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H Liu

Publications and source records attributed to H Liu.

At least 1,045 records · Page 58Linked to original sources
PubMed1994

Brefeldin A inhibits insulin-induced glucose transport stimulation and GLUT4 recruitment in rat adipocytes.

GLUT4, the major insulin-responsive glucose transporter isoform in rat adipocytes, rapidly recycles between an intracellular pool and the plasma membrane in the basal and insulin-stimulated states. To gain insight into the route of this GLUT4 recycling, we studied the effects of brefeldin A (BFA) on glucose transport and glucose transporter subcellular distribution in rat adipocytes in the absence and in the presence of insulin. 3-O-Methyl-D-glucose equilibrium exchange measurements revealed that BFA inhibits insulin-stimulated glucose transport by as much as 80%, whereas the inactive BFA analog, B36, was without effect. The inhibition was reversible and was a saturable function of BFA concentration with an apparent Ki of less than 1 microM. In the absence of insulin, on the other hand, BFA caused a slight (up to 2-fold) increase in glucose transport. Subcellular fractionation and semiquantitative immunoblotting analysis revealed that BFA inhibits insulin-induced redistribution of GLUT4 from microsomes to the plasma membranes, with a dose dependence similar to that for glucose transport inhibition. BFA also caused a slight increase in the plasma membrane GLUT4 level in the absence of insulin. BFA did not affect the subcellular distribution of GLUT1 in these experiments. These findings strongly suggest that GLUT4 recycling in rat adipocytes involves a BFA-sensitive, coat protein-mediated, membrane-budding step, which is distinct between the constitutive and the insulin-induced pathways.

Adipocytes↗
PubMed1994

Preparation of mixed ligand immobilized artificial membranes for predicting drug binding to membranes.

Mixed ligand immobilized artificial membranes (IAMs) are surfaces that contain at least two immobilized membrane phospholipids which are designated as either the primary phospholipid or the secondary phospholipid. The primary immobilized phospholipid refers to the immobilized phospholipid that has the highest surface density. For this work, the primary immobilized phospholipid was a single-chain ether phosphatidylcholine (PC) analog. Four mixed-ligand IAMs were prepared by use of immobilized PC as the primary immobilized phospholipid. The secondary immobilized phospholipid ligand was either phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, or phosphatidic acid. All of these secondary phospholipids are bonded at approximately 6-10 mol % relative to the molar amount of immobilized PC. Each secondary phospholipid contains functional groups in the polar head group region that require protecting groups during the immobilization process. The four-step synthetic strategy to prepare mixed-ligand IAMs involves (i) immobilization of the PC analog at high density to silica propylamine (SPA), (ii) immobilization of the second phospholipid (PL) analog at low density, (iii) end capping residual amines with a long-chain anhydride followed by end capping with a short-chain anhydride, and (iv) deprotection of the polar head group protecting groups. The surface density of the mixed PLs bonded to the silica support was approximately 130 mumol of PLs/g of SPA. High-performance liquid chromatography using these mixed lipid IAMs can be exploited to rapidly predict the membrane binding properties of drugs.

Adrenergic alpha-Agonists↗
PubMed1994

Evidence for presynaptic N-methyl-D-aspartate autoreceptors in the spinal cord dorsal horn.

The N-methyl-D-aspartate (NMDA) receptor has been implicated in a variety of systems that undergo plastic changes in the central nervous system. We used electron microscopic immunocytochemistry with an antibody directed against an alternatively spliced exon near the C terminus of NMDAR1, the essential functional subunit of the NMDA receptor, to study the distribution of the NMDA receptor in the spinal cord and CA1 region of the hippocampus, two regions where NMDA-mediated long-term plasticity has been demonstrated. In CA1, we found that the NMDA receptor is exclusively expressed on postsynaptic structures. By contrast, in the spinal cord we found that in about one-third of labeled synapses, the receptor is located in the presynaptic terminal, immediately adjacent to the vesicle release site at the active zone. Using combined postembedding immunocytochemistry, we also showed that > 70% of the NMDA receptor immunoreactive terminals are glutamate positive, which suggests that the presynaptic NMDA receptor is an autoreceptor. Nerve ligation studies demonstrated that the receptor is transported in dorsal roots and sciatic nerve to the spinal cord and periphery, respectively. These data indicate that an NMDA autoreceptor is located in terminals of primary afferent fibers, where it could facilitate the transmission of inputs to the spinal cord by increasing the release of neurotransmitter from the primary afferent terminal.

