Biomedical subjects

X F Li

Publications and source records attributed to X F Li.

At least 55 records · Page 3Linked to original sources

PubMed1999

Visceral chemical nociception in mice lacking mu-opioid receptors: effects of morphine, SNC80 and U-50,488.

Writhing responses to intraperitoneal acetic acid administration and their modulation by mu-, kappa- and delta-opioid receptor agonists were compared in wild-type and mu-opioid receptor knockout mice. Unpretreated homozygous knockout mice displayed less writhing than wild-type mice. U-50,488 [trans-3,4-dichloro-N-methyl-N-[2-(1-pyrolidinyl)cyclohexyl]-benze neacetamide]) reduced writhing responses in wild-type and knockouts. Morphine and SNC80 [(+)-4-[9-alpha-R)-alpha-(2S,5RO-4-allyl-2,5-dimethyl-1-piperaziny l)-3-methoxybenzyl]-N,N-diethylbenzamide] were effective in wild-type mice but ineffective in knockouts. Mu-opioid receptors appear to play important roles in responses to this visceral nociceptive stimulus and its modulation by mu- and delta-opioid receptor agonists.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗

PubMed1999

Biological effects of rare earth protein complexes: influence of lanthanide ions Eu3+, Tb3+ on secondary structure of calmodulins.

The secondary structure of four kinds of calmodulins (CaMs; i.e., Brassica campestris pollen CaM, bovine brain CaM, earthworm calcium binding protein, and earthworm new calcium binding protein) in thin films are determined by the FTIR resolution enhanced technique and curve fitting. The variation in the secondary structure of CaM upon its binding with Ca2+, Eu3+, and Tb3+, the assay of phosphodiesterase enzyme, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis are also investigated. The effect of lanthanide ions on the conformation of CaM are described.

Animals↗

PubMed1999

Functional characterization and m-RNA expression of 5-HT receptors mediating contraction in human umbilical artery.

5-HT1-like and 5-HT2 receptors have both been described to mediate contractions to 5-HT in the human umbilical artery (HUA). However, the nature of the 5-HT receptor subtypes is unknown. 2 In isometric force studies with ring preparations of HUA alpha-methyl-5-hydroxytryptamine (alpha-Me-5-HT) and 5-hydroxytryptamine (5-HT) contracted HUA with pED50 values of 8.04 and 7.74, respectively. In the presence of a subthreshold concentration of another vasoconstrictor sumatriptan and 5-nonyloxytryptamine elicited concentration-dependent contractions with pEC50 values of 7.21 and 7.67, respectively. In the presence of the selective 5-HT1B/D receptor antagonist GR127935, contractile responses elicited by sumatriptan and 5-nonyloxytryptamine were competitively antagonized (pKB 9.01 and 9.02, respectively). In the experiments with 5-HT, GR127935 appeared to be non-competitive with shallow Schild plot slopes. The data were fitted with two linear regression lines and the calculated pKB of the high affinity component (8.90) was comparable to that expected for GR127935 at the 5-HT1B/1D receptor. Several 5-HT2 selective receptor antagonists (spiperone, cyproheptadine, pirenperone) competitively inhibited responses to 5-HT. The selective 5-HT2A antagonist ketanserin against sumatriptan and 5-nonyloxytryptamine behaved as a weak antagonist while against 5-HT demonstrated a competitive antagonism (pKB 8.56). Using specific primers for human 5-HT1B, 5-HT1D and 5-HT2A receptor genes, the reverse transcriptase-polymerase chain reaction revealed mRNA expression of 5-HT1B and 5-HT2A receptors in the HUA. The results suggest that the HUA has a functional population of 5-HT1B and 5-HT2A receptor subtypes which are involved in the contractile response to 5-HT. Contractions mediated by 5-HT1B receptors can be 'uncovered' by exposure to other vasoactive agents.

Humans↗

PubMed1999

Distribution of angiotensin AT1 and AT2 receptor subtypes in the rat kidney.

