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H Liu

Publications and source records attributed to H Liu.

At least 973 records · Page 54Linked to original sources
PubMed1995

Predicting drug-membrane interactions by HPLC: structural requirements of chromatographic surfaces.

Drug-membrane interactions have recently been studied by immobilized artificial membrane (IAM) chromatography (Pidgeon, C.; et al. J. Med. Chem. 1995, 38, 590-595. Ong, S.; et al. Anal. Chem. 1995, 67, 755-762), and the molecular recognition properties of IAM surfaces toward drug binding/partitioning appear to be remarkably close to the molecular recognition properties of fluid membranes. The structural requirements of chromatography surfaces to emulate biological partitioning are unknown. To begin to elucidate the surface structural requirements needed to predict drug partitioning into membranes, three bonded phases were prepared. The chromatography bonded phases were prepared by immobilizing (i) a single-chain analog containing the phosphocholine (PC) headgroup (IAM.PC.DD), (ii) a long-chain alcohol containing polar OH groups protruding from the surface (12-OH-silica), and (iii) a long-chain fatty acid containing OCH3 groups protruding from the surface (12-MO-silica). The 12-OH-silica surface can be considered as an immobilized "octanol" phase with OH groups protruding from the surface and is therefore a solid phase model of octanol/water partitioning systems. As expected, improved capability of predicting solute-membrane interactions as found for the chromatographic surface containing the PC polar head-group because the PC headgroup is also found in natural cell membranes. For instance, the IAM.PC.DD column predicted drug partitioning into dimyristoylphosphatidylcholine liposomes (r = 0.864) better than 12-OH-silica (r = 0.812), and 12-MO-silica (r = 0.817). IAM. PC.DD columns also predicted intestinal drug absorption (r = 0.788) better than 12-OH-silica (r = 0.590) and 12-MO-silica (r = 0.681); reversed phase octadecylsilica (ODS) columns could not predict intestinal absorption (r = 0.10). Collectively, these results suggest that chromatographic surfaces containing interfacial polar groups, i.e., PC, OH, and OCH3, model drug-membrane interactions, but surfaces lacking interfacial polar functional groups (e.g., ODS surface) are poor models. Most interestingly, drug partitioning into octanol/water systems does not correlate with drug binding to the immobilized octanol phase. However, drug partitioning into immobilized octanol correlates with drug partitioning into liposomes (r = 0.812).

Chromatography, High Pressure Liquid↗
PubMed1995

Modulation of GLUT1 intrinsic activity in clone 9 cells by inhibition of oxidative phosphorylation.

Brief (1-2 h) exposure of Clone 9 cells to inhibitors of oxidative phosphorylation such as azide is known to markedly increase glucose uptake. Clone 9 cells express GLUT1 but not GLUT2, -3, and -4, and the azide effect was not accompanied by any increase in cellular or plasma membrane GLUT1 level. To identify the molecular event underlying this apparent increase in GLUT1 intrinsic activity, we studied the acute effects of azide on the substrate binding activity of GLUT1 in Clone 9 cells by measuring glucose-sensitive cytochalasin B binding. The glucose-displaceable, cytochalasin B binding activity was barely detectable in membranes isolated from Clone 9 cells under control conditions but was readily detectable after a 60-min incubation of cells in the presence of 5 mM azide showing a 3-fold increase in binding capacity with no change in binding affinity. Furthermore, the cytochalasin B binding activity of purified human erythrocyte GLUT1 reconstituted in liposomes was significantly reduced in the presence of cytosol derived from azide-treated Clone 9 cells but not in the presence of cytosol from control cells; this effect was heat-labile and abolished by the presence of the peptide corresponding to the GLUT1 COOH-terminal sequence. These results suggest that a cytosolic protein in Clone 9 cells binds to GLUT1 at its COOH-terminal domain and inhibits its substrate binding and that azide-induced metabolic alteration releases GLUT1 from this inhibitory interaction. Studying the binding of cytosolic proteins derived from 35S-labeled Clone 9 cells to glutathione S-transferase fusion protein containing glucose transporter COOH-terminal sequences, we identified 28- and 70-kDa proteins that bind specifically to the cytoplasmic domain of GLUT1 and GLUT4 in vitro. We also found a 32P-labeled, 85-kDa protein that binds to GLUT4 but not to GLUT1 and only in cytosol derived from azide-treated cells. The roles, if any, of these glucose transporter-binding proteins in the azide-sensitive modulation of GLUT1 substrate binding activity in Clone 9 cells are yet to be determined.

Animals↗
PubMed1995

Stimulatory effect of saponin from Panax ginseng on immune function of lymphocytes in the elderly.

We used the saponin Rg1 extracted from Panax ginseng to study its effects on lymphocytes of 10 young and 19 elderly persons. The proliferative response of lymphocytes cocultured for 72 h with PHA and saponin was measured by using MTT method and the 3H-TdR incorporation procedure. PHA and Rg1 had stimulative effects on the phenotype of lymphocytes (P < 0.001). Rg1 also increased the fluidity of lymphocyte membrane of the aged (P < 0.001). The CD25 and CD45RA positive cells of lymphocytes in the elderly were lower than those of the young people, 8.6% +/- 2.7% vs 10.43% +/- 3.5%; 20.95% +/- 15.5% vs. 50.86% +/- 4.2%, respectively. More CD45RO positive cells (41.5% +/- 13.9%) than CD45RA positive cell lymphocyte populations were seen in the aged. The CD45RO positive cells of the young people were 39.63% +/- 3.2%. We discussed the cause of declined immune function of lymphocytes of aged person and the mechanism of the effect of P. ginseng on lymphocytes.

