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PubMed2004

NMR spectroscopic filtration of polypeptides and proteins in complex mixtures.

Due to the inherent complexity of the natural biological environment, most studies on polypeptides, proteins and nucleic acids have so far been performed in vitro, away from physiologically relevant conditions. Nuclear magnetic resonance is an ideal technique to extend the in vitro analysis of simple model systems to the more complex biological context. This work shows how diffusion-based spectroscopic selection can be combined with isotopic labeling to tackle and optimize the NMR analysis of specific macromolecules in multicomponent mixtures. Typical media include cell-free systems containing overexpressed proteins, lysates and proteolytic mixtures. We present a few variants of diffusion-edited HSQC pulse sequences for the selective spectroscopic detection of protein and polypeptide resonances within complex mixtures containing undesired species of smaller molecular weight. Due to diffusion-based filtering, peak intensities of fast diffusing small molecules are attenuated more than peaks due to large molecules. The basic sequence, denoted as PFGSTE-HSQC, combines translational diffusion-ordering with two dimensional heteronuclear single quantum correlation spectroscopy. The GCSTE-HSQC and BPPSTE-HSQC sequences include bipolar gradients and are therefore suitable for both diffusion-based filtering and determination of diffusion coefficients of individual mixture components. Practical applications range from protein stability/folding investigations in physiologically relevant contexts to prescreening of tertiary fold and resonance assignments in structural genomics studies. A few applications of diffusion-edited HSQC to an E. coli cell lysate containing the (15)N-labeled B domain of streptococcal protein G (GB1), and to a (15)N-labeled N-acetylglycine/apomyoglobin mixture are presented. In addition, we provide specific guidelines for experimental setup and parameter optimization.

Data Interpretation, Statistical↗
PubMed2006

Optimal utilization of donor grafts with extended criteria: a single-center experience in over 1000 liver transplants.

OBJECTIVE: Severely limited organ resources mandate maximum utilization of donor allografts for orthotopic liver transplantation (OLT). This work aimed to identify factors that impact survival outcomes for extended criteria donors (ECD) and developed an ECD scoring system to facilitate graft-recipient matching and optimize utilization of ECDs. METHODS: Retrospective analysis of over 1000 primary adult OLTs at UCLA. Extended criteria (EC) considered included donor age (>55 years), donor hospital stay (>5 days), cold ischemia time (>10 hours), and warm ischemia time (>40 minutes). One point was assigned for each extended criterion. Cox proportional hazard regression model was used for multivariate analysis. RESULTS: Of 1153 allografts considered in the study, 568 organs exhibited no extended criteria (0 score), while 429, 135 and 21 donor allografts exhibited an EC score of 1, 2 and 3, respectively. Overall 1-year patient survival rates were 88%, 82%, 77% and 48% for recipients with EC scores of 0, 1, 2 and 3 respectively (P < 0.001). Adjusting for recipient age and urgency at the time of transplantation, multivariate analysis identified an ascending mortality risk ratio of 1.4 and 1.8 compared to a score of 0 for an EC score of 1, and 2 (P < 0.01) respectively. In contrast, an EC score of 3 was associated with a mortality risk ratio of 4.5 (P < 0.001). Further, advanced recipient age linearly increased the death hazard ratio, while an urgent recipient status increased the risk ratio of death by 50%. CONCLUSIONS: Extended criteria donors can be scored using readily available parameters. Optimizing perioperative variables and matching ECD allografts to appropriately selected recipients are crucial to maintain acceptable outcomes and represent a preferable alternative to both high waiting list mortality and to a potentially futile transplant that utilizes an ECD for a critically ill recipient.

Adult↗
PubMed2005

Rapid optimization of working parameters of microwave-driven multilevel qubits for minimal gate leakage.