Amino Acid Sequence↗
PubMed1994

Structure determination of an Fab fragment that neutralizes human rhinovirus 14 and analysis of the Fab-virus complex.

The crystal structure of Fab17-IA, an antigen-binding fragment from a murine immunoglobulin that neutralizes human rhinovirus 14 (HRV14), has been solved to 2.7 A resolution. Fab17-IA crystallized into three different space groups depending upon the method used to purify the intact antibody. The structure was determined by use of molecular and isomorphous replacement methods. The current model has a crystallographic R-factor of approximately 19% for 10,192 independent reflections between 8 and 2.7 A. Correlation coefficient calculations showed that the Fab17-IA structure can be fit into the Fab17-IA/HRV14 image reconstruction density to within 5 A positional accuracy and to within a few degrees of rotation. The resulting interface of the docked antibody was examined and showed extensive charge and shape complementarity with the virus surface that was supported by site-directed mutagenesis experiments. The success of this approach validates the utility of combining X-ray crystallography with cryo-electron microscopy of complex macromolecular assemblies.

Amino Acid Sequence↗
PubMed1994

Supercritical fluid extraction as a sample preparation technique for the direct isolation of drugs from plasma prior to analysis.

Supercritical carbon dioxide was used for the direct extraction of drugs from plasma prior to analysis. The supercritical fluid was directly passed through plasma samples spiked with either a neutral (flavone) or an acidic (ketorolac) drug. The addition of an antifoam agent to the plasma prior to extraction was required to avoid restrictor plugging caused by denaturation of the plasma proteins by the supercritical fluid. The effluent from the extraction cell was bubbled through a small volume of methanol or into an empty tube to trap the extracted drug. The effect of extraction pressure and time on absolute recovery were examined. The absolute recovery, selectivity, precision and accuracy of the supercritical fluid extraction approach was compared to conventional liquid-liquid extraction using reversed-phase HPLC with ultraviolet detection.

Animals↗
PubMed1994

Fourier transform infrared spectra studies of protein in reverse micelles: effect of AOT/isooctane on the secondary structure of alpha-chymotrypsin.

The amide I region Fourier transform infrared (FTIR) spectra of alpha-chymotrypsin have been studied in deuterium oxide (D2O) solution and also in reverse micellar solution of AOT/isooctane. The Fourier second derivative was applied to all spectra, revealing that the amide I band of alpha-chymotrypsin in D2O and in reverse micellar solution consists of nine components. The band frequencies are assigned to alpha-helix, beta-sheet, random and turn structure. The second derivative spectra of alpha-chymotrypsin have been shifted in the reverse micellar solution of AOT/isooctane in comparison with its spectra in D2O. This shift has also changed the intensity of each band. Through accurate measurement of the band intensities, the relative amount of different structure of alpha-chymotrypsin can be estimated. The comparison of the calculated results obtained in D2O with those obtained in reverse micellar solution provides the possibility to analyze the effect of reverse micellar solution of AOT/isooctane on the secondary structure of alpha-chymotrypsin. The results indicate that the reverse micellar solution has decreased the amount of alpha-helix and beta-sheet structure and increased the amount of random and turn structure in alpha-chymotrypsin. The increase of the amount of random structure might loosen the structure of alpha-chymotrypsin and change the activity of the enzyme.

