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PubMed2001

Reduction in matrix-related signal suppression effects in electrospray ionization mass spectrometry using on-line two-dimensional liquid chromatography.

The effect of liquid chromatographic separation on matrix-related signal suppression in electrospray ionization mass spectrometry (LC-ESI-MS) was investigated. A method incorporating on-line two-dimensional liquid chromatography mass spectrometry (LC/LC-MS) was developed to compensate for matrix effects and signal suppression in qualitative and quantitative analysis. The LC/LC-MS(MS) approach was successfully applied for single-component and multicomponent analysis in a variety of complex matrixes. It was demonstrated that matrix-related signal suppression could be induced solely by (i) column overload, (ii) matrix component-analyte coelution, or a combination of each. Application of on-line orthogonal LC/LC separations can be effective in reducing both causes of matrix-related signal suppression effects i.e., column overload and matrix-analyte coelution for a variety of LCn/MSn applications.

Animals↗
PubMed2002

[Is sufficient femoral head coverage obtained after Pemberton's pericapsular osteotomy? Evaluation by three-dimensional computed tomographic reconstruction].

OBJECTIVES: We evaluated femoral head coverage by three-dimensional computed tomographic reconstruction after Pemberton's pericapsular osteotomy with open reduction in cases with developmental dysplasia of the hip. METHODS: In a prospective design, routine radiographs of 15 consecutive patients were obtained and three-dimensional computed tomographic reconstruction was performed preoperatively and six months after surgery. The mean age at the time of surgery was 26 months (range 18 to 34 months). Twelve patients were girls and three were boys. RESULTS: The mean acetabular index was 38 degrees (range 33 degrees -52 degrees ) preoperatively, and 19 degrees (range 16 degrees -23 degrees ) postoperatively. Comparison between pre- and postoperative three-dimensional computed tomographic reconstruction images revealed increased acetabular adduction and extension, and decreased acetabular anteversion in all cases. Sufficient coverage of the femoral head was documented in all patients. CONCLUSION: Pemberton's pericapsular osteotomy provides successful results in appropriate cases, with sufficient coverage of the femoral head. However, if there is any doubt, utilization of three-dimensional computed tomographic reconstruction images may contribute to a more precise evaluation of the outcome.

Acetabulum↗
PubMed2003

Correlation method for variance reduction of Monte Carlo integration in RS-HDMR.

The High Dimensional Model Representation (HDMR) technique is a procedure for efficiently representing high-dimensional functions. A practical form of the technique, RS-HDMR, is based on randomly sampling the overall function and utilizing orthonormal polynomial expansions. The determination of expansion coefficients employs Monte Carlo integration, which controls the accuracy of RS-HDMR expansions. In this article, a correlation method is used to reduce the Monte Carlo integration error. The determination of the expansion coefficients becomes an iteration procedure, and the resultant RS-HDMR expansion has much better accuracy than that achieved by direct Monte Carlo integration. For an illustration in four dimensions a few hundred random samples are sufficient to construct an RS-HDMR expansion by the correlation method with an accuracy comparable to that obtained by direct Monte Carlo integration with thousands of samples.

Journal Article↗
PubMed2006

Concepts and approaches towards understanding the cellular redox proteome.

The physiological activity of a significant subset of cell proteins is modified by the redox state of regulatory thiols. The cellular redox homeostasis depends on the balance between oxidation of thiols through oxygen and reactive oxygen species and reduction by thiol-disulfide transfer reactions. Novel and improved methodology has been designed during recent years to address the level of thiol/disulfide regulation on a genome-wide scale. The approaches are either based on gel electrophoresis or on chromatographic techniques coupled to high end mass spectrometry. The review addresses diagonal 2D-SDS-PAGE, targeted identification of specific redox-interactions, affinity chromatography with thioredoxins and glutaredoxins, gel-based and non-gel based labelling techniques with fluorophores (such as Cy3, Cy5, ICy), radioisotopes, or with isotope-coded affinity tags (ICAT), differential gel electrophoresis (DIGE) and combined fractional diagonal chromatography (COFRADIC). The extended methodological repertoire promises fast and new insight into the intricate regulation network of the redox proteome of animals, bacteria, and plants.