We propose an effective method to optimize the working parameters (WPs) of microwave-driven quantum gates implemented with multilevel qubits. We show that by treating transitions between each pair of levels independently, intrinsic gate errors due primarily to population leakage to undesired states can be determined by spectroscopic properties of the qubits and minimized by choosing proper WPs. The validity and efficiency of the approach are demonstrated by applying it to optimize the WPs of two coupled rf SQUID flux qubits for controlled-not operation. The result of this independent transition approximation (ITA) is in good agreement with that of dynamic method (DM). The ratio of the speed of ITA to that of DM scales exponentially as 2(n) when the number of qubits n increases.

Journal Article↗
PubMed2003

Lipase-catalyzed methanolysis of triricinolein in organic solvent to produce 1,2(2,3)-diricinolein.

The objective of this study was to find the optimal parameters for lipase-catalyzed methanolysis of triricinolein to produce 1,2(2,3)-diricinolein. Four different immobilized lipases were tested, Candida antarctica type B (CALB), Rhizomucor miehei (RML), Pseudomonas cepacia (PCL), and Penicillium roquefortii (PRL). n-Hexane and diisopropyl ether (DIPE) were examined as reaction media at three different water activities (a(w)), 0.11, 0.53, and 0.97. The consumption of triricinolein and the formation of 1,2(2,3)-diricinolein, methyl ricinoleate, and ricinoleic acid were followed for up to 48 h. PRL gave the highest yield of 1,2(2,3)-diricinolein. Moreover, this lipase showed the highest specificity for the studied reaction, i.e., high selectivity for the reaction with triricinolein but low for 1,2(2,3)-diricinolein. Recoveries of 93 and 88% DAG were obtained using PRL in DIPE at a(w) of 0.11 and 0.53, respectively. Further, NMR studies showed that a higher purity of the 1,2(2,3)-isomer vs. the 1,3-isomer was achieved at higher a(w) (88% at a(w) = 0.53), compared to lower a(w) (71% at a(w) = 0.11). The DAG obtained was acylated by the DAG acyltransferase from Arabidopsis thaliana. Therefore, this enzymatic product is a useful enzyme substrate for lipid biosynthesis. Accordingly, the use of PRL in DIPE at a(w) 0.53 is considered optimal for the synthesis of 1,2(2,3)-diricinolein from triricinolein.

Chromatography, High Pressure Liquid↗
PubMed2005

Designs of a microwave TE011 mode cavity for a space borne H-maser.

Method of Lines and Finite Element Analysis investigations have been performed to optimize parameters in a TE011 mode cavity resonator suitable for a spaceborne hydrogen maser. We report on designs that were explored to find a global maximum in the important design parameters for the microwave cavity used in a hydrogen maser. The criteria sought in this exercise were both the minimization of the total volume of the cavity and the maximization of the product of the z-component of the magnetic energy filling factor and the cavity TE011 mode Q-factor (Q.eta). Different configurations were studied. They were a sapphire tube in a copper cylinder, a sapphire tube in a copper cylinder with Bragg reflectors, and spherical copper cavities both empty and sapphire-lined on the inside cavity surface. At 320 K, the simulations resulted in an optimum product Q.eta = 4.9 x 10(4), with an inner cavity radius of 80 mm and unity aspect ratio. This represents a 54% improvement over an earlier design. The expected increase in the product Q . eta) with the inclusion of Bragg reflectors to the sapphire tube was not achieved. Moreover, the z-component of the magnetic energy filling factor was greatly reduced due to an increase in the radial magnetic field. The sapphire-lined spherical cavity showed no better performance than an equivalent-sized empty copper spherical cavity. For the empty cavity the simulations resulted in the product Q.eta = 4.4 x 10(4). The empty spherical cavity resonator is not suitable for the spaceborne hydrogen maser as the total volume in this case is 33% larger than that of the optimized sapphire tube resonator.

Journal Article↗
PubMed1999

Comparison of the PACE 2 assay, two amplification assays, and Clearview EIA for detection of Chlamydia trachomatis in female endocervical and urine specimens.

