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PubMed2002

Purification and characterization of recombinant malate synthase enzymes from Streptomyces coelicolor A3(2) and S. clavuligerus NRRL3585.

Malate synthases (MS) from Streptomyces coelicolor A3(2) and S. clavuligerus NRRL3585 were cloned by polymerase chain reaction into a glutathione S-transferase (GST) fusion expression vector and heterologously expressed in Escherichia coli. The fusion GST-MS construct improved the soluble expression of MS by approximately 10-fold compared to the soluble expression of nonfusion MS. With the significant improvement in levels of soluble MS, purification and subsequent cleavage of recombinant MS from GST were facilitated in this study. Using purified enzymes, optimized parameters, which achieved maximal specific activity, were established in the enzymatic assay for streptomycete MS. The average purified specific activities of S. coelicolor and S. clavuligerus MS were 26199 and 11821 nmol/mg min, respectively. Furthermore, enzymatic analysis revealed that the two streptomycete MS displayed a similar Km value for acetyl-CoA, but S. coelicolor MS had a Km value for glyoxylate that is approximately sixfold higher than S. clavuligerus MS.

Chromatography, Affinity↗

PubMed2001

Determination of diphenylamine stabilizer and its nitrated derivatives in smokeless gunpowder using a tandem MS method.

A novel method for determination of diphenylamine (DPA) and its nitrated derivatives, which are considered as characteristic components in smokeless powder and gunshot residues, is described. A tandem mass spectrometric method is established and mass spectrometer parameters optimized for each compound to obtain higher sensitivity. Under optimum conditions, quantitative analysis was carried out. The linear ranges are 5.0-200.0, 2.0-200.0 and 5.0-250.0 ng ml-1 and the detection limits are 1.0, 0.5 and 2.5 ng ml-1 for diphenylamine (DPA), N-NO-diphenylamine (N-NO-DPA) and 4-NO2-diphenylamine (4-NO2-DPA), respectively. Intra-assay and inter-assay precision and accuracy of analysis of these three samples were investigated. Based on the regression lines obtained above, smokeless samples were analyzed. It was found that there are 0.952% DPA, 0.384% N-NO-DPA and 0.128% 4-NO2-DPA in smokeless powder. Recovery tests showed that using cotton swabs, 80.3 +/- 4.9% DPA, 79.6 +/- 3.1% N-NO-DPA and 83.1 +/- 5.4% 4-NO2-DPA could be recovered from human hands.

Journal Article↗

PubMed1978

The kinetics and thermodynamics of the reaction of solid-state fully reduced membrane-bound cytochrome oxidase with carbon monoxide as studied by dual-wavelength multichannel spectroscopy and flash photolysis.

1. The results of non-linear optimization studies on the mechanism of reaction of solid-state fully reduced membrane-bound cytochrome oxidase with CO over the 178--203 K range are presented. The analysis is carried out on data obtained by dual-wavelength multichannel spectroscopy at three wavelength pairs (444--463 nm, 590--630 nm and 608--630 nm), which yield three distinct progress curves. The only model that satisfies the triple requirement of a standard deviation within the standard error of the data, a random distribution of residuals and good determination of the optimized parameters is a two-species sequential mechanism: flash photolysis yields unliganded cytochrome oxidase and free CO, which then recombine to form species Ic; Ic is then converted into species IIc, which is identical with the cytochrome oxidase-CO complex existing before flash photolysis. All the thermodynamic parameters describing this model are calculated. 2. On the basis of the data obtained from this paper, together with data from potentiometric studies, magnetic susceptibility measurements and i.r. spectroscopy, the chemical identity of the species is suggested.

Binding Sites↗

PubMed1984

Rapid optimization of immunoadsorbent characteristics.

Immunoaffinity chromatography is employed in many research areas. We have developed an assay system that overcomes some of the tediousness and uncertainty in dealing with immunoadsorbents (IA). The preparation of IA and the effect of various procedures on the dissociation of antigen-antibody complex and the regeneration of IA are rapidly screened by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. Non-covalently bound proteins are dissociated and separated during the electrophoresis from IA beads. A wide variety of associative and dissociative conditions can be tested on small amounts of IA. Information about biospecifially and non-biospecifically adsorbed proteins can also be obtained. By using relatively small volumes of media containing either an antigen or another biospecific molecule, the optimal parameters for affinity chromatography (specificity, binding capacity, efficiency of solvents in dissociation of the complex and their effect on the adsorbent), or even for ion-exchange chromatography, can be determined without first performing several time- and material-consuming chromatographic experiments.

