Inflation of 430-parsec bipolar radio bubbles in the Galactic Centre by an energetic event.

The Galactic Centre contains a supermassive black hole with a mass of four million Suns1 within an environment that differs markedly from that of the Galactic disk. Although the black hole is essentially quiescent in the broader context of active galactic nuclei, X-ray observations have provided evidence for energetic outbursts from its surroundings2. Also, although the levels of star formation in the Galactic Centre have been approximately constant over the past few hundred million years, there is evidence of increased short-duration bursts3, strongly influenced by the interaction of the black hole with the enhanced gas density present within the ring-like central molecular zone4 at Galactic longitude |l| < 0.7 degrees and latitude |b| < 0.2 degrees. The inner 200-parsec region is characterized by large amounts of warm molecular gas5, a high cosmic-ray ionization rate6, unusual gas chemistry, enhanced synchrotron emission7,8, and a multitude of radio-emitting magnetized filaments9, the origin of which has not been established. Here we report radio imaging that reveals a bipolar bubble structure, with an overall span of 1 degree by 3 degrees (140 parsecs × 430 parsecs), extending above and below the Galactic plane and apparently associated with the Galactic Centre. The structure is edge-brightened and bounded, with symmetry implying creation by an energetic event in the Galactic Centre. We estimate the age of the bubbles to be a few million years, with a total energy of 7 × 1052 ergs. We postulate that the progenitor event was a major contributor to the increased cosmic-ray density in the Galactic Centre, and is in turn the principal source of the relativistic particles required to power the synchrotron emission of the radio filaments within and in the vicinity of the bubble cavities.

A novel multifunctional labeling reagent for enhanced protein characterization with mass spectrometry.

Individual peptides with lysine at the C-terminus as well as protein tryptic digests were reacted with 2-methoxy-4,5-dihydro-1H-imidazole, converting lysine residues to their 4,5-dihydro-1H-imidazol-2-yl derivatives. The mass spectra of derivatized digests exhibit a greater number of more intense features than their underivatized counterparts, thus increasing the information obtained in peptide mapping experiments. Additionally, MS/MS spectra of the derivatized peptides are greatly simplified in comparison to their native species, yielding primarily an easily interpretable series of y-ions. Finally, this novel label also enables differential quantitation studies, as a stable isotopic form containing four deuterium atoms can readily be produced.

Kinetic intermediates in the folding of gaseous protein ions characterized by electron capture dissociation mass spectrometry.

Alternative mechanisms propose that protein folding in solution proceeds either through specific obligate intermediates or by a multiplicity of routes in a "folding funnel". These questions are examined in the gas phase by using a new method that provides details of the noncovalent binding of solvent-free protein ions. Capture of an electron by a multiply charged cation causes immediate dissociation (ECD) of a backbone bond, but with negligible excitation of noncovalent bonds; thus ECD of a linear protein ion produces two measurable fragment ions only if these are not held together by noncovalent bonds. Thermal unfolding of 9+ ions of cytochrome c proceeds through the separate unfolding of up to 13 backbone regions (represented by 44 bond cleavages) with melting temperatures of <26 to 140 degrees C. An 0.25 s laser IR pulse induces unfolding of 9+ ions in <4 s in six of these regions, followed by their refolding in 2 min. However, for the 15+ ions a laser IR pulse causes slower unfolding through poorly defined intermediates that leads to far more ECD products (63% increase in bond cleavages) after 1 min, even more than heating to 140 degrees C, with refolding to a more compact conformation in 10 min. Random isomerization appears to produce a dynamic mixture of conformers that folds through a variety of pathways to the most stable conformer(s), consistent with a "folding funnel"; this might also be considered as an extension of the classical view to a system with a far smaller free energy change yielding multiple conformers. As cautions to inferring solution conformational structure from gas-phase data, no structural relationship between these gaseous folding intermediates and those in solution is apparent, consistent with reduced hydrophobic bonding and increased electrostatic repulsion. Further, equilibrium folding of gaseous ions can require minutes, and even momentary unfolding of an intermolecular complex during this time can be irreversible.

Electrospray ionization mass spectrometry analysis of changes in phospholipids in RBL-2H3 mastocytoma cells during degranulation.