Screening for sexually transmitted diseases (STDs) in a greater proportion of sexually active patients has become an accepted protocol by most health care providers. The purpose of this study was to compare the current test methods for detection of Chlamydia trachomatis used at the University of South Alabama, the PACE 2 assay (Gen-Probe) and the Clearview EIA (Wampole Laboratories), with two amplification technologies, the AMP CT (Gen-Probe) and LCx (Abbott) assays. In addition, a number of demographic parameters were ascertained by asking questions at the time of examination as well as for health care provider concerns and preferences. One urine and four endocervical swab specimens were collected in random order from 787 female patients attending one of four obstetrics-gynecology clinics. Eighty-seven percent of patients had no STD-related symptoms. Patients were considered positive for C. trachomatis if three or more assays (swab and/or urine) were positive. Abbott and Gen-Probe confirmed discrepant results by alternate amplified assays. A total of 66 true-positive specimens were detected by use of the combination of endocervical swabs and urine specimens. After discrepant analysis, sensitivities for endocervical swab specimens for the EIA and the PACE 2, LCx, and AMP CT assays were 50, 81, 97, and 100%, respectively. Sensitivities for the LCx and AMP CT assays with urine specimens were 98 and 81%, respectively. The prevalence of C. trachomatis was 8.4%, as determined by amplification technology. Overall, the amplification technologies were the most sensitive methods with either swab (AMP CT assay) or urine (LCx assay) specimens. The PACE 2 assay offered the advantage of a simpler and less expensive assay with acceptable sensitivity. The clearview CT EIA, while yielding a rapid in-office result, had unacceptably low sensitivity. The wide variation in performance with amplification assays with urine specimens as reported in both this study and the literature obviates the need to clarify optimal parameters for this specimen type.

Alabama↗
PubMed1998

Cell-mediated immunity and postpartum thyroid dysfunction: a possibility for the prediction of disease?

Postpartum (pp) thyroid dysfunction (PPTD) is thought to be caused by an autoimmune (AI) destruction of thyroid follicles during the pp period. The chronic thyroid AI process [already present in pregnancy, as shown by the positivity for thyroid peroxidase antibodies (TPO-Ab)] becomes overt disease in the pp period, and one assumes that this exacerbation represents a rebound phenomenon after a general immunosuppression during pregnancy. The presence of TPO-Ab in pregnancy has been suggested as a predictor for later PPTD development. Apart from B cells, e.g. production of autoantibodies, various functions of the cell-mediated immune (CMI) system, including those of peripheral T cells, monocytes, and dendritic cells (DC), are also disturbed in AI states. The objectives of the present study were: determining alterations in various CMI parameters in pregnancies followed by PPTD vs. those not