Mass-spectrometric Analysis of Myelin Proteolipids Reveals New Features of This Family of Palmitoylated Membrane Proteins

Journal of Neurochemistry. May, 2002  |  Pubmed ID: 12065672

In this study, we have investigated the structure of the native myelin proteolipid protein (PLP), DM-20 protein and several low molecular mass proteolipids by mass spectrometry. The various proteolipid species were isolated from bovine spinal cord by size-exclusion and ion-exchange chromatography in organic solvents. Matrix-assisted laser desorption ionization-time of flight-mass spectrometry (MALDI-TOF-MS) of PLP and DM-20 revealed molecular masses of 31.6 and 27.2 kDa, respectively, which is consistent with the presence of six and four molecules of thioester-bound fatty acids. Electrospray ionization-MS analysis of the deacylated proteins in organic solvents produced the predicted molecular masses of the apoproteins (29.9 and 26.1 kDa), demonstrating that palmitoylation is the major post-translational modification of PLP, and that the majority of PLP and DM-20 molecules in the CNS are fully acylated. A series of myelin-associated, palmitoylated proteolipids with molecular masses raging between 12 kDa and 18 kDa were also isolated and subjected to amino acid analysis, fatty acid analysis, N- and C-terminal sequencing, tryptic digestion and peptide mapping by MALDI-TOF-MS. The results clearly showed that these polypeptides correspond to the N-terminal region (residues 1-105/112) and C-terminal region (residues 113/131-276) of the major PLP, and they appear to be produced by natural proteolytic cleavage within the 60 amino acid-long cytoplasmic domain. These proteolipids are not postmortem artifacts of PLP and DM-20, and are differentially distributed across the CNS.

Androgen Deficiency, Meibomian Gland Dysfunction, and Evaporative Dry Eye

Annals of the New York Academy of Sciences. Jun, 2002  |  Pubmed ID: 12114274

We have recently discovered that women with primary and secondary Sjögren's syndrome are androgen-deficient. We hypothesize that this hormone insufficiency contributes to the meibomian gland dysfunction, tear film instability, and evaporative dry eye that are characteristic of this autoimmune disorder. If our hypothesis is correct, we predict: (1) that androgens regulate meibomian gland function, control the quality and/or quantity of lipids produced by this tissue, and promote the formation of the tear film's lipid layer; and (2) that androgen deficiency, due to an attenuation in androgen synthesis (e.g., during Sjögren's syndrome, menopause, aging, complete androgen-insensitivity syndrome [CAIS] and anti-androgen use), will lead to meibomian gland dysfunction and evaporative dry eye. The following studies were designed to test these predictions.

Complete Androgen Insensitivity Syndrome: Effect on Human Meibomian Gland Secretions

Archives of Ophthalmology. Dec, 2002  |  Pubmed ID: 12470144

To determine whether androgen receptors affect the fatty acid profiles of neutral and polar lipids in human meibomian gland secretions.

Molecular Identification of a Danger Signal That Alerts the Immune System to Dying Cells

Nature. Oct, 2003  |  Pubmed ID: 14520412

In infections, microbial components provide signals that alert the immune system to danger and promote the generation of immunity. In the absence of such signals, there is often no immune response or tolerance may develop. This has led to the concept that the immune system responds only to antigens perceived to be associated with a dangerous situation such as infection. Danger signals are thought to act by stimulating dendritic cells to mature so that they can present foreign antigens and stimulate T lymphocytes. Dying mammalian cells have also been found to release danger signals of unknown identity. Here we show that uric acid is a principal endogenous danger signal released from injured cells. Uric acid stimulates dendritic cell maturation and, when co-injected with antigen in vivo, significantly enhances the generation of responses from CD8+ T cells. Eliminating uric acid in vivo inhibits the immune response to antigens associated with injured cells, but not to antigens presented by activated dendritic cells. Our findings provide a molecular link between cell injury and immunity and have important implications for vaccines, autoimmunity and inflammation.

Mass Profiling-directed Isolation and Identification of a Stage-specific Serologic Protein Biomarker of Advanced Prostate Cancer

