Photodissociation tandem mass spectrometry at 266 nm of an aliphatic peptide derivatized with phenyl isothiocyanate and 4-sulfophenyl isothiocyanate

Photodissociation (PD) tandem mass spectra have been obtained at 266 nm for the protonated molecules of a tryptic peptide, ASHLGLAR, and of its phenyl isothiocyanate and 4-sulfophenyl isothiocyanate derivatives, generated by matrix-assisted laser desorption/ionization. Derivatization with the aromatic chromophores greatly reduced the intensity of the laser required for efficient PD. Major fragment ions observed in the three spectra are quite similar. General features of the PD tandem mass spectra and their potential utility for analytical purposes are discussed.     

bPAK-interacting exchange factor may regulate actin cytoskeleton through interaction with actin

p21-activated kinase (PAK)-interacting exchange factor (PIX) is known to be involved in regulation of Cdc42/Rac GTPases and PAK activity. PIX binds to the proline-rich region of PAK, and regulates biological events through activation of Cdc42/Rac GTPase. To further investigate the role of PIX we produced monoclonal antibodies (Mab) against bPIX. Three clones; N-C6 against N-terminal half and C-A3 and C-B7 against C- terminal half of bPIX were generated and characterized. N-C6 Mab detected bPIX as a major band in most cell lines. C-A3 Mab recognizes GIT-binding domain (GBD), but it does not interfere with GIT binding to bPIX. Using C-A3 Mab possible bPIX interaction with actin in PC12 cells was examined. bPIX Mab (C-A3) specifically precipitated actin of the PC12 cell lysates whereas actin Mab failed to immunoprecipitate bPIX. Co-sedimentation of PC12 cell lysates with the polymerized F-actin resulted in the recovery of most of bPIX in the cell lysates. These results suggest that bPIX may not interact with soluble actin but with polymerized F-actin and revealed that bPIX constitutes a functional complex with actin. These data indicate real usefulness of the bPIX Mab in the study of bPIX role(s) in regulation of actin cyoskeleton.     

Effect of interacting nanoparticles on the ordered morphology of block copolymer/nanoparticle mixtures

We investigated the effect of hard additives, that is, magnetic nanoparticles (NPs) and metal NPs, on the ordered morphology of block copolymers by varying the NP concentration. To characterize the structural changes of a block copolymer associated with different NP loadings, small-angle X-ray scattering and transmission electron microscopy were performed. Monodisperse maghemite (γ-Fe₂O₃) NPs (7 nm in diameter) and silver (Ag) NPs (6 nm in diameter) with surfaces modified with oleic acids were synthesized, and a cylinder-forming poly(styrene-block-isoprene) diblock copolymer was used as a structure-directing matrix for the NPs. As the NP concentration increased, domains of NP aggregates were observed for both magnetic and metal NPs. In the case of mixtures of cylinder-forming poly(styrene-block-isoprene) and Ag NPs with weak particle–particle interactions, random aggregates of Ag NPs were observed, and the ordered morphology of the block copolymer lost its long-range order with an increase in the NP concentration. However, regular, latticelike aggregates obtained with γ-Fe₂O₃ NPs, because of the strong interparticle interactions, induced an intriguing morphological transformation from hexagonal cylinders to body-centered-cubic spheres via undulated cylinders, whereas the neat block copolymer did not show such a morphological transition over a wide range of temperatures. The interplay between magnetic NPs and the block copolymer was also tested with magnetic NPs of different sizes. © 2006 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 44: 3571–3579, 2006     

Mimicry of tandem repeat peptides against cell surface carbohydrates

Our approach to multivalent peptide construction relies on tentacle peptides, also known as a multiple antigenic peptides, which contain two and four repeats of a selected peptide. In this communication, we report the results of preliminary studies aimed at (1) the selection of short peptides against the carbohydrate, sLeX, (2) the synthesis of tentacle dimers and tetramers of the selected peptides, and (3) the determination of affinities and specificities of the peptides to several related carbohydrates by using the surface plasmon resonance (SPR) and the equilibrium dialysis techniques. Binding affinity studies, as well as assays of in vitro binding of the peptides to a sLeX-specific cell line, have shown that the tetrameric peptides bind to the cell surface sugars.     

Ionizing radiation induces blockade of c-Jun N-terminal kinase-dependent cell death pathway in a manner correlated with p21(Cip/WAF1) induction in primary cultured normal human fibroblasts

During radiotherapy of cancer, neighboring normal cells may receive sub-lethal doses of radiation. To investigate whether such low levels of radiation modulate normal cell responses to death stimuli, primary cultured human fibroblasts were exposed to various doses of gamma-rays. Analysis of cell viability using an exclusion dye propidium iodide revealed that the irradiation up to 10 Gy killed the fibroblasts only to a minimal extent. In contrast, the cells efficiently lost their viability when exposed to 0.5-0.65 mM H(2)O(2). This type of cell death was accompanied by JNK activation, and was reversed by the use of a JNK-specific inhibitor SP600125. Interestingly, H(2)O(2) failed to kill the fibroblasts when these cells were pre-irradiated, 24 h before H(2)O(2) treatment, with 0.25-0.5 Gy of gamma-rays. These cytoprotective doses of gamma-rays did not enhance cellular capacity to degrade H(2)O(2), but elevated cellular levels of p21(Cip/WAF1), a p53 target that can suppress H(2)O(2)-induced cell death by blocking JNK activation. Consistently, H(2)O(2)-induced JNK activation was dramatically suppressed in the pre-irradiated cells. The overall data suggests that ionizing radiation can impart normal fibroblasts with a survival advantage against oxidative stress by blocking the process leading to JNK activation.     