ANG II contributes importantly to the regulation of renal vascular resistance, glomerular filtration, and tubular epithelial transport, yet there remains a paucity of information regarding the localization of the ANG II type 1 and 2 (AT1 and AT2) receptors within the rat kidney particularly within the vasculature. The present study was designed to localize the transcriptional and translational site(s) of AT1 and AT2 receptor (AT1R and AT2R, respectively) expression within the rat kidney. Using immunohistochemistry, we detected the AT(1)R translational sites throughout the kidney, with the strongest labeling found in the vasculature of the renal cortex and the proximal tubules of the outer medulla. The AT2R protein expression was found throughout the rat kidney, although there was little to no expression found in the glomerulus and medullary thick ascending limbs of Henle (TAL). Gene-specific primers were then designed to distinguish between the receptor subtypes within microdissected renal tubular and vascular segments using RT-PCR. AT1AR, AT1BR, and AT2R mRNA were found within the renal vasculature (afferent arterioles, arcuate artery, and outer medullary descending vasa recta). The mRNA for both the AT1R isoforms was also detected in the glomeruli and the renal tubules (proximal tubules, TAL, and collecting ducts); however, no AT2R mRNA was detected within the glomerulus and was inconsistently found within the medullary TAL (MTAL). Taken together, these data show that mRNA for the AT1R subtypes was located in all of the renal tubular and vascular segments. Evidence for AT2R mRNA was also found in all but two of the vascular and tubular segments, the MTAL, and the glomeruli. These results are consistent with the whole tissue immunohistochemically localized receptors.

Angiotensin I↗

PubMed1999

The dynamic expression of the epidermal growth factor receptor and epidermal growth factor ligand family in a differentiating intestinal epithelial cell line.

The Caco-2 intestinal epithelial cell line differentiates when cultured on plastic or permeable filters, and offers a valuable system to study events associated with enterocytic differentiation in vitro. Little is known as to whether the expression of the epidermal growth factor receptor (EGFR) and its ligands changes as intestinal epithelial cells differentiate. We found that total cellular EGFR protein and mRNA transcript levels were relatively unchanged during Caco-2 cell differentiation, but the expression of surface EGFR and patterns of steady state epidermal growth factor (EGF)-family ligand expression changed significantly. EGFR affinity, surface EGFR expression levels, and the repertoire of expressed EGF-family ligands, were different between Caco-2 cells cultured on plastic and filters. Functionally, EGFR-mediated cell proliferation and tyrosine phosphorylation of the signal transduction protein SHC could be inhibited in Caco-2 cells cultured on filters, but not on plastic. Thus, the substrate on which the cells were grown and the degree of cell differentiation strongly modulate EGFR affinity, EGFR surface expression, the steady state expression of EGF-family ligands, as well as, EGFR-mediated cellular responses. Our results suggest that the EGFR system is regulated during intestinal epithelial cell differentiation primarily at the level of ligand expression.

Amphiregulin↗

PubMed1999

Hypoxia down-regulates placenta growth factor, whereas fetal growth restriction up-regulates placenta growth factor expression: molecular evidence for "placental hyperoxia" in intrauterine growth restriction.

