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W R Shieh

Publications and source records attributed to W R Shieh.

13 recordsLinked to original sources
PubMed1996

Lipid products of phosphoinositide 3-kinase bind human profilin with high affinity.

To gain insight into the physiological function of phosphoinositide 3-kinase (PI 3-kinase) lipid products, this study examines the interactions of the D-3 phosphoinositides with profilin and the consequent effects on actin dynamics and phosphoinositide turnover. Profilin, a ubiquitous actin-regulating protein, plays a putative role in regulating actin assembly and PLC-gamma 1 signaling in light of its unique interactions with actin and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]. Here we raise evidence that the affinity of profilin with the D-3 phosphoinositides is substantially higher than that of PtdIns(4,5)P2. The dissociation constants (Kd) are estimated to be 1.1 microM, 5.7 microM, and 11 microM for phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4)P2], phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3], and PtdIns(4,5)P2, respectively. Spectroscopic data show that while all these phosphoinositides alter the tryptophan fluorescence of profilin in a similar fashion, the respective conformational effect on profilin is vastly different. Based on CD data, the alpha-helical contents of profilin in the presence of 8 molar equiv of PtdIns(4,5)P2, PtdIns(3,4,5)P3, and PtdIns(3,4)P2 are 17.4%, 11.5%, and 1.4%, respectively, vis-a-vis 9.4% for profilin alone. In contrast, no appreciable change in the fluorescence and CD spectra is observed when related inositol phosphates such as Ins(1,4,5)P3, Ins(1,3,4,5)P4, or Ins(1,3,4)P3 at comparable concentrations are tested. Evidence suggests that this differential recognition bears functional significance concerning the intricate roles of profilin and inositol lipids in modulating actin polymerization and PtdIns(4,5)P2 turnover. The relative potency of individual phosphoinositides in offsetting the inhibitory effect of profilin on actin assembly is PtdIns(3,4)P2 > PtdIns(3,4,5)P3 > PtdIns(4,5)P2, consistent with their relative binding affinity with profilin. Moreover, the inhibitory effect of profilin on PLC-gamma 1-mediated PtdIns(4,5)P2 hydrolysis is overcome by PtdIns(3,4)P2 and PtdIns(3,4,5)P3 through a combined effect of PLC-gamma 1 activation and preferential profilin binding. This D-3 phosphoinositide-mediated regulation may represent a new mechanism for controlling PtdIns(4,5)P2 turnover by PLC-gamma 1.

Actins↗
PubMed1996

Antagonistic effect of inositol pentakisphosphate on inositol triphosphate receptors.

We present evidence that inositol 1,3,4,5,6-pentakisphosphate acts as an antagonist of inositol triphosphate receptors in vitro by dampening the activity of Ca(2+)-mobilizing inositol phosphates. This antagonism bears physiological significance as cells maintain substantial quantities of this phosphoinositol in the cytosol, especially during cell differentiation or proliferation. Over a concentration range from 30 to 150 microM, this pentakisphosphate causes a two- to five-fold increase in the EC50 values of inositol 1,4,5-trisphosphate and other active phosphoinositols in effecting Ca2+ efflux from brain microsomes. This specific inhibition results from competitive receptor binding, and is not noted with inositol hexaphosphate or related metabolites at comparable concentrations. Thus, inositol 1,3,4,5,6-pentakisphosphate in concert with other allosteric modulators may constitute an integral component for the receptor regulation.

Animals↗
PubMed1995

Preparation and characterization of a D-myo-inositol 1,4,5-trisphosphate-specific antibody.

Antibodies against Ins(1,4,5)P3 were raised by immunizing rabbits with two types of InsP3-BSA conjugates which were synthesized by covalently coupling Ins(1,4,5)P3 to the carrier protein via alkyl linkages. The anti-Ins(1,4,5)P3 antibody was detected by a novel ELISA using Ins(1,4,5)P3-immobilized microtitre plates. Both antiserum preparations showed specific binding with Ins(1,4,5)P3, with titres of 1:4000. Most inositol phosphates, including Ins1P, Ins(4,5)P2, Ins(1,3,4)P3, Ins(1,5,6)P3, Ins(1,2,5,6)P1, Ins(3,4,5,6)P4, Ins(1,3,4,5,6)P5, InsP6, and PtdIns(4,5)P2, did not exhibit significant molecular interactions with the antibodies. Ins(1,3,4,5)P4, however, cross-reacted with these antibodies with one-third of the affinity as that of Ins(1,4,5)P3, in part due to the largely shared structural motifs. The differential affinity was significantly improved by affinity purification on Ins(1,4,5)P3-agarose. The affinity-purified antibody displayed IC50 values of 12 nM and 730 nM for Ins(1,4,5)P3 and Ins(1,3,4,5)P4 respectively, according to a competitive ELISA; these values are in line with those reported for the Ins(1,4,5)P3 receptor. The modes of ligand recognition at the binding sites of these two types of biomolecules are, however, different. Moreover, although the ligand binding was interfered with by multivalent anions such as ATP4-, HPO4(3-) and SO4(2-) at high concentrations, no inhibition was noted with heparin, an antagonist of the Ins(1,4,5)P3 receptor.