Cell Respiration↗
PubMed1995

[Three-dimensional imaging of acetabular fractures using spiral CT].

Seven adult patients with acetabular fractures were investigated with conventional X-rays, axial spiral computed tomographic (CT) images and three-dimensional (3D) surface rendered reconstructions. Spiral CT has the advantage that it acquires the data much faster than incremental CT, resulting in less motion artefacts. A semi-automatic three-dimensional environment for segmentation has been developed in our institution. It created significant reduction of user interaction and improved visualization of acetabular and femoral joint surfaces. The main advantages of three-dimensional visualization of acetabular fractures included: creation of unlimited and unique views by which fracture location, fracture extension, fragment shape and fragment position became more clear, better visualization of acetabular dome and quadrilateral plate and more simple and easier interpretation of these complex fracture patterns. On the other hand, 3D surface reconstructions do not accurately demonstrate undisplaced fractures, intra-articular fragments may be obscured, congruence assessment is uneasy, and soft tissue injuries are not shown. Therefore, we consider 3D images of acetabular fractures as being complementary to and not a substitute to plain radiographs and axial CT scans.

Acetabulum↗
PubMed2003

Simple model for drag reduction.

Direct numerical simulations established that the finite-extension nonlinear-elasticity-Peterlin (FENE-P) model of viscoelastic flows exhibits the phenomenon of turbulent drag reduction which is caused in experiments by dilute polymeric additives. To gain analytic understanding of the phenomenon, we introduce in this paper a simple one-dimensional model of the FENE-P equations. We demonstrate drag reduction in the simple model, and explain analytically the main observations which include (i) reduction of velocity gradients for fixed throughput and (ii) increase of throughput for fixed dissipation.

Journal Article↗
PubMed1988

The role of cytochrome c diffusion in mitochondrial electron transport.

We have compared the modes and rates of cytochrome c diffusion to the rates of cytochrome c-mediated electron transport in isolated inner membranes and in whole intact mitochondria. For inner membranes, an increasing ionic strength results in an increasing rate of cytochrome c diffusion, a decreasing concentration (affinity) of cytochrome c near the membrane surface as well as near its redox partners, and an increasing rate of electron transport. For intact mitochondria, an increasing ionic strength results in a parallel, increasing rate of cytochrome c-mediated electron transport. In both inner membranes and intact mitochondria the rate of cytochrome c-mediated electron transport is highest at physiological ionic strength (100-150 mM), where the diffusion rate of cytochrome c is highest and its diffusion mode is three-dimensional. In intact mitochondria, succinate and duroquinol-driven reduction of endogenous cytochrome c is greater than 95% at all ionic strengths, indicating that cytochrome c functions as a common pool irrespective of its diffusion mode. Using a new treatment to obtain bimolecular diffusion-controlled collision frequencies in a heterogenous diffusion system, where cytochrome c diffuses laterally, pseudo-laterally, or three-dimensionally while its redox partners diffuse laterally, we determined a high degree of collision efficiency (turnover/collisions) for cytochrome c with its redox partners for all diffusion modes of cytochrome c. At physiological ionic strength, the rapid diffusion of cytochrome c in three dimensions and its low concentration (affinity) near the surface of the inner membrane mediate the highest rate of electron transport through maximum collision efficiencies. These data reveal that the diffusion rate and concentration of cytochrome c near the surface of the inner membrane are rate-limiting for maximal (uncoupled) electron transport activity, approaching diffusion control.

Animals↗
PubMed2002

Three-dimensional color Doppler sonography and uterine artery arteriography of fibroids: assessment of changes in vascularity before and after embolization.