Antigen-Antibody Complex↗

PubMed1994

A rapid and simple method for the purification of transglutaminase from Streptoverticillium mobaraense.

Transglutaminase from Streptoverticillium mobaraense was partially purified by ion-exchange chromatography on a weak acid material and hydrophobic chromatography. The separation with a strong acid ion-exchanger produces homogeneous transglutaminase, in a single step and with high yields, directly from the centrifuged and filtered culture fluid of the micro-organism. The procedure reproduced several times could be also carried out on a larger scale with the optimized parameters of the laboratory isolations. The purified enzyme demonstrated good storage stability.

Chromatography, Ion Exchange↗

PubMed2005

Effect of slow-acting wide dynamic range compression on measures of intelligibility and ratings of speech quality in simulated-loss listeners.

The purpose of this study was twofold: (a) to determine the extent to which 4-channel, slow-acting wide dynamic range amplitude compression (WDRC) can counteract the perceptual effects of reduced auditory dynamic range and (b) to examine the relation between objective measures of speech intelligibility and categorical ratings of speech quality for sentences processed with slow-acting WDRC. Multiband expansion was used to simulate the effects of elevated thresholds and loudness recruitment in normal hearing listeners. While some previous studies have shown that WDRC can improve both speech intelligibility and quality, others have found no benefit. The current experiment shows that moderate amounts of compression can provide a small but significant improvement in speech intelligibility, relative to linear amplification, for simulated-loss listeners with small dynamic ranges (i.e., flat, moderate hearing loss). This benefit was found for speech at conversational levels, both in quiet and in a background of babble. Simulated-loss listeners with large dynamic ranges (i.e., sloping, mild-to-moderate hearing loss) did not show any improvement. Comparison of speech intelligibility scores and subjective ratings of intelligibility showed that listeners with simulated hearing loss could accurately judge the overall intelligibility of speech. However, in all listeners, ratings of pleasantness decreased as the compression ratio increased. These findings suggest that subjective measures of speech quality should be used in conjunction with either objective or subjective measures of speech intelligibility to ensure that participant-selected hearing aid parameters optimize both comfort and intelligibility.

Acoustic Stimulation↗

PubMed2001

Purification of Purkinje cells by fluorescence-activated cell sorting from transgenic mice that express green fluorescent protein.

The cerebellar Purkinje cell has been the focus of numerous studies involving the analysis of development and information processing in the nervous system. Purkinje cells represent less than 0.1% of the total cell content of the cerebellum. To facilitate studies of molecules that are expressed in such a small proportion of neurons, we have established procedures for the purification of these cells. Transgenic mice were developed in which the expression of green fluorescent protein (GFP) was controlled by the L7 promoter. In adult cerebellum, GFP fluorescence was only detected in Purkinje cells, where it filled dendrites, soma and axons. GFP fluorescence was detected in Purkinje cells as early as embryonic day 17 and increased during development in vivo and in dissociated cerebellar culture. Mirroring endogenous L7 expression, high levels of GFP were observed in retinal rod bipolar cells. Lower levels of GFP were seen in olfactory periglomerular cells, neurons in the interpeduncular nucleus, and superior colliculus neurons. Cerebella from transgenic mice were dissociated by mild enzymatic treatment and Purkinje cells were isolated by fluorescence-activated cell sorting (FACS). By selecting optimal parameters, a fraction of viable Purkinje cells that was 94% pure was obtained. These results indicate that FACS is a powerful tool for isolating Purkinje cells from postnatal L7-GFP transgenic mice. GFP-positive neurons will also be useful in the real-time observation of dendritic morphogenesis and axonal outgrowth during development, or after neuronal activity in vitro.

Animals↗

PubMed2003

Impact of parturition on iron status in nonanaemic iron deficiency.