followed by PPTD; and determining the usefulness of these parameters in the prediction of PPTD. In a prospective study (region: Kempenland, southeast Netherlands), a random sample of 291 women were tested at 12 and 32 weeks gestation and 4 weeks pp for TPO-Ab. Women were followed until 9 months pp, for developing PPTD. PPTD was defined as both: an abnormal TSH, and fT4 pp women developing PPTD and/or being positive for TPO-Ab (n = 26); and thyroidological uneventful control women of the same cohort, matched for age and parity (n = 21), were tested for thyroid-stimulating antibodies, percentages of peripheral blood lymphocyte subsets using fluorescence-activated cell sorter analysis (CD3, CD4, CD8, CD16, CD56, major histocompatibility complex-class II), for monocyte polarization, and for cluster capability of monocyte-derived DC. Results were: 1) 31 women (10.7%) were positive for TPO-Ab (TPO-Ab+) in gestation (12 and/or 32 weeks); 2) 15 women (5.2%) developed PPTD, of whom 10 were TPO-Ab+ in gestation; 3) pregnancy-related CMI alterations consisted of low percentages of CD16+CD56+ natural killer (NK), cells and a low DC cluster capability at 12 weeks gestation (these functions were normalized at 32 weeks gestation); 4) the TPO-Ab+ PPTD+ women (4 hyper, 5 hypo, and 1 hyper/hypo) were characterized by a persistently low percentage of NK cells, a lowered monocyte polarization, and a raised percentage of major histocompatibility complex-class II+CD3+ T cells; 5) the TPO-Ab- PPTD+ women (all 5 hyper) had neither thyroid-stimulating antibodies nor CMI alterations, apart from those normally seen in pregnancy; 6) 21 women were positive for TPO-Ab in pregnancy but did not develop PPTD (they had the same lowered NK cell percentages and monocyte polarization as the TPO-Ab+ PPTD+ cases, but they had normal percentages of activated peripheral T cells and a lower titer of TPO-Ab); 7) determination of the number of NK cells and monocyte polarization hardly contributed to the prediction of PPTD (as compared with TPO-Ab status), because of strong interindividual variation and close association with the presence of TPO-Ab; and 8) combining TPO-Ab assays with testing for activated T cells was the most optimal parameter for the prediction of TPO-Ab+ cases of PPTD in our small test set. We conclude that TPO-Ab+ pregnant women who develop PPTD show several CMI abnormalities other than those seen in normal pregnant women, such as persistently lower percentage of NK cells, a lowered monocyte polarization, and a raised percentage of activated T cells. The latter seems rather specific for the actual PPTD development and is not found in TPO-Ab+ (but PPTD) uncomplicated pregnancies. TPO-Ab- (but PPTD+) women had no signs of CMI abnormalities (apart from those specific for the pregnancy state). Although studied cases are low in number, our data are suggestive for the existence of two forms of PPTD: a TPO-Ab+ (AI) form (two-thirds of patients, classical PPTD pattern); and a TPO-Ab- (non-AI) form (one-third of patients, only hyper). Such assumption implies that, at best, two