OBJECTIVE: To assess the accuracy of three-dimensional color Doppler sonography and uterine artery arteriography in depicting changes in fibroid vascularity before and after embolization. METHODS: Preembolization and postembolization three-dimensional color Doppler sonography and selective uterine artery arteriography were retrospectively compared in 15 patients who underwent uterine artery embolization for treatment of symptomatic fibroids. Three-dimensional color Doppler sonography was performed by using a scanner with color power angiographic imaging capability. Vascularity was quantified by using an estimation of power-weighted pixel density as described by our group in previously published studies. Uterine artery arteriography was performed by using a standard selective microcatheter embolization technique. For purposes of comparison, fibroids were classified as either hypervascular or hypovascular relative to myometrial vascularity before and minutes to several hours after uterine artery embolization. Changes in fibroid vascularity (i.e., from hypervascular to hypovascular) as depicted by three-dimensional color Doppler sonography were compared with those shown on uterine artery arteriography and classified as being in agreement or disagreement. RESULTS: In 13 (87%) of 15 patients there was agreement; in 2 (13%) of 15 there was disagreement. In both cases of disagreement, three-dimensional color Doppler sonography showed collateral flow not depicted by uterine artery arteriography The mean reduction in quantitated vascularity after uterine artery embolization was 44% (range, 19%-78%). CONCLUSIONS: Three-dimensional color Doppler sonography accurately depicts fibroid vascularity and in some cases can reveal collateral flow not depicted by uterine artery arteriography.

Adult↗
PubMed2001

Alkylation kinetics of proteins in preparation for two-dimensional maps: a matrix assisted laser desorption/ionization-mass spectrometry investigation.

All existing protocols for protein separation by two-dimensional (2-D) gel electrophoresis require the full reduction, denaturation, and alkylation as a precondition for an efficient and meaningful separation of such proteins. Existing literature provides a strong evidence to suggest that full reduction and denaturation can be achieved in a relatively short time; the same thing, however, can not be said for the alkylation process, which the present study shows that more than 6 h are required for a complete alkylation. We have used matrix assisted laser desorption/ionisation-time of flight-mass spectrometry (MALDI-TOF-MS) to monitor protein alkylation by iodoacetamide over the period 0-24 h at pH 9. The present, fast and specific MS method provided clear indication on the extent and speed of alkylation which reached approximately 70% in the first 2 min, yet the remaining 30% resisted complete alkylation up to 6 h. The use of sodium dodecyl sulfate (SDS) during the alkylation step resulted in a strong quenching of this reaction, whereas 2% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) exerted a much reduced inhibition. The implications of the present measurements on 2-D gel analysis in particular and proteomics in general are discussed.

Alkylation↗
PubMed1990

Special considerations in the elderly patient.

Special considerations in the treatment of the elderly hypertensive include altered pathophysiology, the increasing likelihood of coexisting disease, and the question of whether reduction of left ventricular (LV) mass should be a therapeutic goal. The objective of our study was to compare the ability of a calcium blocker, verapamil, and a beta-blocker, atenolol, to induce reduction of LV mass and to determine the effects of any such reduction on LV filling and contractile performance. Forty-two hypertensive patients over 60 years of age were randomized to progressively increasing doses of long-acting verapamil or atenolol in a double-blind fashion and followed over a six-month period. Blood pressure decreased in both groups but atenolol-treated subjects more frequently required the addition of chlorthalidone. Despite similar blood pressure reductions, mean LV mass index, determined by two-dimensional echocardiography, was significantly reduced in the verapamil group magnitude of [104 +/- 5 g/m2 to 85 +/- 5 g/m2 (P less than 0.01)] but was not changed in the atenolol group. Following withdrawal of therapy, gated blood pool scan determined peak filling rate and the ratio of peak filling to peak ejection significantly increased in the group evidencing reduction (2.42 +/- 0.2 to 3.31 +/- 0.4 EDV/sec and 0.61 +/- 0.03 to 0.85 +/- 0.05, respectively) but did not change in the group which showed no reduction. Cardiac output and ejection fraction were maintained, both at rest and during mild upright exercise in the group evidencing reduction. Reduction of LV mass occurs more frequently with verapamil than with atenolol therapy in elderly hypertensives. Reduction improves diastolic filling and does not impair contractile performance.

Age Factors↗
PubMed2004

Thioredoxin targets of developing wheat seeds identified by complementary proteomic approaches.

