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L Liu

Publications and source records attributed to L Liu.

At least 793 records · Page 44Linked to original sources
PubMed1997

Determination of montelukast (MK-0476) and its S-enantiomer in human plasma by stereoselective high-performance liquid chromatography with column-switching.

A steoreoselective high-performance liquid chromatographic method was developed for the quantification of montelukast (free acid of Singulair, or MK-0476), a potent and selective leukotriene D4 (cysLT1) receptor antagonist, and it S-enantiomers (L-768,232). The method involves protein precipitation and fluorescence detection. Chromatographic separation of the enantiomers from endogenous components in plasma and chiral resolution of the enantiomers are achieved by using column switching HPLC and an alpha-acid glycoprotein chiral column. The assay is linear in the range of 28.9-386 ng ml-1 of free acids of montelukast and L-768,232. The intraday precision (% relative standard deviation) values of this method were in the range of 2.5-9.1% for montelukast, and 2.4-6.8% for L-768,232, while the intraday accuracy values were in the range of 97-103% for montelukast and 96-104% for L-768,232. The interday precision values of this method at 48.2 and 193 ng ml-1 were 5.3 and 3.6%, respectively, for montelukast, and 4.2 and 3.7%, respectively, for L-768,232, while the interday accuracy values at these concentrations were 97 and 103%, respectively, for montelukast and 99 and 102%, respectively, for L-768,232. The utility of the methodology was demonstrated by analysis of plasma samples from a study in which healthy volunteers received 10 mg per day of montelukast orally for 7 days. Results of this study indicate that there is no apparent bioinversion of montelukast to its S-enantiomer in humans.

Acetates↗
PubMed1997

Expression of a functional non-ribosomal peptide synthetase module in Escherichia coli by coexpression with a phosphopantetheinyl transferase.

BACKGROUND: Non-ribosomal peptide synthetases (NRPSs) found in bacteria and fungi are multifunctional enzymes that catalyze the synthesis of a variety of biologically important peptides. These enzymes are composed of modular units, each responsible for the activation of an amino acid to an aminoacyl adenylate and for the subsequent formation of an aminoacyl thioester with the sulfhydryl group of a 4'-phosphopantetheine moiety. Attempts to express these modules in Escherichia coli have resulted in recombinant proteins deficient in 4'-phosphopantetheine. The recent identification of a family of phosphopantetheinyl transferases (P-pant transferases) associated with NRPS have led us to investigate whether coexpression of NRPS modules with P-pant transferases in E. coli would lead to the incorporation of 4'-phosphopantetheine. RESULTS: A truncated module of gramicidin S synthetase, PheAT(His6), was expressed as a His6 fusion protein in E. coli with and without Gsp, the P-pant transferase associated with gramicidin S synthetase. Although PheAT(His6) expressed alone in E. coli catalyzed Phe-AMP formation from Phe and ATP, <1% was converted to the Phe thioester. In contrast, >80% of the PheAT(His6) that was coexpressed with Gsp could form the Phe thioester in the presence of Phe and ATP. CONCLUSIONS: Our finding indicates the presence of an almost equimolar amount of 4'-phosphopantetheine covalently bound to the NRPS module PheAT(His6), and that the functional expression of NRPS modules in E. coli is possible, provided that they are coexpressed with an appropriate P-pant transferase.

Amino Acid Isomerases↗
PubMed1997

The modular organization of multifunctional peptide synthetases.

Gramicidin S synthetase 2 from B. brevis was affinity labeled at its valine thiolation center with the thiol reagent N-[3H]ethylmaleimide. From a tryptic digest of the enzyme-inhibitor complex a radioactive fragment was isolated in pure form by two reversed-phase HPLC steps. It was identified by liquid-phase N-terminal sequencing in combination with electrospray mass spectrometry (ESI-MS) as a hexadecapeptide containing the thiolation motif LGG(H/D)S(L/I). By ESI-MS it was demonstrated that a 4'-phosphopantetheine cofactor was attached to this fragment at its reactive serine. These results are consistent with the "Multiple Carrier Model" of nonribosomal peptide biosynthesis. Site-specific mutagenesis has been performed in thiolation, elongation, and epimerization motifs of some of the modules of surfactin synthetase from B. subtilis to clarify the function of prominent conserved amino acid residues in the intermediate steps of peptide biosynthesis. The modular structure of multifunctional peptide synthetases is discussed.

