Search

Exercise has been shown to improve function of the left ventricle (LV) following myocardial infarction (MI). The mechanisms to explain this benefit have not been fully delineated, but may involve improved mechanics resulting in unloading effects and increased endothelial nitric oxide synthase levels [1,2]. Accordingly, the goal of this study was to determine how the LV infarct proteome is altered by a post-MI exercise regimen. Sprague-Dawley rats underwent ligation of the left descending coronary artery to induce MI. Exercise training was initiated four weeks post-MI and continued for 8 weeks in n=12 rats. Compared with the sedentary MI group (n=10), the infarct region of rats receiving exercise showed 20 protein spots with altered intensities in two-dimensional gels (15 increased and 5 decreased; p<0.05). Of 52 proteins identified in 20 spots, decreased levels of voltage-dependent anion-selective channel 2 and increased levels of glutathione perioxidase and manganese superoxide were confirmed by immunoblotting. Cardiac function was preserved in rats receiving exercise training, and the beneficial effect was linked with changes in these 3 proteins. In conclusion, our results suggest that post-MI exercise training increases anti-oxidant levels and decreases ion channel levels, which may explain, in part, the improved cardiac function seen with exercise., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Rieske proteins are a class of electron transport proteins that are intricately involved in respiratory and photosynthetic processes. One unique property of Rieske proteins is that the reduction potential is pH-dependent. The ionizable groups responding to changes in pH have recently been shown to be the two histidine residues that ligate the [2Fe-2S] cluster. To probe the chemical reactivity toward and the accessibility of the ligating histidines to small molecules, akin to the substrate quinol and the inhibitor stigmatellin, the Thermus thermophilus Rieske protein was reacted with diethyl pyrocarbonate (DEPC) over a range of pH values. The modification was followed by UV-visible, circular dichroism, and EPR spectroscopies and the end product analyzed by mass spectrometry. The ligating His154, as well as the two nonligating histidines and surface-exposed lysines, were modified. Interestingly, modification of the protein by DEPC was also found to reduce the metal cluster. The ability to control the redox state was examined by the addition of oxidants and reductants and removal of the DEPC-histidine adduct by sodium hydroxide. Characterization of the DEPC-modified Rieske protein, which remains redox active, offers a probe to analyze the effects of small molecules that inhibit the function of the bc(1) complex and that have also been shown to interact with the ligating histidines of the Rieske [2Fe-2S] cluster in crystal structures of the complex.

Matrix metalloproteinase-9 (MMP-9) deletion has been shown to improve remodeling of the left ventricle post-myocardial infarction (MI), but the mechanisms to explain this improvement have not been fully elucidated. MMP-9 has a broad range of in vitro substrates, but relevant in vivo substrates are incompletely defined. Accordingly, we evaluated the infarct regions of wild-type (wt) and MMP-9 null (null) mice using a proteomic strategy. Wt and null groups showed similar infarct sizes (48+/-3 in wt and 45+/-3% in null), indicating that both groups received an equal injury stimulus. Left ventricle infarct tissue was homogenized and analyzed by 2-DE and MS. Of 31 spot intensity differences, the intensities of 9 spots were higher and 22 spots were lower in null mice compared to wt (all p<0.05). Several extracellular matrix proteins were identified in these spots by MS, including fibronectin, tenascin-C, thrombospondin-1, and laminin. Fibronectin was observed on the gels at a lower than expected molecular weight in the wt group, which suggested substrate cleavage, and the lower molecular weight spot was observed at lower intensity in the MMP-9 null group, which suggested cleavage by MMP-9. Immunoblotting confirmed the presence of fibronectin cleavage products in the wt samples and lower levels in the absence of MMP-9. In conclusion, examining infarct tissue from wt and MMP-9 null mice by proteomic analysis provides a powerful and unique method to identify in vivo candidate MMP substrates.

An isolation procedure utilizing ammonium sulfate fractionation and affinity chromatography was used to purify an elastase inhibitor present in large amounts in marama beans (Tylosema esculentum). The protein appeared to be heterogeneous due to carbohydrate differences, demonstrating two bands on SDS gels with molecular weights of 17.8 kDa and 20 kDa. Partial sequence, derived from mass spectrometry, indicated that the protein is a Kunitz-type inhibitor distinct from other known plant serine protease inhibitors. The marama bean inhibitor is specific for elastase, with very low K(i) for both pancreatic and neutrophil elastase. The quantity of elastase inhibitor present in marama beans is many times greater than in soybean or any other bean or nut source reported to date. This raises the question of why a bean found in an arid corner of the Kalahari Desert would be so rich in a very potent elastase inhibitor.

Matrix metalloproteinase-7 (MMP-7) deletion has been shown to improve survival after myocardial infarction (MI). MMP-7 has a large array of in vitro substrates, but in vivo substrates for MMP-7 following MI have not been fully identified. Accordingly, we evaluated the infarct regions of wild-type (WT; n = 12) and MMP-7 null (null; n = 10) mice using a proteomic strategy. Seven days post-MI, infarct regions of the left ventricles were excised, homogenized, and protein extracts were analyzed by two-dimensional gel electrophoresis and mass spectrometry. Of 13 spots that showed intensity differences between WT and null, the intensities of eight spots were higher and those of five spots were lower in the null group (p < 0.05). Fibronectin and tenascin-C, known in vitro substrates of MMP-7, were identified in spots that showed lower intensity in the null. Immunoblotting and in vitro cleavage assays confirmed reduced fibronectin and tenascin-C fragment generation in the null, and this effect was restored by exogenous administration of MMP-7. Lower levels of full-length peroxiredoxin-1 and -2 and higher levels of the full-length peroxiredoxin-3 were detected in the null group, suggesting MMP-7 deletion may also indirectly regulate protein levels through nonenzymatic mechanisms. In conclusion, this is the first study to identify fibronectin and tenascin-C as in vivo MMP-7 substrates in the infarcted left ventricle using a proteomic approach.

The transient receptor potential vanilloid 1 (TRPV1) channel is the principal detector of noxious heat in the peripheral nervous system. TRPV1 is expressed in many nociceptors and is involved in heat-induced hyperalgesia and thermoregulation. The precise mechanism or mechanisms mediating the thermal sensitivity of TRPV1 are unknown. Here, we have shown that the oxidized linoleic acid metabolites 9- and 13-hydroxyoctadecadienoic acid (9- and 13-HODE) are formed in mouse and rat skin biopsies by exposure to noxious heat. 9- and 13-HODE and their metabolites, 9- and 13-oxoODE, activated TRPV1 and therefore constitute a family of endogenous TRPV1 agonists. Moreover, blocking these substances substantially decreased the heat sensitivity of TRPV1 in rats and mice and reduced nociception. Collectively, our results indicate that HODEs contribute to the heat sensitivity of TRPV1 in rodents. Because oxidized linoleic acid metabolites are released during cell injury, these findings suggest a mechanism for integrating the hyperalgesic and proinflammatory roles of TRPV1 and linoleic acid metabolites and may provide the foundation for investigating new classes of analgesic drugs.

Pseudomonas chlororaphis phage 201 phi 2-1 produces a large structurally complex virion, including the products of 89 phage genes. Many of these proteins are modified by proteolysis during virion maturation. To delineate the proteolytic maturation process, 46 slices from an SDS-polyacrylamide gel were subjected to tryptic digestion and then HPLC-electrospray ionization-tandem mass spectrometry analysis. The scale of the experiment allowed high sequence coverage and detection of mass spectra assigned to peptides with one end produced by trypsin and the other end derived from a maturation cleavage (semitryptic peptides). Nineteen cleavage sites were detected in this way. From these sites, a cleavage motif was defined and used to predict the remaining cleavages required to explain the gel mobility of the processed polypeptide species. Profiling the gel with spectrum counts for specific polypeptide regions was found to be helpful in deducing the patterns of proteolysis. A total of 29 cleaved polypeptides derived from 19 gene products were thus detected in the mature 201 phi 2-1 virion. When combined with bioinformatics analyses, these results revealed the presence of head protein-encoding gene modules. Most of the propeptides that were removed from the virion after processing were acidic, whereas the mature domain remaining in the virion was nearly charge-neutral. For four of these processed virion proteins, the portions remaining in the mature virion were mutually homologous. Spectrum counts were found to overestimate the relative quantity of minor polypeptide species in the virion. The resulting sensitivity for minor species made it possible to observe a small amount of general proteolysis that also affected the virions.