The role of thioredoxin in wheat starchy endosperm was investigated utilizing two proteomic approaches. Thioredoxin targets were isolated from total KCl-soluble extracts of endosperm and flour and separated by 2-DE following (1) reduction of the extract by the NADP/thioredoxin system and labeling the newly generated sulfhydryl (SH) groups with monobromobimane (mBBr), and, in parallel, (2) trapping covalently interacting proteins on an affinity column prepared with mutant thioredoxin h in which one of the active site cysteines was replaced by serine. The two procedures were complementary: of the total targets, one-third were observed with both procedures and one-third were unique to each. Altogether 68 potential targets were identified; almost all containing conserved cysteines. In addition to confirming known interacting proteins, we identified 40 potential thioredoxin targets not previously described in seeds. A comparison of the results obtained with young endosperm (isolated 10 days after flowering) to those with mature endosperm (isolated 36 days after flowering) revealed a unique set of proteins functional in processes characteristic of each developmental stage. Flour contained 36 thioredoxin targets, most of which have been found in the isolated developing endosperm.

Bridged Bicyclo Compounds↗
PubMed2003

Effects of total contact insoles on the plantar stress redistribution: a finite element analysis.

OBJECTIVE: To investigate the effects of total contact insoles on the plantar stress redistribution using three-dimensional finite element analysis. DESIGN: The efficacies of stress reduction and redistribution of two total contact insoles with different material combinations were compared with those of a regular flat insole used as a baseline condition. BACKGROUND: Many specially designed total contact insoles are currently used to reduce the high plantar pressure in diabetic patients. However, the design of total contact insoles is mostly empirical and little scientific evidence is available to provide a guideline for persons who prescribe such insoles. METHODS: To use three-dimensional finite element models of the foot together with insoles to investigate the effects of total contact insoles on the foot plantar pressure redistributions. Nonlinear foam material properties for the different insole materials and the contact behavior in the foot-insole interface were considered in the finite element analysis. RESULTS: Results showed that the peak and the average normal stresses were reduced in most of the plantar regions except the midfoot and the hallux region when total contact insoles were worn compared with that of the flat insole condition. The reduction ratios of the peak normal stress ranged from 19.8% to 56.8%. CONCLUSIONS: Finite element analysis results showed that the two sets of total contact insoles used in the current study can both reduce high pressures at regions such as heel and metatarsal heads and can redistribute the pressure to the midfoot region when compared with the flat insole condition. RELEVANCE: It is possible to simulate foot deformities, change in material properties, different ambulatory loading conditions, and different orthotic conditions by altering the finite element model in a relatively easy manner and these may be of interests to the medical professionals who treat foot-related problems.

Adult↗
PubMed1993

Identifying proteins from two-dimensional gels by molecular mass searching of peptide fragments in protein sequence databases.

A rapid method for the identification of known proteins separated by two-dimensional gel electrophoresis is described in which molecular masses of peptide fragments are used to search a protein sequence database. The peptides are generated by in situ reduction, alkylation, and tryptic digestion of proteins electroblotted from two-dimensional gels. Masses are determined at the subpicomole level by matrix-assisted laser desorption/ionization mass spectrometry of the unfractionated digest. A computer program has been developed that searches the protein sequence database for multiple peptides of individual proteins that match the measured masses. To ensure that the most recent database updates are included, a theoretical digest of the entire database is generated each time the program is executed. This method facilitates simultaneous processing of a large number of two-dimensional gel spots. The method was applied to a two-dimensional gel of a crude Escherichia coli extract that was electroblotted onto poly(vinylidene difluoride) membrane. Ten randomly chosen spots were analyzed. With as few as three peptide masses, each protein was uniquely identified from over 91,000 protein sequences. All identifications were verified by concurrent N-terminal sequencing of identical spots from a second blot. One of the spots contained an N-terminally blocked protein that required enzymatic cleavage, peptide separation, and Edman degradation for confirmation of its identity.

Amino Acid Sequence↗
PubMed2004

Bimetallic (Ag)Au nanoparticles prepared by the seed growth method: two-dimensional assembling, characterization by energy dispersive X-ray analysis, X-ray photoelectron spectroscopy, and surface enhanced raman spectroscopy, and proposed mechanism of growth.