Early placental development occurs in an environment of relative hypoxia. Hypoxia promotes angiogenesis and up-regulates vascular endothelial growth factor (VEGF) expression while it down-regulates placenta growth factor (PIGF) that possess 53% homology with VEGF. Morphological studies show poor placental vascular development and an increase in the mitotic index of cytotrophoblasts in intrauterine growth restriction (IUGR). We hypothesized that the reported relatively high oxygen level in the intervillous space in contact with IUGR placental villi will limit angiogenesis by changes in VEGF and PIGF expression and function. Western immunoblot analysis demonstrates a diametric expression of PIGF and VEGF proteins throughout pregnancy with PIGF levels increasing and VEGF levels decreasing, consistent with placental oxygenation. In IUGR placentae, the ratio of PIGF/GAPDH mRNA was increased by 2.3-fold (p < 0.03) and PIGF protein levels were also increased, (p < 0.05) as compared with gestationally-matched normal placentae. PIGF mRNA and protein were localized to the trophoblast bilayer and villous mesenchyme of the human placenta throughout gestation. In vitro studies demonstrated that increasing oxygen tension (hyperoxia) up-regulated PIGF protein in term placental villous explants, whereas hypoxic culture of a term trophoblast choriocarcinoma cell line (BeWo) down-regulated PIGF mRNA and protein and VEGFR-1 (Flt-1) autophosphorylation. The addition of PIGF-1 to a spontaneously transformed first trimester cytotrophoblast cell line stimulated DNA synthesis while PIGF-2 had little effect. VEGF and PIGF exert their biological actions by means of a common receptor VEGFR-1. In the first trimester trophoblast cells, PIGF-1 increased the association of phosphorylated extracellular signal-related kinase (ERK) with VEGFR-1 immunoprecipitates while both PIGF-1 and PIGF-2 also potentiated endogenous VEGF mediated association of phosphorylated extracellular related kinase (ERK) with VEGFR-2 (KDR). More importantly, the addition of PIGF-1 had little effect while PIGF-2 inhibited cell growth in cultured endothelial cells derived from human umbilical vein. Nitric oxide (NO) is reported to promote angiogenesis and PIGF-2 inhibited the basal release of NO from the first trimester trophoblast. The tissue expression and functional studies support the hypothesis of "placental hyperoxia" in early-onset IUGR because hypoxia down-regulates trophoblast PIGF levels, PIGF expression is increased in IUGR, and PIGF-2 inhibits endothelial cell growth. Taken together, these changes provide a cellular explanation for the observed poor angiogenesis in the pathogenesis of IUGR and show that the two PIGF isoforms may modulate trophoblast and endothelial cell function differently, possibly through potentiation of VEGF mediated activation of VEGF-2.

Cell Line↗

PubMed1998

Anti-autolysis of trypsin by modification of autolytic site Arg117.

In order to improve the stability of trypsin, an approach to knock out the autolytic site has been carried out in this investigation. Compared with trypsins from other species, the autolytic site Arg117-Val118 of rat trypsin is the most interesting candidate to work on. The Arg117 residue was designed to be deleted or replaced by other amino acid residues to destroy the autolytic site. With DNA site-directed mutagenesis method, one deletion mutant and several replacement mutants were selected. After expression and purification, the kinetic and anti-autolytic properties of mutant trypsins were studied. No net charge difference of the trypsin molecules was observed by native PAGE analysis. Kinetic studies show that the activities of mutants vary from one another. R117L gives 32 times the activity of wild type trypsin while R117C has no detective activity. Among 8 selected mutants with characteristic properties, 7 of them give prolonged half life during anti-autolytic assay with the exception of R117M which is more sensitive to autolysis.

Amino Acid Substitution↗

PubMed1998

Cocaine reward models: conditioned place preference can be established in dopamine- and in serotonin-transporter knockout mice.

Cocaine and methylphenidate block uptake by neuronal plasma membrane transporters for dopamine, serotonin, and norepinephrine. Cocaine also blocks voltage-gated sodium channels, a property not shared by methylphenidate. Several lines of evidence have suggested that cocaine blockade of the dopamine transporter (DAT), perhaps with additional contributions from serotonin transporter (5-HTT) recognition, was key to its rewarding actions. We now report that knockout mice without DAT and mice without 5-HTT establish cocaine-conditioned place preferences. Each strain displays cocaine-conditioned place preference in this major mouse model for assessing drug reward, while methylphenidate-conditioned place preference is also maintained in DAT knockout mice. These results have substantial implications for understanding cocaine actions and for strategies to produce anticocaine medications.

Animals↗

PubMed1998

Molecular cloning of a novel potassium-dependent sodium-calcium exchanger from rat brain.