Evidence that in vivo bacteriophage T3 DNA packaging includes capsid hyper-expansion that is triggered by lengthening of incompletely packaged DNA (ipDNA) is presented here. This evidence includes observation that some of the longer ipDNAs in T3-infected cells are packaged in ipDNA-containing capsids with hyper-expanded outer shells (HE ipDNA-capsids). In addition, artificially induced hyper-expansion is observed for the outer shell of a DNA-free capsid. Detection and characterization of HE ipDNA-capsids are based on two-dimensional, non-denaturing agarose gel electrophoresis, followed by structure determination with electron microscopy and protein identification with SDS-PAGE/mass spectrometry. After expulsion from HE ipDNA-capsids, ipDNA forms sharp bands during gel electrophoresis. The following hypotheses are presented: (1) T3 has evolved feedback-initiated, ATP-driven capsid contraction/hyper-expansion cycles that accelerate DNA packaging when packaging is slowed by increase in the packaging-resisting force of the ipDNA and (2) each gel electrophoretic ipDNA band reflects a contraction/hyper-expansion cycle.

MS was used to characterize the 24 kDa human growth hormone (hGH) glycoprotein isoform and determine the locus of O-linked oligosaccharide attachment, the oligosaccharide branching topology, and the monosaccharide sequence. MALDI-TOF/MS and ESI-MS/MS analyses of glycosylated 24 kDa hGH tryptic peptides showed that this hGH isoform is a product of the hGH normal gene. Analysis of the glycoprotein hydrolysate by high-performance anion-exchange chromatography with pulsed amperometric detection and HPLC with fluorescent detection for N-acetyl neuraminic acid (NeuAc) yielded the oligosaccharide composition (NeuAc(2), N-acetyl galactosamine(1), Gal(1)). After beta-elimination to release the oligosaccharide from glycosylated 24 kDa hGH, collision-induced dissociation of tryptic glycopeptide T6 indicated that there had been an O-linked oligosaccharide attached to Thr-60. The sequence and branching structure of the oligosaccharide were determined by ESI-MS/MS analysis of tryptic glycopeptide T6. The mucin-like O-oligosaccharide sequence linked to Thr-60 begins with N-acetyl galactosamine and branches in a bifurcated topology with one appendage consisting of galactose followed by NeuAc and the other consisting of a single NeuAc. The oligosaccharide moiety lies in the high-affinity binding site 1 structural epitope of hGH that interfaces with both the growth hormone and the prolactin receptors and is predicted to sterically affect receptor interactions and alter the biological actions of hGH.

Our earlier reports documented that calreticulin, a multifunctional Ca2+-binding protein in endoplasmic reticulum lumen, possessed protein acetyltransferase function termed Calreticulin Transacetylase (CRTAase). The autoacetylation of purified human placental CRTAase concomitant with the acetylation of receptor proteins by a model acetoxycoumarin, 7,8-Diacetoxy-4-methylcoumarin, was observed. Here, we have examined the autoacetylation property of CRTAase by immunoblotting and mass spectrometry. Ca2+ was found to inhibit CRTAase activity. The inhibition of both autoacetylation of CRTAase as well as acetylation of the receptor protein was apparent when Ca2+) was included in the reaction mixture as visualized by interaction with anti-acetyl lysine antibody. The acetylation of lysines residues: -48, -62, -64, -153, and -159 in N-domain and -206, -207, -209, and -238 in P-domain of CRTAase were located by high-performance liquid chromatography-electronspray ionization tandem mass spectrometry. Further, computer assisted protein structure modeling studies were undertaken to probe the effect of autoacetylation of CRTAase. Accordingly, the predicted CRTAase 3D model showed that all the loop regions of both N- and P-domain bear the acetylated lysines. Energy minimization of the acetylated residues revealed charge neutralization of lysines due to the N-epsilon-acetylation which may facilitate the interaction of CRTAase with the protein substrate and the subsequent transacetylase action.

The tightly packaged double-stranded DNA (dsDNA) genome in the mature particles of many tailed bacteriophages has been shown to form multiple concentric rings when reconstructed from cryo-electron micrographs. However, recent single-particle DNA packaging force measurements have suggested that incompletely packaged DNA (ipDNA) is less ordered when it is shorter than approximately 25% of the full genome length. The study presented here initially achieves both the isolation and the ipDNA length-based fractionation of ipDNA-containing T3 phage capsids (ipDNA-capsids) produced by DNA packaging in vivo; some ipDNA has quantized lengths, as judged by high-resolution gel electrophoresis of expelled DNA. This is the first isolation of such particles among the tailed dsDNA bacteriophages. The ipDNA-capsids are a minor component (containing approximately 10(-4) of packaged DNA in all particles) and are initially detected by nondenaturing gel electrophoresis after partial purification by buoyant density centrifugation. The primary contaminants are aggregates of phage particles and empty capsids. This study then investigates ipDNA conformations by the first cryo-electron microscopy of ipDNA-capsids produced in vivo. The 3-D structures of DNA-free capsids, ipDNA-capsids with various lengths of ipDNA, and mature bacteriophage are reconstructed, which reveals the typical T=7l icosahedral shell of many tailed dsDNA bacteriophages. Though the icosahedral shell structures of these capsids are indistinguishable at the current resolution for the protein shell (approximately 15 A), the conformations of the DNA inside the shell are drastically different. T3 ipDNA-capsids with 10.6 kb or shorter dsDNA (<28% of total genome) have an ipDNA conformation indistinguishable from random. However, T3 ipDNA-capsids with 22 kb DNA (58% of total genome) form a single DNA ring next to the inner surface of the capsid shell. In contrast, dsDNA fully packaged (38.2 kb) in mature T3 phage particles forms multiple concentric rings such as those seen in other tailed dsDNA bacteriophages. The distance between the icosahedral shell and the outermost DNA ring decreases in the mature, fully packaged phage structure. These results suggest that, in the early stage of DNA packaging, the dsDNA genome is randomly distributed inside the capsid, not preferentially packaged against the inner surface of the capsid shell, and that the multiple concentric dsDNA rings seen later are the results of pressure-driven close-packing.

Although atypical antipsychotics are widely known to induce alterations in lipid and glucose metabolism, the mechanisms by which these alterations occur remain unknown. Several recent studies have shown that atypical antipsychotics induce oxidative stress and oxidative cell injury by increasing levels of lipid and protein oxidation. In this study, a novel proteomic approach was used to identify specific proteins oxidized after clozapine treatment. Differentiated neuroblastoma SKNSH cells were treated with 0, 5 or 20 mum clozapine for 24 h and protein extracts were labelled with 6-iodoacetamide fluorescein (6-IAF). The lack of incorporation of 6-IAF to cysteine residues is an indicator of protein oxidation. Labelled proteins were exposed to 2D electrophoresis, and differential protein labelling was assessed. Increased oxidation after clozapine treatment was observed in 10 protein spots (p<0.05), although only four of them remained significant after correcting for analysis with two drug concentrations. Five proteins, corresponding to nine of the spots, were identified by HPLC-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) as mitochondrial ribosomal protein S22, mitochondrial malate dehydrogenase, calumenin, pyruvate kinase and 3-oxoacid CoA transferase. The latter four proteins play important roles in energy metabolism. These results suggest that oxidative stress may be a mechanism by which antipsychotics increase the risk for metabolic syndrome and diabetes.