BACKGROUND: Iron-deficient nonanaemic parturients risk underdiagnosis as a result of the reliance on postpartum ferritin and haemoglobin as markers of iron status. Ferritin is an acute-phase protein whose levels increase during the inflammatory response, as occurs after delivery. Our aims were to evaluate the impact of parturition on iron status, erythropoiesis and the inflammatory response, and identify the optimal parameters and timing for diagnosing iron deficiency in the presence of postpartum inflammation. MATERIALS AND METHODS: Conventional parameters of iron status, erythropoiesis and the inflammatory response (serum ferritin, serum iron, transferrin saturation, C-reactive protein) were compared with more recent parameters [soluble transferrin receptors (sTfR), hypochromic red cells, reticulocyte indices] within 48 h either side of delivery in 64 iron-deficient nonanaemic women (defined by a prepartum serum ferritin < or =15 microg L(-1), and a pre- and postpartum haemoglobin of > or =11.0 g dL(-1) and > or =10.0 g dL(-1), respectively). RESULTS: Mean sTfR decreased pre to postpartum from 7.3 to 5.8 microg mL(-1) (P<0.01), while mean serum ferritin increased from 9.7 to 16.9 microg L(-1) (P<0.01). Serum ferritin did not correlate with haemoglobin pre or postpartum (r=0.04, P=0.7; r=0.2, P=0.09), but a correlation persisted postpartum between hypochromic red blood cells and haemoglobin (r=-0.26; P<0.05). The percentage of hypochromic red cells remained virtually unchanged pre- and postpartum (4.0% vs. 3.8%; NS). Postpartum mean reticulocyte haemoglobin content (CHr) was 27.1 +/- 1.6 pg. CONCLUSION: Iron status should be tested prepartum, in the absence of an inflammatory response, rather than in the early postpartum. A valuable additional parameter, where available, might be the hypochromic red cell percentage, which is virtually uninfluenced by the inflammatory response. Furthermore, hypochromic red cell percentage, CHr and sTfR can be helpful to differentiate between functional iron deficiency and depleted iron stores.

Adult↗

PubMed2003

Growth and beta-galactosidase activity in cultures of Kluyveromyces marxianus under increased air pressure.

AIMS: To investigate the effect of total air pressure raise on cell growth and intracellular beta-galactosidase activity in batch cultures of Kluyveromyces marxianus CBS 7894. METHODS AND RESULTS: A pressurized bioreactor was used for K. marxianus batch cultivation under increased air pressure from 1.2 to 6 bar. Under these conditions no inhibition of cell growth was observed. Moreover, the improvement of the oxygen transfer rate (OTR) from the gas to the culture medium by pressurization led to an enhancement of the cell growth rate obtained at atmospheric pressure without aeration. The specific beta-galactosidase productivity increased from 5.8 to 17.0 U gCD-1 h-1 using a 6-bar air pressure instead of air at atmospheric pressure. The antioxidant enzyme superoxide dismutase (SOD) was slightly induced by the air pressure raise, which indicates that the defensive mechanisms of the cells can cope with an air pressure up to 6 bar. CONCLUSIONS: These experiments showed that the increase of air pressure up to 6 bar is an alternative to other methods of preventing the oxygen limitation and can be applied in the beta-galactosidase production by K. marxianus. SIGNIFICANCE AND IMPACT OF THE STUDY: The results here reported proved that, in what biological aspects are concerned, it is possible to use the air pressure increase as an optimization parameter of beta-galactosidase production in high-density cell cultures of K. marxianus strains.

Air Pressure↗

PubMed2002

The Vienna functional electrical stimulation system for restoration of walking functions in spastic paraplegia.

An eight-channel stimulation system, currently intended for stimulation of lower extremities, was developed and is introduced. The major development goals were easy handling, modularity to make the system easily adaptable for other functional electrical stimulation (FES) applications, and a wide stimulation parameter range for application-specific parameter optimization. For paraplegic stepping, the system worn by the patient consists of 2 four-channel stimulation modules, a central unit holding the battery and circuitry for power management and communication control, a wireless remote control unit, and a palmtop computer as the main control and input device. A software package for Microsoft Windows supports the design and optimization of stimulation sequences in the rehabilitation center. First tests with patients familiar with FES showed smoother movements during stepping and acceptable good handling. In combination with the PC software, the required stimulation sequences could be created in a very short time.

Computer Systems↗

PubMed2002

The Acute Dialysis Quality Initiative--part VII: fluid composition and management in CRRT.