Amino Acid Isomerases↗
PubMed1997

Role of alpha 2-adrenoceptors in the regulation of intestinal water transport.

1. The influence of the sympathetic nervous system on intestinal fluid transport by the jejunum and ileum of the anaesthetized rat was investigated under basal conditions and during active secretion induced by intra-arterial infusion of vasoactive intestinal peptide (VIP). 2. Intra-arterial infusion of noradrenaline (3, 10, 30 nmol min-1, i.a.) and i.v. injection of the selective alpha 2-adrenoceptor agonist UK 14,304 (1 mumol kg-1, i.v.) increased the rate of basal fluid absorption. The effect of UK 14,304 was blocked by yohimbine (10 mumol kg-1, i.v.). However, the selective alpha 1-adrenoceptor agonist phenylephrine (5 mumol kg-1, i.v.) did not alter either the jejunal or ileal absorption rate. 3. The alpha 2-adrenoceptor antagonists yohimbine (0.3, 1.0, 3 and 10 mumol kg-1, i.v.) and rauwolscine (10 mumol kg-1, i.v.) decreased the basal absorption rate, while the alpha 1-adrenoceptor antagonist prazosin (3 mumol kg-1, i.v.) was without effect. Intracerebroventricular injection of yohimbine (3 mumol kg-1) caused a significant antiabsorptive effect in the jejunum but not ileum. 4. Peripheral chemical sympathectomy induced by pretreating animals with 6-hydroxydopamine (150 mg kg-1, i.p., total dose) induced a trend towards impaired absorption in the jejunum and ileum. 5. The findings provide evidence that the sympathetic nervous system exerts tonic control on intestinal fluid transport and that the effect is mainly through peripheral alpha 2-adrenoceptors. 6. The subtype determination of alpha 2-adrenoceptors in modulating intestinal fluid transport was assessed by determining the effects of alpha 2-adrenoceptor agents on intestinal fluid secretion induced by i.a. infusion of VIP (0.8 microgram min-1). 7. Intravenous administration of UK 14,304 caused a dose-dependent reversal of the secretory phase of the VIP-induced response, but failed to restore fluid transport to the control level of net absorption. EC50 values were 0.17 mumol kg-1 in the jejunum and 0.22 mumol kg-1 in the ileum. 8. The effect of UK 14,304 was blocked by the selective alpha 2A/D antagonist BRL 44408 and the nonselective alpha 2 antagonist yohimbine (each 10 mumol kg-1). The selective alpha 2B/C antagonist ARC 239 (10 mumol kg-1) did not affect the antisecretory action of UK 14,304. It is suggested that the alpha 2-adrenoceptors in the rat intestinal epithelium are the alpha 2D or alpha 2A-like subtype.

Adrenergic alpha-2 Receptor Agonists↗
PubMed1997

Nucleotide sequence of a gene encoding an organophosphorus nerve agent degrading enzyme from Alteromonas haloplanktis.

Organophosphorus acid anhydrolases (OPAA) catalyzing the hydrolysis of a variety of toxic organophosphorus cholinesterase inhibitors offer potential for decontamination of G-type nerve agents and pesticides. The gene (opa) encoding an OPAA was cloned from the chromosomal DNA of Alteromonas haloplanktis ATCC 23821. The nucleotide sequence of the 1.7 -kb DNA fragment contained the opa gene (1.3 kb) and its flanking region. We report structural and functional similarity of OPAAs from A. haloplanktis and Alteromonas sp JD6.5 with the enzyme prolidase that hydrolyzes dipeptides with a prolyl residue in the carboxyl-terminal position. These results corroborate the earlier conclusion that the OPAA is a type of X-Pro dipeptidase, and that X-Pro could be the native substrate for such an enzyme in Alteromonas cells.