An effective method was developed for isolation and analysis of bovine heart complex I subunits. The method uses C18 reversed-phase high-performance liquid chromatography (HPLC) and a water/acetonitrile gradient containing 0.1% trifluoroacetic acid. Employing this system, 36 of the 45 complex I subunits elute in 28 distinct chromatographic peaks. The 9 subunits that do not elute are B14.7, MLRQ, and the 7 mitochondrial-encoded subunits. The method, with ultraviolet (UV) detection, is suitable for either analytical (<50 microg protein) or preparative (>250 microg protein) applications. Subunits eluting in each chromatographic peak were initially determined by matrix-assisted laser desorption/ionization-time-of-flight/mass spectrometry (MALDI-TOF/MS) with subsequent positive identification by reversed-phase HPLC-electrospray ionization (ESI)/tandem mass spectrometry (MS/MS) analysis of tryptic digests. In the latter case, subunits were identified with a 99% probability using Mascot for database searching and Scaffold for assessment of protein identification probabilities. The reversed-phase HPLC subunit analysis method represents a major improvement over previous separation methods with respect to resolution, simplicity, and ease of application.

A common request of proteomics core facilities is protein identification. However, in some instances primary sequence information for the protein in question is not present in public databases. In other cases, the amino acid sequence of a protein may differ in some way from the sequence predicted from the gene sequence in a database as a result of gene mutation, gene splicing, and/or multiple posttranslational modifications. Thus, it may be necessary to determine the sequence of one or more peptides de novo in order to identify and/or adequately characterize the protein of interest. The primary goal of this study was to give participating laboratories an opportunity to evaluate their proficiency in sequencing unknown peptides that are not included in any published database. Samples containing 3-6 pmol each of five synthetic peptides with amino acid sequences that were not present in public databases were sent to 106 laboratories. One nonstandard amino acid was present in one of the peptides. From a comparison of the results obtained by different strategies, participating laboratories will be able to gauge their own capabilities and establish realistic expectations for the approaches that can be used for this determination.

Pseudomonas chlororaphis phage 201varphi2-1 is a relative of Pseudomonas aeruginosa myovirus phiKZ. Phage 201 phi2-1 was examined by complete genomic sequencing (316,674 bp), by a comprehensive mass spectrometry survey of its virion proteins and by electron microscopy. Seventy-six proteins, of which at least 69 have homologues in phiKZ, were identified by mass spectrometry. Eight proteins, in addition to the major head, tail sheath and tail tube proteins, are abundant in the virion. Electron microscopy of 201 phi2-1 revealed a multitude of long, fine fibers apparently decorating the tail sheath protein. Among the less abundant virion proteins are three homologues to RNA polymerase beta or beta' subunits. Comparison between the genomes of 201 phi2-1 and phiKZ revealed substantial conservation of the genome plan, and a large region with an especially high rate of gene replacement. The phiKZ-like phages exhibited a two-fold higher rate of divergence than for T4-like phages or host genomes.

Sulfhydryl groups of platelet surface proteins are important in platelet aggregation. While p-chloromercuribenzene sulphonate (pCMBS) has been used in most studies on platelet surface thiols, the specific thiol-proteins that pCMBS reacts with to inhibit aggregation have not been well defined. Since the thiol-containing P2Y(12) ADP receptor is involved in most types of platelet aggregation, we used the ADP scavenger apyrase and the P2Y(12) receptor antagonist 2-MeSAMP to examine thiol-dependent reactions in the absence of contributions from this receptor. We provide evidence for a non-P2Y(12) thiol-dependent reaction near the final alphaIIbbeta3-dependent events of aggregation. We then used 3-(N-maleimidylpropionyl)biocytin (MPB) and pCMBS to study thiols in alphaIIbbeta3. As previously reported, disruption of the receptor was required to obtain labelling of thiols with MPB. Specificity of labelling for thiols in the alphaIIb and beta3 subunits was confirmed by identification of the purified proteins by mass spectrometry and by inhibition of labelling with 5,5'-dithiobis-(2-nitrobenzoic acid). In contrast to MPB, pCMBS preferentially reacted with thiols in alphaIIbbeta3 and blocked aggregation under physiological conditions. Similarly, pCMBS preferentially inhibited signalling-independent activation of alphaIIbbeta3 by Mn(2+). Our results suggest that the thiols in alphaIIbbeta3 that are blocked by pCMBS are important in the activation of this integrin.

Background: Electron micrographs of bacteriophage T7 reveal a tail shorter than needed to reach host cytoplasm during infection-initiating injection of a T7 DNA molecule through the tail and cell envelope. However, recent data indicate that internal T7 proteins are injected before the DNA molecule is injected. Thus, bacteriophage/host adsorption potentially causes internal proteins to become external and lengthen the tail for DNA injection. But, the proposed adsorption-induced tail lengthening has never been visualized., Findings: In the present study, electron microscopy of particles in T7 lysates reveals a needle-like capsid extension that attaches partially emptied bacteriophage T7 capsids to non-capsid vesicles and sometimes enters an attached vesicle. This extension is 40-55 nm long, 1.7-2.4x longer than the T7 tail and likely to be the proposed lengthened tail. The extension is 8-11 nm in diameter, thinner than most of the tail, with an axial hole 3-4 nm in diameter. Though the bound vesicles are not identified by microscopy, these vesicles resemble the major vesicles in T7 lysates, found to be E. coli outer membrane vesicles by non-denaturing agarose gel electrophoresis, followed by mass spectrometry., Conclusion: The observed lengthened tail is long enough to reach host cytoplasm during DNA injection. Its channel is wide enough to be a conduit for DNA injection and narrow enough to clamp DNA during a previously observed stalling/re-starting of injection. However, its outer diameter is too large to explain formation by passing of an intact assembly through any known capsid hole unless the hole is widened.

Yeast mutants lacking mitochondrial NAD(+)-specific isocitrate dehydrogenase (idhDelta) or aconitase (aco1Delta) were found to share several growth phenotypes as well as patterns of specific protein expression that differed from the parental strain. These shared properties of idhDelta and aco1Delta strains were eliminated or moderated by co-disruption of the CIT1 gene encoding mitochondrial citrate synthase. Gas chromatography/mass spectrometry analyses indicated a particularly dramatic increase in cellular citrate levels in idhDelta and aco1Delta strains, whereas citrate levels were substantially lower in idhDeltacit1Delta and aco1Deltacit1Delta strains. Exogenous addition of citrate to parental strain cultures partially recapitulated effects of high endogenous levels of citrate in idhDelta and aco1Delta strains. Finally, effects of elevated cellular citrate in idhDelta and aco1Delta mutant strains were partially alleviated by addition of iron or by an increase in pH of the growth medium, suggesting that detrimental effects of citrate are due to elevated levels of the ionized form of this metabolite.

Changes in protein abundance in skeletal muscle are central to a large number of metabolic and other disorders, including, and perhaps most commonly, insulin resistance. Proteomics analysis of human muscle is an important approach for gaining insight into the biochemical basis for normal and pathophysiological conditions. However, to date, the number of proteins identified by this approach has been limited, with 107 different proteins being the maximum reported so far. Using a combination of one-dimensional gel electrophoresis and high performance liquid chromatography electrospray ionization tandem mass spectrometry, we identified 954 different proteins in human vastus lateralis muscle obtained from three healthy, nonobese subjects. In addition to a large number of isoforms of contractile proteins, we detected all proteins involved in the major pathways of glucose and lipid metabolism in skeletal muscle. Mitochondrial proteins accounted for 22% of all proteins identified, including 55 subunits of the respiratory complexes I-V. Moreover, a number of enzymes involved in endocrine and metabolic signaling pathways as well as calcium homeostasis were identified. These results provide the most comprehensive characterization of the human skeletal muscle proteome to date. These data hold promise for future global assessment of quantitative changes in the muscle proteome of patients affected by disorders involving skeletal muscle.