Autoantibodies↗
PubMed2005

Aerobic exercise interventions for adults living with HIV/AIDS.

BACKGROUND: The profile of HIV infection is constantly changing. Although once viewed as an illness progressing to death, among those with access to antiretroviral therapy, HIV can now present as a disease with an uncertain natural history, perhaps a chronic manageable disease for some. This increased chronicity of HIV infection has been mirrored by increased prevalence of disablement in the HIV-infected population (Rusch 2004). Thus, the needs of these individuals have increasingly included the management of impairments (problems with body function or structure as a significant deviation or loss, such as pain or weakness), activity limitations (difficulties an individual may have in executing activities, such as inability to walk) and participation restrictions (problems an individual may experiences in involvement in life situations, such as inability to work) (WHO 2001). Exercise is a key strategy employed by people living with HIV/AIDS and by rehabilitation professionals to address these issues. Exercise has been shown to improve strength, cardiovascular function and psychological status in seronegative populations (Bouchard 1993), but what are the effects of exercise for adults living with HIV? If the risks and benefits of exercise for people living with HIV are better understood, appropriate exercise may be undertaken by those living with HIV/AIDS and appropriate exercise prescription may be practiced by healthcare providers. If effective and safe, exercise may enhance the effectiveness of HIV management, thus improving the overall outcome for adults living with HIV. OBJECTIVES: To examine the safety and effectiveness of aerobic exercise interventions on immunological/virological, cardiopulmonary and psychological parameters in adults living with HIV/AIDS. SEARCH STRATEGY: To identify the appropriate studies, we conducted a search using MEDLINE, EMBASE, SCIENCE CITATION INDEX, AIDSLINE, CINAHL, HEALTHSTAR, PSYCHLIT, SOCIOFILE, SCI, SSCI, ERIC and DAI. We also reviewed both published and unpublished abstracts and proceedings from major international and national HIV/AIDS conferences such as the Intersciences Conference on Antimicrobial Agents and Chemotherapy (ICAAC), the Conference on Retroviruses and Opportunistic Infections (CROI), the Infectious Diseases Society of America Conference (IDSA) and the International AIDS Conference (IAC). Reference lists from pertinent articles and books were reviewed and personal contacts with authors were used, as well as Collaborative Review Group databases. Targeted journals were handsearched for relevant articles. There were no language restrictions. Searches for the original review covered the period from 1980 to July 1999. The first update of this review included an additional search of the literature, followed by identification of included studies that met the inclusion criteria from August 1999 to January 2001. For the second update, we conducted a search to identify additional studies published from February 2001 to August 2003. SELECTION CRITERIA: Studies were included if they were randomized controlled trials (RCTs) comparing aerobic exercise interventions with no aerobic exercise interventions or another exercise or treatment modality, performed at least three times per week for at least four weeks among adults (18 years of age or older) living with HIV/AIDS. DATA COLLECTION AND ANALYSIS: Data on study design, participants, interventions, outcomes and methodological quality were abstracted from studies that met the inclusion criteria onto specifically designed data collection forms by at least two reviewers. Meta-analysis was conducted using RevMan 4.2 computer software on outcomes whenever possible. MAIN RESULTS: A total of 10 studies (six from the original search, two from the first updated search and two from this second updated search) met the inclusion criteria for this review. Main results indicated that performing constant or interval aerobic exercise, or a combination of constant aerobic exercise and progressive resistive exercise for at least 20 minutes, at least three times per week for four weeks appears to be safe and may lead to significant reductions in depressive symptoms and potentially clinically important improvements in cardiopulmonary fitness. These findings are limited to those participants who continued to exercise and for whom there was adequate follow-up data. AUTHORS' CONCLUSIONS: Aerobic exercise appears to be safe and may be beneficial for adults living with HIV/AIDS. These findings are limited by the small sample sizes and large withdrawal rates of the included studies. Future research would benefit from an increased attention to participant follow-up and intention-to-treat analysis. Further research is required to determine the optimal parameters of aerobic exercise and stage of disease in which aerobic exercise may be most beneficial for adults living with HIV.

Adult↗
PubMed2001

Two-stage continuous process development for the production of medium-chain-length poly(3-hydroxyalkanoates).

Pseudomonas oleovorans forms medium-chain-length poly(3-hydroxyalkanoate) (PHA) most effectively at growth rates below the maximum specific growth rate. Under adequate conditions, PHA accumulates in inclusion bodies in cells up to levels higher than half of the cell mass, which is a time-consuming process. For PHA production, a two-stage continuous cultivation system with two fermentors connected in series is a potentially useful system. It offers production of cells at a specific growth rate in a first compartment at conditions that lead cells to generate PHA at higher rates in a second compartment, with a relatively long residence time. In such a system, dilution rates of 0.21 h(-1) in the first fermentor (D(1)) and 0.16 h(-1) in the second fermentor (D(2)) were found to yield the highest volumetric PHA productivity. Transient-state experiments allowed investigation of D(1) and D(2) over a wide dilution rate range at high resolution in time-saving experiments. Furthermore, the influence of temperature, pH, nutrient limitation, and carbon source on PHA productivity was investigated and results similar to optimum conditions in single-stage chemostat cultivations of P. oleovorans were found. With all culture parameters optimized, a volumetric PHA productivity of 1.06 g L(-1) h(-1) was determined. Under these conditions, P. oleovorans cells contained 63% (dry weight) PHA in the effluent of the second fermentor. This is the highest PHA productivity and PHA content reported thus far for P. oleovorans cultures grown on alkanes.

Alkanes↗
PubMed2003

Potential energy functions: from consistent force fields to spectroscopically determined polarizable force fields.