Amides↗
PubMed1994

Characterization of the effect of cholecystokinin (CCK) on neurons in the periaqueductal gray of the rat: immunocytochemical and in vivo and in vitro electrophysiological studies.

The periaqueductal gray (PAG) is an important integration site for pain, autonomic functions, vocalization, fear and anxiety. Cholecystokinin (CCK) is a major neurotransmitter in the PAG and CCK receptors are heterogeneously distributed within the PAG. Since CCK antagonists are anxiolytic and potentiate morphine analgesia, it is possible that these effects of CCK are mediated through alteration of neuronal activities in the PAG. The goals of this study were to examine the anatomical and physiological properties of the PAG CCK containing systems. The distribution of CCK-containing axons and boutons in PAG was examined using immunohistochemical procedures. These studies show that CCK-like immunoreactive (CCK-LIR) fibers and terminals are present throughout PAG, but are particularly heavily concentrated in a focal column that runs longitudinally throughout the rostrocaudal axis of dorsolateral PAG and in nucleus cuneiformis which represents a caudolateral extension of PAG. The physiological effects of CCK on PAG neurons were examined in both in vivo and in vitro preparations. In the in vivo experiments multibarreled electrodes were used to record from PAG neurons and to apply CCK and the CCK antagonists, CR1409 and proglumide. Of 37 neurons recorded in vivo, CCK caused excitation in 25 cells, inhibited 7 cells and had no effect on 5 cells. The excitatory effect was blocked by CR1409 in 11/11 cells tested. Proglumide blocked the excitatory response of CCK in 12/14 cells. Proglumide blocked the inhibitory effect in 2 of 7 cells, but CR1409 had no effect on CCK-evoked inhibition in 7 cells tested. Extracellular, conventional intracellular and whole cell patch clamping procedures were used to study CCK actions in the in vitro slice preparation. In the extracellular recording experiments, responses of PAG cells to CCK were measured in slices that were maintained at 22 degrees C (room temperature) and at 32 degrees C. CCK excited 40/56, inhibited 7/56 and had no effect on 9/56 cells; excitatory responses were blocked by CR1409 in 32/36 cells and by proglumide in 25/27 cells tested. Inhibitory responses to CCK were unaffected by CR1409, but were blocked in 3/7 cells by proglumide. Conventional intracellular recordings were made from 13 cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1994

Maternal hypothyroidism during early pregnancy and intellectual development of the progeny.

OBJECTIVE: To investigate whether maternal hypothyroidism before the onset of fetal thyroid function influences mental development of the offspring. DESIGN: We examined IQs in children in whom the mothers had been hypothyroid during early pregnancy (group 1). The IQs were compared with those of siblings who were not exposed to maternal hypothyroidism during gestation (group 2). PATIENTS: Group 1 consisted of eight children. Mothers were examined for thyroid status during the fifth to 10th gestational weeks and were found to have distinctly low thyroxine levels and high thyrotropin levels; the levels became normal after thyroxine supplementation by 13 to 28 weeks of gestation. Seven of the eight children had nine siblings who had not been exposed to maternal hypothyroidism during gestation (group 2). Ages at examination were 4 to 10 years in group 1 and 4 to 15 years in group 2. RESULTS: All children in group 1 showed normal IQs. There was no significant difference in the mean IQ between the children in group 1 who had siblings (112 +/- 11) and their siblings in group 2 (106 +/- 8). Even the subject whose mother had had the lowest thyroxine level (free thyroxine, 2.3 pmol/L) had an IQ similar to that of his sibling. CONCLUSION: These data provide evidence against the presence of adverse effects of maternal hypothyroidism during early pregnancy on the subsequent mental development of the offspring.

Adolescent↗
PubMed1994

Changes in glucose transport and transporter isoforms during the activation of human peripheral blood lymphocytes by phytohemagglutinin.

