Biomedical subjects

Q S Li

Publications and source records attributed to Q S Li.

At least 19 recordsLinked to original sources

PubMed2001

Residue size at position 87 of cytochrome P450 BM-3 determines its stereoselectivity in propylbenzene and 3-chlorostyrene oxidation.

We report here oxidation of propylbenzene and 3-chlorostyrene by wild-type cytochrome P450 BM-3 with high turnover (479 nmol 1-phenyl-1-propanol/min/nmol P450 and 300 nmol 3-chlorostyrene oxide/min/nmol P450). Furthermore, the residue size at position 87 of P450 BM-3 was found to play critical roles in determining stereoselectivity in oxidation of propylbenzene and 3-chlorostyrene. Replacement of Phe87 with Val, Ala and Gly resulted in decreases in optical purity of produced (R)-(+)-1-phenyl-1-propanol from 90.0 to 37.4, 26.0 and -15.6% e.e., respectively, and in increases in those of produced (R)-(+)-3-chlorostyrene oxide from -61.0 to -38.0, 67.0 and 94.6% e.e., respectively.

Amino Acid Sequence↗

PubMed2001

Stochastic resonance in delayed two-coupled oscillators without common perturbations.

Stochastic resonance (SR) in a two-coupled oscillator system with time delay is investigated. The system shows multistability of a desynchronized state and two synchronized states with different collective frequencies, which may be interpreted as multistable perception of ambiguous or reversible figures. To model the situations where the two oscillators exist in different environments, periodic signals and noises at their inputs are not uniformly given. SR in an individual oscillator, characterized by the output signal-to-noise ratio, is examined based on numerical simulations. We find that phase shift between the signals at inputs of different oscillators weakens SR, the oscillator with only an input signal does not show SR, and the oscillator with input noise shows SR irrespective of it having an input signal or not. The results have implications in the area of information transmission in biological systems.

Journal Article↗

PubMed2001

Alpha(2) adrenoceptor agonists as potential analgesic agents. 3. Imidazolylmethylthiophenes.

A series of imidazolylmethylthiophenes has been prepared and evaluated as ligands for the alpha(2) adrenoceptor. These compounds were tested in two animal models that are predictive of analgesic activity in humans. The 3-thienyl compounds were generally the most potent, particularly those with substitution in the 4-position. A subset of the most active compounds was further evaluated for adverse cardiovascular effects in the anesthetized rat model. In addition to excellent binding at the alpha(2D) adrenoceptor, the 4-bromo analogues 20e and 21e were very active in the rat abdominal irritant test (RAIT) with ED(50) doses of 0.38 and 0.31 mg/kg, respectively. We constructed a pharmacophore model based on the biological activity of the present series, dexmedetomidine (1), and conformationally restrained analogues 3 and 4.

Adrenergic alpha-Agonists↗

PubMed2001

Critical role of the residue size at position 87 in H2O2- dependent substrate hydroxylation activity and H2O2 inactivation of cytochrome P450BM-3.

Replacement of phenylalanine 87 with alanine or glycine (mutant F87A or F87G) greatly increased the H2O2-supported substrate hydroxylation activity of cytochrome P450BM-3, whose original H2O2-supported activity is hardly detectable. On the other hand, replacement of phenylalanine 87 with valine (mutant F87V) did not. In the oxidation of p-nitrophenoxydodecanoic acid (12-pNCA), the turnover numbers of the mutant F87A in the presence of NADPH and O2, or H2O2 were 493 and 162 nmol/min/nmol, respectively. The H2O2-supported F87A hydroxylation activity was further confirmed with free fatty acids as substrates. Moreover, the stability of F87A in H2O2 solutions also largely increased. The order of the stability of the wild type (WT), F87A, and their substrate (12-pNCA)-binding complexes in H2O2 solutions listed from high to low was F87A, WT, F87A substrate-binding complex, and WT substrate-binding complex. We propose that the free space size in the vicinity of the heme iron significantly influences P450BM-3 H2O2-supported activity and H2O2 inactivation.