Biological membranes contain an extraordinary diversity of lipids. Phospholipids function as major structural elements of cellular membranes, and analysis of changes in the highly heterogeneous mixtures of lipids found in eukaryotic cells is central to understanding the complex functions in which lipids participate. Phospholipase-catalyzed hydrolysis of phospholipids often follows cell surface receptor activation. Recently, we demonstrated that granule fusion is initiated by addition of exogenous, nonmammalian phospholipases to permeabilized mast cells. To pursue this finding, we use positive and negative mode Fourier-transform ion cyclotron resonance mass spectrometry (FTICR-MS) to measure changes in the glycerophospholipid composition of total lipid extracts of intact and permeabilized RBL-2H3 (mucosal mast cell line) cells. The low energy of the electrospray ionization results in efficient production of molecular ions of phospholipids uncomplicated by further fragmentation, and changes were observed that eluded conventional detection methods. From these analyses we have spectrally resolved more than 130 glycerophospholipids and determined changes initiated by introduction of exogenous phospholipase C, phospholipase D, or phospholipase A2. These exogenous phospholipases have a preference for phosphatidylcholine with long polyunsaturated alkyl chains as substrates and, when added to permeabilized mast cells, produce multiple species of mono- and polyunsaturated diacylglycerols, phosphatidic acids, and lysophosphatidylcholines, respectively. The patterns of changes of these lipids provide an extraordinarily rich source of data for evaluating the effects of specific lipid species generated during cellular processes, such as exocytosis.

Charge/radical site initiation versus coulombic repulsion for cleavage of multiply charged ions. Charge solvation in poly(alkene glycol) ions.

Electrospray ionization of poly(ethylene glycol) (PEG) followed by separation with Fourier-transform mass spectrometry traps (PEG100 + nH)n+ ions. Both collisionally activated dissociation (CAD) and electron capture dissociation (ECD) of these ions (n = 5, 6, 7) produce PEGx fragment ions in which the x values correspond closely to those for an equal distribution of charges in the linear polymer ion, e.g., for n = 7, near x = 1, 17, 34, 50, 67, 83, and 100. However, positions intermediate between these charges should represent the maximum coulombic repulsion, so this is not a specific driving force for fragmentation, which is instead consistent with charge site (CAD) or radical site (ECD) initiation. These conclusions were confirmed by studies of a variety of other poly(alkene glycol) polymers. For these, the ECD spectra of the protonated species are consistent with the predicted charge solvation by the ion's oxygen atoms.

Electron capture dissociation of gaseous multiply charged ions by Fourier-transform ion cyclotron resonance.

Fourier-transform ion cyclotron resonance instrumentation is uniquely applicable to an unusual new ion chemistry, electron capture dissociation (ECD). This causes nonergodic dissociation of far larger molecules (42 kDa) than previously observed (<1 kDa), with the resulting unimolecular ion chemistry also unique because it involves radical site reactions for similarly larger ions. ECD is highly complementary to the well known energetic methods for multiply charged ion dissociation, providing much more extensive protein sequence information, including the direct identification of N- versus C-terminal fragment ions. Because ECD only excites the molecule near the cleavage site, accompanying rearrangements are minimized. Counterintuitively, cleavage of backbone covalent bonds of protein ions is favored over that of noncovalent bonds; larger (>10 kDa) ions give far more extensive ECD if they are first thermally activated. This high specificity for covalent bond cleavage also makes ECD promising for studying the secondary and tertiary structure of gaseous protein ions caused by noncovalent bonding.

Phosphopeptide/phosphoprotein mapping by electron capture dissociation mass spectrometry.

Of methods for dissociation of multiply charged peptide and protein ions, electron capture dissociation (ECD) has the advantages of cleaving between a high proportion of amino acids, without loss of such posttranslational modifications as glycosylation and carboxylation. Here this capability is successfully extended to phosphorylation, for which collisionally activated dissociation (CAD) can cause extensive loss of H3PO4 and HPO3. As shown here, these losses are minimal in ECD spectra, an advantage for measuring the degree of phosphorylation. For phosphorylated peptides, ECD and CAD spectra give complementary backbone cleavages for identifying modification sites. For a 24-kDa heterogeneous phosphoprotein, bovine beta-casein, activated ion ECD cleaved 87 of 208 backbone bonds that identified a phosphorylation site at Ser-15, and localized three more among Ser-17,-18, -19, and -22 and Thr-24, and the last among four other sites. This is the first direct site-specific characterization of this key post-translational modification on a protein without its prior degradation, such as proteolysis.

