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PubMed2002

Two-dimensional separations with electrospray ionization ambient pressure high-resolution ion mobility spectrometry/quadrupole mass spectrometry.

The coupling of ion mobility spectrometry (IMS) instruments with mass spectrometers has been described since early in IMS development, most commonly with quadrupole mass analyzers. The recent development of IMS with time-of-flight (TOF) instruments has demonstrated that the time compatibility (IMS milliseconds and TOFMS microseconds) of the two techniques enables rapid two-dimensional separations to be performed, theoretically in the order of seconds for a complete analysis. This study presents a unique way to operate a traditional IMS/QMS system to attain separations similar to those achieved with IMS/TOF. For this new approach, the quadrupole was slowly scanned in the single-ion monitoring mode while IMS spectra were continually embedded in each m/z step. In this way, two-dimensional separations (IMS drift times and m/z) were obtained using the traditional IMS/QMS arrangement. An example of a five amino acid separation (quadrupole scan of 40 m/z values at a rate of approximately 7 steps/min) led to a complete two-dimensional analysis within 6 min, comparable to rapid chromatographic separations with mass spectrometry. Proposed approaches to reduce the analysis time are discussed and a reduction in the analysis time to less than 1 min is feasible when the IMS/QMS separation conditions are optimized.

Algorithms↗
PubMed2006

Structure elucidation of NeuAc, NeuGc and Kdn-containing O-glycans released from Triturus alpestris oviductal mucins. Characterization of the poly LacdiNAc sequence: HSO3(4)(GalNAcbeta1-4GlcNAcbeta1-3)1-3GalNAcbeta1-4(GlcNAcbeta1-3)0-1GlcNAcbeta1-6GalNAc-ol.

Eggs from Amphibia are surrounded by several layers of jelly that are needed for proper fertilization. Jelly coat is composed of highly glycosylated mucin-type glycoproteins containing up to 60% of carbohydrates, which display a remarkable species-specificity. This material obtained from Triturus alpestris was submitted to reductive beta-elimination and the released oligosaccharide-alditols were further fractionated by HPLC. Structural characterization was performed through a combination of two dimensional (1)H-(1)H and (1)H-(13)C NMR and ESI-MS/MS analysis. Numerous carbohydrate chains are characterized by the presence of the Cad (Sd(a)) determinant, including respectively NeuAc, NeuGc or Kdn as a sialic acid. But the most significant O-glycan sequences which mark the difference between the jelly of T. alpestris and other studies amphibian jellies are polymers of GalNAc(beta 1-4)GlcNAc (LacdiNAc) which form part of the following sequence: HSO(3)(4)(GalNAcbeta 1-4GlcNAcbeta 1-3)(1-3)GalNAcbeta 1-4(GlcNAcbeta 1-3)(0-1)GlcNAcbeta 1-6GalNAc-ol.

Animals↗
PubMed2006

Changes in disulfide bond content of proteins in a yeast strain lacking major sources of NADPH.

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.

Acetic Acid↗
PubMed1999

A new method for the placement of brachytherapy probes in paranasal sinus and nasopharynx neoplasms.

PURPOSE: In afterloading therapy, the exact placement of the applicator is crucial for the result of radiotherapy. A deviation of few millimeters from the target leads to a significant reduction of radiation energy to the tumor. METHODS AND MATERIALS: We present a new method using a computer-assisted device for three-dimensional placing of afterloading probes based on CT scans that allows for the exact placement. RESULTS: Target points have been reached with an accuracy of 0.6 mm. Since 1991 we have used this technique for 24 clinical applications in 11 patients with recurrent neoplasms in the region of the paranasal sinuses and nasopharynx for palliative treatment. CONCLUSIONS: The presented method is a reliable and precise procedure for the positioning of afterloading probes, avoiding the inaccuracy of the traditional methods.

Adult↗
PubMed2003

Glutamine starvation of monocytes inhibits the ubiquitin-proteasome proteolytic pathway.