We have explored the mechanism of stimulation of glucose transport during PHA stimulation of human peripheral blood lymphocytes (HPBT) enriched in T cells. Equilibrium exchange flux of 3-O-methyl glucose (3-O-MG) was stimulated two- and fourfold at 24 and 48 h after PHA stimulation, respectively. The increase was transient in that the flux rate returned to control (unstimulated) levels by 96 h. Immunoblotting and immunoprecipitation using specific Abs revealed that resting HPBT expresses glucose transporter isoforms GLUT-2 and GLUT-3 but not GLUT-1. After PHA stimulation, GLUT-1 expression was induced predominantly in the plasma membrane, whereas GLUT-3 expression was simultaneously down-regulated. GLUT-1 expression was detectable at 24 h, peaked at 48 h, and disappeared at 96 h. The total number of glucose transporters per cell measured as the total capacity of D-glucose displaceable cytochalasin B binding did not change significantly at any time after PHA stimulation. PHA stimulation also caused expression of high affinity IL-2R and secretion of IL-2. The IL-2 secretion was transient, which peaked at 24 h, slightly preceding the GLUT-1 expression peak and disappeared at 72 h. In PHA-activated HPBT cells synchronized at G0-G1, GLUT-1 was not expressed but was rapidly induced by exposure to IL-2. This induction did not occur in the presence of cyclosporin A, which inhibits IL-2 secretion. Based on these observations, we conclude that PHA stimulation increases glucose transport partly by inducing the expression of GLUT-1 instead of GLUT-3 and that GLUT-1 expression is induced by signals generated by IL-2 binding to its high affinity receptors.

Cells, Cultured↗
PubMed1994

Synaptic relationship between substance P and the substance P receptor: light and electron microscopic characterization of the mismatch between neuropeptides and their receptors.

Light microscopic studies have demonstrated significant mismatches in the location of neuropeptides and their respective binding sites in the central nervous system. In the present study we used an antiserum raised against a synthetic peptide corresponding to the carboxyl-terminal tail of the substance P (SP) receptor (SPR) to further explore the relationship between a neuropeptide and its receptor. Light microscopy revealed an excellent correlation between the patterns of SPR immunoreactivity and of 125I-labeled SPR-binding sites in the central nervous system. The SPR appeared to be exclusively expressed by neurons; in fact, the SPR decorates the somatic and dendritic surface of neurons, producing Golgi-like images. Electron microscopic analysis in cortex, striatum, and spinal cord revealed that approximately 70% of the surface membrane of immunoreactive neurons is SPR laden. Simultaneous electron microscopic labeling of SP and SPR demonstrated significant mismatch at the synaptic level. Although some SP terminals contacted SPR-immunoreactive membrane, no more than 15% of the SPR-laden membrane apposed synaptic terminals. These results suggest that in contrast to more "classical" central and peripheral nervous system synapses, wherein the receptor immediately apposes the site of neurotransmitter storage and release, much of the surface of SPR-expressing neurons can be targeted by SP that diffuses a considerable distance from its site of release.

Animals↗
PubMed1994

Signalling the molecular stress response to nephrotoxic and mutagenic cysteine conjugates: differential roles for protein synthesis and calcium in the induction of c-fos and c-myc mRNA in LLC-PK1 cells.

Nephrotoxic and mutagenic cysteine conjugates (NCC) are activated by the enzyme cysteine conjugate, beta-lyase, to reactive acylating species which bind covalently to cellular macromolecules. We now show that an early event after treatment of LLC-PK1 cells with NCC is the induction of mRNA for both c-fos and c-myc. Treatment with S-(1,2-dichlorovinyl)-L-cysteine (DCVC) induced c-fos (53-fold) and c-myc mRNA (20-fold) and increased transcription about 3-fold for both genes. Covalent binding was required for induction of both mRNAs. Dithiothreitol partially prevented induction of both c-fos and c-myc RNA. Buffering the DCVC-induced increase in cytosolic free calcium had no effect on c-fos mRNA, but partially blocked c-myc mRNA induction. Cycloheximide blocked the induction of c-myc mRNA in the absence of an effect on c-fos induction. The data suggest that the increase in c-fos mRNA is a primary response to DCVC toxicity and occurs without a requirement for protein synthesis or an increase in intracellular free calcium. In contrast, c-myc induction requires protein synthesis, suggesting that the presence of another primary response factor may regulate induction either transcriptionally or posttranscriptionally. The data suggest that different signalling pathways regulate induction of c-fos and c-myc mRNA in response to stress caused by reactive acylating species.