Proteomics. Jul, 2005  |  Pubmed ID: 15952230

Carcinoma of the prostate (CaP) is the second leading cause of cancer-related mortality among American men. While high cure rates are associated with localized CaP, no cure exists for advanced recurrent disease. At present there are no known serologic biomarkers specific to this stage of the disease. Several groups have used mass spectrometry (MS) based mass profiling (MP) combined with multivariate analysis to identify diagnostically predictive protein peaks for CaP in serum and tissues. Nevertheless, an appreciable level of skepticism exists for MP attributed primarily to a lack of definitive protein characterization. To address this problem, we have applied an approach that combines MP with a whole-protein based top-down separation strategy for the identification of a stage-specific marker in a group comprising 16 patients with CaP (metastatic and localized disease) and 15 healthy individuals. MP, combined with multivariate analysis, yielded 17 serum proteins specific to metastatic disease. A single protein detected at m/z 7771 was found to be significantly decreased in the sera of all the metastatic CaP patients but not in localized CaP or healthy individuals. This protein was therefore chosen as the primary candidate for further analysis. The complex nature of the serologic proteome necessitated an isolation strategy that included a C18 prefractionation, followed by multidimensional liquid chromatography and, finally, two-dimensional gel electrophoresis. The separation process was monitored by UV-Vis and matrix-assisted laser desorption/ionization-time of flight MS analysis. This strategy was found to greatly facilitate subsequent MS characterization of the unknown protein, which was identified as platelet factor 4, a chemokine with prothrombolytic and antiangiogenic activities. Confirmation was achieved using both Western blot analysis and enzyme-linked immunosorbent assay. With the growing interest in using MP for patient classification and diagnosis, our approach and its variations should be powerful in the separation and characterization of proteins following MP.

MutS Inhibits RecA-mediated Strand Transfer with Methylated DNA Substrates

Nucleic Acids Research. , 2005  |  Pubmed ID: 15972855

DNA mismatch repair (MMR) sensitizes human and Escherichia coli dam cells to the cytotoxic action of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) while abrogation of such repair results in drug resistance. In DNA methylated by MNNG, MMR action is the result of MutS recognition of O6-methylguanine base pairs. MutS and Ada methyltransferase compete for the MNNG-induced O6-methylguanine residues, and MMR-induced cytotoxicity is abrogated when Ada is present at higher concentrations than normal. To test the hypothesis that MMR sensitization is due to decreased recombinational repair, we used a RecA-mediated strand exchange assay between homologous phiX174 substrate molecules, one of which was methylated with MNNG. MutS inhibited strand transfer on such substrates in a concentration-dependent manner and its inhibitory effect was enhanced by MutL. There was no effect of these proteins on RecA activity with unmethylated substrates. We quantified the number of O6-methylguanine residues in methylated DNA by HPLC-MS/MS and 5-10 of these residues in phiX174 DNA (5386 bp) were sufficient to block the RecA reaction in the presence of MutS and MutL. These results are consistent with a model in which methylated DNA is perceived by the cell as homeologous and prevented from recombining with homologous DNA by the MMR system.

Functional Modulation of IFT Kinesins Extends the Sensory Repertoire of Ciliated Neurons in Caenorhabditis Elegans

The Journal of Cell Biology. Feb, 2006  |  Pubmed ID: 16492809

The diversity of sensory cilia on Caenorhabditis elegans neurons allows the animal to detect a variety of sensory stimuli. Sensory cilia are assembled by intraflagellar transport (IFT) kinesins, which transport ciliary precursors, bound to IFT particles, along the ciliary axoneme for incorporation into ciliary structures. Using fluorescence microscopy of living animals and serial section electron microscopy of high pressure-frozen, freeze-substituted IFT motor mutants, we found that two IFT kinesins, homodimeric OSM-3 kinesin and heterotrimeric kinesin II, function in a partially redundant manner to build full-length amphid channel cilia but are completely redundant for building full-length amphid wing (AWC) cilia. This difference reflects cilia-specific differences in OSM-3 activity, which serves to extend distal singlets in channel cilia but not in AWC cilia, which lack such singlets. Moreover, AWC-specific chemotaxis assays reveal novel sensory functions for kinesin II in these wing cilia. We propose that kinesin II is a "canonical" IFT motor, whereas OSM-3 is an "accessory" IFT motor, and that subtle changes in the deployment or actions of these IFT kinesins can contribute to differences in cilia morphology, cilia function, and sensory perception.

A Study of the Efficacy of Various Home Filtration Substrates in the Removal of Microcystin-LR from Drinking Water

Journal of Water and Health. Mar, 2006  |  Pubmed ID: 16604842

This study was conducted to determine whether common water filtration and purification systems bought by consumers and used in the home would remove cyanotoxins from water. Commonly used universal filter housings and filter sizes were utilized to identify filter media that may be effective in the removal of microcystin-LR in deionized water. Results suggest that the efficacy of home filtration devices in removing microcystin-LR varies considerably with the type of device being used. Carbon filters successfully removed microcystin-LR allowing only 0.05-0.3% of the toxin load to pass through the filter. On the other hand, pleated paper and string wound filters allowed > 90% of microcystin-LR present in the sample to pass through the filters. Theoretically, the use of carbon home filtration devices tested in this study may provide protection against human exposure to cyanotoxin in addition to protection provided by water treatment methodologies utilized in water treatment facilities. Further studies need to be done to assess the efficacy of home filtration devices for various cyanotoxins and for other filtering conditions such as increased toxin load, the presence of other contaminants in drinking water, and the repetitive use of the same filter over longer time intervals.