Middle-aged and old left ventricles (LVs) are structurally and functionally very similar. Compared to a young LV, both show increased wall thickness and increased cavity size, with preserved cardiac function. However, when a stressor such as myocardial infarction occurs, striking differences are revealed between young and old LVs and there is a marked reduction in survival rates for the old group. The objective of this study was to investigate the proteomic basis of age-related changes in the LV of male mice in order to identify proteins that are differentially expressed between middle-aged and old groups and to gain mechanistic insight into effects of aging on the unstressed heart. Young (3 months old; n = 6), middle-aged (MA; 15 months old; n = 6), and old (23 months old; n = 5) LVs were examined by echocardiography, homogenized, and separated into soluble and insoluble protein fractions using differential extraction. We found that the LV mass-to-tibia ratio increased from 6.4 +/- 0.2 mg/mm in young to 11.0 +/- 0.6 and 10.1 +/- 0.7 mg/mm in MA and old, respectively (both p < 0.05 vs young), which was caused by increases in both LV wall thickness and volume. Using two-dimensional gel electrophoresis, we detected age-related alterations in the levels of 73 proteins (all p < 0.05). Among these proteins were mortalin, peroxiredoxin 3, epoxide hydrolase, and the superoxide dismutases SOD-1 (Cu/ZnSOD) and SOD-2 (MnSOD), which have been previously associated with aging and/or cardiovascular disease. Together, these results reveal proteomic changes that occur in the LV with age. The proteins identified here may be useful markers of cardiac aging and may help in deducing mechanisms to explain the inability of the old heart to withstand challenge.

To investigate the apparent genomic complexity of long-genome bacteriophages, we have sequenced the 218,948-bp genome (6479-bp terminal repeat), and identified the virion proteins (55), of Bacillus thuringiensis bacteriophage 0305phi8-36. Phage 0305phi8-36 is an atypical myovirus with three large curly tail fibers. An accurate mode of DNA pyrosequencing was used to sequence the genome and mass spectrometry was used to accomplish the comprehensive virion protein survey. Advanced informatic techniques were used to identify classical morphogenesis genes. The 0305phi8-36 genes were highly diverged; 19% of 247 closely spaced genes have similarity to proteins with known functions. Genes for virion-associated, apparently fibrous proteins in a new class were found, in addition to strong candidates for the curly fiber genes. Phage 0305phi8-36 has twice the virion protein coding sequence of T4. Based on its genomic isolation, 0305phi8-36 is a resource for future studies of vertical gene transmission.

The determination of differences in relative protein abundance is a critical aspect of proteomics research that is increasingly used to answer diverse biological questions. The Association of Biomolecular Resource Facilities Proteomics Research Group 2006 study was a quantitative proteomics project in which the aim was to determine the identity and the relative amounts of eight proteins in two mixtures. There are numerous methodologies available to study the relative abundance of proteins between samples, but to date, there are few examples of studies that have compared these different approaches. For the 2006 Proteomics Research Group study, there were 52 participants who used a wide variety of gel electrophoresis-, HPLC-, and mass spectrometry-based methods for relative quantitation. The quantitative data arising from this study were evaluated along with several other experimental details relevant to the methodologies used.

Objective: Research has focused on insulin receptor substrate (IRS)-1 as a locus for insulin resistance. Tyrosine phosphorylation of IRS-1 initiates insulin signaling, whereas serine/threonine phosphorylation alters the ability of IRS-1 to transduce the insulin signal. Of 1,242 amino acids in IRS-1, 242 are serine/threonine. Serine/threonine phosphorylation of IRS-1 is affected by many factors, including insulin. The purpose of this study was to perform global assessment of phosphorylation of serine/threonine residues in IRS-1 in vivo in humans., Research Design and Methods: In this study, we describe our use of capillary high-performance liquid chromotography electrospray tandem mass spectrometry to identify/quantify site-specific phosphorylation of IRS-1 in human vastus lateralis muscle obtained by needle biopsy basally and after insulin infusion in four healthy volunteers., Results: Twenty-two serine/threonine phosphorylation sites were identified; 15 were quantified. Three sites had not been previously identified (Thr495, Ser527, and S1005). Insulin increased the phosphorylation of Ser312, Ser616, Ser636, Ser892, Ser1101, and Ser1223 (2.6 +/- 0.4, 2.9 +/- 0.8, 2.1 +/- 0.3, 1.6 +/- 0.1, 1.3 +/- 0.1, and 1.3 +/- 0.1-fold, respectively, compared with basal; P < 0.05); phosphorylation of Ser348, Thr446, Thr495, and Ser1005 decreased (0.4 +/- 0.1, 0.2 +/- 0.1, 0.1 +/- 0.1, and 0.3 +/- 0.2-fold, respectively; P < 0.05)., Conclusions: These results provide an assessment of IRS-1 phosphorylation in vivo and show that insulin has profound effects on IRS-1 serine/threonine phosphorylation in healthy humans.

To bypass limitations of ensemble averaging biochemical analysis, microscopy-based detection and tracking are needed for single protein molecules that are diffusing in aqueous solution. Confining the molecules to a planar zone dramatically assists tracking. Procedures of microscopy should be routine enough so that effort is focused on the biochemistry. Fluorescence microscopy and partial planar confinement of single, untethered, aqueous protein molecules have been achieved here by use of a routine procedure. With this procedure, multiple thermally diffusing Alexa 488-stained bovine serum albumin molecules were observed during partial confinement to a thin aqueous zone next to a cover slip. The procedure produces confinement by partial re-swelling of a previously dried agarose gel on the microscope slide. Confinement was confirmed through analysis that revealed thermal motion lower in the third dimension than it was in the plane of observation.

A single tryptophan (W(334(I))) within the mitochondrial-encoded core subunits of cytochrome c oxidase (CcO) is selectively oxidized when hydrogen peroxide reacts with the binuclear center. W(334(I)) is converted to hydroxytryptophan as identified by reversed-phase HPLC-electrospray ionization tandem mass spectrometry analysis of peptides derived from the three SDS-PAGE purified subunits. Total sequence coverage of subunits I, II and III was limited to 84%, 66% and 54%, respectively. W(334(I)) is located on the surface of CcO at the membrane interface. Two other surface tryptophans within nuclear-encoded subunits, W(48(IV)) and W(19(VIIc)), are also oxidized when hydrogen peroxide reacts with the binuclear center (Musatov et al. (2004) Biochemistry 43, 1003-1009). Two aromatic-rich networks of amino acids were identified that link the binuclear center to the three oxidized tryptophans. We propose the following mechanism to explain these results. Electron transfer through the aromatic networks moves the free radicals generated at the binuclear center to the surface-exposed tryptophans, where they produce hydroxytryptophan.

A yeast mutant lacking the two major cytosolic sources of NADPH, glucose-6-phosphate dehydrogenase (Zwf1p) and NADP+-specific isocitrate dehydrogenase (Idp2p), has been demonstrated to lose viability when shifted to medium with acetate or oleate as the carbon source. This loss in viability was found to correlate with an accumulation of endogenous oxidative by-products of respiration and peroxisomal beta-oxidation. To assess effects on cellular protein of endogenous versus exogenous oxidative stress, a proteomics approach was used to compare disulfide bond-containing proteins in the idp2Deltazwf1Delta strain following shifts to acetate and oleate media with those in the parental strain following similar shifts to media containing hydrogen peroxide. Among prominent disulfide bond-containing proteins were several with known antioxidant functions. These and several other proteins were detected as multiple electrophoretic isoforms, with some isoforms containing disulfide bonds under all conditions and other isoforms exhibiting a redox-sensitive content of disulfide bonds, i.e., in the idp2Deltazwf1Delta strain and in the hydrogen peroxide-challenged parental strain. The disulfide bond content of some isoforms of these proteins was also elevated in the parental strain grown on glucose, possibly suggesting a redirection of NADPH reducing equivalents to support rapid growth. Further examination of protein carbonylation in the idp2Deltazwf1Delta strain shifted to oleate medium also led to identification of common and unique protein targets of endogenous oxidative stress.