Amino Acid Sequence↗
PubMed1997

SHIP, a new player in cytokine-induced signalling.

We recently purified and cloned the cDNAs for the murine and human forms of a novel 145 kDa inositol polyphosphate 5-phosphatase (5-ptase) that becomes tyrosine phosphorylated and associated with Shc following stimulation of hemopoietic cells with multiple cytokines. Unlike most 5-ptases which hydrolyze phosphatidylinositol-4,5-P2-bisphosphate (PI-4,5-P2) and/or inositol-1,4,5-trisphosphate (I-1,4,5-P3), this enzyme selectively hydrolyzes the 5'-phosphate from inositol-1,3,4,5-tetraphosphate (I-1,3,4,5-P4) and phosphatidylinositol-3,4,5-trisphosphate (PI-3,4,5-P3), two inositol polyphosphates recently implicated in growth factor-mediated signalling. This 5-ptase is also unique among 5-ptases in that it is the only one to date to possess an SH2 domain. In this review we discuss the cloning, the Shc binding and the potential role of this protein, which we call SHIP, for SH2-containing inositol 5-phosphatase, in cell proliferation, differentiation and apoptosis.

Adaptor Proteins, Signal Transducing↗
PubMed1997

Surface-related triggering of the neutrophil respiratory burst. Characterization of the response induced by IgG adsorbed to hydrophilic and hydrophobic glass surfaces.

Hydrophilic and hydrophobic glass surfaces precoated with human albumin, fibrinogen, or IgG were investigated with respect to their ability to activate the neutrophil NADPH-oxidase. We found that IgG-coated surfaces induced a substantial and prolonged neutrophil production of reactive oxygen species (ROS). When a hydrophilic surface was used to support protein binding, a somewhat lower neutrophil response (around 35%) was obtained, compared with the response induced by IgG on a hydrophobic surface. The production of ROS was completely eliminated when cytochalasin B was added to the measuring system, suggesting the involvement of the cell cytoskeleton in the activation process. The relation between the intra- and extracellular generation of ROS was further assessed, and we found that most of the ROS produced were released from the cells, in agreement with a model in which the activating surfaces induce a 'frustrated' phagocytic response. Serum totally inhibited 'frustrated' phagocytosis provided that the IgG molecules were sticking to a hydrophilic surface.

Albumins↗
PubMed1997

Cloning and expression of the glutamate racemase gene of Bacillus pumilus.

A glutamate racemase gene (murI) was found in Bacillus pumilus cells and cloned into Escherichia coli WM335, a D-glutamate auxotroph, by means of a genetic complement method. MurI of B. pumilus encodes a 272-amino acid protein with an unusual initiation codon, TTG. The deduced amino acid sequence shows significant similarity with those of glutamate racemases from E. coli (ratio of identical residues, 28%), Pediococcus pentosaceus (44%), and Staphylococcus haemolyticus (49%). B. pumilus MurI was expressed as a fusion protein connected to the N-terminal 12 residues of beta-galactosidase; the fusion protein showed glutamate racemase activity, and resembled the enzyme of P. pentosaceus in physicochemical and enzymological properties.

Amino Acid Isomerases↗
PubMed1997

Molecular characterization of a subgroup I geminivirus from a legume in South Africa.