Amino Acid Substitution↗

PubMed2001

Rational evolution of a medium chain-specific cytochrome P-450 BM-3 variant.

The single mutant F87A of cytochrome P-450 BM-3 from Bacillus megaterium was engineered by rational evolution to achieve improved hydroxylation activity for medium chain length substrates (C8-C10). Rational evolution combines rational design and directed evolution to overcome the drawbacks of these methods when applied individually. Based on the X-ray structure of the enzyme, eight mutation sites (P25, V26, R47, Y51, S72, A74, L188, and M354) were identified by modeling. Sublibraries created by site-specific randomization mutagenesis of each single site were screened using a spectroscopic assay based on omega-p-nitrophenoxycarboxylic acids (pNCA). The mutants showing activity for shorter chain length substrates were combined, and these combi-libraries were screened again for mutants with even better catalytic properties. Using this approach, a P-450 BM-3 variant with five mutations (V26T, R47F, A74G, L188K, and F87A) that efficiently hydrolyzes 8-pNCA was obtained. The catalytic efficiency of this mutant towards omega-p-nitrophenoxydecanoic acid (10-pNCA) and omega-p-nitrophenoxydodecanoic acid (12-pNCA) is comparable to that of the wild-type P-450 BM-3.

Bacillus megaterium↗

PubMed2001

The CRP-cAMP complex and downregulation of the glnAp2 promoter provides a novel regulatory linkage between carbon metabolism and nitrogen assimilation in Escherichia coli.

In Escherichia coli, glnA (encoding glutamine synthetase) is transcribed from two promoters (glnAp1 and glnAp2). The glnAp1 is a sigma(70)-dependent promoter that is activated by the cAMP receptor protein (CRP). Under nitrogen-deficient growth conditions, glnAp1 is repressed by NtrC-phosphate. The downstream glnAp2 promoter is sigma(54)-dependent and is activated by NtrC-phosphate. Here, we show that glnAp2 expression is affected by different carbon sources and that the CRP-cAMP complex inhibits the glnAp2 promoter activity. Primer extension and KMnO4 footprinting analysis indicate that the inhibitory effect is at the transcriptional level in vivo. When glnAp2 is activated by NifA, a similar inhibitory effect by CRP-cAMP is observed. Site-directed mutagenesis and deletion analysis indicate that the characterized and putative CRP-binding sites located in the upstream region of the glnAp2 promoter are not essential for the inhibitory effect. CRP-cAMP inhibits sigma(54)-dependent glnAp2 strongly, by 21-fold. By activating glnAp1 and downregulating glnAp2, the overall effect of CRP-cAMP on glnA expression is an approximately fourfold reduction, which correlates with the reduction of gamma-glutamyl transferase activities in the cells. We propose therefore that a physiological role of CRP-cAMP activation of glnAp1 is to partially compensate for CRP-cAMP downregulation of glnAp2, allowing a low but non-negligible level of expression of the important genes transcribed from it. A novel regulatory linkage between carbon and nitrogen regulons is proposed.

Carbon↗

PubMed2001

Exact solutions for free vibration of shear-type structures with arbitrary distribution of mass or stiffness.

In this paper, shear-type structures such as frame buildings, etc., are treated as nonuniform shear beams (one-dimensional systems) in free-vibration analysis. The expression for describing the distribution of shear stiffness of a shear beam is arbitrary, and the distribution of mass is expressed as a functional relation with the distribution of shear stiffness, and vice versa. Using appropriate functional transformation, the governing differential equations for free vibration of nonuniform shear beams are reduced to Bessel's equations or ordinary differential equations with constant coefficients for several functional relations. Thus, classes of exact solutions for free vibrations of the shear beam with arbitrary distribution of stiffness or mass are obtained. The effect of taper on natural frequencies of nonuniform beams is investigated. Numerical examples show that the calculated natural frequencies and mode shapes of shear-type structures are in good agreement with the field measured data and those determined by the finite-element method and Ritz method.