We review our methodology for producing physically accurate potential energy functions, particularly relevant in the context of Lifson's goal of including frequency agreement as one of the criteria of a self-consistent force field. Our spectroscopically determined force field (SDFF) procedure guarantees such agreement by imposing it as an initial constraint on parameter optimization, and accomplishes this by an analytical transformation of ab initio "data" into the energy function format. After describing the elements of the SDFF protocol, we indicate its implementation to date and then discuss recent advances in our representation of the force field, in particular those required to produce an SDFF for the peptide group.

Acetamides↗
PubMed2003

On the nature of the forces controlling selectivity in the high performance capillary electrochromatographic separation of peptides.

In this minireview, the nature of the forces controlling selectivity in the high performance capillary electrochromatographic (HP-CEC) separation of peptides has been examined. For uncharged and charged peptides, a synergistic interplay occurs in HP-CEC systems between adsorptive/partitioning events and electrokinetically driven motion. Moreover, at high field strengths, both bulk electrophoretic migration and surface electrodiffusion occur. Thus, the migration behavior of peptides in different HP-CEC systems can be rationalized in terms of the combined consequences of these various processes. Moreover, in HP-CEC, the buffer electrolyte interacts with both the peptide analytes and the sorbent as bulk phenomena. These buffer-mediated processes control the solvational characteristics, ionization status and conformational behavior of the peptides as well as regulate the double-layer properties of the sorbent, and the ion flux and electro-osmotic flow characteristics of the HP-CEC system per se. These buffer electrolyte effects mediate mutual interactions between the peptide and the sorbent, irrespective of whether the interaction occurs at the surface of microparticles packed into a capillary, at the surface of a contiguous monolithic structure formed or inserted within the capillary or at the walls of the capillary as is the case with open tubular HP-CEC. Diverse molecular and submolecular forces thus coalesce to provide the basis for the different experimental modes under which HP-CEC can be carried out. As a consequence of this interplay, experimental parameters governing the separation of peptides in HP-CEC can be varied over a wide range of conditions, ensuring numerous options for enhanced selectivity, speed, and resolution of peptides. The focus of the peptide separation examples presented in this minireview has been deliberately restricted to the use of HP-CEC capillaries packed with n-alkyl-bonded silicas or mixed-mode strong ion exchange sorbents, although other types of sorbent chemistries can be employed. From these examples, several conclusions have been drawn related to the use of HP-CEC in the peptide sciences. These observations confirm that variation of a specific parameter, such as the pH or the content of the organic solvent modifier in the buffer electrolyte, simultaneously influences all other physicochemical aspects of the specific HP-CEC separation. Peptide selectivity in HP-CEC thus cannot be fine-tuned solely through the use of single parameter optimization methods. In this context, HP-CEC differs significantly from the analogous reverse phase high performance liquid chromatography (RP-HPLC) procedures with peptides. Rather, more sophisticated multiparameter optimization procedures, involving knowledge of (a) the field strength polarity, (b) its contour and flux characteristics, (c) effects of buffer electrolyte composition and pH, (e) the influence of the temperature, and (f) the impact of the sorbent characteristics, are required if the full capabilities offered by HP-CEC procedures are to be exploited. In this minireview, the HP-CEC migration behavior of several different sets of synthetic peptides has been examined, and general guidelines elaborated from these fundamental considerations to facilitate the interpretation and modulation of peptide selectivity in HP-CEC.

Chromatography, Micellar Electrokinetic Capillary↗
PubMed2003

Cell affinity separations using magnetically stabilized fluidized beds: erythrocyte subpopulation fractionation utilizing a lectin-magnetite support.