Peripheral blood monocytes utilize free glutamine (Gln) in addition to glucose as an important energy substrate. Although this demand increases upon activation, monocytes are commonly confronted with decreased plasma Gln during critical illness and thus suffer from Gln-starvation. Here we investigate the influence of Gln-starvation on protein stability and its effects on the monocyte proteome. Gln-starvation caused a reduction of protein degradation which was accompanied by an accumulation of ubiquitin-protein conjugates and a reduction of intracellular ATP. Similar effects were observed under ATP-reducing conditions and in the presence of a proteasome inhibitor. Using two-dimensional gel electrophoresis we identified the IL-1beta precursor protein (pIL-1beta) as the, by far, most induced protein in endotoxin-treated monocytes. The degradation of the short-lived pIL-1beta was strongly reduced during Gln-starvation, while the degradation of the long-lived, constitutively expressed beta-actin was less affected. This indicates that although Gln-starvation reduces protein breakdown on the overall proteasome level, it leads to differential changes in the stability of specific proteins. This selective effect is likely to contribute to the immunocompromised state of monocytes commonly observed during critical illness.

Actins↗
PubMed2005

Probing heme propionate involvement in transmembrane proton transfer coupled to electron transfer in dihemic quinol:fumarate reductase by 13C-labeling and FTIR difference spectroscopy.

Quinol:fumarate reductase (QFR) is the terminal enzyme of anaerobic fumarate respiration. This membrane protein complex couples the oxidation of menaquinol to menaquinone to the reduction of fumarate to succinate. Although the diheme-containing QFR from Wolinella succinogenes is known to catalyze an electroneutral process, its three-dimensional structure at 2.2 A resolution and the structural and functional characterization of variant enzymes revealed locations of the active sites that indicated electrogenic catalysis. A solution to this apparent controversy was proposed with the so-called "E-pathway hypothesis". According to this, transmembrane electron transfer via the heme groups is strictly coupled to a parallel, compensatory transfer of protons via a transiently established pathway, which is inactive in the oxidized state of the enzyme. Proposed constituents of the E-pathway are the side chain of Glu C180 and the ring C propionate of the distal heme. Previous experimental evidence strongly supports such a role of the former constituent. Here, we investigate a possible heme-propionate involvement in redox-coupled proton transfer by a combination of specific (13)C-heme propionate labeling and Fourier transform infrared (FTIR) difference spectroscopy. The labeling was achieved by creating a W. succinogenes mutant that was auxotrophic for the heme-precursor 5-aminolevulinate and by providing [1-(13)C]-5-aminolevulinate to the medium. FTIR difference spectroscopy revealed a variation on characteristic heme propionate vibrations in the mid-infrared range upon redox changes of the distal heme. These results support a functional role of the distal heme ring C propionate in the context of the proposed E-pathway hypothesis of coupled transmembrane electron and proton transfer.

Base Sequence↗
PubMed1994

N-acetylaspartate as an in vivo marker of neuronal viability in kainate-induced status epilepticus: 1H magnetic resonance spectroscopic imaging.

N-acetylaspartate (NAA) has been proposed as a marker of neuronal density. Therefore, regional measurement of NAA by magnetic resonance spectroscopic imaging (MRSI) may provide a sensitive method for detection of selective neuronal loss, in contrast to conventional imaging techniques such as magnetic resonance imaging (MRI). To test this hypothesis, we produced selective neuronal injury by kainate-induced status epilepticus. Three days later three-dimensional 1H-MRSI was obtained and compared with conventional T2-weighted MRI and histological findings in normal and kainate-treated rats. Reduction of NAA determined by MRSI in piriform cortex, amygdala, and hippocampus correlated well with neuronal injury determined from histology. Changes of NAA, without any MRI changes in hippocampus, indicated greater sensitivity of MRSI for detection of neuronal injury. These results are consistent with the hypothesis that reduction of NAA measured by MRSI may be a sensitive marker of neuronal injury in vivo in a variety of disease states.

Animals↗
PubMed2000

Statistical properties of actions of periodic orbits.