Animals↗
PubMed1994

2-Phenylphenanthridinone and related compounds: aryl hydrocarbon receptor agonists and suicide inactivators of P4501A1.

The effects of several 2-substituted phenanthridinones (2-nitro-, 2-t-butyl-, 2-bromo-, 2-phenyl-, 2-ethyl-, 2-methoxy-, 2-iodo-, 2-n-butyl-, 2-chloro-, 2-trifluoromethyl-, 2-fluoro-, 2-isopropyl-, and 2-methyl) and phenanthridinone as ligands for the rat liver cytosolic aryl hydrocarbon (Ah) receptor were determined using a competitive binding assay and 2,3,7,8-[3H]tetrachlorodibenzo-p-dioxin (TCDD) as the radioligand. The competitive binding IC50 values varied from 317 (2-trifluoromethyl-) to 5870 nM (2-methoxyphenanthridinone); the relative low Ah receptor binding affinities for these compounds were paralleled by their weak activity as inducers of ethoxyresorufin O-deethylase (EROD) activity in rat hepatoma H4IIE cells; however, there was not a correlation between their structure-binding and structure-induction relationship. In cells cotreated with 1 nM TCDD plus different concentrations (0.01-10 microM) of the 2-substituted phenanthridinones, several of these compounds inhibited induction of EROD activity by TCDD; 2-t-butyl- and 2-phenylphenanthridinone (2-PP) were the most active compounds, causing a > 80% reduction in the induced response. 2-PP was selected as a model to further investigate the mechanism of this inhibitory response. The results of interactive studies in rat hepatoma H4IIE cells cotreated with 2-PP plus TCDD or [3H]TCDD showed that 2-PP did not inhibit formation of the nuclear Ah receptor complex or induction of CYP1A1 mRNA levels or CYP1A1 protein. In contrast, incubation of 2-PP with either rat hepatoma H4IIE cells treated with TCDD or hepatic microsomes from TCDD-treated rats resulted in a rapid loss of EROD activity. In parallel experiments, [3H]2-PP was incubated with hepatic microsomes from TCDD-treated rats and analysis by denaturing electrophoresis showed that [3H]2-PP formed a covalent adduct with a 50- to 55-kDa protein and thus acted as a suicide inactivator of CYP1A1.

Animals↗
PubMed1994

Effect of dimethylsulfoxide and hydroxyethyl starch in the preservation of fractionated human marrow cells.

Fractionated human bone marrow cells, isolated by density centrifugation, could be well cryopreserved in both a DMSO/HES/albumin mixture and the conventional cryoprotectant (DMSO/serum) with either a programmed freezer or a -80 degrees C freezer. A lower initial temperature rise and a delayed plateau phase in the mixture in comparison with those in the DMSO/serum were observed using a programmed procedure without heat fusion compensation. Plateau phase in the mixture could also be shortened with increased liquid nitrogen to minimize the transition phase duration. However, the method of mononuclear cells immersed in a -80 degrees C freezer and stored in liquid nitrogen is simple and easily standardized. Additionally, widely used lactated Ringer's solution instead of Normosol-R can be employed to prepare the mixture for the preservation of fractionated cells.