Activated ion electron capture dissociation for mass spectral sequencing of larger (42 kDa) proteins.

In previous studies, electron capture dissociation (ECD) has been successful only with ionized smaller proteins, cleaving between 33 of the 153 amino acid pairs of a 17 kDa protein. This has been increased to 99 cleavages by colliding the ions with a background gas while subjecting them to electron capture. Presumably this ion activation breaks intramolecular noncovalent bonds of the ion's secondary and tertiary structure that otherwise prevent separation of the products from the nonergodic ECD cleavage of a backbone covalent bond. In comparison to collisionally activated dissociation, this "activated ion" (AI) ECD provides more extensive, and complementary, sequence information. AI ECD effected cleavage of 116, 60, and 47, respectively, backbone bonds in 29, 30, and 42 kDa proteins to provide extensive contiguous sequence information on both termini; AI conditions are being sought to denature the center portion of these large ions. This accurate "sequence tag" information could potentially identify individual proteins in mixtures at far lower sample levels than methods requiring prior proteolysis.

Automated de novo sequencing of proteins by tandem high-resolution mass spectrometry.

A de novo sequencing program for proteins is described that uses tandem MS data from electron capture dissociation and collisionally activated dissociation of electrosprayed protein ions. Computer automation is used to convert the fragment ion mass values derived from these spectra into the most probable protein sequence, without distinguishing Leu/Ile. Minimum human input is necessary for the data reduction and interpretation. No extra chemistry is necessary to distinguish N- and C-terminal fragments in the mass spectra, as this is determined from the electron capture dissociation data. With parts-per-million mass accuracy (now available by using higher field Fourier transform MS instruments), the complete sequences of ubiquitin (8.6 kDa) and melittin (2.8 kDa) were predicted correctly by the program. The data available also provided 91% of the cytochrome c (12.4 kDa) sequence (essentially complete except for the tandem MS-resistant region K(13)-V(20) that contains the cyclic heme). Uncorrected mass values from a 6-T instrument still gave 86% of the sequence for ubiquitin, except for distinguishing Gln/Lys. Extensive sequencing of larger proteins should be possible by applying the algorithm to pieces of approximately 10-kDa size, such as products of limited proteolysis.

Automated reduction and interpretation of high resolution electrospray mass spectra of large molecules.

Here a fully automated computer algorithm is applied to complex mass spectra of peptides and proteins. This method uses a subtractive peak finding routine to locate possible isotopic clusters in the spectrum, subjecting these to a combination of the previous Fourier transform/Patterson method for primary charge determination and the method for least-squares fitting to a theoretically derived isotopic abundance distribution for m/z determination of the most abundant isotopic peak, and the statistical reliability of this determination. If a predicted protein sequence is available, each such m/z value is checked for assignment as a sequence fragment. A new signal-to-noise calculation procedure has been devised for accurate determination of baseline and noise width for spectra with high peak density. In 2 h, the program identified 824 isotopic clusters representing 581 mass values in the spectrum of a GluC digest of a 191 kDa protein; this is >50% more than the number of mass values found by the extremely tedious operator-applied methodology used previously. The program should be generally applicable to classes of large molecules, including DNA and polymers. Thorough high resolution analysis of spectra by Horn (THRASH) is proposed as the program's verb.

Electron capture dissociation for structural characterization of multiply charged protein cations.

For proteins of < 20 kDa, this new radical site dissociation method cleaves different and many more backbone bonds than the conventional MS/MS methods (e.g., collisionally activated dissociation, CAD) that add energy directly to the even-electron ions. A minimum kinetic energy difference between the electron and ion maximizes capture; a 1 eV difference reduces capture by 10(3). Thus, in an FTMS ion cell with added electron trapping electrodes, capture appears to be achieved best at the boundary between the potential wells that trap the electrons and ions, now providing 80 +/- 15% precursor ion conversion efficiency. Capture cross section is dependent on the ionic charge squared (z2), minimizing the secondary dissociation of lower charge fragment ions. Electron capture is postulated to occur initially at a protonated site to release an energetic (approximately 6 eV) H. atom that is captured at a high-affinity site such as -S-S- or backbone amide to cause nonergodic (before energy randomization) dissociation. Cleavages between every pair of amino acids in mellitin (2.8 kDa) and ubiquitin (8.6 kDa) are represented in their ECD and CAD spectra, providing complete data for their de novo sequencing. Because posttranslational modifications such as carboxylation, glycosylation, and sulfation are less easily lost in ECD than in CAD, ECD assignments of their sequence positions are far more specific.