In the last several years, interest has increased significantly about the endocannabinoids anandamide and 2-arachidonylglycerol, two lipid messengers that activate cannabinoid receptors. Quantification of these compounds in biological samples presents numerous technical challenges. Because of their low abundance, endocannabinoids are usually quantified by isotope dilution assays using mass spectrometry coupled to either gas chromatography or high-performance liquid chromatography. Although endocannabinoid levels in biological fluids, such as plasma and cerebrospinal fluid, can be directly determined by these techniques, the complex lipid profile of brain tissue samples mandates purification of lipid extracts before GC/MS analysis; this step is not necessary when using HPLC/MS. We have found that when silica gel chromatography is used for endocannabinoid purification, poor recovery and loss of deuterium from the internal standards lead to inaccurate estimation of endocannabinoid levels. By contrast, purification strategies using C(18) solid-phase extraction permits precise and reproducible GC/MS quantification of endocannabinoids in tissue samples.

Mutations in DJ-1 cause an autosomal recessive, early onset familial form of Parkinson disease (PD). However, little is presently known about the role of DJ-1 in the more common sporadic form of PD and in other age-related neurodegenerative diseases, such as Alzheimer disease (AD). Here we report that DJ-1 is oxidatively damaged in the brains of patients with idiopathic PD and AD. By using a combination of two-dimensional gel electrophoresis and mass spectrometry, we have identified 10 different DJ-1 isoforms, of which the acidic isoforms (pI 5.5 and 5.7) of DJ-1 monomer and the basic isoforms (pI 8.0 and 8.4) of SDS-resistant DJ-1 dimer are selectively accumulated in PD and AD frontal cortex tissues compared with age-matched controls. Quantitative Western blot analysis shows that the total level of DJ-1 protein is significantly increased in PD and AD brains. Mass spectrometry analyses reveal that DJ-1 is not only susceptible to cysteine oxidation but also to previously unsuspected methionine oxidation. Furthermore, we show that DJ-1 protein is irreversibly oxidized by carbonylation as well as by methionine oxidation to methionine sulfone in PD and AD. Our study provides new insights into the oxidative modifications of DJ-1 and indicates association of oxidative damage to DJ-1 with sporadic PD and AD.

Serine/threonine phosphorylation of insulin receptor substrate-1 (IRS-1) regulates the function and subsequent insulin signaling of this protein. Human IRS-1 has 1242 amino acid residues, including 182 serines and 60 threonines. The size, complexity, and relatively low abundance of this protein in biological samples make it difficult to map and quantify phosphorylation sites by conventional means. A mass spectrometry peak area based quantification approach has been developed and applied to assess the relative abundance of IRS-1 phosphorylation in the absence or presence of stimuli. In this method, the peak area for a phosphopeptide of interest is normalized against the average of peak areas for six selected representative IRS-1 peptides that serve as endogenous internal standards. Relative quantification of each phosphopeptide is then obtained by comparing the normalized peak area ratios for untreated and treated samples. Two non-IRS-1 peptides were added to each digest for use as HPLC retention time markers and additional standards as well as references to the relative quantity of IRS-1 in different samples. This approach does not require isotopic or chemical labeling and can be applied to various cell lines and tissues. Using this method, we assessed the relative changes in the quantities of two tryptic phosphopeptides isolated from human IRS-1 expressed in L6 cells incubated in the absence or presence of insulin or tumor necrosis factor-alpha. Substantial increases of phosphorylation were observed for Thr(446) upon stimulation. In contrast, no obvious change in the level of phosphorylation was observed for Ser(1078). This mass spectrometry based strategy provides a powerful means to quantify changes in the relative phosphorylation of peptides in response to various stimuli in a complex, low-abundance protein.

Subunits located near the cardiolipin binding sites of bovine heart cytochrome c oxidase (CcO) were identified by photolabeling with arylazido-cardiolipin analogues and detecting labeled subunits by reversed-phase HPLC and HPLC-electrospray ionization mass spectrometry. Two arylazido-containing cardiolipin analogues were synthesized: (1) 2-SAND-gly-CL with a nitrophenylazido group attached to the polar headgroup of cardiolipin (CL) via a linker containing a cleavable disulfide; (2) 2',2''-bis-(AzC12)-CL with two of the four fatty acid tails of cardiolipin replaced by 12-(N-4-azido-2-nitrophenyl) aminododecanoic acid. Both arylazido-CL derivatives were used to map the cardiolipin binding sites within two types of detergent-solubilized CcO: (1) intact 13-subunit CL-containing CcO (three to four molecules of endogenous CL remain bound per CcO monomer); (2) 11-subunit CL-free CcO (subunits VIa and VIb are missing because they dissociate during CL removal). Upon the basis of these photolabeling studies, we conclude that (1) subunits VIIa, VIIc, and possibly VIII are located near the two high-affinity cardiolipin binding sites, which are present in either form of CcO, and (2) subunit VIa is located adjacent to the lower affinity cardiolipin binding site, which is only present in the 13-subunit form of CcO. These data are consistent with the recent CcO crystal structure in which one cardiolipin is located near subunit VIIa and a second is located near subunit VIa (PDB ID code referenced in Tomitake, T. et al. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 15304-15309). However, we propose that a third cardiolipin is bound between subunits VIIa and VIIc near the entrance to the D-channel. Cardiolipin bound at this location could potentially function as a proton antenna to facilitate proton entry into the D-channel. If true, it would explain the CcO requirement of bound cardiolipin for full electron transport activity.

Using headspace gas chromatography-mass spectrometry, we detected significant amounts of nitrous oxide in the reaction products of the monooxygenase reaction catalyzed by neuronal nitric oxide synthase. Nitrous oxide is a dimerization product of nitroxyl anion; its presence in the reaction products indicates that the nitroxyl anion is a product of the neuronal nitric oxide synthase-catalyzed reaction.

Insulin receptor substrate 1 (IRS-1), an intracellular substrate of the insulin receptor tyrosine kinase, also is heavily phosphorylated on serine and threonine residues, and several serine phosphorylation sites alter the function of IRS-1. Because of the large number of serine/threonine residues, position-by-position analysis of these potential phosphorylation sites by mutagenesis is difficult. To circumvent this, we have employed matrix-assisted laser desorption/ionization time-of-flight and HPLC-electrospray ionization tandem mass spectrometry techniques to scan for serine and threonine residues that are phosphorylated in full-length human IRS-1 ectopically expressed in cells using an adenoviral vector. This approach revealed 12 phosphorylation sites on serine or threonine residues, 10 of which were novel sites. Seven of these sites were in proline-directed motifs, whereas five were in arginine-directed sites. Sequence inspection suggested that phosphorylation of Ser1223 might alter the interaction of IRS-1 with the protein tyrosine phosphatase Src homology domain 2 (SH2)-containing phosphatase-2 (SHP-2). Mutation of Ser1223 to alanine to prevent phosphorylation resulted in increased association of SHP-2 with IRS-1, decreased insulin-stimulated tyrosine phosphorylation of IRS-1 in CHO/IR cells, and decreased insulin-stimulated association of the p85 regulatory subunit of phosphatidylinositol-3-kinase with IRS-1. This mutation had no effect on association of IRS-1 with the insulin receptor. Sequence analysis showed the Ser1223 region to be widely conserved evolutionarily. These data suggest that phosphorylation of Ser1223 dampens association of IRS-1 with SHP-2, thereby increasing net insulin-stimulated tyrosine phosphorylation.

