Faculty Bibliography

Germ plasm, a specialized cytoplasm present at the posterior of the early Drosophila embryo, is necessary and sufficient for germ cell formation. Germ plasm is rich in mitochondria and contains electron dense structures called polar granules. To identify novel polar granule components we isolated proteins that associate in early embryos with Vasa (VAS) and Tudor (TUD), two known polar granule associated molecules. We identified Maternal expression at 31B (ME31B), eIF4A, Aubergine (AUB) and Transitional Endoplasmic Reticulum 94 (TER94) as components of both VAS and TUD complexes and confirmed their localization to polar granules by immuno-electron microscopy. ME31B, eIF4A and AUB are also present in processing (P) bodies, suggesting that polar granules, which are necessary for germ line formation, might be related to P bodies. Our recovery of ER associated proteins TER94 and ME31B confirms that polar granules are closely linked to the translational machinery and to mRNP assembly

Most metastatic melanoma patients fail to respond to available therapy, underscoring the need for novel approaches to identify new effective treatments. In this study, we screened 2,000 compounds from the Spectrum Library at a concentration of 1 micromol/L using two chemoresistant melanoma cell lines (M-14 and SK-Mel-19) and a spontaneously immortalized, nontumorigenic melanocyte cell line (melan-a). We identified 10 compounds that inhibited the growth of the melanoma cells yet were largely nontoxic to melanocytes. Strikingly, 4 of the 10 compounds (mebendazole, albendazole, fenbendazole, and oxybendazole) are benzimidazoles, a class of structurally related, tubulin-disrupting drugs. Mebendazole was prioritized to further characterize its mechanism of melanoma growth inhibition based on its favorable pharmacokinetic profile. Our data reveal that mebendazole inhibits melanoma growth with an average IC(50) of 0.32 micromol/L and preferentially induces apoptosis in melanoma cells compared with melanocytes. The intrinsic apoptotic response is mediated through phosphorylation of Bcl-2, which occurs rapidly after treatment with mebendazole in melanoma cells but not in melanocytes. Phosphorylation of Bcl-2 in melanoma cells prevents its interaction with proapoptotic Bax, thereby promoting apoptosis. We further show that mebendazole-resistant melanocytes can be sensitized through reduction of Bcl-2 protein levels, showing the essential role of Bcl-2 in the cellular response to mebendazole-mediated tubulin disruption. Our results suggest that this screening approach is useful for identifying agents that show promise in the treatment of even chemoresistant melanoma and identifies mebendazole as a potent, melanoma-specific cytotoxic agent

The fibrillar peptide amyloid-beta (A beta) has a chief function in the pathogenesis of Alzheimer's disease. Interleukin 1 beta (IL-1 beta) is a key cytokine in the inflammatory response to A beta. Insoluble materials such as crystals activate the inflammasome formed by the cytoplasmic receptor NALP3, which results in the release of IL-1 beta. Here we identify the NALP3 inflammasome as a sensor of A beta in a process involving the phagocytosis of A beta and subsequent lysosomal damage and release of cathepsin B. Furthermore, the IL-1 beta pathway was essential for the microglial synthesis of proinflammatory and neurotoxic factors, and the inflammasome, caspase-1 and IL-1 beta were critical for the recruitment of microglia to exogenous A beta in the brain. Our findings suggest that activation of the NALP3 inflammasome is important for inflammation and tissue damage in Alzheimer's disease

Calpains are calcium-dependent enzymes that determine the fate of proteins through regulated proteolytic activity. Calpains have been linked to the modulation of memory and are key to the pathogenesis of Alzheimer disease (AD). When abnormally activated, calpains can also initiate degradation of proteins essential for neuronal survival. Here we show that calpain inhibition through E64, a cysteine protease inhibitor, and the highly specific calpain inhibitor BDA-410 restored normal synaptic function both in hippocampal cultures and in hippocampal slices from the APP/PS1 mouse, an animal model of AD. Calpain inhibition also improved spatial-working memory and associative fear memory in APP/PS1 mice. These beneficial effects of the calpain inhibitors were associated with restoration of normal phosphorylation levels of the transcription factor CREB and involved redistribution of the synaptic protein synapsin I. Thus, calpain inhibition may prove useful in the alleviation of memory loss in AD