Layered core-shell bimetallic silver-gold nanoparticles were prepared by overdeposition of Au over Ag seeds by the seed-growth method using tetrachloroauric acid, with hydroxylamine hydrochloride as the reductant. The effects of pH, reduction rate, and seeding conditions on the morphology and surface plasmon extinction of the bimetallic nanoparticles were investigated. Nanoparticles prepared by a rapid reduction in the neutral ambient and assembled into two-dimensional nanoparticulate films by adsorption of 2,2'-bipyridine were characterized by energy-dispersive X-ray analysis, X-ray photoelectron spectroscopy, surface-enhanced Raman scattering spectroscopy, and transmission electron microscopy. The results are consistent with Ag core and Ag/Au-alloyed shell composition of the nanoparticles. Evidence of the presence of Ag on the surface of the nanoparticles, of enrichment of the Ag/Au alloy shell by Ag toward or at the nanoparticle surface, and of modification of the nanoparticle surface by adsorbed chlorides is also provided. Reduction of the size of the Ag seeds, alloying of Ag and Au in the shell of the nanoparticles, and modification of their surfaces by adsorbed chlorides are tentatively attributed to positive charging of the nanoparticles during the electrocatalytic overdeposition of Au over Ag seeds.

Journal Article↗
PubMed1988

Calcaneal fractures: three-dimensional treatment.

A series of 22 calcaneal fractures operated over 4 yr is presented. Radiographic evaluation of these fractures using axial, lateral, anteroposterior, and oblique medial projection (Broden's view) with varying tube angulation toward the head and computerized axial tomography in two planes, coronal and transverse, were done. This repeatedly demonstrated intra-articular splitting fractures along the axis of the os calcis through the posterior facet, extending to the calcaneocuboid joint. These fractures were surgically reduced as described by McReynolds with sequential medial and lateral approaches. Intraoperative X-rays demonstrate the three-dimensional nature of this injury. Bohler's angle may be reconstituted with apparent reduction of the posterior facet when projected laterally; however, Broden's and axial views show persistent widening and split of the posterior facet. The 22 patients have been followed from 12 to 46 mo. All have heels of normal width and wear standard shoes. The first two cases, done only medially, have less than anatomic reconstruction of the subtalar joint. Functional results have been encouraging. Of six active duty members so treated, three returned to full duty, a second who was medically retired as a result of other injuries had minimal heel problems, and fifth had a contralateral open pilon fracture which hampers him, and the sixth patient is now 4 mo postbilateral calcaneal fractures with return to duty anticipated. Most of the 22 have some decrease in subtalar motion without apparent gait abnormality. No cases of medial or lateral impingement or tendinitis are present. Fractures of the calcaneus are amendable to open reduction and internal fixation. Sequential medial and anterolateral exposure allow anatomic reconstruction in three dimensions, avoiding pitfalls of two-dimensional analysis and treatment.

Activities of Daily Living↗
PubMed1995

Visualization of endothelial clefts and nuclei in living microvessels with combined reflectance and fluorescence confocal microscopy.

OBJECTIVE: The cellular basis for spatial heterogeneity along postcapillary venules in their response to inflammatory mediators is not understood. To study permeability regulatory processes on an individual cell basis in intact microvessels, we developed methods to delineate individual endothelial cells within living, single perfused microvessels. METHODS: Individual postcapillary microvessels in the mesenteries of frogs and hamsters were perfused with chloride-free Ringer's solutions containing AgNO3 (0.1 g per 100 ml) for 5--10 s. Vessels were immediately flushed with Ringer's solution containing serum albumin and some vessels subsequently perfused with the fluorescent nucleic acid stain YO-PRO-1 (1 microM) for 10-15 min. Vessels were imaged in situ using laser scanning confocal microscopy. A reflectance image from silver precipitate in the endothelial clefts and a fluorescence image of the nuclei were simultaneously recorded. Stacks of confocal images were merged and used to reconstruct the three-dimensional orientation of endothelial cells. Hydraulic conductivity of some frog mesenteric venular vessels, measured before and after perfusion with AgNO3, was used to assess the integrity of the vessels. RESULTS: The endothelial cell clefts were delineated with a very fine, readily imaged precipitate of silver. Hydraulic conductivity measured after AgNO3 perfusion in frog mesenteric venular microvessels was not significantly different from control. Vessels showed a slight reduction in reactivity to inflammatory stimuli. The three-dimensional pattern of endothelial cells in the living vessels was imaged. CONCLUSIONS: Combining confocal microscopy with silver staining in single perfused microvessels was shown to be an effective means to delineate the three-dimensional pattern of endothelial cells forming microvessel walls. It enables further study of the vascular structure and function relationships at the individual cell level in intact microvessels.