ART2, a T Cell Surface Mono-ADP-ribosyltransferase, Generates Extracellular Poly(ADP-ribose)

The Journal of Biological Chemistry. Nov, 2006  |  Pubmed ID: 16931513

NAD functions in multiple aspects of cellular metabolism and signaling through enzymes that covalently transfer ADP-ribose from NAD to acceptor proteins, thereby altering their function. NAD is a substrate for two enzyme families, mono-ADP-ribosyltransferases (mARTs) and poly(ADP-ribose) polymerases (PARPs), that covalently transfer an ADP-ribose monomer or polymer, respectively, to acceptor proteins. ART2, a mART, is a phenotypic marker of immunoregulatory cells found on the surface of T lymphocytes, including intestinal intraepithelial lymphocytes (IELs). We have shown that the auto-ADP-ribosylation of the ART2.2 allelic protein is multimeric. Our backbone structural alignment of ART2 (two alleles of the rat art2 gene have been reported, for simplicity, the ART2.2 protein investigated in this study will be referred to as ART2) and PARP suggested that multimeric auto-ADP-ribosylation of ART2 may represent an ADP-ribose polymer, rather than multiple sites of mono-ADP-ribosylation. To investigate this, we used highly purified recombinant ART2 and demonstrated that ART2 catalyzes the formation of an ADP-ribose polymer by sequencing gel and by HPLC and MS/MS mass spectrometry identification of PR-AMP, a breakdown product specific to poly(ADP-ribose). Furthermore, we identified the site of ADP-ribose polymer attachment on ART2 as Arg-185, an arginine in a crucial loop of its catalytic core. We found that endogenous ART2 on IELs undergoes multimeric auto-ADP-ribosylation more efficiently than ART2 on peripheral T cells, suggesting that these distinct lymphocyte populations differ in their ART2 surface topology. Furthermore, ART2.2 IELs are more resistant to NAD-induced cell death than ART2.1 IELs that do not have multimeric auto-ADP-ribosylation activity. The data suggest that capability of polymerizing ADP-ribose may not be unique to PARPs and that poly(ADP-ribosylation), an established nuclear activity, may occur extracellularly and modulate cell function.

Influence of Aging on the Polar and Neutral Lipid Profiles in Human Meibomian Gland Secretions

Archives of Ophthalmology. Sep, 2006  |  Pubmed ID: 16966624

To determine whether aging is associated with significant alterations in the polar and neutral lipid profiles in human meibomian gland secretions.

M1 Muscarinic Receptors Inhibit L-type Ca2+ Current and M-current by Divergent Signal Transduction Cascades

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Nov, 2006  |  Pubmed ID: 17093080

Ion channels reside in a sea of phospholipids. During normal fluctuations in membrane potential and periods of modulation, lipids that directly associate with channel proteins influence gating by incompletely understood mechanisms. In one model, M(1)-muscarinic receptors (M(1)Rs) may inhibit both Ca(2+) (L- and N-) and K(+) (M-) currents by losing a putative interaction between channels and phosphatidylinositol-4,5-bisphosphate (PIP(2)). However, we found previously that M(1)R inhibition of N-current in superior cervical ganglion (SCG) neurons requires loss of PIP(2) and generation of a free fatty acid, probably arachidonic acid (AA) by phospholipase A(2) (PLA(2)). It is not known whether PLA(2) activity and AA also participate in L- and M-current modulation in SCG neurons. To test whether PLA(2) plays a similar role in M(1)R inhibition of L- and M-currents, we used several experimental approaches and found unanticipated divergent signaling. First, blocking resynthesis of PIP(2) minimized M-current recovery from inhibition, whereas L-current recovered normally. Second, L-current inhibition required group IVa PLA(2) [cytoplasmic PLA(2) (cPLA(2))], whereas M-current did not. Western blot and imaging studies confirmed acute activation of cPLA(2) by muscarinic stimulation. Third, in type IIa PLA(2) [secreted (sPLA(2))](-/-)/cPLA(2)(-/-) double-knock-out SCG neurons, muscarinic inhibition of L-current decreased. In contrast, M-current inhibition remained unaffected but recovery was impaired. Our results indicate that L-current is inhibited by a pathway previously shown to control M-current over-recovery after washout of muscarinic agonist. Our findings support a model of M(1)R-meditated channel modulation that broadens rather than restricts the roles of phospholipids and fatty acids in regulating ion channel activity.