In this article, the formation of prokaryotic and eukaryotic cardiolipin is reviewed in light of its biological function. I begin with a detailed account of the structure of cardiolipin, its stereochemistry, and the resulting physical properties, and I present structural analogs of cardiolipin that occur in some organisms. Then I continue to discuss i) the de novo formation of cardiolipin, ii) its acyl remodeling, iii) the assembly of cardiolipin into biological membranes, and iv) the degradation of cardiolipin, which may be involved in apoptosis and mitochondrial fusion. Thus, this article covers the entire metabolic cycle of this unique phospholipid. It is shown that mitochondria produce cardiolipin species with a high degree of structural uniformity and molecular symmetry, among which there is often a dominant form with four identical acyl chains. The subsequent assembly of cardiolipin into functional membranes is largely unknown, but the analysis of crystal structures of membrane proteins has revealed a first glimpse into the underlying principles of cardiolipin-protein interactions. Disturbances of cardiolipin metabolism are crucial in the pathophysiology of human Barth syndrome and perhaps also play a role in diabetes and ischemic heart disease

Adrenergic receptors and their downstream effector molecules are expressed in all cell types in the skin, and it is only recently that functionality of the catecholamine agonist activated signaling in the cutaneous repair process has been revealed. In addition to responding to systemic elevations in catecholamines (as in stress situations) or to pharmacologically administered adrenergic agonists, epidermal keratinocytes themselves can synthesize catecholamine ligands. They also respond to these systemic or self-generated agonists via receptor mediated signaling, resulting in altered migration, and changes in wound re-epithelialization. Endothelial cells, inflammatory cells, dermal fibroblasts, and mesenchymal stem cells, all cells that contribute to the wound repair process, express multiple subtypes of adrenergic receptors and exhibit responses that can be either contribute or impair healing-and occasionally, depending on the species and assay conditions, results can be conflicting. There is still much to be uncovered regarding how this self-contained autocrine and paracrine signaling system contributes to cutaneous wound repair

Gag-CD8+ T cell responses are associated with immune control of HIV infection. Since during HIV infection Nef impairs T cell responses, we evaluated whether deletion of nef from a non-infectious HIV DNA vaccine (Delta4 Nef+), creating Delta5 Nef(-), would affect its immunogenicity. When compared with Delta4, mice injected with Delta5 developed significantly lower CD8+ T cell responses to Gag, but no significant change in the responses to Env was observed. In vitro, deletion of Nef abrogated the induced cell death, production of virus-like particles and release of Gag from transfected cells. Thus, the effect of Nef in causing extrusion of Gag might adjuvant the CD8+ T cell responses to Gag in DNA vaccine.

Thirteen different hexa-peri-hexabenzocoronenes (HBCs) I-III were newly synthesized, and their self-assembling behaviors were investigated. Taking into account also the reported behaviors of amphiphilic HBCs, some structural parameters of HBC essential for the tubular assembly were revealed. Points to highlight include (1) the importance of two phenyl groups attached to one side of the HBC unit, (2) essential roles of long paraffinic side chains on the other side of the phenyl groups, and (3) no necessity of hydrophilic oligo(ethylene glycol) side chains. The hierarchical nanotubular structure, rendered by virtue of a synchrotron radiation technique, was virtually identical to our previous proposal, where the nanotubes are composed of helically coiled bilayer tapes with a tilting angle of approximately 45 degrees. Each tape consists of pi-stacked HBC units, where the inner and outer HBC layers are connected by interdigitation of paraffinic side chains. The coiled structure is most likely caused by a steric congestion of the phenyl groups attached to the HBC unit, whose tilting direction may determine the handedness of the helically chiral nanotube.