Fluid composition and management are important parts of continuous renal replacement therapy (CRRT). Most commercially available CRRT solutions are able to reestablish electrolyte homeostasis provided some phosphate supplementation is given. Supraphysiologic glucose concentrations should be avoided. Predilution fluid replacement allows higher ultrafiltration rates and can be considered as an adjunct to the anticoagulation regimen. Lactate is an effective buffer in most CRRT patients. Bicarbonate is preferred in patients with lactic acidosis and/or liver failure. When citrate is used as anticoagulant, frequent monitoring of pH is required. The clinical consequences of CRRT-induced decreases of body temperature are not clear. Substitution fluid should be sterile, but the bacteriologic requirements for CRRT dialysate are less clear. There is no consensus on the optimal parameters to monitor fluid management. Integrated balancing systems have theoretical advantages over adaptive use of intravenous fluid pumps. Although there is evidence that volume overload is associated with adverse outcome, there is no evidence that fluid removal per se improves outcome in critically ill patients with or without acute renal failure.

Buffers↗

PubMed1978

[The protective effect of magnesium-aspartate-procaine cardioplegia on the hypertrophied left ventricle of the mini-pig (author's transl)].

After induction of left ventricular hypertrophy by supravalvar constriction of the ascending aorta in mini pigs (ATP and lactate) were measured under different cardioplegic conditions. In normothermia and plain anoxic arrest ATP decrease and lactate increase were significantly slower in hypertrophied myocardium compared to normal myocardium. Injection cardioplegia using magnesium-aspartate-procaine at 37 degrees C did not influence the ATP decrease and lactate increase in the hypertrophied ventricle, whereas in the normal heart it showed some protection according to these parameters. Optimal ATP preservation and the lowest lactate increase rate were achieved in left ventricular hypertrophy by combined application of magnesium-aspartate-procaine and hypothermia of 25 degrees C. We conclude that normothermic injection cardioplegia has no protective effect on the hypertrophied left ventricle, whereas additional hypothermia can improve magnesium-aspartate-procaine cardioplegia significantly.

Adenosine Triphosphate↗

PubMed2006

[Digital ultrasonic diagnosis of lower leg venous thrombosis using harmonic imaging and cross beam technology].

PURPOSE: To evaluate the extent to which Tissue Harmonic Imaging (THI) and spatial averaging (Sono-CT, Cross Beam) facilitate the diagnosis of lower leg venous thrombosis. MATERIALS AND METHODS: In 100 cases of lower leg venous thrombosis, the conventional B-scan, THI, and ultrasonic CT technology (Cross Beam) in examinations using a multi-frequency transducer head (9 - 14 MHz, Logiq 9, GE) were compared during follow-up studies using compression sonography over at least 6 weeks. Two independent examiners performed digital image documentation (PACS connection) without modifying the gray level parameters optimized in the B-scan. A thrombosis was considered to be proven sonographically if at least one ultrasonic modality yielded findings reproducible in follow-up checks at a comparable site in accordance with both examiners. RESULTS: Thromboses in the V. tibialis posterior category (97 %) and the fibular category (81 %) were found most often, followed by muscular venous thromboses (48 %). Thromboses in the V. tibialis anterior category were rare (12 %). The procedure comparison showed an advantage for ultrasonic CT technology in terms of the demarcation of thromboses of the deeper veins (sensitivity 98 %), as compared to THI (sensitivity 87 %, specificity 91 %, PPV: 99 %) and the B-scan (sensitivity 81 %). CONCLUSION: If a high-resolution linear transducer is used, THI and ultrasonic CT-Technology can facilitate the diagnosis of a lower leg venous thrombosis.

Adult↗

PubMed1996

Experimental evaluation of the optimal in-vitro conditions for bipolar electrocoagulation in the bile duct.