A South African geminivirus for which we propose the name bean yellow dwarf virus (BeYDV) has been isolated from French bean (Phaseolus vulgaris cv. Bonus) showing stunting, chlorosis and leaf curl symptoms. A full-length cloned copy of the viral genome produced characteristic symptoms of the disease when reintroduced into French bean by agroinoculation, and was systemically infectious in Nicotiana benthamiana, N. tabacum, Lycopersicon esculentum, Datura stramonium and Arabidopsis thaliana. BeYDV resembles subgroup I geminiviruses which infect monocotyledonous plants in having a single DNA component, two non-overlapping virion-sense (V1 and V2) and two overlapping complementary-sense (C1 and C2) coding regions, and an intron within the complementary-sense coding regions that is excised to produce a C1C2 fusion protein. It is most closely related to tobacco yellow dwarf virus from Australia, the only subgroup I geminivirus previously known to infect dicotyledonous plants, although it is sufficiently dissimilar (65% nucleotide sequence identity) to be considered a distinct virus.

Cloning, Molecular↗
PubMed1997

Localization of human intestinal defensin 5 in Paneth cell granules.

Antibiotic peptides of higher animals include the defensins, first discovered in phagocytic cells but recently also found to be produced by epithelial cells. We biosynthesized recombinant human intestinal defensin 5 (rHD-5) using the baculovirus-insect cell expression system. Since insect cells process defensin incompletely and secrete the precursor proHD-5, we substituted a methionine for an alanine at a likely processing site to allow selective chemical cleavage with cyanogen bromide, and rHD-5 was used to elicit polyclonal antibodies. By the immunoperoxidase-staining technique, the antibodies selectively stained Paneth cells of the normal adult small intestine. Immunogold electron microscopy further localized HD-5 to the Paneth cell secretory granules. Since some defensins exert activity cytotoxic to mammalian cells, we assayed the effect of rHD-5 on the human intestinal cell lines Caco2 and Int407. proHD-5 did not exert cytotoxic activity, and rHD-5 showed only minimal activity against Int407 and was inert against Caco2. Since Paneth cells release their granules adjacent to the mitotic cells of the intestinal crypts, HD could protect this cell population against invasion and parasitization by microbes.

Adult↗
PubMed1997

A possible role for acetylated intermediates in diaminopimelate and tabtoxinine-beta-lactam biosynthesis in Pseudomonas syringae pv. tabaci BR2.024.

The deduced product of an open reading frame (ORF3) located in the tabtoxinine-beta-lactam (T beta L) biosynthetic region of Pseudomonas syringae pv. tabaci BR2.024 (BR2.024) has significant sequence homology to the dapD products of other bacteria. dapD encodes L-2,3,4,5-tetrahydrodipicolinate succinyl coenzyme A succinyltransferase (THDPA-ST), an enzyme in the diaminopimelate (DAP) and lysine biosynthetic pathway. Complementation studies, in vitro transcription-translation experiments, and enzymatic assays indicated that ORF3 encodes a product with THDPA-ST activity in Escherichia coli dapD mutant beta 274. However, a BR2.024 mutant with an insert in ORF3 was prototrophic, and only basal THDPA-ST activity was detected in extracts of both parent and mutant. This finding suggested that ORF3 was not required for DAP biosynthesis and that it did not encode a product with THDPA-ST activity. The results of enzymatic studies, indicating that BR2.024 uses acetylated intermediates for DAP biosynthesis, are consistent with the hypothesis that BR2.024 does not need THDPA-ST for DAP biosynthesis. The ORF3 mutant produced reduced levels of tabtoxin, indicating that ORF3 may have a role in T beta L biosynthesis. We have named the gene tabB and have proposed a possible function for the gene product.

Acetylation↗
PubMed1997

Characterization of dapB, a gene required by Pseudomonas syringae pv. tabaci BR2.024 for lysine and tabtoxinine-beta-lactam biosynthesis.