Journal Article↗

PubMed2001

Vibratory characteristics of multistep nonuniform orthotropic shear plates with line spring supports and line masses.

A new exact approach for free-vibration analysis of multistep nonuniform orthotropic shear plates with line spring supports and line masses is presented. The governing differential equation for free vibrations of an orthotropic shear plate with variably distributed mass and stiffness is established. It is proved that it is possible to separate a shear plate as two independent shear beams for free-vibration analysis. The jkth natural frequency of a shear plate is equal to the square root of the square sum of the jth natural frequency of a shear beam and the kth natural frequency of another shear beam. The jkth mode shape of the shear plate is the product of the jth mode shape of a shear beam and the kth mode shape of another shear beam. In this paper, the function for describing the distribution of mass of each step plate can be selected as an arbitrary one, and the distribution of shear stiffness is expressed as a functional relation with the mass distribution, and vice versa. The exact solutions of one-step shear plates with varying cross section are obtained first for eight cases. Then, the derived exact solutions are used to establish the frequency equation of a multistep nonuniform orthotropic shear plate with spring supports and line masses using the transfer matrix method and the recurrence method developed in this paper. The numerical example shows that the calculated results are in good agreement with the experimental data, and the proposed procedure is an exact and efficient method.

Journal Article↗

PubMed2001

Engineering cytochrome P450 BM-3 for oxidation of polycyclic aromatic hydrocarbons.

Cytochrome P450 BM-3, a self-sufficient P450 enzyme from Bacillus megaterium that catalyzes the subterminal hydroxylation of long-chain fatty acids, has been engineered into a catalyst for the oxidation of polycyclic aromatic hydrocarbons. The activities of a triplet mutant (A74G/F87V/L188Q) towards naphthalene, fluorene, acenaphthene, acenaphthylene, and 9-methylanthracene were 160, 53, 109, 287, and 22/min, respectively. Compared with the activities of the wild type towards these polycyclic aromatic hydrocarbons, those of the mutant were improved by up to 4 orders of magnitude. The coupling efficiencies of the mutant towards naphthalene, fluorene, acenaphthene, acenaphthylene, and 9-methylanthracene were 11, 26, 5.4, 15, and 3.2%, respectively, which were also improved several to hundreds fold. The high activities of the mutant towards polycyclic aromatic hydrocarbons indicate the potential of engineering P450 BM-3 for the biodegradation of these compounds in the environment.

Bacillus megaterium↗

PubMed2000

Directed evolution of the fatty-acid hydroxylase P450 BM-3 into an indole-hydroxylating catalyst.

The self-sufficient cytochrome P450 BM-3 enzyme from Bacillus megaterium catalyzes subterminal hydroxylation of saturated long-chain fatty acids and structurally related compounds. Since the primary structure of P450 BM-3 is homologous to that of mammalian P450 type II, it represents an excellent model for this family of enzymes. During studies on the directed evolution of P450 BM-3 into a medium-chain fatty-acid hydroxylase, several mutants, in particular the triple mutant Phe87Val, Leu188Gln, Ala74Gly, were observed to hydroxylate indole, producing indigo and indirubin at a catalytic efficiency of 1365 M(-1)s(-1) (kcat=2.73 s(-1) and Km=2.0 mM). Both products were unequivocally characterized by NMR and MS analysis. Wild-type P450 BM-3 is incapable to hydroxylate indole. These results demonstrate that an enzyme can be engineered to catalyze the transformation of substrates with structures widely divergent from those of its native substrate.

Bacillus megaterium↗

PubMed2000

Antithrombotic properties of RWJ-50353, a potent and novel thrombin inhibitor.