We investigate statistical properties of unstable periodic orbits, especially actions for two simple linear maps (p-adic Baker map and sawtooth map). The action of periodic orbits for both maps is written in terms of symbolic dynamics. As a result, the expression of action for both maps becomes a Hamiltonian of one-dimensional spin systems with the exponential-type pair interaction. Numerical work is done for enumerating periodic orbits. It is shown that after symmetry reduction, the dyadic Baker map is close to generic systems, and the p-adic Baker map and sawtooth map with noninteger K are also close to generic systems. For the dyadic Baker map, the trace of the quantum time-evolution operator is semiclassically evaluated by employing the method of Phys. Rev. E 49, R963 (1994). Finally, using the result of this and with a mathematical tool, it is shown that, indeed, the actions of the periodic orbits for the dyadic Baker map with symmetry reduction obey the uniform distribution modulo 1 asymptotically as the period goes to infinity. (c) 2000 American Institute of Physics.

Journal Article↗
PubMed1978

An additional dimension to grating perception.

Visual acuity (A) for a two-dimensional multiplicative sinusoidal contrast grid (sinusoidal chessboard) was measured as a function of spatial frequency (omega) of modulation in one of the dimensions. The reduction in acuity as frequency was increased was well described by the equation: A = -2pikomega + c, where k and c are constants. At suprathreshold contrasts, the frequency of modulation appeared to be doubled relative to the underlying modulation frequency. This doubling is not related to the frequency of the harmonic components of the chessboard, but suggests the existence of a perceptual mechanism sensitive to areas of sinusoidal modulation which are seen at twice the frequency of the variation in contrast.

Depth Perception↗
PubMed2002

Identification by redox proteomics of glutathionylated proteins in oxidatively stressed human T lymphocytes.

Formation of mixed disulfides between glutathione and the cysteines of some proteins (glutathionylation) has been suggested as a mechanism through which protein functions can be regulated by the redox status. The aim of this study was to identify the proteins of T cell blasts that undergo glutathionylation under oxidative stress. To this purpose, we radiolabeled cellular glutathione with (35)S, exposed T cells to oxidants (diamide or hydrogen peroxide), and performed nonreducing, two-dimensional electrophoresis followed by detection of labeled proteins by phosphorimaging and their identification by mass spectrometry techniques. We detected several proteins previously not recognized to be glutathionylated, including cytoskeletal proteins (vimentin, myosin, tropomyosin, cofilin, profilin, and the already known actin), enzymes (enolase, aldolase, 6-phosphogluconolactonase, adenylate kinase, ubiquitin-conjugating enzyme, phosphoglycerate kinase, triosephosphate isomerase, and pyrophosphatase), redox enzymes (peroxiredoxin 1, protein disulfide isomerase, and cytochrome c oxidase), cyclophilin, stress proteins (HSP70 and HSP60), nucleophosmin, transgelin, galectin, and fatty acid binding protein. Based on the presence of several protein isoforms in control cells, we suggest that enolase and cyclophilin are heavily glutathionylated under basal conditions. We studied the effect of glutathionylation on some of the enzymes identified in the present study and found that some of them (enolase and 6-phosphogluconolactonase) are inhibited by glutathionylation, whereas the enzymatic activity of cyclophilin (peptidylprolyl isomerase) is not. These findings suggest that protein glutathionylation might be a common mechanism for the global regulation of protein functions.

Cells, Cultured↗
PubMed2004

Mitochondrial dysfunction and oxidative damage in parkin-deficient mice.

Loss-of-function mutations in parkin are the predominant cause of familial Parkinson's disease. We previously reported that parkin-/- mice exhibit nigrostriatal deficits in the absence of nigral degeneration. Parkin has been shown to function as an E3 ubiquitin ligase. Loss of parkin function, therefore, has been hypothesized to cause nigral degeneration via an aberrant accumulation of its substrates. Here we employed a proteomic approach to determine whether loss of parkin function results in alterations in abundance and/or modification of proteins in the ventral midbrain of parkin-/- mice. Two-dimensional gel electrophoresis followed by mass spectrometry revealed decreased abundance of a number of proteins involved in mitochondrial function or oxidative stress. Consistent with reductions in several subunits of complexes I and IV, functional assays showed reductions in respiratory capacity of striatal mitochondria isolated from parkin-/- mice. Electron microscopic analysis revealed no gross morphological abnormalities in striatal mitochondria of parkin-/- mice. In addition, parkin-/- mice showed a delayed rate of weight gain, suggesting broader metabolic abnormalities. Accompanying these deficits in mitochondrial function, parkin-/- mice also exhibited decreased levels of proteins involved in protection from oxidative stress. Consistent with these findings, parkin-/- mice showed decreased serum antioxidant capacity and increased protein and lipid peroxidation. The combination of proteomic, genetic, and physiological analyses reveal an essential role for parkin in the regulation of mitochondrial function and provide the first direct evidence of mitochondrial dysfunction and oxidative damage in the absence of nigral degeneration in a genetic mouse model of Parkinson's disease.

