors of infection and trigger the immune responses through ligand-specific signaling pathways. These ligands are pathogen-associated products, such as components of bacterial walls and viral nuclear acids. A common response to such ligands is the activation of mitogen-activated protein kinase p38, whereas double-stranded viral RNA additionally induces the phosphorylation of eukaryotic translation initiation factor 2α (eIF2α). Here we have shown that p38 and eIF2α phosphorylation represent two biochemical markers of the effects induced by N-(3-oxo-acyl)homoserine lactones, the secreted products of a number of Gram-negative bacteria, including the human opportunistic pathogen Pseudomonas aeruginosa. Furthermore, N-(3-oxo-dodecanoyl)homoserine lactone induced distension of mitochondria and the endoplasmic reticulum as well as c-jun gene transcription. These effects occurred in a wide variety of cell types including alveolar macrophages and bronchial epithelial cells, requiring the structural integrity of the lactone ring motif and its natural stereochemistry. These findings suggest that N-(3-oxo-acyl)homoserine lactones might be recognized by receptors of the innate immune system. However, we provide evidence that N-(3-oxo-dodecanoyl)homoserine lactone-mediated signaling does not require the presence of the canonical innate immune system receptors, Toll-like receptors, or two members of the NLR/Nod/Caterpillar family, Nod1 and Nod2. These data offer a new understanding of the effects of N-(3-oxo-dodecanoyl)homoserine lactone on host cells and its role in persistent airway infections caused by P. aeruginosa.

Structural insights into Nox4 and Nox2: motifs involved in function and cellular localization.

ABSTRACT Regulated generation of reactive oxygen species (ROS) is primarily accomplished by NADPH oxidases (Nox). Nox1 to Nox4 form a membrane-associated heterodimer with p22phox, creating the docking site for assembly of the activated oxidase. Signaling specificity is achieved by interaction with a complex network of cytosolic components. Nox4, an oxidase linked to cardiovascular disease, carcinogenesis, and pulmonary fibrosis, deviates from this model by displaying constitutive H2O2 production without requiring known regulators. Extensive Nox4/Nox2 chimera screening was initiated to pinpoint structural motifs essential for ROS generation and Nox subcellular localization. In summary, a matching B loop was crucial for catalytic activity of both Nox enzymes. Substitution of the carboxyl terminus was sufficient for converting Nox4 into a phorbol myristate acetate (PMA)-inducible phenotype, while Nox2-based chimeras never gained constitutive activity. Changing the Nox2 but not the Nox4 amino terminus abolished ROS generation. The unique heterodimerization of a functional Nox4/p22phox Y121H complex was dependent on the D loop. Nox4, Nox2, and functional Nox chimeras translocated to the plasma membrane. Cell surface localization of Nox4 or PMA-inducible Nox4 did not correlate with O2− generation. In contrast, Nox4 released H2O2 and promoted cell migration. Our work provides insights into Nox structure, regulation, and ROS output that will aid inhibitor design.

Synaptic regeneration and glial reactions in the transected spinal cord of the lamprey

SummaryWe have examined axonal growth and synaptic regeneration in identified giant neurons of the transected lamprey spinal cord using intracellular injection of horseradish peroxidase. Wholemounts together with serial section light and electron microscopy, show that axons from identified Müller and Mauthner reticulospinal neurons grow across the lesion and regenerate new synaptic contacts. Relatively normal swimming returns in these animals by 3–4 weeks after spinal transection. This occurs despite the formation of regenerated synapses in regions of the cord that are not usually occupied by these neurons.The regenerating axons branch profusely in contrast to their unbranched state in the normal animal. In addition to showing the two synaptic configurations found normally, synapses may be formed by slender sprouts from the growing giant axon. These ‘sprout’ type synaptic contacts appear unique to the regenerating neuron. Only regenerated chemical synapses were seen; the morphologically mixed chemical and electrical (gap junction) synaptic complex common in the normal animal was not observed at regenerated synapses.The site of spinal transection in the functionally recovered animal shows an increase in the number of ependymal and glial cells. Ependymal-like cells appear in regions away from the central canal. The expanded ependymal and glial processes covering the peripheral surface of the injured cord become convoluted, in contrast to their normal smooth configuration. There is no collagen within the cord at the site of transection but a considerable deposition is seen external to the cord surface.Axonal growth across a spinal lesion and subsequent synaptic regeneration can be examined in single identifiable giant interneurons in the spinal cord of the larval lamprey. This preparation may be used as an assay to investigate factors that could contribute to functional recovery following central nervous system injury in the higher vertebrates.