The antithrombotic, anticoagulant, and kinetic properties of RWJ-50353, a novel, reversible, active-site-directed thrombin inhibitor, were evaluated. RWJ-50353 inhibited the catalytic activity of human alpha-thrombin with a K(i) of 0.19+/-0.02 nM. It showed a 16-fold selectivity relative to inhibition of trypsin and at least 330-fold selectivity relative to inhibition of other biologically important serine proteases. In a gel-filtered platelet preparation, RWJ-50353 inhibited alpha-thrombin-induced aggregation with an IC(50) of 32+/-6 nM. In a canine arteriovenous shunt antithrombotic model, RWJ-50353 demonstrated a significant dose-related (0.1-1.0 mg/kg, i.v.) reduction in thrombus formation with 50% inhibition (ID(50)) obtained at 0.46+/-0.1 mg/kg. In a rabbit deep vein thrombosis model, RWJ-50353 dose-dependently (0.1-1. 0 mg/kg, i.v.) reduced thrombus formation with an ID(50) of 0.25+/-0. 03 mg/kg. The antithrombotic activity in both of these models was associated with only mild prolongations in bleeding time and coagulation parameters. These results demonstrate that RWJ-50353 is a potent, selective thrombin inhibitor that is an effective antithrombotic agent after intravenous administration in models of arterial and venous thrombosis and may be useful in the management of various thrombotic disorders.

Animals↗

PubMed2000

Activation-induced T cell death occurs at G1A phase of the cell cycle.

Peripheral negative selection of cycling T cells after TCR engagement and deletion of activated T cells after an immune response occur by an apoptotic process termed activation-induced cell death (AICD). The cross-linking of TCR-CD3 complex with anti-CD3 monoclonal antibody led to significant apoptotic cell death in peripheral blood T cells. To further define cell cycle restriction points for triggering AICD in T cells, we evaluated the association between cell cycle progression and death signal transduction. Simultaneous DNA / RNA quantification analysis revealed that T cells entering G1A phase of the cell cycle may acquire sensitivity to AICD. The activation of caspase-3 was induced when T cells entered G1A phase. Up-regulation of cyclin-dependent kinases (Cdk4 and Cdk6) and cyclin D3 was initiated in TCR-stimulated T cells entering G1A phase and expression of these markers steadily increased as T cells progressed from G1A into G1B phase. Interestingly, caspase-3 inhibitors could inhibit the up-regulation of these G1 cell cycle regulators and induce G0 / G1A arrest as well as the inhibition of AICD. On the basis of these results, AICD signals are most likely transduced into TCR-stimulated T cells entering G1A phase. T cells that fail to progress from G1A into G1B phase undergo AICD.

Apoptosis↗

PubMed2000

Intracellular cytokine profile of T cells from children with acute lymphoblastic leukemia.

PURPOSE: During an ongoing immune response, cytokines produced by T helper types 1 (Th1) and 2 (Th2) together with T cytotoxic types 1 (Tc1) and 2 (Tc2) are critical to the effectiveness of that response. Dysregulated expansion of one or the other subset may contribute to the impaired function of the T-cell-mediated immune system in cancer patients. In the present study we have investigated whether such dysregulation might exist in children with acute lymphoblastic leukemia (ALL). METHODS: We analyzed 61 blood samples from 45 children with B cell precursor ALL and 16 healthy children. Interleukin(IL)-2, IL-4, and interferon gamma (IFN gamma) production of their respective purified CD4+ and CD8+ T cells were assessed at the single-cell level by intracellular-cytokine-staining flow cytometry. RESULTS: At the time of diagnosis, IL-2-producing cell populations in CD4+ and CD8+ T cells were reduced below the normal range in 31 of 44 (70.5%) and 23 of 38 (60.5%) cases respectively. Similarly, IFN gamma-producing cell populations in CD4+ and CD8+ T cells decreased in 17 of 44 (38.6%) and 18 of 38 (47.4%) cases respectively. Conversely cell populations capable of IL-4 production in CD4+ and CD8+ T cell subsets were increased in 13 of 30 (43.3%) and 15 of 30 (50.0%) cases respectively. Therefore, the Th1-to-Th2 and Tc1-to-Tc2 ratios (1.6 +/- 2.2 and 7.7 +/- 6.7 respectively) were significantly lower in peripheral blood T cells of ALL patients (n = 30) than those (6.0 +/- 2.9 and 20.1 +/- 10.3 respectively) in 15 healthy controls (P < 0.0001). Although both CD45RA+/CD4+ and CD45RA+/CD8+ cells significantly increased in 43 ALL patients (P < 0.05), there existed no apparent correlation between CD45 isoform expression and cytokine (IL-2 and IFN gamma) production. Interestingly, the ability to produce both IL-2 and IFN gamma was recovered in 8 cases examined, after complete remission had been achieved. CONCLUSION: These observations suggest that, in both CD4+ and CD8+ T cells of ALL patients, there is a dysregulation in the functionality of Th1 (Tc1) and Th2 (Tc2) cells with a gross reduction of Th1 (Tc1) cell populations and an expansion in Th2 (Tc2).