We have isolated a novel cDNA clone from rat cerebral cortex encoding a protein of 670 amino acids (NCKX2) that has significant similarity to the 1199-amino acid-long Na/Ca-K exchanger of bovine rod outer segment (NCKX1). NCKX2 transcripts are 10.5 kilobase pairs in length and are expressed abundantly in neurons throughout the brain and with much lower abundance in selected other tissues. The predicted topology of the rat NCKX2 protein is very similar to that of bovine NCKX1, beginning with a solitary transmembrane segment (M0), which is removed as a "signal peptide" in bovine NCKX1, an extracellular loop, a cluster of five transmembrane spanning segments (M1 to M5), a long cytoplasmic loop, and a final hydrophobic cluster (M6 to M11). Within the hydrophobic clusters, rat NCKX2 shares 80% identity and 91% similarity with bovine NCKX1. The two larger hydrophilic loops are much shorter in NCKX2 than in NCKX1, accounting largely for the difference in length between the two proteins, and are dissimilar in sequence except for a 32-amino acid stretch with 69% identity in the cytosolic loop. NCKX2 was epitope-tagged in the extracellular domain and was shown to be expressed at the surface of transfected HEK cells. Analysis of NCKX2 function by fluorescent imaging of fura-2-loaded transfected cells demonstrated that NCKX2 is a potassium-dependent sodium/calcium exchanger.

Amino Acid Sequence↗

PubMed1998

Nonaqueous capillary electrophoretic separation and thermo-optical absorbance detection of five tricyclic antidepressants and metabolism of amitriptyline by Cunninghamella elegans.

We developed a technique based on nonaqueous capillary electrophoresis and laser-based thermo-optical absorbance detection to assay five antidepressants with similar structures and mass-to-charge ratios. A mixture of methanol and acetonitrile with ammonium acetate was essential to achieve baseline resolution of these compounds. We investigated the effects of ammonium acetate concentration, temperature, applied voltage, and capillary length on separation efficiency. The nonaqueous capillary electrophoresis and laser-based thermo-optical absorbance detection technique was used to study the metabolism of amitriptyline by Cunninghamella elegans. Sample preparation procedures were simplified for fast screening of the parent drug and its metabolites. Reproducible electropherograms were obtained from replicate cultures of C. elegans growing in the presence of amitriptyline.

Amitriptyline↗

PubMed1998

On-line nonaqueous capillary electrophoresis and electrospray mass spectrometry of tricyclic antidepressants and metabolic profiling of amitriptyline by Cunninghamella elegans.

An on-line nonaqueous capillary electrophoresis-electrospray mass spectrometry (ESI-MS) technique was developed using a commercial ion spray interface. The nonaqueous capillary electrophoresis ESI-MS system was used to profile tricyclic antidepressants of similar structures and mass-to-charge ratios. We found that pure methanol can be used as a sheath liquid to obtain stable ion spray from nonaqueous capillary electrophoresis. The flow rate of the coaxial nebulizing gas affected baseline signals, separation efficiency, and migration times. Other nonaqueous capillary electrophoresis operating conditions and electrospray parameters were optimized for enhanced baseline separation and high sensitivity detection. The effect of sample stacking on separation and detection was evaluated. The calculated detection limits were approximately 3 pg injected onto the capillary. ESI mass spectra of tricyclic antidepressants from a single quadrupole MS were obtained and elucidated. The information was used to propose fragmentation pathways of the tricyclic antidepressants. The method was also used to analyze the metabolites of amitriptyline produced by the fungus Cunninghamella elegans. Sixteen metabolites were detected and most of them were tentatively identified as demethylated and/or hydroxylated, and/or N-oxidized products.

Amitriptyline↗

PubMed1998

Ontogeny of hepatocyte growth factor (HGF) and its receptor (c-met) in human placenta: reduced HGF expression in intrauterine growth restriction.