Assessing Potential Removal of Low-head Dams in Urban Settings: an Example from the Ottawa River, NW Ohio

Environmental Management. Jan, 2007  |  Pubmed ID: 17122999

This is a study of the scientific component of an effort to restore an urban river by removing a low-head dam. The Secor Dam is owned by a local government entity near Toledo, Ohio. The proposed removal of the last structure impeding flow on the Ottawa River has broad appeal, but the owner is concerned about liability issues, particularly potential changes to the flood regime, the presence of contaminated sediments behind the dam, and possible downstream transport of reservoir sediments. Assessing sediment contamination involved sediment sampling and analysis of trace metals and organic contaminants. Forecasting sediment transport involved field methods to determine the volume and textural properties of reservoir and upstream sediment and calculations to determine the fate of reservoir sediments. Forecasting changes in the flood regime involved HEC-RAS hydrological models to determine before and after dam removal flood scenarios using LiDAR data imported into an ArcGIS database. The resulting assessment found potential sediment contamination to be minor, and modeling showed that the removal of the dam would have minimal impacts on sediment transport and flood hazards. Based on the assessment, the removal of the dam has been approved by its owners.

Oocyte CD9 is Enriched on the Microvillar Membrane and Required for Normal Microvillar Shape and Distribution

Developmental Biology. Apr, 2007  |  Pubmed ID: 17239847

Microvilli are found on the surface of many cell types, including the mammalian oocyte, where they are thought to act in initial contact of sperm and oocyte plasma membranes. CD9 is currently the only oocyte protein known to be required for sperm-oocyte fusion. We found CD9 is localized to the oocyte microvillar membrane using transmission electron microscopy (TEM). Scanning electron microscopy (SEM) showed that CD9 null oocytes, which are unable to fuse with sperm, have an altered length, thickness and density of their microvilli. One aspect of this change in morphology was quantified using TEM by measuring the radius of curvature at the microvillar tips. A small radius of curvature is thought to promote fusibility and the radius of curvature of microvillar tips on CD9 wild-type oocytes was found to be half that of the CD9 null oocytes. We found that oocyte CD9 co-immunoprecipitates with two Ig superfamily cis partners, EWI-2 and EWI-F, which could have a role in linking CD9 to the oocyte microvillar actin core. We also examined latrunculin B-treated oocytes, which are known to have reduced fusion ability, and found altered microvillar morphology by SEM and TEM. Our data suggest that microvilli may participate in sperm-oocyte fusion. Microvilli could act as a platform to concentrate adhesion/fusion proteins and/or provide a membrane protrusion with a low radius of curvature. They may also have a dynamic interaction with the sperm that serves to capture the sperm cell and bring it into close contact with the oocyte plasma membrane.

Mass Spectrometry Analysis of HIV-1 Vif Reveals an Increase in Ordered Structure Upon Oligomerization in Regions Necessary for Viral Infectivity

Proteins. Nov, 2007  |  Pubmed ID: 17598142

HIV-1 Vif, an accessory protein in the viral genome, performs an important role in viral pathogenesis by facilitating the degradation of APOBEC3G, an endogenous cellular inhibitor of HIV-1 replication. In this study, intrinsically disordered regions are predicted in HIV-1 Vif using sequence-based algorithms. Intrinsic disorder may explain why traditional structure determination of HIV-1 Vif has been elusive, making structure-based drug design impossible. To characterize HIV-1 Vif's structural topology and to map the domains involved in oligomerization we used chemical cross-linking, proteolysis, and mass spectrometry. Cross-linking showed evidence of monomer, dimer, and trimer species via denaturing gel analysis and an additional tetramer via western blot analysis. We identified 47 unique linear peptides and 24 (13 intramolecular; 11 intermolecular) noncontiguous, cross-linked peptides, among the noncross-linked monomer, cross-linked monomer, cross-linked dimer, and cross-linked trimer samples. Almost complete peptide coverage of the N-terminus is observed in all samples analyzed, however reduced peptide coverage in the C-terminal region is observed in the dimer and trimer samples. These differences in peptide coverage or "protections" between dimer and trimer indicate specific differences in packing between the two oligomeric forms. Intramolecular cross-links within the monomer suggest that the N-terminus is likely folded into a compact domain, while the C-terminus remains intrinsically disordered. Upon oligomerization, as evidenced by the intermolecular cross-links, the C-terminus of one Vif protein becomes ordered by wrapping back on the N-terminal domain of another. In addition, the majority of the intramolecular cross-links map to regions that have been previously reported to be necessary for viral infectivity. Thus, this data suggests HIV-1 Vif is in a dynamic equilibrium between the various oligomers potentially allowing it to interact with other binding partners.

Electron Crystallography of Membrane Proteins

Methods in Molecular Biology (Clifton, N.J.). , 2007  |  Pubmed ID: 17656758

Electron crystallography studies the structure of two-dimensional crystals of membrane proteins or other crystalline arrays. This method has been used to determine the atomic structures of six membrane proteins and tubulin, as well as several other structures at a slightly lower resolution, where secondary structure motifs could be identified. To preserve the high-resolution structure of 2D crystals, the meticulous sample preparation for electron crystallography is of outmost importance. Charge-induced specimen drift and lack of specimen flatness can severely affect the resolution of images for tilted samples. However, sample preparations that sandwich the two-dimensional crystals between symmetrical carbon films reduce the charge-induced specimen drift, and the flatness of the preparations can be optimized by the choice of the grid material and the preparation protocol. Data collection in the cryoelectron microscope using either the imaging or the electron diffraction mode has to be performed after low-dose procedures. Spot scanning further reduces the charge-induced specimen drift.