Two-dimensional mass spectrometry of biomolecules at the subfemtomole level.

Multiple dimensions of unique molecular structure information can now be obtained from proteins and DNA using mass spectrometry. Less than 10(-16) mol of the active major histocompatibility complex signaling peptide in a mixture of thousands can be identified. For large proteins (> 40 kDa), the high resolving power (> 10(5) and 10(-17) mol sensitivity of Fourier-transform mass spectrometry provide exact molecular weight values (+/- 1 or 2 Da) for mixture components, indicating error or modifications compared with the predicted DNA sequence. Selecting a specific molecular species, its two-dimensional spectrum indicates the part of the molecule that is modified; a three-dimensional spectrum of that fragment further isolates the modification site.

Fialuridine is phosphorylated and inhibits DNA synthesis in isolated rat hepatic mitochondria.

Fialuridine (FIAU) is a thymidine analog effective against hepatitis B virus. Toxicity associated with FIAU treatment included clinical signs consistent with mitochondrial dysfunction, including severe lactic acidosis. To understand further the mechanism of FIAU toxicity, we examined the effect of FIAU on DNA synthesis in mitochondria. Mitochondria isolated from livers of naive rats were treated in vitro with concentrations of FIAU or FIAU triphosphate (FIAU-TP) ranging from 0.1 to 200 microM. A 14 or 32% decrease in mitochondrial DNA synthesis compared to controls was observed when isolated mitochondria were treated with 25 microM FIAU or FIAU-TP, respectively. Since it is thought that nucleosides must be phosphorylated to inhibit DNA polymerase, studies were conducted to determine whether isolated rat mitochondria could phosphorylate FIAU. Results using lanthanum chloride precipitation and HPLC analysis showed that enzymes present in a mitochondrial lysate were capable of phosphorylating FIAU to form FIAU monophosphate.

Dose-dependent induction of GST-P+ staining foci by the rat hepatocarcinogen methapyrilene in the medium-term bioassay.

Previous studies have demonstrated that methapyrilene hydrochloride (MP) is a rat-specific nongenotoxic carcinogen which induces liver tumors in a dose-dependent manner following chronic exposure in the diet. This study was conducted to determine the dose response of MP in the medium-term bioassay and to compare the response to tumor incidence. Two weeks following a single initiating dose of diethylnitrosamine (DEN), male F344 rats were administered MP at doses of 0, 62.5, 125, 250, or 1000 ppm in the diet for 6 weeks. A 2/3 partial hepatectomy was performed 3 weeks post-DEN. At termination, sections from the remaining three lobes were stained with GST-P antibody. Number and size of foci were measured using an image analysis system with a digitizing board. MP induced a dose-dependent increase in the number of GST-P+ foci/cm2 (0 ppm = 0.85 foci/cm2; 62.5 ppm = 1.29 foci/cm2; 125 ppm = 1.59 foci/cm2; 250 ppm = 6.55 foci/cm2; 1000 ppm = 28.23 foci/cm2). A significantly greater number of foci were observed in the caudate lobe than in the anterior and posterior lobes. The size of individual foci was largely unaffected. This study demonstrates a strong correlation between foci induction and tumor incidence and suggests that this assay may have utility in predicting dose responses for the chronic bioassay.

Incorporation of fialuridine (FIAU) into mitochondrial DNA and effects of FIAU on the morphology of mitochondria in human hepatoblastoma cells.

Fialuridine (FIAU), a thymidine nucleoside analogue with anti-hepatitis B virus activity, showed clinical toxicity consistent with mitochondrial dysfunction. In vitro methods were used to understand further this toxicity. Using a sensitive and specific radioimmunoassay, FIAU was found to be present in nuclear DNA of human hepatoblastoma cells incubated for 6 days in 10 or 50 n M drug, at a level of 1 residue per 63 or 39 thymidines, respectively, and was present in mitochondrial DNA at a level of 1 residue per 2139 or 1696 thymidines, respectively. Human hepatoblastoma cells were incubated for 6 days in increasing concentrations of FIAU or, for comparative purposes, the nucleoside analogue dideoxycytidine (ddC), after which time the cells were examined by electron microscopy. At 10 mum and higher concentrations, both compounds induced morphological changes in the ultrastructure of mitochondria characterized by marked mitochondrial swelling, loss of internal cristae and dissolution of the internal matrix. These results, considered along with previously published studies, indicate that FIAU has deleterious effects in vitro on mitochondrial function and structure that occur relatively quickly but without an apparent decrease in the abundance of mitochondrial DNA.