Severe intrauterine growth restriction (IUGR) is characterized by abnormal placentation. Mouse gene knockout studies show that an absence of either hepatocyte growth factor (HGF) or its receptor, c-met, leads to intrauterine death secondary to severe IUGR with deficient placentation. In this study, immunocytochemistry localized HGF protein throughout placental villi across gestation, whereas c-met protein was localized only to the perivillous trophoblast and vascular endothelium. Within the IUGR placentae, a reduction in HGF immunostaining within the villous stroma was observed. HGF mRNA was strongly expressed in the perivascular tissue around the stem villous arteries throughout gestation, with weaker expression within the villous stroma and the terminal villi. c-met mRNA expression was limited to the perivillous trophoblast, particularly in the first trimester, with only a faint hybridization signal from the villous stroma. Placental mRNA expression was examined quantitatively using a ribonuclease protection assay: HGF and c-met mRNA expression increased from the first to the second trimester, reaching a zenith before decreasing again through the third trimester to term. HGF mRNA levels were significantly reduced in the IUGR placentae (P = 0.036), whereas c-met mRNA expression was within the normal range for gestation. These findings suggest that HGF derived from the perivascular tissue of stem villous arteries may play an important role in controlling normal villous development. Whereas reduced expression of HGF within IUGR placentae does not prove a causative link with abnormal villous development, the association lends support to this possibility.

Adult↗

PubMed1998

Localization, quantification, and activation of platelet-activating factor receptor in human endometrium during the menstrual cycle: PAF stimulates NO, VEGF, and FAKpp125.

Implantation is characterized by an inflammatory-like response with expansion of extracellular fluid volume, increased vascular permeability, and vasodilatation. These effects are believed to be mediated at the paracrine level by prostaglandin E2 and platelet-activating factor (PAF), but the cellular mechanism (or mechanisms) remains largely unknown. We demonstrate that PAF receptor (PAF-R) immunoreactivity and mRNA are detected in proliferative and secretory endometrial glands, however, the responsiveness of endometrium to physiological concentrations of PAF is confined predominantly to the secretory endometrium. Semiquantitative reverse transcription-polymerase chain reaction revealed that PAF-R transcript levels were highest in the mid-late proliferative and late secretory phases of the cycle. Interaction of PAF with its receptor resulted in the rapid release of nitric oxide (NO), increased expression of vascular endothelial growth factor (VEGF), and activation of FAKpp125, a focal adhesion kinase, demonstrating that the PAF-R is functionally active. Inhibition of NO synthesis by NG-monomethyl-L-arginine produced dose-dependent attenuation of PAF-evoked NO release, indicating NOS activation; the dependency of PAF-evoked NO release on PKC and extracellular Ca2+ was confirmed by PKC inhibitor Ro 31-8220 and by the removal of extracellular Ca2+. PAF up-regulated VEGF gene expression in a concentration- and time-dependent fashion in human endometrial epithelial cell lysates. Transcription of VEGF was rapidly followed by secretion of the protein. These data support our premise that this autocoid acts as an angiogenic mediator in the regeneration of the endometrium after menses and as a vasodilator to promote blastocyst attachment during the implantation process.

Cell Adhesion Molecules↗

PubMed1998

Differential signaling and regulation of apical vs. basolateral EGFR in polarized epithelial cells.

Overexpression of the epidermal growth factor receptors (EGFR) in polarized kidney epithelial cells caused them to appear in high numbers at both the basolateral and apical cell surfaces. We utilized these cells to look for differences in the regulation and signaling of apical vs. basolateral EGFR. Apical and basolateral EGFR were biologically active and mediated EGF-induced cell proliferation to similar degrees. Receptor downregulation and endocytosis were less efficient at the apical surface, resulting in prolonged EGF-induced tyrosine kinase activity at the apical cell membrane. Tyrosine phosphorylation of EGFR substrates known to mediate cell proliferation, Src-homologous and collagen protein (SHC), extracellularly regulated kinase 1 (ERK1), and ERK2 could be induced similarly by activation of apical or basolateral EGFR. Focal adhesion kinase was tyrosine phosphorylated more by basolateral than by apical EGFR; however, beta-catenin was tyrosine phosphorylated to a much greater degree following the activation of mislocalized apical EGFR. Thus EGFR regulation and EGFR-mediated phosphorylation of certain substrates differ at the apical and basolateral cell membrane domains. This suggests that EGFR mislocalization could result in abnormal signal transduction and aberrant cell behavior.