Plasmodium-induced Inflammation by Uric Acid

PLoS Pathogens. Mar, 2008  |  Pubmed ID: 18369465

Infection of erythrocytes with the Plasmodium parasite causes the pathologies associated with malaria, which result in at least one million deaths annually. The rupture of infected erythrocytes triggers an inflammatory response, which is induced by parasite-derived factors that still are not fully characterized. Induced secretion of inflammatory cytokines by these factors is considered a major cause of malaria pathogenesis. In particular, the inflammatory cytokine tumor necrosis factor (TNF) is thought to mediate most of the life-threatening pathologies of the disease. Here we describe the molecular characterization of a novel pathway that results in the secretion of TNF by host cells. We found that erythrocytes infected by Plasmodium accumulate high concentrations of hypoxanthine and xanthine. Degradation of Plasmodium-derived hypoxanthine/xanthine results in the formation of uric acid, which triggers the secretion of TNF. Since uric acid is considered a "danger signal" released by dying cells to alert the immune system, Plasmodium appears to have co-evolved to exploit this warning system. Identifying the mechanisms used by the parasite to induce the host inflammatory response is essential to develop urgently needed therapies against this disease.

Vaccinia Peptides Eluted from HLA-DR1 Isolated from Virus-infected Cells Are Recognized by CD4+ T Cells from a Vaccinated Donor

Journal of Proteome Research. Jul, 2008  |  Pubmed ID: 18507432

Class II MHC proteins bind peptides and present them to CD4 (+) T cells as part of the immune system's surveillance of bodily tissues for foreign and pathogenic material. Antigen processing and presentation pathways have been characterized in detail in normal cells, but there is little known about the actual viral peptides that are presented to CD4 (+) T cells that signal infection. In this study, two-dimensional LC-MS/MS was used to identify vaccinia virus-derived peptides among the hundreds to thousands of peptide antigens bound to the human class II MHC protein HLA-DR1 on the surface of vaccinia virus-infected cells. The peptides, derived from the I6L, D6R, and A10L viral proteins, were 15 residues in length, bound efficiently to HLA-DR1 as synthetic peptides, and were recognized by vaccinia-specific CD4 (+) T cells obtained from an immunized donor.

Differential Proteomics in the Aging Noble Rat Ventral Prostate

Proteomics. Jul, 2008  |  Pubmed ID: 18546156

Incidence of prostatic diseases increases dramatically with age which may be related to a decline in androgen support. However, the key mechanisms underlying prostate aging remain unclear. In the present study, we investigated the aging process in the ventral prostate (VP) of Noble rats by identifying differentially expressed prostate proteins between 3- and 16-month-old animals using ICAT and MS. In total, 472 proteins were identified with less than a 1% false positive rate, among which 34 were determined to have a greater than two-fold increase or 1.7-fold decrease in expression in the aged VPs versus their younger counterparts. The majority of the differentially expressed proteins identified have not been previously reported to be associated with prostate aging, and they fall into specific functional categories, including oxidative stress/detoxification, chaperones, protein biosynthesis, vesicle transport, and intracellular trafficking. The expression of GST, ferritin, clusterin, kininogen, oxygen regulated protein 150, spermidine synthase, ADP ribosylation factor, and cyclophilin B was verified by Western blot analyses on samples used for the ICAT study, as well as on those obtained from an independent group of animals comprised of three age groups. To the best of our knowledge, this is the first study on the proteome of the aging rat prostate.

Low-dose Aberration Corrected Cryo-electron Microscopy of Organic Specimens

Ultramicroscopy. Nov, 2008  |  Pubmed ID: 18703285

Spherical aberration (C(s)) correction in the transmission electron microscope has enabled sub-angstrom resolution imaging of inorganic materials. To achieve similar resolution for radiation-sensitive organic materials requires the microscope to be operated under hybrid conditions: low electron dose illumination of the specimen at liquid nitrogen temperature and low defocus values. Initial images from standard inorganic and organic test specimens have indicated that under these conditions C(s)-correction can provide a significant improvement in resolution (to less than 0.16nm) for direct imaging of organic samples.

In Vitro Evidence That Commercial Influenza Vaccines Are Not Similar in Their Ability to Activate Human T Cell Responses

Vaccine. Jan, 2009  |  Pubmed ID: 18977404

We evaluated three commercial trivalent inactivated vaccines (TIVs) from the 2007-2008 season in terms of their ability to elicit in vitro T cell responses. T cell-mediated immunity may offer a more cross-reactive vaccine approach for the prevention of pandemic or epidemic influenza. Human cytotoxic T cell lines demonstrated differences in matrix protein 1 and nucleocapsid protein recognition of autologous target cells. Peripheral blood mononuclear cells stimulated with each of the TIVs showed statistically significant differences between the vaccines in the numbers of IFNgamma producing cells activated. These data suggest that TIV vaccines are not similar in their ability to activate human T cell responses.