Animals↗
PubMed2003

Molecular analysis of microbial communities in nitrification and denitrification reactors treating high ammonia leachate.

Molecular analysis of microbial populations in two bench-scale nitrification and denitrification reactors fed with high ammonia landfill leachate was conducted in this study by using DGGE, cloning, and FISH techniques in addition to classical efficiency control parameters. Nitrification tank was operated with a computer-controlled alkalinity dosing system to supply the alkalinity intermittently as consumed on the basis of on-line pH monitoring. By keeping the pH at 7.0 with this system, 99% nitrification efficiency and rates of about 0.14-0.18 mgNH4+-N/mgVSSday were obtained. Meanwhile, as ammonia oxidizing bacteria Nitrosomonas and Nitrosococcus mobilis-like cells and as nitrite oxidizing bacteria Nitrobacter-related cells were intensively indicated. Moreover, some aerobic denitrifiers as Thauera species were also identified. After the termination of pH adjustment in the preceding anaerobic reactors, nitrification tank was loaded with more biodegradable COD as a result of reduced COD removal in anaerobic reactors. Microbial diversity was immediately affected from this alteration and heterotrophic carbonaceous bacteria and aerobic denitrifiers have dominated. To provide the former high efficiencies, retention time has increased from 24 to 48 h and a second pump dosing HCl was included to the automatic control system. Subsequent to these precautions, numbers of ammonia (Nso190) and nitrite oxidizing bacteria (NIT3) were comparatively increased. In denitrification system, about 98% denitrification efficiencies were obtained at 2000 mg/L NOx-N concentrations if sodium acetate was supplied as carbon source. Meanwhile, with 20 gVSS/l biomass concentration, denitrification rates of about 1.34 mgNOx-N/mgVSSday were obtained. All sludge samples have represented similar DGGE patterns and Paraccoccus-related species were identified as dominant denitrifying bacteria.

Ammonia↗
PubMed1981

Expression of keratin and vimentin intermediate filaments in rabbit bladder epithelial cells at different stages of benzo[a]pyrene-induced neoplastic progression.

Rabbit bladder epithelium, grown on collagen gels and exposed to the chemical carcinogen benzo[a]pyrene, produced nontumorigenic altered foci as well as tumorigenic epithelial cell lines during 120-180 d in culture. Immunofluorescence studies revealed extensive keratin filaments in both primary epithelial cells and benzo[a]pyrene-induced altered epithelial foci but showed no detectable vimentin filaments. The absence of vimentin expression in these cells was confirmed by two-dimensional gel electrophoresis. In contrast, immunofluorescence staining of the cloned benzo[a]pyrene-transformed rabbit bladder epithelial cell line, RBC-1, revealed a reduction in filamentous keratin concomitant with the expression of vimentin filaments. The epithelial nature of this cell line was established by the observation that cells injected into nude mice formed well-differentiated adenocarcinomas. Frozen sections of such tumors showed strong staining with antikeratins antibodies, but no detectable staining with antivimentin antibodies. These results demonstrated a differential expression of intermediate filament type in cells at different stages of neoplastic progression and in cells maintained in different growth environments. It is apparent that the expression of intermediate filaments throughout neoplastic progression is best studied by use of an in vivo model system in parallel with culture studies.

Animals↗
PubMed1999

Delayed Detonation at a Single Point in Exploding White Dwarfs.