Albumins↗
PubMed1994

Induction of Alzheimer-like beta-amyloid immunoreactivity in the brains of rabbits with dietary cholesterol.

beta-amyloid and ALZ-50 immunocytochemical reactivity were determined in the brains of rabbits fed either a control or 2% cholesterol diet. Control rabbits demonstrated no accumulation of intracellular immunolabeled beta-amyloid within 3 min after death. In animals fed the experimental diet for 4, 6, and 8 weeks (postmortem interval < 3 min), there was an increasingly mild-to-moderate-to-severe accumulation of intracellular immunolabeled beta-amyloid. Whether or not beta-amyloid is causally linked to processes leading to dementia, it is related in some way to the prime cause of human death; heart disease. Hypercholesterolemic rabbits may provide an animal model to study altered beta-APP metabolism leading to Alzheimer-like beta-amyloid accumulation xe03and extracellular deposition in brain.

Amyloid beta-Peptides↗
PubMed1994

Purification and properties of uricase from Candida sp. and its application in uric acid analysis in serum.

The purification of uricase from Candida sp. was carried out by precipitation with ammonium sulfate then further proceeded with Sephadex G200, and DEAE-cellulose DE52 chromatographies. The specific activity of the enzyme was enhanced from 0.05-12 (U/mg protein). The purity of the enzyme was judged to be homogeneous by SDS-PAGE. Some of the general properties of enzyme were investigated. The optimum reaction pH and temperature were 8.5 and 30 degrees C, respectively. The enzyme was stable at a pH range from 8.5-9.5 and at temperatures lower than 35 degrees C. The apparent Km value of the enzyme was calculated to be about 5.26 x 10(-6) mol/L. The molecular weight was determined to be 70,000-76,000 by the gel filtration and SDS-PAGE techniques. The isoelectric point was determined to be pH 5.6. The effects of some metallic ions on enzyme activity and stability were discussed. The partial purified uricase was used in serum uric acid determination. The within-batch imprecision percentage ranged from 2.16-2.63 and the between-batch imprecision percentage ranged from 2.4-3.6. The recovery ratio were from 96-101%. The correlation among this method and Boehringer, Roche, or Biotrol enzymatic kits were Y = 1.086x-0.50 (r = 0.981), Ya = 0.959x-0.29 (r = 0.97), and Yb = 1.110x-0.45 (r = 0.956), respectively. A linear calibration curve was obtained at 2.5-15 mg/dL uric acid. The stability of reagents and the effects of some substances in serum were also surveyed.

Candida↗
PubMed1994

Neural coding of aversive and appetitive gustatory stimuli: interactions in the hamster brain stem.

There is increasing evidence, both electrophysiological and behavioral, that bitter and sweet stimuli drive parallel pathways in the gustatory brainstem. Here we report two lines of investigation that suggest significant interactions among these parallel systems. First, responses recorded from single cells in the hamster's parabrachial nuclei (PbN) show that quinine hydrochloride (QHCl) produces a substantial suppression (> 40%) of the responses of PbN cells to sucrose. Sucrose stimulation has a reciprocal suppressive effect on the response to QHCl. These results imply that aversive and appetitive stimuli produce mutual inhibition in the gustatory system; studies of the chorda tympani nerve response suggest that this inhibition likely arises within the brainstem. A second line of investigation, using both an in vitro brainstem slice preparation and in vivo pharmacological manipulations of cells in the hamster NST, has demonstrated an inhibitory network within the rostral NST that plays a role in the modulation of taste activity. Patch-clamp and extracellular recording studies in vitro show that cells within the rostral central subdivision of the NST are inhibited by gamma-aminobutyric acid (GABA); this mediation is largely through the GABAA receptor subtype. Here we show that responses to taste stimulation recorded extracellularly from NST cells in vivo can be inhibited by local microinjections of GABA; this inhibition is blocked by the GABAA receptor antagonist bicuculline methiodide. Responses to sucrose are significantly more inhibited than those to NaCl or KCl. These combined lines of evidence show that appetitive and aversive stimuli activate mutually inhibitory systems within the brainstem and suggest that the basis for this interaction is a GABAergic inhibitory network within the NST.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