Biochemical Characterization of Membrane Fractions in Murine Sperm: Identification of Three Distinct Sub-types of Membrane Rafts

Journal of Cellular Physiology. Mar, 2009  |  Pubmed ID: 19006178

Despite enormous interest in membrane raft micro-domains, no studies in any cell type have defined the relative compositions of the raft fractions on the basis of their major components--sterols, phospholipids, and proteins--or additional raft-associating lipids such as the ganglioside, G(M1). Our previous localization data in live sperm showed that the plasma membrane overlying the acrosome represents a stabilized platform enriched in G(M1) and sterols. These findings, along with the physiological requirement for sterol efflux for sperm to function, prompted us to characterize sperm membrane fractions biochemically. After confirming limitations of commonly used detergent-based approaches, we utilized a non-detergent-based method, separating membrane fractions that were reproducibly distinct based on sterol, G(M1), phospholipid, and protein compositions (both mass amounts and molar ratios). Based on fraction buoyancy and biochemical composition, we identified at least three highly reproducible sub-types of membrane raft. Electron microscopy revealed that raft fractions were free of visible contaminants and were separated by buoyancy rather than morphology. Quantitative proteomic comparisons and fluorescence localization of lipids suggested that different organelles contributed differentially to individual raft sub-types, but that multiple membrane micro-domain sub-types could exist within individual domains. This has important implications for scaffolding functions broadly associated with rafts. Most importantly, we show that the common practice of characterizing membrane domains as either "raft" or "non-raft" oversimplifies the actual biochemical complexity of cellular membranes.

Disruption of the M80-Fe Ligation Stimulates the Translocation of Cytochrome C to the Cytoplasm and Nucleus in Nonapoptotic Cells

Proceedings of the National Academy of Sciences of the United States of America. Feb, 2009  |  Pubmed ID: 19196960

Native cytochrome c (cyt c) has a compact tertiary structure with a hexacoordinated heme iron and functions in electron transport in mitochondria and apoptosis in the cytoplasm. However, the possibility that protein modifications confer additional functions to cyt c has not been explored. Disruption of methionine 80 (M80)-Fe ligation of cyt c under nitrative stress has been reported. To model this alteration and determine if it confers new properties to cyt c, a cyt c mutant (M80A) was constitutively expressed in cells. M80A-cyt c has increased peroxidase activity and is spontaneously released from mitochondria, translocating to the cytoplasm and nucleus in the absence of apoptosis. Moreover, M80A models endogenously nitrated cyt c because nitration of WT-cyt c is associated with its translocation to the cytoplasm and nucleus. Further, M80A cyt c may up-regulate protective responses to nitrative stress. Our findings raise the possibility that endogenous protein modifications that disrupt the M80-Fe ligation (such as tyrosine nitration) stimulate nuclear translocation and confer new functions to cyt c in nonapoptotic cells.

Natural Lipid Ligands Associated with Human CD1d Targeted to Different Subcellular Compartments

Journal of Immunology (Baltimore, Md. : 1950). Apr, 2009  |  Pubmed ID: 19342656

CD1d is an MHC class I-like membrane glycoprotein that presents lipid Ags to NKT cells. Despite intensive biochemical, genetic, and structural studies, the endogenous lipids associated with CD1d remain poorly defined because of the biochemical challenges posed by their hydrophobic nature. In this study, we report the generation of a protease-cleavable CD1d variant with a similar trafficking pattern to wild-type CD1d that can be purified in the absence of detergent and allows the characterization of the naturally associated lipids. In addition, we used soluble variants of CD1d that are secreted or retained in the endoplasmic reticulum (ER) to survey their acquired lipids. By using multiple mass spectrometry methods, we found that CD1d retained in the ER is predominantly loaded with the most abundant phospholipid in the cell, phosphatidyl choline, while the protease cleavable version of CD1d contains bound sphingomyelin and lysophospholipids in addition to phosphatidyl choline. The secreted soluble version of CD1d, in contrast, lacks detectable phosphatidyl choline and the only detectable associated lipid is sphingomyelin. The data suggest that, in the absence of infection or stress, CD1d molecules survey the ER, the secretory pathway, and the endocytic pathway, and accumulate the most abundantly available lipids present in these compartments.

Proteomics Analysis of Perilymph and Cerebrospinal Fluid in Mouse

The Laryngoscope. May, 2009  |  Pubmed ID: 19358201

Proteins in perilymph may alter the delivery profile of implantable intracochlear drug delivery systems through biofouling. Knowledge of protein composition will help anticipate interactions with delivered agents.