The dapB gene, which encodes L-2,3-dihydrodipicolinate reductase, the second enzyme of the lysine branch of the aspartic amino acid family, was cloned and sequenced from a tabtoxin-producing bacterium, Pseudomonas syringae pv. tabaci BR2.024. The deduced amino acid sequence shared 60 to 90% identity to known dapB gene products from gram-negative bacteria and 19 to 21% identity to the dapB products from gram-positive bacteria. The consensus sequence for the NAD(P)H binding site [(V/I)(A/G)(V/I)XGXXGXXG)] and the proposed substrate binding site (HHRHK) were conserved in the polypeptide. A BR2.024 dapB mutant is a diaminopimelate auxotroph and tabtoxin negative. The addition of a mixture of L-,L-, D,D-, and meso-diaminopimelate to defined media restored growth but not tabtoxin production. Cloned DNA fragments containing the parental dapB gene restored the ability to grow in defined media and tabtoxin production to the dapB mutant. These results indicate that the dapB gene is required for both lysine and tabtoxin biosynthesis, thus providing the first genetic evidence that the biosynthesis of tabtoxin proceeds in part along the lysine biosynthetic pathway. These data also suggest that L-2,3,4,5-tetrahydrodipicolinate is a common intermediate for both lysine and tabtoxin biosynthesis.

Amino Acid Sequence↗
PubMed1997

Sequence-mediated regulation of adenovirus gene expression by repression of mRNA accumulation.

Gene expression in complex transcription units can be regulated at virtually every step in the production of mature cytoplasmic mRNA, including transcription initiation, elongation, termination, pre-mRNA processing, nucleus-to-cytoplasm mRNA transport, and alterations in mRNA stability. We have been characterizing alternative poly(A) site usage in the adenovirus major late transcription unit (MLTU) as a model for regulation at the level of pre-mRNA 3'-end processing. The MLTU contains five polyadenylation sites (L1 through L5). The promoter proximal site (L1) functions as the dominant poly(A) site during the early stage of adenovirus infection and in plasmid transfections when multiple poly(A) sites are present at the 3' end of a reporter plasmid. In contrast, stable mRNA processed at all five poly(A) sites is found during the late stage of adenovirus infection, after viral DNA replication has begun. Despite its dominance during early infection, L1 is a comparatively poor substrate for 3'-end RNA processing both in vivo and in vitro. In this study we have investigated the basis for the early L1 dominance. We have found that mRNA containing an unprocessed L1 poly(A) site is compromised in its ability to enter the steady-state pool of stable mRNA. This inhibition, which affects either the nuclear stability or nucleus-to-cytoplasm transport of the pre-mRNA, requires a cis-acting sequence located upstream of the L1 poly(A) site.

Adenoviruses, Human↗
PubMed1997

Fatty acid modulation of K+ channels in taste receptor cells: gustatory cues for dietary fat.

In an attempt to determine the chemosensory cues, if any, provided by fats in the oral cavity, we have performed patch-clamp recordings on isolated rat taste receptor cells during application of free fatty acids. Cis-polyunsaturated fatty acids, when applied extracellularly, inhibit delayed-rectifying K+ channels. In a subset of cells, these fatty acids also enhance inwardly rectifying K+ currents. Saturated, monounsaturated, and trans-polyunsaturated fatty acids have no significant effect on K+ currents. These effects do not involve activation of G protein-mediated pathways, including protein kinase C and protein kinase A, lipoxygenase pathways, cyclooxygenase pathways, or cytochrome P-450 pathways, consistent with direct effects on these ion channels or closely associated proteins. The net effect of fatty acids is to prolong stimulus-induced depolarizations of taste receptor cells, and we propose the effects on K+ channels represent the mechanism by which fats are detected by receptor cells in the oral cavity.

Animals↗
PubMed1997

Differing sympathetic and vagal effects on atrial fibrillation in dogs: role of refractoriness heterogeneity.