Animals↗
PubMed1994

Molecular interactions of endogenous D-myo-inositol phosphates with the intracellular D-myo-inositol 1,4,5-trisphosphate recognition site.

A systematic effort was made to elucidate the mode of recognition at the inositol 1,4,5-trisphosphate-specific receptor. Eleven D-myo-inositol phosphates were synthesized and tested for Ca(2+)-mobilizing and receptor-binding activities, which included Ins(1,3,4,5,6)P5, Ins(1,2,5,6)P4, Ins(1,3,4,5)P4, Ins(1,3,4,6)P4, Ins(1,4,5,6)P4, Ins(3,4,5,6)P4, Ins(1,3,4)P4, Ins(1,4,5)P3, Ins(1,5,6)P3, Ins(1,4)P2, and Ins(4,5)P2. Of these, Ins(1,4,5)P3, Ins(1,3,4,6)P4, Ins(1,3,4,5)P4, Ins(1,4,5,6)P4, and Ins(4,5)P2 were able to elicit Ca2+ release from rat brain microsomes. Binding experiments suggest that the ability of these polyphosphates to effect Ca2+ mobilization arises from interactions with the Ins(1,4,5)P3-specific receptor. Accordingly, a model accounting for the ligand recognition is proposed. The Ins(1,4,5)P3-binding site is presumably composed of two domains. The anchoring domain binds the 4,5-bisphosphate 6-hydroxy motif. Disruption of this structural feature abolishes the agonist activity. The auxiliary domain exerts long-range interactions with the 1-phosphate, thus enhancing the binding affinity. The stereochemical requirement for this electrostatic interaction is, however, less stringent. Evidence suggests that Ca(2+)-mobilizing inositol phosphates are able to effect productive binding by assuming conformations displaying or mimicking these essential structural features.

Adenosine↗
PubMed1994

D-myo-inositol 1,4,5-trisphosphate analogues as useful tools in biochemical studies of intracellular calcium mobilization.

Two types of structural variants of D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] were prepared by a chemoenzymatic route. These 6-O-substituted analogues retained the biological activity of Ins(1,4,5)P3, and were able to elicit Ca2+ release from porcine brain microsomes. Moreover, these derivatives allowed the preparation of Ins(1,4,5)P3-based immunogens and affinity matrix which were successfully applied to the preparation and purification of antibodies against Ins(1,4,5)P3. These antibodies displayed discriminative affinity towards Ins(1,4,5)P3, and provide a useful tool to study intracellular Ca2+ mobilization.

Animals↗
PubMed1993

Purification and characterization of novel "2-arylpropionyl-CoA epimerases" from rat liver cytosol and mitochondria.

Investigation on the biochemical isomerization of ibuprofen led us to the successful purification of "2-arylpropionyl-CoA epimerase" from rat liver cytosol and mitochondria. The purified enzymes from both subcellular fractions exhibit similar physical and catalytic properties and are distinctly different from rat liver methylmalonyl-CoA epimerase. Both are monomeric proteins with an apparent molecular mass of 42 kDa and show similar contents of most amino acids. Their UV spectra gave no indication of any bound cofactors, and their enzyme activities were not affected by exposure to EDTA or metal ions (except Cu2+). These results suggest that the cytosolic and mitochondrial epimerases may be structurally related. The purified enzymes catalyze the epimerization of various 2-arylpropionyl-CoAs with some degree of stereochemical differentiation. For 2-(4-isobutylphenyl)propionyl-CoA, the equilibrium constant was estimated to be 1.5 in favor of the R-isomer. Evidence indicated that the proton exchange may be mediated by a 2-base mechanism and that a carboxylic residue in the active site may serve as a general base for proton abstraction.

Amino Acids↗
PubMed1993

A 13C NMR study on ibuprofen metabolism in isolated rat liver mitochondria.

A 13C NMR approach was taken to study the biotransformation of (R)- and (S)-[2-13C,2-2H]ibuprofen-CoA in isolated rat liver mitochondria. The 13C chemical shift induced by 2H substitution for the C-2 of [2-13C,2-2H]-ibuprofen-CoA is about 0.5 ppm relative to the corresponding 1H-bearing thioester. The C-2 resonances for [2-13C]- and [2-13C,2-2H]ibuprofen-CoA are 5 ppm downfield with respect to their acid counterparts. Consequently, this technique permits real time analyses of the metabolic fate of the doubly labeled substrate in a quantitative manner. The initial rate of the epimerization of (S)-[2-13C,2-2H]ibuprofen-CoA relative to the (R)-counterpart by intact mitochondria was estimated to be 1.5, which is in line with the equilibrium constant determined with the purified epimerase. Contrary to the reports by other groups, this 13C NMR study did not find spontaneous hydrolysis of the CoA-thioester upon being exposed to intact mitochondria. Current focus of this investigation is to extend this NMR methodology to the levels of whole cells and live animals to gain a better understanding of the enantioselective metabolism of ibuprofen.