Serine phosphorylation of insulin receptor substrate-1 (IRS-1) can regulate tyrosine phosphorylation of IRS-1 and subsequent insulin signaling. The 182 serine and 60 threonine residues in IRS-1 make position-by-position analysis of potential phosphorylation sites by mutagenesis difficult. Tandem mass spectrometry provides a more efficient way to identify phosphorylated residues in IRS-1. Toward this end, we overexpressed glutathione S-transferase-IRS-1 fusion proteins in E. coli and treated them in vitro with various kinases followed by identification of phosphorylation sites using high-performance liquid chromatography-electrospray ionization tandem mass spectrometry. Nine phosphorylation sites were detected in the tryptic digests of middle and C-terminal regions of IRS-1 treated with protein kinase A or extracellular signal-regulated kinase 2. Of these sites, five have not previously been detected by any method and provide novel candidates for identification in cells or in vivo.

Grb10 is a Src-homology 2 (SH2) and Pleckstrin-homology (PH) domain-containing protein that binds to several autophosphorylated receptor tyrosine kinases including the insulin receptor (IR). Our previous studies showed that Grb10 underwent insulin-stimulated serine phosphorylation, yet the kinase(s) responsible for phosphorylation and the sites of the phosphorylation remain unknown. In this report, we show that Grb10 is a direct substrate of the p42/44 mitogen-activated protein kinase (MAPK). In addition, we found that inhibition of the MAPK signaling pathway reduced Grb10 phosphorylation in cells. Using site-directed mutagenesis, phosphopeptide mapping, and capillary HPLC-electrospray-tandem mass spectrometry analysis, we identified Ser(150), Ser(418), and Ser(476) of human Grb10zeta as MAPK-mediated in vitro phosphorylation sites. In vivo labeling and two-dimensional phosphopeptide mapping studies revealed that Ser(150) and Ser(476) of human Grb10zeta are phosphorylated in intact cells. Replacing Ser(150) and Ser(476) with alanines reduced the inhibitory effect of human Grb10zeta on insulin-stimulated IRS1 tyrosine phosphorylation. Taken together, our findings suggest that phosphorylation of the adaptor protein may provide a feedback inhibitory mechanism by which Grb10 regulates insulin signaling.

Although oxidative stress has been strongly implicated in the pathogenesis of Alzheimer disease (AD) and Parkinson disease (PD), the identities of specific protein targets of oxidative damage remain largely unknown. Here, we report that Cu,Zn-superoxide dismutase (SOD1), a key antioxidant enzyme whose mutations have been linked to autosomal dominant neurodegenerative disorder familial amyotrophic lateral sclerosis (ALS), is a major target of oxidative damage in AD and PD brains. By using a combination of two-dimensional gel electrophoresis, immunoblot analysis, and mass spectrometry, we have identified four human brain SOD1 isoforms with pI values of 6.3, 6.0, 5.7, and 5.0, respectively. Of these, the SOD1 pI 6.0 isoform is oxidatively modified by carbonylation, and the pI 5.0 isoform is selectively accumulated in AD and PD. Moreover, Cys-146, a cysteine residue of SOD1 that is mutated in familial ALS, is oxidized to cysteic acid in AD and PD brains. Quantitative Western blot analyses demonstrate that the total level of SOD1 isoforms is significantly increased in both AD and PD. Furthermore, immunohistochemical and double fluorescence labeling studies reveal that SOD1 forms proteinaceous aggregates that are associated with amyloid senile plaques and neurofibrillary tangles in AD brains. These findings implicate, for the first time, the involvement of oxidative damage to SOD1 in the pathogenesis of sporadic AD and PD. This work suggests that AD, PD, and ALS may share a common or overlapping pathogenic mechanism(s) that could potentially be targeted by similar therapeutic strategies.

Alzheimer's disease (AD) is a progressive amnestic dementia that involves post-translational hyperphosphorylation, enzymatic cleavage, and conformational alterations of the microtubule-associated protein tau. The truncation state of tau influences many of its pathologic characteristics, including its ability to assume AD-related conformations and to assemble into filaments. Cleavage also appears to be an important marker in AD progression. Although C-terminal truncation of tau at D421 has recently been attributed to the apoptotic enzyme caspase-3, N-terminal processing of the protein remains mostly uncharacterized. Here, we report immunohistochemical staining in a cohort of 35 cases ranging from noncognitively impaired to early AD with a panel of three N-terminal anti-tau antibodies: Tau-12, 5A6, and 9G3-pY18. Of these three, the phosphorylation-independent epitope of 5A6 was the earliest to emerge in the pathological lesions of tau, followed by the appearance of the Tau-12 epitope. The unmasking of the Tau-12 epitope in more mature 5A6-positive tangles was not correlated with tau phosphorylation at tyrosine 18 (9G3-pY18). Still, later in the course of tangle evolution, the extreme N terminus of tau was lost, correlating temporally with the appearance of a C-terminal caspase-truncated epitope lacking residues 422-441. In addition, caspase-6 cleaved the N terminus of tau in vitro, preventing immunoreactivity with both Tau-12 and 5A6. Mass spectrometry confirmed that the in vitro caspase-6 truncation site is D13, a semicanonical and hitherto undescribed caspase cleavage site in tau. Collectively, these results suggest a role for caspase-6 and N-terminal truncation of tau during neurofibrillary tangle evolution and the progression of Alzheimer's disease.

We have examined oxidized proteins in the brain regions of wild-type (WT) and ApoE-knockout (KO) animals. Total protein oxidation in the hippocampus of young-KO (6 month) animals was approximately 2-fold greater than that of young-WT (6 month) animals and was similar to that of old-WT (18 month) and old-KO (18 month) animals. In the cortex of the same animals, the levels of total protein oxidation in all four groups were not significantly different. Two-dimensional electrophoresis (2-DE) coupled with immunostaining for protein carbonylation revealed six specific oxidation-sensitive proteins, the oxidation levels of which were increased in young-KO, old-WT, and old-KO mice compared with young-WT mice. These six oxidation-sensitive proteins were identified by mass spectrometry as glial fibrillary acidic protein, creatine kinase BB, disulfide isomerase, chaperonin subunit 5, dihydropyrimidase-related protein 2, and mortalin. These results indicate that the ApoE gene product offers protection against age-associated oxidative damage in the brain. Moreover, two of these proteins, creatine kinase and dihydropyrimidase-related protein 2, have recently been found to be oxidized in the brains of human subjects with Alzheimer's disease [Aksenov et al. J. Neurochem. 74: 2520-2527; 2000; Castegna et al. J. Neurochem. 82: 1524-1532; 2002]. These data suggest that the ApoE-knockout mouse serves as an appropriate model for studying pathogenic oxidative mechanisms influencing risk and progression of Alzheimer's disease.

Alzheimer's disease (AD) and Parkinson's disease (PD) are the two most common neurodegenerative diseases that occur either in relatively rare, familial forms or in common, sporadic forms. The genetic defects underlying several monogenic familial forms of AD and PD have recently been identified, however, the causes of other AD and PD cases, particularly sporadic cases, remain unclear. To gain insights into the pathogenic mechanisms involved in AD and PD, we used a proteomic approach to identify proteins with altered expression levels and/or oxidative modifications in idiopathic AD and PD brains. Here, we report that the protein level of ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1), a neuronal de-ubiquitinating enzyme whose mutation has been linked to an early-onset familial PD, is down-regulated in idiopathic PD as well as AD brains. By using a combination of two-dimensional gel electrophoresis and mass spectrometry, we have identified three human brain UCH-L1 isoforms, a full-length form and two amino-terminally truncated forms. Our proteomic analyses reveal that the full-length UCH-L1 is a major target of oxidative damage in AD and PD brains, which is extensively modified by carbonyl formation, methionine oxidation, and cysteine oxidation. Furthermore, immunohistochemical studies show that prominent UCH-L1 immunostaining is associated with neurofibrillary tangles and that the level of soluble UCH-L1 protein is inversely proportional to the number of tangles in AD brains. Together, these results provide evidence supporting a direct link between oxidative damage to the neuronal ubiquitination/de-ubiquitination machinery and the pathogenesis of sporadic AD and PD.