Dose-responses in rat hepatic protein modification and expression following exposure to the rat hepatocarcinogen methapyrilene.

Dose-related effects of methapyrilene (MP) on protein modification and expression were examined using two-dimensional gel electrophoresis (2-D PAGE) coupled with computer analysis. Methapyrilene was administered ad libitum at doses of 0, 62.5, 125, 250 and 1000 p.p.m. to male F-344 rats for 12 weeks beginning at 8 weeks of age. Following treatment, livers were removed and frozen for 2-D PAGE analysis. Separation of hepatic proteins was conducted using ISO-DALT technology. Changes in abundance and modification of hepatic proteins were determined using the Kepler software package. Covalent modifications of three specific mitochondrial proteins were quantified using a charge modification index. Dose-response relationships were analyzed using Tukey's trend test. Results demonstrated that covalent modification of the three mitochondrial proteins was linearly related to dose and that a dose effect could be found at all dose levels in 2 out of 3 proteins. Two forms of change in protein expression were observed: (i) a dose-dependent change with effects at all doses and (ii) a change only at the toxic dose of 1000 p.p.m. MP. These results demonstrate a molecular effect of MP at doses that do not produce cellular responses including toxicity or increases in cell replication suggesting that these specific mitochondrial modifications are molecular dosimeters but are probably not direct factors and/or sufficient factors in carcinogenesis. This study also demonstrates the potential use of 2-D PAGE electrophoresis to delineate the effect of dose on expression of specific proteins.

H-ras 61st codon activation in archival proliferative hepatic lesions isolated from female B6C3F1 mice exposed to the leukotriene D4-antagonist, LY171883.

LY171883, a peroxisome proliferator and leukotriene D4-antagonist, induced a statistically significant increase in the number of hepatic lesions in B6C3F1 female mice in a 2 year oncogenicity study at dietary doses of 0.0225% and 0.075%. The mutation frequency and spectrum of the 61st codon of H-ras was determined for 64 independent, archived lesions from the LY171883 2 year oncogenicity study using the polymerase chain reaction (PCR), allele specific oligo hybridization (ASO) and DNA sequencing. Results showed 41 (64%) of these lesions had mutations at the 61st codon (16/21 hepatocellular carcinomas, 4/10 hepatocellular adenomas, 19/26 focal hepatocellular hyperplasias and 2/7 focal hepatocellular atypia). These mutations consisted of 18 C-A transversions, 16 A-G transitions and seven A-T transversions. Compared to the mutation frequency for spontaneously occurring archival B6C3F1 hepatic lesions (41%), the frequency of LY171883 lesions (64%) was significantly higher (P < 0.01). The frequencies of H-ras 61st codon mutations among the LY171883 lesion types (hepatocellular carcinomas 76%, hepatocellular adenomas 40%, focal hepatocellular hyperplasias 73% and hepatocellular atypia 29%) were also significantly different (P = 0.035). In contrast, spontaneous lesions showed no statistical difference in the frequencies of mutation among lesion types (P > 0.5). The mutation spectrum of the LY171883 lesions was not significantly different from the spontaneous spectra. It may be concluded that based on the similarity in mutation spectrum and the increase in mutation frequency, LY171883 may selectively promote spontaneous hepatic lesions containing H-ras 61st codon mutations. In addition, the difference in mutation frequency among lesion types does not support a linear progression of all LY171883 lesions through focal atypia, focal hepatocellular hyperplasias, hepatocellular adenomas and hepatocellular carcinomas.

Expression of TRPM-2 during involution and regeneration of the rat liver.

Increased message levels of testosterone-repressed prostate message-2 (TRPM-2) have been associated with programmed cell death in many tissues. To study its involvement in the apoptotic elimination of hepatocytes during liver involution and regeneration, levels of TRPM-2 message were evaluated in situ and by the ribonuclease protection assay. Although significant increases in apoptotic bodies were observed in rats 96 h following treatment with lead nitrate and ethylene dibromide, an increase in TRPM-2 message was not detected. Therefore, the expression of TRPM-2 mRNA may be a poor indicator of the extent to which apoptosis occurs during liver involution.