Delayed detonation in an exploding white dwarf, which propagates from an off-center transition point rather than from a spherical transition shell, is described and simulated. The differences between the results of two-dimensional simulations and the one-dimensional case are presented and discussed. The two-dimensional effects become significant in transition density below 3rho7, where the energetics, the production of Fe group elements, and the symmetry of the explosion are all affected. In the two-dimensional case, the explosion is less energetic and less Ni is produced in the detonation phase of the explosion. For low transition density, the reduction in Ni mass can reach 20%-30%. The asymmetry in abundances between regions close to the transition point and regions far from that point is large and could be a source of polarization patterns in the emitted light. We conclude that the spatial and temporal distribution of transition locations is an important parameter that must be included in delayed detonation models for Type Ia supernovae.

Journal Article↗
PubMed2006

Many-body quantum dynamics of polarization squeezing in optical fibers.

We report new experiments that test quantum dynamical predictions of polarization squeezing for ultrashort photonic pulses in a birefringent fiber, including all relevant dissipative effects. This exponentially complex many-body problem is solved by means of a stochastic phase-space method. The squeezing is calculated and compared to experimental data, resulting in excellent quantitative agreement. From the simulations, we identify the physical limits to quantum noise reduction in optical fibers. The research represents a significant experimental test of first-principles time-domain quantum dynamics in a one-dimensional interacting Bose gas coupled to dissipative reservoirs.

Journal Article↗
PubMed1998

Effects of magnets with non-unit magnetic permeability on an elliptically polarizing undulator.

This study employs the three-dimensional magnetostatic code TOSCA to assess numerically the effects of NdFeB magnets with non-unit magnetic permeability on an elliptically polarizing undulator. A reduction of a few percent of the on-axis magnetic field strength is predicted. In addition, a deviation of +/-100 G cm uncompensated dipole steering is predicted in a phase shift of 180 degrees for the elliptically polarizing undulator EPU5.6 (having a period length of 56 mm) at the minimum gap of 18 mm, which is related primarily to the configuration of the device end scheme. Results presented herein demonstrate that implementing an active compensation mechanism is a prerequisite for minimizing the orbit distortion during phase-shift adjustment, particularly for operating such a polarizing undulator in a third-generation machine having a median energy similar to that of the 1.5 GeV storage ring at SRRC.

Journal Article↗
PubMed1990

Pathogenesis of monilethrix: computer stereography and electron microscopy.

The plucked hairs and biopsied hair follicles of the scalp were obtained from a female patient with monilethrix. By scanning electron microscopy, the plucked hairs showed a typical moniliform feature composed of alternated nodes and internodes. By computer stereography, reconstructed three-dimensional models of in vivo hair structures showed that the diameter of hair shaft was partially reduced in the keratogenous zone and that the reduction was severe in the hair cortex and cuticle layers but mild in the inner root sheath. By transmission electron microscopy, a significant degeneration of hair matrix cells was found, and a zig-zag disarray of cortical tonofibrils and invaginations of the hair cuticle cells into the cortex were noticed in the suprabulbar portion. In some hairs, the hair bulbs showed no degeneration, but a degeneration of cortical cells and invaginations of hair cuticle were seen in some portions in the developing zone of the cortex. This suggests that not every hair matrix is damaged in a synchronized fashion, but individual hairs are affected independently. Furthermore, in all the hair and hair follicles examined, cytoplasmic vacuolations were always seen in the various layers, and abnormal formation of tonofibrils was often observed in the cortex. In monilethrix, a cell abnormality may inherently be present in the hair tissue and, when such abnormality occurs severely, the cortical cells are particularly affected in the hair matrix. This seems to result in a decrease in number of cortical cells and thinning of the hair shaft.

Child, Preschool↗
PubMed1999

Dual function of the selenoprotein PHGPx during sperm maturation.

The selenoprotein phospholipid hydroperoxide glutathione peroxidase (PHGPx) changes its physical characteristics and biological functions during sperm maturation. PHGPx exists as a soluble peroxidase in spermatids but persists in mature spermatozoa as an enzymatically inactive, oxidatively cross-linked, insoluble protein. In the midpiece of mature spermatozoa, PHGPx protein represents at least 50 percent of the capsule material that embeds the helix of mitochondria. The role of PHGPx as a structural protein may explain the mechanical instability of the mitochondrial midpiece that is observed in selenium deficiency.

Animals↗