Although sympathetic activation is believed to promote atrial fibrillation (AF), the effects of sympathetic stimulation on AF have not been systematically studied. In seven morphine-chloralose-anesthetized dogs, autonomic decentralization increased atrial effective refractory period (ERP) and reentrant wavelength (WL) and decreased the duration of AF induced by burst atrial pacing. Graded bilateral stellate ansa stimulation decreased ERP and WL to values similar to those before decentralization but did not return AF duration to predecentralization values. Sympathetic and bilateral vagal stimulation were adjusted in six additional dogs to produce similar effects on ERP and WL. Despite comparable effects on mean WL, the duration of AF was increased by vagal stimulation from 14 +/- 5 (control) to 372 +/- 96 s (P < 0.001) but was not altered significantly (26 +/- 10 s) by sympathetic stimulation. Vagal stimulation increased the variability in atrial refractoriness, as indicated by the standard deviation of ERP at seven atrial sites and of activation frequency during AF at 112 recording sites, whereas sympathetic stimulation had no significant effect on these indexes of ERP heterogeneity. We conclude that sympathetic stimulation is much less effective than vagal stimulation in promoting AF and that heterogeneity in atrial ERP may be important in determining the ability to sustain AF.

Animals↗
PubMed1997

Expression of glycogen phosphorylase isozymes in developing rat lung.

Glycogen accumulates to significant levels in epithelial cells of the developing respiratory tract. Mobilization of glycogen stores is regulated differentially along the respiratory epithelium such that glycogenolysis in the alveolar epithelium (the site of surfactant synthesis) precedes that in the bronchial and bronchiolar epithelium. The initial step in glycogen degradation is catalyzed by glycogen phosphorylase, which exists as three genetically distinct isozymes referred to as muscle, liver, and brain isoforms. The goal of this study was to characterize the temporal and spatial expression of each of the glycogen phosphorylase isozymes in developing lung to determine which isoform(s) was associated with glycogen mobilization in the fetal type II epithelial cell. RNA levels encoding glycogen phosphorylase were assessed by ribonuclease protection assay using isoform-specific antisense probes. RNAs encoding the brain and liver isozymes were detected in isolated day 20 fetal type II epithelial cells and at lower levels in adult type II cells. The muscle isoform RNA was barely detectable in fetal type II cells and was undetectable in adult type II cells. Expression of brain and liver isoform RNAs was higher in whole fetal lung than in fetal type II cells. Consistent with this result, in situ hybridization studies demonstrated widespread expression of the brain and liver isoforms in developing lung tissues; in contrast, expression of the muscle isoform was restricted to the pulmonary vein. Glycogen phosphorylase enzyme activity corresponding to the brain isoform was clearly detected in isolated fetal type II cells; however, the majority of enzyme activity migrated as two bands with distinct electrophoretic mobilities that may have been the result of isoform heterodimerization. Collectively, these results suggest that the brain and liver isoforms of glycogen phosphorylase may be involved in mobilization of type II cell glycogen during late fetal lung development.

Animals↗
PubMed1997

Expression of the insulin-like growth factor system in the hypokalemic rat kidney.

We studied the renal expression of the insulin-like growth factor (IGF) system to gain a better perspective of its potential role in the hyperplastic adaptation of the distal nephron to potassium deficiency. Rats were pair fed 1% or 0.002% potassium diets for periods up to 10 days. IGF-I mRNA was diminished in potassium-deficient rats within 4 days, whereas mRNA for IGF binding protein-1 (IGFBP-1), a collecting duct-associated protein, was increased by day 7. At day 10 mRNA for IGFBP-1 in potassium-deficient animals averaged 2.07 +/- 0.53 (mean +/- SD, relative densitometry units) compared with 0.89 +/- 0.26 in control rats (n = 4, P = 0.002). Conversely, IGFBP-3, a binding protein whose mRNA has been localized to the interstitial compartment, averaged 2.40 +/- 0.02 in potassium-deficient rats and 4.77 +/- 0.05 in controls (n = 4, P < 0.03) at day 10 of treatment. Immunohistochemistry performed using a specific IGFBP-1 antibody revealed hyperplasia of distal nephron segments along with an increase in IGFBP-1 in potassium-depleted rats. These data suggest that IGFBP-1 may play an important role in the control of cellular adaptations in the hypokalemic rat kidney either directly by influencing cell migration or indirectly by localizing IGF-I to the distal nephron.

Animals↗