Uric Acid is a Mediator of the Plasmodium Falciparum-induced Inflammatory Response

PloS One. , 2009  |  Pubmed ID: 19381275

Malaria triggers a high inflammatory response in the host that mediates most of the associated pathologies and contributes to death. The identification of pro-inflammatory molecules derived from Plasmodium is essential to understand the mechanisms of pathogenesis and to develop targeted interventions. Uric acid derived from hypoxanthine accumulated in infected erythrocytes has been recently proposed as a mediator of inflammation in rodent malaria.

Long-term Statin Therapy and CSF Cholesterol Levels: Implications for Alzheimer's Disease

Dementia and Geriatric Cognitive Disorders. , 2009  |  Pubmed ID: 19478483

It is not yet established whether statins (lipophilic or hydrophilic) reduce the risk of Alzheimer's disease and, if so, by differentially modifying brain lipid levels. Our aim was to assess changes in brain cholesterol metabolism as reflected in the cerebrospinal fluid (CSF) before and after treatment with either atorvastatin or simvastatin.

Electron Microscopy and Spectroscopy on the Ultrafast Timescale

Chemphyschem : a European Journal of Chemical Physics and Physical Chemistry. Mar, 2010  |  Pubmed ID: 20148430

HPLC/MS(N) Analysis of Retinoids

Methods in Molecular Biology (Clifton, N.J.). , 2010  |  Pubmed ID: 20552427

This protocol describes a highly sensitive and selective method to quantify retinoids using normal-phase HPLC with online APCI MS(N). The retinoids are key regulators of gene expression, retinol being oxidized via a retinaldehyde intermediate to retinoic acid (RA) which activates specific nuclear receptors, the signalling of which is turned off by oxidative inactivation of the ligand to 4-oxo-RA and other metabolites. Many of these retinoids are present only transiently at low concentrations in tissues and during analysis are labile to heat, light, and oxygen. HPLC with online APCI MS(N) provides a rapid technique to quantify these retinoids simultaneously. Techniques to extract the retinoids and prevent their degradation are described, with an emphasis on transcriptionally active RA. RA controls patterning of gene expression in the embryo, organizing embryonic morphology in the central nervous system. Similarly, a patterned distribution of RA controls function of the adult CNS, a tissue particularly difficult to analyse for RA because of its high lipid content. To understand how these patterns are organized in the brain and change over time, it is essential to determine the concentration of RA in small areas of tissues, and techniques of exquisite sensitivity are indispensable.

A Method for MS(E) Differential Proteomic Analysis of Archival Formalin-fixed Celloidin-embedded Human Inner Ear Tissue

Hearing Research. Dec, 2010  |  Pubmed ID: 20708670

Proteomic analysis of cadaveric formalin-fixed, celloidin-embedded (FFCE) temporal bone tissue has the potential to provide new insights into inner ear disorders. We have developed a liquid chromatography-mass spectrometry (LC-MS) method for tissue sections embedded with celloidin. Q-TOF (Quadrupole-time of flight mass spectrometry) MS(E) (mass spectrometry where E represents collision energy) and Identity(E)™ were used in conjunction with nano-UPLC (capillary ultrahigh pressure liquid chromatography) for robust identification and quantification of a large number of proteins. Formalin-fixed paraffin-embedded (FFPE) mouse liver sections were used to evaluate formalin de-cross-linking by five different methods. Unfixed fresh mouse liver tissue was used as a control. Five different methods for preparation of FFPE tissue for MS analysis were compared, as well as four methods for celloidin removal with FFCE mouse liver tissue. The methods judged best were applied to FFCE 20 μm sections of mouse inner ear samples, and FFCE 20 μm human inner ear and human otic capsule bone sections. Three of the five-tissue extraction methods worked equally in detecting peptides and proteins from FFPE mouse liver tissue. The modified Liquid Tissue kit protocol was chosen for further studies. Four different celloidin removal methods were compared and the acetone removal method was chosen for further analysis. These two methods were applied to the analysis of FFCE inner ear and otic capsule sections. Proteins from all major cellular components were detected in the FFCE archival human temporal bone sections. This newly developed technique enables the use of FFCE tissues for proteomic studies.

Haptoglobin As an Early Serum Biomarker of Virus-induced Autoimmune Type 1 Diabetes in Biobreeding Diabetes Resistant and LEW1.WR1 Rats

Experimental Biology and Medicine (Maywood, N.J.). Nov, 2010  |  Pubmed ID: 20975081