Alexa dye staining of proteins is used for the fluorescence microscopy of single particles that are sometimes multimolecular protein complexes. To characterize the staining, post-staining determination must be made of which protein(s) in a complex have been Alexa-stained. The present communication describes the use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for performing this determination. The Alexa-stained proteins are observed directly in gels by illumination with an ultraviolet transilluminator. The test multimolecular particle is bacteriophage T7. The protein capsid of T7 is a multimolecular complex that has both external and internal proteins. SDS-PAGE of Alexa-stained bacteriophage T7 produces fluorescent capsid proteins each of which usually comigrates with an unstained protein. However, one Alexa-induced modification of protein migration was observed by SDS-PAGE. Mass spectrometry shows that the protein with modified migration is the major protein of the outer shell of the T7 capsid. The procedures used are generally applicable. The distribution of Alexa staining among T7 capsid proteins depends on the size of the dye molecule used. The larger the dye molecule is, the greater the preference for external proteins.

Hydrogen peroxide does more than react with the binuclear center of oxidized bovine cytochrome c oxidase and generate the well-characterized "peroxy" and "ferryl" forms. Hydrogen peroxide also inactivates detergent-solubilized cytochrome c oxidase in a time- and concentration-dependent manner. There is a 70-80% decrease of electron-transport activity, peroxidation of bound cardiolipin, modification of two nuclear-encoded subunits (IV and VIIc), and dissociation of approximately 60% of subunits VIa and VIIa. Modification of subunit VIIc and dissociation of subunit VIIa are coupled events that probably are responsible for the inactivation of cytochrome c oxidase. When cytochrome c oxidase is exposed to 500 microM hydrogen peroxide for 30 min at pH 7.4 and room temperature, subunits IV (modified up to 20%) and VIIc (modified up to 70%) each have an increased mass of 16 Da as detected by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and electrospray ionization mass spectrometry. In each case, the increased mass is caused by oxidation of a tryptophan (Trp19 within subunit VIIc and Trp48 within subunit IV), almost certainly due to formation of hydroxytryptophan. We conclude that hydrogen peroxide-induced oxidation of tryptophan and cardiolipin proceeds via the binuclear center since both modifications are prevented if the binuclear center is first blocked with cyanide. Bound cardiolipin and oxidized tryptophans are localized relatively far from the binuclear center (30-60 A); therefore, oxidation probably occurs by migration of a free radical generated at the binuclear center to these distal reaction sites.

The anxiolytic-like effects of Ginkgo biloba extract (GBE) and its four terpenoid components (ginkgolide-A, ginkgolide-B, ginkgolide-C, and bilobalide) were assessed using the elevated plus-maze test in mice. Administration of GBE as a single oral dose (0.5 or 1 g/kg, po) caused a state of suppressed motor activity and, thus, shortened the time spent in the open-sided arms. However, when GBE (0.063-1 g/kg, po) was administered daily for 7 days and the plus-maze test was carried out 24 h after the final administration, the time spent in the open-sided arms was prolonged, with the peak anxiolytic-like effect at 0.125 g/kg. A combination of seven-day administration of GBE (0.125 g/kg) and a single dose of diazepam (1 mg/ kg, po, 10 min before testing) enhanced the anxiolytic-like effect. Flumazenil (0.3 mg/kg, ip, 10 min before testing) blocked the effect of diazepam, but not of GBE. Daily administration of ginkgolide-A (1 or 2 mg/kg, po) resulted in an anxiolytic-like effect by the third treatment, with the maximal effect observed after the fifth administration. Neither ginkgolide-B, ginkgolide-C, nor bilobalide produced any anxiolytic-like effects. At doses higher than 0.5 g/kg, GBE not only inhibited motor activity but also suppressed active avoidance behavior, reduced caffeine-induced stimulation, and enhanced pentobarbital-induced sleep, while ginkgolide-A (up to 20 mg/kg) did not exhibit these effects. Diazepam (1 mg/kg) is known to enhance pentobarbital-induced sleep. These results suggest that GBE produces a significant anxiolytic-like effect following repeated administration and that ginkgolide-A is most likely responsible for this effect. There are also indications that although GBE exerts a sedative effect at comparatively higher doses, ginkgolide-A has a relatively weak tendency to produce benzodiazepine-like side effects.

Purpose: To conduct a phase 1 trial of rebeccamycin analog (NSC #655649) in children with solid tumors to establish the dose-limiting toxicity (DLT) and maximum-tolerated dose (MTD); to establish the pharmacokinetic profile in children, and to document any evidence of antitumor activity., Methods: A 1-hour infusion of NSC #655649 was administered every 21 days to 17 patients younger than 21 years who had malignant tumors refractory to conventional therapy. Doses ranged from 450 mg/m2 to 760 mg/m2. Pharmacokinetics were done in at least three patients per dose level. The first course was used to determine DLT and MTD., Results: Sixteen patients on three dose levels were assessable for toxicities. At 760 mg/m2, four patients had dose-limiting neutropenia and thrombocytopenia. Pharmacokinetics were assessable in 13 patients. Plasma concentrations declined triexponentially and concentrations above the range of in vitro antitumor activity were maintained for 3 days. Analysis of urine extracts revealed the presence of an N-de-ethylated metabolite and probable involvement of cytochrome P450 isoenzyme(s) in the metabolism of NSC #655649. Pharmacodynamic studies showed a relationship between the area under the curve and percentage change in absolute neutrophil count in the E(max) model (r2 = 0.56, P = 0.001)., Conclusions: The recommended phase 2 dose of NSC #655649 administered as a 1-hour infusion every 21 days to children with solid tumors is 585 mg/m2. Both neutropenia and thrombocytopenia were found to be dose-limiting toxicities.

We have examined the effects of the beta-amyloid peptide (Abeta(25-35)) on fibroblasts derived from subjects with Alzheimer's disease (AD) and from age-matched controls. The peptide was significantly more cytotoxic to the AD-derived fibroblasts. The level of protein oxidation was also greater in the cells from AD subjects. Two-dimensional electrophoresis (2-DE) coupled with immunostaining for protein carbonylation revealed specific oxidation-sensitive proteins (OSPs) in both the control and AD-derived cells. Two specific OSPs were identified by mass spectrometry as heat shock protein 60 (HSP 60) and vimentin. Exposure of the cells to Abeta(25-35) resulted in a twofold increase in the level of oxidation of these two OSPs in the cells derived from controls, but a ninefold increase in their level of oxidation in the fibroblasts from AD subjects. These observations are of particular interest because of the proposed anti-apoptotic roles of both HSP 60 and vimentin and our recent observation that these same two proteins are particularly susceptible to oxidation in neuronally derived cells.

Oxidative damage to neuronal proteins appears to be central to the toxicity associated with a number of neuropathologies, including Alzheimer's disease. We have examined this by using oxidative stress to induce apoptosis in a mouse hippocampal neuronal cell line (HT-22). Oxidatively modified proteins were measured by high-resolution two-dimensional gel electrophoresis coupled with oxidation-specific immunostains. Under these conditions the oxidatively stressed cells undergo apoptosis, and specific proteins are oxidized. The three proteins that appeared to be most susceptible to oxidation were identified by mass spectrometry. Those oxidized proteins are heat shock protein 60 and vimentin, both believed to function as antiapoptotic proteins, and a third protein with sequence homology to hemoglobin alpha-chain. When the cells were pretreated with vitamin E, these proteins were not oxidized and the cells did not undergo apoptosis.