BACKGROUND AND STUDY AIMS: Although there have been many reports concerning the therapeutic use and potential of laser and monopolar electrocoagulation therapy, little has been published on the effects of bipolar electrocoagulation on biliary tissue. Although it has been reported that endoscopic recanalization of malignant stent obstruction using bipolar electrocoagulation is an advantageous form of treatment, the optimal parameters for this type of treatment have not yet been determined. The purpose of this prospective study was therefore to evaluate, under in-vitro conditions, the maximum applicable energy required; the influence of probe angle and duration time on the extent of the coagulation achieved; and whether multiple intermittent short pulses show any significant differences in the extent of coagulation achieved, in comparison with the use of a single continuous pulse. PATIENTS AND METHODS: In this in-vitro study, experiments were carried out in 84 common bile ducts from freshly slaughtered swine. The extension of tissue injury after electrocoagulation treatment was measured at 60 combinations of varying levels of power output, probe angles, duration times, and pulse patterns. All of the results were correlated with the diameter of the untreated and normal bile duct walls under in-vitro conditions. RESULTS: When energy was applied at levels up to a maximum of 16 J, the extension of tissue coagulation was about 58% of the untreated bile duct wall diameter. Energy settings exceeding 16 J involved the whole wall diameter in most cases, often including the periductal fatty tissue. Additional energy applications higher than 16 J, 32 J, and 100 J, showed undesirable tissue changes, such as an increasing diameter in the treated bile duct walls of up to 56.8% of the wall diameter of control specimens. CONCLUSIONS: The study evaluated the standard settings in an in-vitro model. The results indicate that protective and effective use of bipolar electrocoagulation on normal porcine bile duct tissue requires: the use of a low power setting (less than 16 J); the use of continuous short pulse patterns; placement of the probe tip at an obtuse angle (0 degree); and the use of short application times.

Animals↗

PubMed1991

[Digital luminescence radiography (DLR) for skeletal diagnosis in traumatology].

170 patients who had suffered trauma to bones or soft tissues were examined by a digital and a conventional film/screen technique. In addition to "standard" and edge-enhanced images, post-processing was carried out and compared with conventional radiographs. Four radiologists and two surgeons evaluated the compacta, spongiosa, soft tissues, fractures and osteo-synthetic materials as shown on the two systems. The "standard" digital images are inferior to film images, but edge-enhanced images showed definite advantages for demonstrating soft tissues. Evaluation of osteo-synthetic material is limited by the creation of artifacts. Recognition of detail can be significantly improved by using post-processing with optimized parameters.

Adolescent↗

PubMed2004

Bohmian dynamics on subspaces using linearized quantum force.

In the de Broglie-Bohm formulation of quantum mechanics the time-dependent Schrodinger equation is solved in terms of quantum trajectories evolving under the influence of quantum and classical potentials. For a practical implementation that scales favorably with system size and is accurate for semiclassical systems, we use approximate quantum potentials. Recently, we have shown that optimization of the nonclassical component of the momentum operator in terms of fitting functions leads to the energy-conserving approximate quantum potential. In particular, linear fitting functions give the exact time evolution of a Gaussian wave packet in a locally quadratic potential and can describe the dominant quantum-mechanical effects in the semiclassical scattering problems of nuclear dynamics. In this paper we formulate the Bohmian dynamics on subspaces and define the energy-conserving approximate quantum potential in terms of optimized nonclassical momentum, extended to include the domain boundary functions. This generalization allows a better description of the non-Gaussian wave packets and general potentials in terms of simple fitting functions. The optimization is performed independently for each domain and each dimension. For linear fitting functions optimal parameters are expressed in terms of the first and second moments of the trajectory distribution. Examples are given for one-dimensional anharmonic systems and for the collinear hydrogen exchange reaction.

Journal Article↗

PubMed2004

Ground and excited states of NH4: Electron propagator and quantum defect analysis.

Vertical excitation energies of the Rydberg radical NH4 are inferred from ab initio electron propagator calculations on the electron affinities of NH4+. The adiabatic ionization energy of NH4 is evaluated with coupled-cluster calculations. These predictions provide optimal parameters for the molecular-adapted quantum defect orbital method, which is used to determine Einstein emission coefficients and radiative lifetimes. Comparisons with spectroscopic data and previous calculations are discussed.

Journal Article↗

PubMed2005

Partial averaging and the centroid virial estimator for stereographic projection path-integral simulations in curved spaces.

We develop and test three different partial averaging theories for the stereographic projection path integral in curved spaces. Additionally, we adapt and test the centroid virial estimator for the kinetic energy in curved spaces. We tested both a confining as well as a nonconfining potential for the particle in a ring. All three partial averaging theories are demonstrated to converge linearly in the asymptotic region with k(-2)max, the number of Fourier coefficients. We use an error estimator to determine the optimal parameters for an extrapolation to infinite kmax. We verify that the centroid virial estimator (derived from the primitive DeWitt path-integral formula) converges to the kinetic energy for both confining and nonconfining potentials.

Journal Article↗