Animals↗
PubMed1993

Two-dimensional 1H nuclear magnetic resonance studies of the half-saturated (Ca2+)1 state of calbindin D9k. Further implications for the molecular basis of cooperative Ca2+ binding.

Calbindin D9k exhibits cooperative binding of two calcium ions, hence study of the half-saturated states of the protein is critical to understanding the binding process. However, the half-saturated states are not significantly populated under equilibrium conditions. To circumvent this problem, an absolutely conserved glutamic acid residue in the C-terminal binding site (site II) has been mutated to glutamine (E65Q), causing a substantial reduction in calcium affinity and permitting detailed two-dimensional 1H NMR analysis of calbindin D9k with a calcium ion bound only in the N-terminal EF-hand. Complete 1H resonance assignments have been obtained for (Ca2+)1 E65Q, as well as near complete assignments for the apo and (Ca2+)2 states. A value of 1.1(+/- 0.2) x 10(3) M-1 has been determined for the calcium binding constant in site II, from an analysis of the chemical shift changes in response to titration with calcium. The elements of secondary structure and global folding patterns were identified from nuclear Overhauser effects, backbone spin-spin coupling constants and the exchange rates of backbone amide protons. Although the mutation has only very small effects on the secondary structure and global fold of the protein, it so drastically lowers affinity for Ca2+ in the C-terminal site that (Ca2+)2 E65Q does not correspond to a standard (Ca2+)2 state. From the analysis of the half-saturated state, it is apparent that some reorganization of the structure and changes in the internal dynamics of calbindin D9k does occur for each step of the apo-->(Ca2+)1(I)-->(Ca2+)2 binding pathway. When the first ion is bound to the N-terminal EF-hand, that half of the molecule adopts a conformation and dynamic state similar to the fully calcium-loaded protein state, whereas only minor changes occur in the C-terminal EF-hand. It is only upon binding of the second calcium ion that the C-terminal EF-hand switches over to the fully calcium-loaded state. Together with the results from our earlier study of the apo-->(Ca2+)1(II)-->(Ca2+)2 binding pathway, these findings indicate that changes in protein conformation and dynamics associated with Ca2+ binding contribute to the observed positive cooperativity, and that the molecular details of the cooperative binding events are different for the two binding pathways.

Amides↗
PubMedDate not supplied

Sonographic evaluation of gallbladder kinetics: in vitro and in vivo comparison of different methods to assess gallbladder emptying.

In an in vitro study, 10 gallbladders of adult pigs and 6 gallbladders of lambs, all removed immediately after slaughtering, were stimulated in a water bath by electric means to induce active contraction. Gallbladder emptying was followed by ultrasonography employing five measurement procedures: (1) gallbladder width, (2) longitudinal planimetry, (3) transverse planimetry, (4) ellipsoid method, and (5) sum of cylinders method. In an in vivo investigation, gallbladder emptying of 30 volunteers (12 healthy subjects, 18 diabetics) was evaluated in the same way after ingestion of a fatty meal. Gallbladder width was found to be unsuitable to estimate the decrease in gallbladder volume due to a nonlinear relation of the parameters. Longitudinal planimetry tended to be less valid than transverse planimetry in assessing gallbladder volume reduction. The most valid estimation of gallbladder volume decreases was obtained by the two three-dimensional procedures. However, in neither in vitro nor in vivo could a significant difference between the sum of cylinders method and the ellipsoid method in determining relative volume reduction be established. We conclude that a three-dimensional measurement procedure should be used for valid assessments of gallbladder motility. However, according to our data there is no advantage in using the time-consuming sum of cylinders method compared to the simple ellipsoid method.

Adult↗