Bovine heart cytochrome c oxidase (CcO) was inactivated by the lipid peroxidation product 4-hydroxy-2-nonenal (HNE) in a time- and concentration-dependent manner with pseudo-first-order kinetics. Cytochrome c oxidase electron transport activity decreased by as much as 50% when the enzyme was incubated for 2 h at room temperature with excess HNE (300-500 microM). HNE-modified CcO subunits were identified by two mass spectrometric methods: electrospray ionization mass spectrometry (ESI/MS) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS). All of the experimentally determined molecular masses were in excellent agreement with published sequence values with an accuracy of approximately 1 part per 10000 mass units for subunits smaller than 20 kDa and approximately 1 part per 1000 mass units for the three subunits larger than 20 kDa. Both MS methods detected six CcO subunits with an increased mass of 156 Da after reaction with HNE (subunits II, IV, Vb, VIIa, VIIc, and VIII); this result indicates a single Michael-type reaction site on either a lysine or histidine residue within each subunit. Reaction of HNE with either subunit VIIc or subunit VIII (modified approximately 30% and 50-75%, respectively) must be responsible for CcO inhibition. None of the other subunits were modified more than 5% and could not account for the observed loss of activity. Reaction of HNE with His-36 of subunit VIII is most consistent with the approximately 50% inhibition of CcO: (1) subunit VIII is modified more than any other subunit by HNE; (2) the time dependence of subunit VIII modification is consistent with the percent inhibition of CcO; (3) His-36 was identified as the HNE-modified amino acid residue within subunit VIII by tandem MS analysis.

Previous studies in this laboratory revealed that dihydrohonokiol-B (DHH-B; 3'-(2 propenyl)-5-propyl-(1,1'-biphenyl)-2,4'-diol), a partially reduced derivative of honokiol, was an effective anxiolytic-like agent in mice at an oral dose of 0.04 mg kg(-1), and at higher doses, when evaluated by the elevated plus-maze test. The aim of this study was to further confirm the anxiolytic-like effect of DHH-B using an additional behavioural procedure (Vogel's conflict test in mice) and a biochemical assessment (in-vitro determination of muscimol-stimulated 36Cl- uptake into mouse cortical synaptoneurosomes). As in earlier experiments, DHH-B (0.04-1 mg kg(-1), p.o.) was shown to prolong the time spent in the open-sided arms of the elevated plus-maze in a dose-dependent manner. Moreover, in the Vogel's conflict test, DHH-B (5 mg kg(-1), p.o.) significantly increased punished water intake. In tests with mouse cerebral cortical synaptoneurosomes, 10 and 30 microM of DHH-B significantly increased 36Cl- influx in the absence of muscimol. In the presence of 25 microM muscimol, the addition of 1 microM DHH-B led to significant enhancement of 36Cl- uptake, while 30 microM DHH-B was required to further stimulate the 36Cl- uptake induced by 250 microM muscimol. The results of these studies confirm that DHH-B is a potent anxiolytic-like agent and that GABA(A) receptor-gated Cl(-)-channel complex is involved in the anxiolytic-like efficacy of DHH-B.

A potential new metabolic pathway of melatonin biotransformation is described in this investigation. Melatonin was found to directly scavenge hydrogen peroxide (H(2)O(2)) to form N(1)-acetyl-N(2)-formyl-5-methoxykynuramine and, thereafter this compound could be enzymatically converted to N(1)-acetyl-5-methoxykynuramine by catalase. The structures of these kynuramines were identified using proton nuclear magnetic resonance, carbon nuclear magnetic resonance, and mass spectrometry. This is the first report to reveal a possible physiological association between melatonin, H(2)O(2), catalase, and kynuramines. Melatonin scavenges H(2)O(2) in a concentration-dependent manner. This reaction appears to exhibit two distinguishable phases. In the rapid reaction phase, the interaction between melatonin and H(2)O(2) reaches equilibrium rapidly (within 5 s). The rate constant for this phase was calculated to be 2.3 x 10(6) M(-1)s(-1). Thereafter, the relative equilibrium of melatonin and H(2)O(2) was sustained for roughly 1 h, at which time the content of H(2)O(2) decreased gradually over a several hour period, identified as the slow reaction phase. These observations suggest that melatonin, a ubiquitously distributed small nonenzymatic molecule, might serve to directly detoxify H(2)O(2) in living organisms. H(2)O(2) and melatonin are present in all subcellular compartments; thus, presumably, one important function of melatonin may be complementary in function to catalase and glutathione peroxidase in keeping intracellular H(2)O(2) concentrations at steady-state levels.

Labeled peptides synthesized by core facilities are frequently used by researchers for following trafficking of a peptide, for binding studies, to determine substrate specificity, and for receptor cross-linking studies.The membership of the Association of Biomolecular Resource Facilities was asked to participate in a study focusing on synthesis of a biotin-labeled peptide, and it was suggested that a new strategy, using Rink amide 4-methylbenzhydrylamine resin coupled with Fmoc-Lys(Dde)-OH, be used.This strategy can be used for addition of a variety of labels other than biotin and should prove useful to core facilities. Comparison of the new strategy to other strategies was performed. Biotin labeling has long been assumed to be routine and specific. Despite the assumed routine nature of synthesizing biotinylated peptides, 9 of the 34 samples submitted did not contain any of the correct product. Although synthesis using Fmoc-Lys(Dde)-OH plus biotin generally gave the highest yields, other approaches also yielded a high percentage of the correct product.Therefore, the various strategies are generally comparable. The major advantage of this new approach is that other labels such as fluorescein, dansyl groups, methyl coumarin, and potentially fluorophores and quenchers used for fluorescence resonance energy transfer (FRET) can be directly incorporated into peptides.

Honokiol has previously been shown to be an effective anxiolytic-like agent in mice when administered for 7 days at 0.2 mg/kg/day prior to evaluation in an elevated plus-maze, while 20 mg/kg is required for efficacy as a single oral dose. The aim of this study was to find analogs of honokiol that are more effective for acute administration. Among the eight analogs evaluated, one partially reduced derivative of honokiol [3'-(2-propenyl)-5-propyl-(1,1'-biphenyl)-2,4'-diol] exhibited significant anxiolytic-like activity at 0.04 mg/kg. Following oral administration of 1 mg/kg of this analog, anxiolytic-like activity was clearly evident at 1 h, peaked at 3 h, and remained significant for longer than 4 h after treatment. Combined administration of the derivative with diazepam led to enhanced anxiolytic-like efficacy. Moreover, as with diazepam, the anxiolytic-like effect of the analog was reduced by flumazenil. In contrast, bicuculline, a GABA(A) antagonist, had no effect on the activity of the derivative. Taken together, these results suggest that this analog of honokiol acts at the benzodiazepine recognition site of the GABA(A)-benzodiazepine receptor complex.

Platelet-activating factor is the term used to denote a class of extremely potent lipid mediators that consist predominantly of 1-O-alkyl- and 1-O-acyl-2-acetyl-sn-glycero-3-phosphocholines. A method has been devised for rapid isolation of these acetylated phospholipids by solid-phase extraction prior to direct derivatization with pentafluorobenzoic anhydride and analysis by gas chromatography (GC)/electron-capture mass spectrometry. Recovery through the entire method (lipid isolation, derivatization, and purification) typically ranged from 70% to 85%. Using the direct derivatization procedure described here, the practical limit of detection for each of the standard alkyl- and acyl-platelet-activating factor homologs was 1 fmol injected into the GC. Results from the application of the method to the analysis of alkyl and acyl homologs of platelet-activating factor isolated from stimulated human umbilical vein endothelial cells are presented, exhibiting excellent accuracy and precision for a wide range of tissue levels of this class of potent autacoids.