A magnetically stabilized fluidized bed is used to separate erythrocyte subpopulations. Binding specificity was obtained by immobilizing the lectin Helix pomatia Agglutinin (HpA) or Griffonia simplicifolia I (GSI) onto a magnetite-containing support. Separation of type A and type O erythrocytes with the lectin HpA was particularly effective, leading to a 94% purity of retained type A erythrocytes. A 3.1 +/- 0.6 log removal of type A erythrocytes was also accomplished leading to a 99.7% +/- 0.4% purity and 95% +/- 7% yield of type O erythrocytes in the collected effluent. Elution of the purified cells was accomplished using fluidization in the presence of a sugar competing for the lectin-erythrocyte binding site. A mathematical model based on the depth filtration model of Putnam and Burns (Chem Eng Sci 1997;52(1):93-105) was extended to include multicomponent cell adhesion. This filtration model is the first to take into account the finite binding capacity of the chromatographic support and is used to characterize the cell binding behavior and to determine optimal parameters and conditions that lead to high capacities and selectivities. Model parameter values and observations from in situ adsorption studies suggest that the non-spherical shape of the magnetite-based support allows for a more efficient utilization of the support surface area than the spherical shape. Using a 1.5-cm diameter laboratory column and realistic parameter values, the processing rates of the system are predicted to be at least an order of magnitude greater than the 10(8)/h cells that can typically be processed in packed bed cell affinity chromatography (CAC) systems.

ABO Blood-Group System↗
PubMed2005

Nonconventional hydrolytic dehalogenation of 1-chlorobutane by dehydrated bacteria in a continuous solid-gas biofilter.

Rhodococcus erythropolis NCIMB 13064 and Xanthobacter autotrophicus GJ10 are able to catalyze the conversion of halogenated hydrocarbons to their corresponding alcohols. These strains are attractive biocatalysts for gas phase remediation of polluted gaseous effluents because of their complementary specificity for short or medium and for mono-, di-, or trisubstituted halogenated hydrocarbons (C2-C8 for Rhodococcus erythropolis and C1-C4 for Xanthobacter autotrophicus). After dehydration, these bacteria can catalyze the hydrolytic dehalogenation of 1-chlorobutane in a nonconventional gas phase system under a controlled water thermodynamic activity (a(w)). This process makes it possible to avoid the problems of solubility and bacterial development due to the presence of water in the traditional biofilters. In the aqueous phase, the dehalogenase activity of Rhodococcus erythropolis is less sensitive to thermal denaturation and the apparent Michaelis-Menten constants at 30 degrees C were 0.4 mM and 2.40 micromol min(-1) g(-1) for Km and Vmax, respectively. For Xanthobacter autotrophicus they were 2.8 mM and 0.35 micromol min(-1) g(-1). In the gas phase, the behavior of dehydrated Xanthobacter autotrophicus cells is different from that observed with Rhododcoccus erythropolis cells. The stability of the dehalogenase activity is markedly lower. It is shown that the HCl produced during the reaction is responsible for this low stability. Contrary to Rhodococcus erythropolis cells, disruption of cell walls does not increase the stability of the dehalogenase activity. The activity and stability of lyophilized Xanthobacter autotrophicus GJ10 cells are dependant on various parameters. Optimal dehalogenase activity was determined for water thermodynamic activity (a(w)) of 0.85. A temperature of 30 degrees C offers the best compromise between activity and stability. The pH control before dehydration plays a role in the ionization state of the dehalogenase in the cells. The apparent Michaelis-Menten constants Km and Vmax for the dehydrated Xanthobacter autotrophicus cells were 0.07 (1-chlorobutane thermodynamic activity) and 0.08 micromol min(-1) g(-1) of cells, respectively. A maximal transformation capacity of 1.4 g of 1-chlorobutane per day was finally obtained using 1g of lyophilized Xanthobacter autotrophicus GJ10 cells.

Biodegradation, Environmental↗
PubMed2005

Metabolic pathways reconstruction by frequency and amplitude response to forced glycolytic oscillations in yeast.