Animals↗

PubMed1997

Domain movement in rabbit muscle adenylate kinase might involve proline isomerization.

The fluorescence probe, 8-anilino-1-naphthalenesulfonic acid (ANS), was used to monitor the induced-fit conformational movement in rabbit muscle adenylate kinase. In 50 mM Tris-HCl buffer (pH 8.1), the time course of ANS binding to rabbit muscle adenylate kinase is a biphasic process. The fast phase completes within the dead-time of the stopped-flow equipment used (about 15 ms), while the slow phase ends in about 10 minutes. In the presence of 2.0 microM peptidyl prolyl cis/trans-isomerase, the rate constant of the slow phase reaction is accelerated about 2.4-fold, suggesting that the domain movement during ANS binding to rabbit muscle adenylate kinase may involve proline isomerization. The activation energy of the slow phase was determined to be 74.6 kJ/mol, which is comparable to the activation energy of proline cis/trans-isomerization (about 80 kJ/mol).

Adenylate Kinase↗

PubMed1997

Double coupling Edman chemistry for high-sensitivity automated protein sequencing.

Capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) is a promising new method for the analysis of protein sequencing products. It gives 10 zmol (1 zmol = 10(-21) mol) limits of detection (3 sigma) for fluorescein thiohydantoin (FTH) amino acids. We have developed a separation for the (FTH)-amino acid products generated from 18 of the 20 coded amino acids. The extremely low volume requirement associated with CE-LIF makes it incompatible with commercial sequencers. For this reason, we have also been developing a miniaturized sequencer that can be more easily coupled to our detection system. Both the CE-LIF system and the miniaturized sequencer are described.

Amino Acid Sequence↗

PubMed1997

Association of DNA with nuclear matrix in in vitro assembled nuclei induced by rDNA from Tetrahymena shanghaiensis in Xenopus egg extracts.

The nuclei assembled from exogenous DNA or chromatin in egg extracts resemble their in vivo counterparts in many aspects. However, the distribution pattern of DNA in these nuclei remains unknown. We introduced rDNA from the macronuclei of Tetrahymena into Xenopus cell-free extracts to examine the association of specific DNA sequences with nuclear matrix (NM) in the nuclei assembled in vitro. Our previous works showed the 5'NTS (non-transcription sequences) of the rDNA specifically bind to the NM system in the macronuclei. We show now the rDNA could induce chromatin assembly and nuclear formation in Xenopus cell-free system. When we extracted the NM system and compared the binding affinity of different regions of rDNA with the NM system, we found that the 5'NTS still hold their binding affinity with insoluble structure of the assembled nuclei in the extracts of Xenopus eggs.

Animals↗

PubMed1997

Autolytic site mutant R105C of rat trypsin.

The autolytic site Arg105 of rat trypsin was replaced with Cys by DNA site-directed mutagenesis method. Comparison of expression and purification of R105C trypsin along with the wild type and some other Arg105 mutants indicates that R105C trypsin could be expressed as well but with a lower expression level. It is unexpected that R105C trypsin has no detectable activity toward trypsin substrate TAME, quite different from the wild type and other Arg105 mutants. Native gel electrophoresis analysis indicates that R105C trypsin has a similar mobility rate to that of wild type trypsin. FPLC also gives similar retaining time. The loss of activity of R105C trypsin may result from the conformational change around active site, but not the dimer formation.

Animals↗