Targeted lipidomics using electron capture atmospheric pressure chemical ionization mass spectrometry

There is an increasing need to be able to conduct quantitative lipidomics analyses as a complement to proteomics studies. The highest specificity for proteomics analysis can be obtained using methodology based on electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI) coupled with liquid chromatography/tandem mass spectrometry (LC/MS/MS). For lipidomics analysis it is often necessary to be able to separate enantiomers and regioisomers. This can be very challenging when using methodology based on conventional reversed-phase chromatography. Normal-phase chromatography using chiral columns can provide dramatic improvements in the resolution of enantiomers and regioisomers. However, conventional ESI- and APCI-MS/MS has limited sensitivity, which makes it difficult to conduct studies in cell culture systems where only trace amounts of non-esterified bioactive lipids are present. The use of electron capture APCI-MS/MS overcomes this problem. Enantiomers and regioisomers of diverse bioactive lipids can be quantified using stable isotope dilution methodology coupled with normal-phase chiral chromatography and electron capture APCI-MS/MS. This methodology has allowed a lipidomics profile from rat epithelial cells maintained in culture to be delineated and allowed the effect of a non-selective lipoxygenase inhibitor to be assessed.     

Delta-secretase cleavage of Tau mediates its pathology and propagation in Alzheimer’s disease

Alzheimer’s disease (AD) is a progressive neurodegenerative disease with age as a major risk factor. AD is the most common dementia with abnormal structures, including extracellular senile plaques and intraneuronal neurofibrillary tangles, as key neuropathologic hallmarks. The early feature of AD pathology is degeneration of the locus coeruleus (LC), which is the main source of norepinephrine (NE) supplying various cortical and subcortical areas that are affected in AD. The spread of Tau deposits is first initiated in the LC and is transported in a stepwise manner from the entorhinal cortex to the hippocampus and then to associative regions of the neocortex as the disease progresses. Most recently, we reported that the NE metabolite DOPEGAL activates delta-secretase (AEP, asparagine endopeptidase) and triggers pathological Tau aggregation in the LC, providing molecular insight into why LC neurons are selectively vulnerable to developing early Tau pathology and degenerating later in the disease and how δ-secretase mediates the spread of Tau pathology to the rest of the brain. This review summarizes our current understanding of the crucial role of δ-secretase in driving and spreading AD pathologies by cleaving multiple critical players, including APP and Tau, supporting that blockade of δ-secretase may provide an innovative disease-modifying therapeutic strategy for treating AD.Subject terms: Neurodegeneration, Alzheimer''s disease     

Quantitative analysis of shape-specific interactions of Rev response element with a positively charged Rev peptide by capillary electrophoresis

Human immunodeficiency virus type 1 (HIV-1) Rev protein is known to regulate the expression of proteins via binding to an RNA site termed the HIV Rev response element (RRE) presumably with a defined shape, mediated mainly by electrostatic interactions. We have developed a quantitative method based on CE-LIF detection for a systematic evaluation of interactions between a truncated RRE (tRRE) RNA and an HIV-1 Rev peptide. Employing a fluorescently labeled HIV-1 Rev protein fragment (RevF) as a probe, buffers were evaluated for the separation and detection as well as for the RNA shape-specific formation of the complex. Selection of an optimal buffer condition allowed us to perform quantitation of the tRRE-RevF complex formation and determine its dissociation constant. In addition, competitive inhibitions of the RNA-peptide interaction by some aminoglycosides were evaluated quantitatively by monitoring the complex peak, resulting in determination of IC(50) values. This sensitive and reliable CE-LIF-based method would be of interest in developing various screening systems for RNA interference in drug discovery.     

Notable coordination effects of 2-pyridinesulfonamides leading to efficient aziridination and selective aziridine ring opening

[reaction: see text] We have developed, on the basis of a chelation-strategy, an efficient copper-catalyzed aziridination protocol with the use of 5-methyl-2-pyridinesulfonamide and PhI(OAc)(2). The reaction proceeds smoothly under mild conditions to give aziridines in moderate to good yields in the absence of external ligands or bases. The coordination-assisted approach offers the additional benefits that efficient deprotection of the N-substituent and selective aziridine ring-opening are effectively achieved.     

Surface behavior of amphiphilic heteroarm star-block copolymers with asymmetric architecture

We study the surface behavior of the asymmetric amphiphilic heteroarm poly(ethylene oxide) (PEO)/polystyrene (PS) star polymer on solid substrate. These star polymers differ in both architecture (four- and three-arm molecules, PEO-b-PS(3) and PEO-b-PS(2)) and in the length of PS chains (molecular weight from about 10 000 up to 24 000). We observed that, for a given chemical composition with a predominant content of hydrophobic blocks, the compression behavior of the PS domain structure controls the surface behavior and the final morphology of the monolayers. New features of the surface behavior of star-block copolymers are high stretching of the PS arms from the interface and enhanced stability of the circular PS domain structure, even at high compression. We suggest that for asymmetric star-block copolymers both architecture and chemical composition heavily favor the formation of highly curved interfaces and, thus, more stable circular domain structure with stretched PS arms.