Adolescent↗

PubMed1999

Improvement of the performance of H2O2 oxidation at low working potential by incorporating TTF-TCNQ into a platinum wire electrode for glucose determination.

A micro-biosensor was constructed by incorporating the organic conducting salt tetrathiafulvalene-tetracyanoquinodimethane (TTF-TCNQ) into a platinized platinum (Pt) wire and further covering with the electrochemical polymerical heteropolypyrrole film, in which glucose oxidase (GOx) was entrapped. The enzyme electrode can sensitively determine glucose at a low working potential, mainly based on the oxidation of H2O2. The incorporated TTF-TCNQ can significantly improve the oxidation of H2O2 on the electrode, although a part of the TTF-TCNQ functions as a mediator. Compared with the same electrode prepared without TTF-TCNQ incorporated, the TTF-TCNQ modified electrode had better performance characteristics at a working potential of 200 mV (versus SCE). The response time to 90% of the steady value was shortened from about 40 s to less than 10 s, the lower limit of the linear response was greatly extended from about 1.6 mM to 10 microM, the linear range was shifted from 1.6-10.0 to 0.01-5 mM and the sensitivity was increased from about 1 to 1.5 microA/mM. The electrode was quite stable. For continuous operation, the electrode could work for about 5 weeks and only lost 60% of its original sensitivity. Stored at 4 degrees C for intermittent determinations, the electrode kept 80% sensitivity for over 6 months. Due to covering the electrode with a non-conductive heteropolypyrrole film, ascorbate, urate and 4-acetamidophenol caused only negligible current response at an applied potential of 200 mV.

Biosensing Techniques↗

PubMed1999

mRNA expression of variant Fas molecules in acute leukemia cells.

Fas (Apo-1/CD95) is a cell membrane receptor involved in apoptotic cell death. Soluble variant forms (sFas) lacking the transmembrane domain due to alternative splicing have been identified. Up-regulation of sFas expression is reportedly implicated in prereceptorial blockage of Fas-induced apoptosis in a dose-dependent manner. We examined mRNA expression of Fas and sFas in fresh leukemia cells. All leukemia cells expressed both mRNAs of full-length Fas (FasFull) and sFas with deletion of exon6 (FasDel6). The ratio of FasFull/FasDel6 mRNA expression was not always correlated with Fas-mediated growth inhibition. Interestingly, in a 6-year-old boy with acute myelogenous leukemia, blast cells obtained at onset and at the time of bone marrow relapses expressed distinct amounts of FasDel6 mRNA. Furthermore, the level of FasDel6 expression appeared to be correlated with Fas-resistance in leukemia blasts. In addition, sFas protein levels were elevated in patients' sera at onset with subsequent return to normal levels after complete remission. These results indicated that sFas could be synthesized and released by leukemia blasts and suggested that up-regulation of Fas variant transcript might render leukemia blasts resistant to Fas-mediated growth inhibition in certain cases.

Acute Disease↗

PubMed1999

Method for comparison of the hemodynamic effects of equi-antinociceptive oral doses of drugs in anesthetized rats.