Proteomic profiling of serum is a powerful technique to identify differentially expressed proteins that can serve as biomarkers predictive of disease onset. In this study, we utilized two-dimensional (2D) gel analysis followed by matrix-assisted-laser desorption/ionization time-of-flight mass spectrometry analysis to identify putative serum biomarkers for autoimmune type 1 diabetes (T1D) in biobreeding diabetes resistant (BBDR) rats induced to express the disease. Treatment with toll-like receptor 3 ligand, polyinosinic:polycytidilic acid (pIC), plus infection with Kilham rat virus (KRV), a rat parvovirus, results in nearly 100% of young BBDR rats becoming diabetic within 11-21 d. Sera collected from prediabetic rats at early time points following treatment with pIC + KRV were analyzed by 2D gel electrophoresis and compared with sera from control rats treated with phosphate-buffered saline, pIC alone or pIC + H1, a non-diabetogenic parvovirus. None of the latter three control treatments precipitates T1D. 2D gel analysis revealed that haptoglobin, an acute phase and hemoglobin scavenger protein, was differentially expressed in the sera of rats treated with pIC + KRV relative to control groups. These results were confirmed by Western blot and enzyme-linked immunosorbent assay studies, which further validated haptoglobin levels as being differentially increased in the sera of pIC + KRV-treated rats relative to controls during the first week following infection. Early elevations in serum haptoglobin were also observed in LEW1.WR1 rats that became diabetic following infection with rat cytomegalovirus. The identification and validation of haptoglobin as a putative serum biomarker for autoimmune T1D in rats now affords us the opportunity to test the validity of this protein as a biomarker for human T1D, particularly in those situations where viral infection is believed to precede the onset of disease.

Prevention of Alzheimer's Disease in High Risk Groups: Statin Therapy in Subjects with PSEN1 Mutations or Heterozygosity for Apolipoprotein E Epsilon 4

Alzheimer's Research & Therapy. , 2010  |  Pubmed ID: 21062519

Controlled Growth of Nanoparticles from Solution with in Situ Liquid Transmission Electron Microscopy

Nano Letters. Jul, 2011  |  Pubmed ID: 21619024

Direct visualization of lead sulfide nanoparticle growth is demonstrated by selectively decomposing a chemical precursor from a multicomponent solution using in situ liquid transmission electron microscopy. We demonstrate reproducible control over growth mechanisms that dictate the final morphology of nanostructures while observing growth in real-time with subnanometer spatial resolution. Furthermore, while an intense electron beam can initiate nanoparticle growth, it is also shown that a laser can trigger the reaction independently of the imaging electrons.

Tumor Cell Selective Cytotoxicity and Apoptosis Induction by an Herbal Preparation from Brucea Javanica

North American Journal of Medicine & Science. Apr, 2011  |  Pubmed ID: 21654932

The plant Brucea javanica has shown impressive efficacy for treating various diseases including cancer. However, the mechanism by which B. javanica acts is poorly understood. We have established tissue culture assays to study the effects of B. javanica on cervical and several other cancer cells. Our results demonstrated that the aqueous extract from B. javanica is selectively toxic to cancer cells. Induction of apoptosis by B. javanica appears to be a possible mechanism by which it kills cancer cells. Interestingly, a significant increase of p53 protein level was observed in these apoptotic cells. Our studies indicated that both p53-dependent and p53-independent activities contributed to herb-induced cell death. These results imply that further studies with B. javanica may lead to the development of novel anti-cancer drugs.

Conformational Lability in the Class II MHC 310 Helix and Adjacent Extended Strand Dictate HLA-DM Susceptibility and Peptide Exchange

Proceedings of the National Academy of Sciences of the United States of America. Nov, 2011  |  Pubmed ID: 22084083

HLA-DM is required for efficient peptide exchange on class II MHC molecules, but its mechanism of action is controversial. We trapped an intermediate state of class II MHC HLA-DR1 by substitution of αF54, resulting in a protein with increased HLA-DM binding affinity, weakened MHC-peptide hydrogen bonding as measured by hydrogen-deuterium exchange mass spectrometry, and increased susceptibility to DM-mediated peptide exchange. Structural analysis revealed a set of concerted conformational alterations at the N-terminal end of the peptide-binding site. These results suggest that interaction with HLA-DM is driven by a conformational change of the MHC II protein in the region of the α-subunit 3(10) helix and adjacent extended strand region, and provide a model for the mechanism of DM-mediated peptide exchange.

Re-Electrospraying Splash-Landed Proteins and Nanoparticles

Analytical Chemistry. Feb, 2012  |  Pubmed ID: 22283555

FITC-albumin, Lsr-F, or fluorescent polystyrene latex particles were electrosprayed from aqueous buffer and subjected to dispersion by differential electrical mobility at atmospheric pressure. A resulting narrow size cut of singly charged molecular ions or particles was passed through a condensation growth tube collector to create a flow stream of small water droplets, each carrying a single ion or particle. The droplets were splash landed (impacted) onto a solid or liquid temperature controlled surface. Small pools of droplets containing size-selected particles, FITC-albumin, or Lsr-F were recovered, re-electrosprayed, and, when analyzed a second time by differential electrical mobility, showed increased homogeneity. Transmission electron microscopy (TEM) analysis of the size-selected Lsr-F sample corroborated the mobility observation.