Animals↗
PubMed1992

Incidence of toxigenic vibrios in foods available in Taiwan.

A total of 1088 vibrios and related species were isolated from seafood and aquacultured foods available in Taiwan. They were identified as Vibrio alginolyticus, V. cholerae, V. fluvialis I, V. fluvialis II, V. parahaemolyticus, V. mimicus, Aeromonas caviae, A. hydrophila, A. sobria and other species. Incidence of these Vibrio and Aeromonas species in these foods was high. Vibrio parahaemolyticus was frequently found in seawater and in foods of freshwater origin. The Vibrio isolates were examined for enzymatic and toxigenic activities. Most of them showed strong lipase or protease activities. Haemolytic activities of V. cholerae, V. fluvialis I and V. fluvialis II isolates were mostly strong. About 49% showed cytotoxic activity and 5% cytotonic activity in Chinese hamster ovary cell culture assay. Nevertheless, only three non-O1 V. cholerae (2.07%) and two V. parahaemolyticus isolates (1.65%) produced cholera toxin and thermostable direct haemolysin activity, respectively. Various toxigenic vibrios may be important food-borne pathogens in this region because of their high incidence in foods.

Aeromonas↗
PubMed1991

Metabolic stereoisomeric inversion of ibuprofen in mammals.

Studies on the mechanism and enzymology of metabolic ibuprofen isomerization constituted the focus of this investigation. Comparative in vivo studies revealed that this biotransformation proceeded via a proton abstraction mechanism in all tested species of mammals, which is in agreement with the previous reports. Direct evidence supporting this conclusion stemmed from the in vitro epimerization of ibuprofen-CoA thioester in rat liver homogenates. Chemically synthesized (R)-ibuprofen-CoA thioester was rapidly transformed to its (S)-counterpart by subcellular hepatic preparations. Examination of this epimerase activity in various rat tissue homogenates indicated that this enzyme was highly tissue specific. This biochemical reaction mainly took place in the liver and kidney, whereas low levels of enzyme activity were associated with other tissues. Nevertheless, the liver and kidney homogenates failed to invert (R)-ibuprofen directly even in the presence of all the necessary cofactors. Presumably, the failure to characterize this bioconversion was due to the lack of enzymatic acyl-CoA synthesis in these homogenates. It is noteworthy that the '2-arylpropionyl-CoA epimerase' catalyzed the transformation from either direction and with high turnover rates. The catalytic efficiency of (S)-ibuprofen CoA epimerization appeared to be greater than that of the (R)-counterpart. These in vitro findings suggest that the step of acyl-CoA formation assume a pivotal role in controlling the stereoselectivity and efficiency of the in vivo metabolism. As the responsible acyl-CoA synthetase(s) in different species of animals may exert the reaction with different degrees of enantiomeric preference and efficiency, the resulting stereochemical outcome and metabolic rates of this bioinversion vary accordingly. Consequently, in guinea pigs, this biotransformation proceeds in both directions with nearly equal efficiency, whereas it is virtually unidirectional and slow in humans. Currently, the purification and characterization of this novel '2-arylpropionyl-CoA epimerase' from rat livers constitute the focus of this investigation.

Adult↗
PubMed1990

Metabolic stereoisomeric inversion of 2-arylpropionic acids. On the mechanism of ibuprofen epimerization in rats.

Kinetic and mechanistic studies are described for the metabolic stereoisomeric inversion of R-ibuprofen in rats. After oral administration of R-ibuprofen to rats, the plasma levels and enantiomeric compositions of ibuprofen and its major metabolite were monitored. Although individual animals exhibited great variations in metabolic rates, the concentration ratios of the S- and R-enantiomers of ibuprofen and also its metabolite remained roughly unchanged within 90 min. Even though it is generally believed that this bioconversion is strictly stereospecific in nature, chromatographic analysis revealed that S-ibuprofen also underwent metabolic inversion, however, at a much slower rate, than its R counterpart. The inversion mechanism was assessed by monitoring the loss of labeled deuterium from specifically deuterated ibuprofen. No significant isotope effect was observed for the metabolism of these deuterated derivatives. One deuterium atom was lost in the S-ibuprofen resulted from R-[2-2H]ibuprofen metabolism, whereas all the three deuterium atoms were retained when R-[3,3,3-2H3]ibuprofen was used as the substrate. These results reinforce the proposed mechanism that the inversion proceeds via a thioester carbanion intermediate. The pKa of the alpha-methine proton of ibuprofen N-acetylcysteamine thioester was shown to be 10.34 +/- 0.06, which excludes the possibility that ibuprofen may undergo inversion through the nonenzymatic isomerization of its acyl thioester.

Animals↗