In a typical flowchart for discovery of novel analgesic (or other) agents, a critical path often involves maximization of the separation of the therapeutic endpoint from known adverse-effect (AE) endpoint(s). Although strategies can easily be designed for in vitro paradigms such as high-throughput screening, extension to in vivo testing can represent a major obstacle to the rapid progression to the next step in development. The problem can be particularly acute when the assessment is required for oral dosing, and when it is not known if the therapeutic and AE mechanism(s) of action are the same. As a case in point, alpha(2)-adrenoceptor (alpha(2)-AR) agonists have potential therapeutic use as analgesics, but they also produce cardiovascular (CV) effects. However, whether the two effects are inexorably linked has not been resolved, particularly for oral administration. The present study used a novel method for comparing the CV effects produced by alpha(2)-AR agonists given by intraduodenal administration to anesthetized rats at fixed ratios of the oral antinociceptive ED(50) dose of each agonist. The technique provided a useful screen of compounds. In addition,there was no correlation between CV endpoints and alpha(2A)-AR affinity, suggesting that oral alpha(2)-AR-mediated analgesia and CV effects might be separable or that other mechanisms might be involved.

Adrenergic alpha-2 Receptor Agonists↗

PubMed1999

Epitopes and functional responses defined by a panel of anti-Fas (CD95) monoclonal antibodies.

Fas (CD95) is a cell surface glycoprotein that mediates apoptotic cell death when cross-linked with agonistic anti-Fas monoclonal antibodies (MAbs) or the endogenous Fas ligand. In this study, we investigated the in vitro biological properties of a panel of anti-human Fas MAbs. We found that five anti-Fas MAbs of IgG1 subclass (B.E28, B.G30, B.L25, DX2, and B.G34) induced marked apoptotic cell death in Fas-expressing leukemia cells, although this killing was delayed when compared to the cytolytic effect mediated by the prototypic anti-Fas MAb of IgM subclass (clone CH-11). On the other hand, four clones (ZB4, B.G27, B.D29, and B.K14) efficiently blocked apoptotic cell death induced by the CH-11 MAb or Fas ligand. The ability of these MAbs to inhibit cell death appeared to correlate with their relative affinity for the Fas molecule. Furthermore, different clones recognized the same epitope and elicited different effects (induction or inhibition of cell killing); conversely, different clones elicited the same effect but recognized different epitopes. These results suggest that the different biological effects of anti-Fas MAbs would not be mediated in an epitope-restricted manner. The relative binding affinity might correlate to some extent with the biological properties of the MAb.

Animals↗

PubMed1999

Hyperproduction of L-glutamate oxidase in submerged fermentation of Streptomyces sp. N1 with culture pH control and calcium addition.

Production of L-glutamate oxidase (GluOx) by Streptomyces sp. N1 was investigated by controlling culture pH at 6.2, 6.7, 7.0, and 7.3 in a 5-L stirred fermentor. The corresponding GluOx activities obtained were 2.8, 4.2, 6.0, and 5.3 U/mL, respectively. Microbial growth was inhibited by increasing the medium pH from 6.2 to 7.0. The inhibitory effect was also observed in plate colony growth under incubation with a different initial pH value. The effect of calcium on GluOx production was also studied in the pH-controlled bioreactor. When the culture pH was controlled at 6.2 or 7.0, GluOx production could not be improved or was only improved slightly by initial addition of calcium to the medium. However, when the culture pH was kept at 6.7, initial Ca2+ addition (60 mM) conspicuously enhanced GluOx production up to 9.3 U/mL, which was about twofold of that without Ca2+ addition. The enzyme production level was the highest ever reported in the literature. During fermentation the inhibition of cell growth by Ca2+ addition was observed. For the morphological changes, the cells mostly existed as pellets in the medium without Ca2+ addition, whereas few pellets were found and almost all the cells were dispersed mycelia in the broth with Ca2+ addition.

Journal Article↗