The hypothesis that frequency and amplitude response can be used in a complicated metabolic pathway kinetics model for optimal parameter estimation, as speculated by its successful prior usage for a mechanical oscillator and a heterogeneous chemical system, is tested here. Given the complexity of the glycolysis model of yeast chosen, this question is limited to three kinetics parameters of the 87 in the in vitro model developed in the literature. The direct application of the approach, used with the uninformed selection of operating conditions for the oscillation of external glucose concentration, led to miring the data assimilation process in local minima. Application of linear systems theory, however, identified two natural resonant frequencies that, when excited by external forced oscillations of the same frequency, result in the expression of many harmonics in the Fourier spectra, that is, information-rich experiments. A single such information-rich experiment at one of the resonant frequencies was sufficient to break away from the local minima to find the optimum kinetics parameter estimates. The resonant frequencies themselves represent oscillation modes in glycolysis akin to those previously observed. Furthermore, operation of the bioreactor with large amplitude oscillations of glucose feed (25%) leads to enhanced ethanol average yield by 1.6% at the resonant frequency.

Bioreactors↗
PubMed2003

Mechanistic aspects of on-line electrochemical tagging of free L-cysteine residues during electrospray ionisation for mass spectrometry in protein analysis.

The mechanistic details behind an electrochemically induced tagging of L-cysteine residues in peptides and proteins have been unravelled using cyclic voltammetry. It was found that when hydroquinone is oxidised in the medium used in electrospray ionisation mass spectrometry (ESI-MS) a protonated form of benzoquinone is produced that acts as an efficient electrophile for free L-cysteine residues. Upon substitution of L-cysteine the reduced form of the adduct is formed, which may be further oxidised leading to further substitution of L-cysteine. Digital simulations of the cyclic voltammograms corroborated the mechanism and allowed a determination of the homogeneous second order rate constant corresponding to the addition of L-cysteine onto the protonated form of benzoquinone. The selectivity of the tagging process was confirmed using ESI-MS, which showed that a protein without L-cysteine residues does not react with benzoquinone dissolved in the medium. Finally, the kinetic information obtained in this investigation is used to discuss the optimal parameters for a nanospray capable of quantitative tagging of L-cysteine residues.

Animals↗
PubMed2006

From pentalene to dicyclopenta[b,g]naphthalene, or the change towards delocalized structures.

Using triples-corrected coupled-cluster methods as well as other high-level theoretical approximations, the optimized parameters and isomerization barriers of the family of compounds cyclopentadiene-(benzene)x-cyclopentadiene (x = 0, 1, 2) are computed. In contrast to previous studies, s-indacene presents a localized C(2h) geometry. Also, the localized structure of pentalene is found to be the most stable, but when two benzene rings are intercalated between the five-member rings of pentalene, the resulting molecule preferably adopts a delocalized D(2h) conformation.

Journal Article↗
PubMed2003

Direct identification of all oncogenic mutants in KRAS exon 1 by cycling temperature capillary electrophoresis.

Over the past few decades, advances in genetics and molecular biology have revolutionized our understanding of cancer initiation and progression. Molecular progression models outlining genetic events have been developed for many solid tumors, including colon cancer. Previous reports in the literature have shown a relationship between different KRAS mutations and prognosis and response to medical treatment in colon cancer patients. Furthermore, the presence of a mutated KRAS has been correlated with different clinicopathological variables including age and gender of patients and tumor location. To our knowledge, few institutions screen for KRAS mutations on regular basis in colon cancer patients despite such evidence that knowledge of KRAS exon 1 status is informative. Here, we report on a mutation analysis method adapted to a 96-capillary electrophoresis instrument that allows identification of all 12 oncogenic mutations in KRAS exon 1 under denaturing conditions. To determine the optimal parameters, a series of DNA constructs generated by site-directed mutagenesis was analyzed and the migration times of all mutant peaks were measured. A classification tree was then made based on the differences in migration time between the mutants and an internal standard. A randomized series of 500 samples constructed with mutagenesis as well as 60 blind samples from sporadic colon carcinomas was analyzed to test the method. No wild-type samples were scored as mutants and all mutants were correctly identified. Post polymerase chain reaction (PCR) analysis time of 96 samples was performed within 40 min.

Base Sequence↗