Aged↗
PubMed1995

Evidence for class-specific discrimination of a semiconserved base pair by tRNA synthetases.

Aminoacyl-tRNA synthetases have been divided into two classes based on the existence of two structurally distinct active sites. To date, few class-specific tRNA recognition features have been elucidated. High-resolution X-ray structures of representative class I and class II synthetases complexed to cognate tRNA substrates have been solved. In these structures, the class I enzyme approaches the end of the tRNA acceptor stem from the minor-groove side, while the class II synthetase approaches its cognate tRNA from the major-groove side. This distinction is reflected in the different initial sites (2'- or 3'-OH) of amino acid attachment. The role that the semiconserved G1.C72 terminal base pair plays in the aminoacylation of Escherichia coli tRNAs is probed in this in vitro study. We show here that class II alanyl-, prolyl-, and histidyl-tRNA synthetases are sensitive to changes at position 1 x 72. Previous work on class I synthetases and new data presented here with the valine-specific enzyme indicate that class I enzymes show little sensitivity to replacements of G1.C72. This work provides new evidence for class-specific differences in tRNA acceptor stem interactions that appear to be reflected not only in the initial site of aminoacylation but also in the mode of synthetase interaction with the semiconserved G1.C72 base pair proximal to the amino acid attachment site.

Alanine-tRNA Ligase↗
PubMed1995

Association of native Ca2+ channel beta subunits with the alpha 1 subunit interaction domain.

beta Subunits of voltage-dependent Ca2+ channels play an important role in regulating Ca2+ channel function. The sites of alpha 1-beta subunit interaction have been localized recently to cytoplasmic domains of both subunits. The alpha 1 subunit interaction domain (AID) is an 18-amino-acid conserved motif located between repeats I and II on all alpha 1 subunits which is essential for the binding of beta subunits. In order to further study the interaction of beta subunits with AID, we have expressed a 50-amino-acid glutathione S-transferase (GST) fusion protein from the alpha 1A subunit that contains the AID. Mutant GST fusion proteins that contain a single amino acid change (Y392S, Y392F, and Y392W) in the AIDA along with control GST were coupled to glutathione-Sepharose beads to form affinity beads. Binding assays using these affinity beads with in vitro synthesized 35S-labeled beta 2 and beta 3 subunits demonstrate that the hydroxyl group on tyrosine 392 of AIDA is critical for binding to beta subunits. The affinity bead assay was also used to identify and characterize native beta subunits from detergent extracts of different tissues. The AIDA affinity beads, but not the control or Y392S beads, specifically bind beta subunits from detergent extracts of skeletal muscle, cardiac muscle, and brain. Immunoblot analyses demonstrate the presence of beta 1a in skeletal muscle, beta 2 and beta 3 in cardiac muscle, and beta 1b, beta 3, and beta 4 in brain. The assays also demonstrate the AIDA beads bind to beta subunits from tissue homogenates extracted with low salt and no detergent suggesting the existence of a pool of beta subunits which is not always associated with alpha 1 subunits. Also, beta subunits from solubilized skeletal muscle triads can be affinity-purified using AIDA CNBr-Sepharose. Our data demonstrate that the AID binds to native beta subunits from detergent and non-detergent tissue extracts illustrating that this domain on the alpha 1 subunit is the major anchoring site for the beta subunit.

Amino Acid Sequence↗
PubMed1995

Binding of cephalothin and cefotaxime to D-ala-D-ala-peptidase reveals a functional basis of a natural mutation in a low-affinity penicillin-binding protein and in extended-spectrum beta-lactamases.

Two clinically-important beta-lactam antibiotics, cephalothin and cefotaxime, have been observed by X-ray crystallography bound to the reactive Ser62 of the D-alanyl-D-alanine carboxypeptidase/transpeptidase of Streptomyces sp. R61. Refinement of the two crystal structures produced R factors for 3 sigma (F) data of 0.166 (to 1.8 A) and 0.170 (to 2.0 A) for the cephalothin and cefotaxime complexes, respectively. In each complex, a water molecule is within 3.1 and 3.6 A of the acylated beta-lactam carbonyl carbon atom, but is poorly activated by active site residues for nucleophilic attack and deacylation. This apparent lack of good stereochemistry for facile hydrolysis is in accord with the long half-lives of cephalosporin intermediates in solution (20-40 h) and the efficacy of these beta-lactams as inhibitors of bacterial cell wall synthesis. Different hydrogen binding patterns of the two cephalosporins to Thr301 are consistent with the low cefotaxime affinity of an altered penicillin-binding protein, PBP-2x, reported in cefotaxime-resistant strains of Streptococcus pneumoniae, and with the ability of mutant class A beta-lactamases to hydrolyze third-generation cephalosporins.

Amino Acid Sequence↗
PubMed1995

Induction of nitric oxide synthase and motoneuron death in newborn and early postnatal rats following spinal root avulsion.

It is well established that immature motoneurons are more vulnerable to axonal injury than the adult ones. Many previous studies used distal axonal injury, such as sciatic nerve transection, to examine the response of immature motoneurons to injury. In the present study, a more severe injury, spinal root avulsion, was performed in newborn and early postnatal rats, and the response of immature spinal motoneurons to the injury was observed. In newborn rats, root avulsion causes motoneuron loss which is similar to that of previous studies by distal axonal injury. During the early postnatal development, motoneuron loss in immature rats is greater following root avulsion when compared to cell loss following distal axonal injury at any given age. Following root avulsion, nitric oxide synthase (NOS) is induced in injured immature motoneurons. The induction and accumulation of NOS in injured motoneurons is much more rapid in the immature animals than in the adult, which is coincident with the more rapid motoneuron loss in the immature animals. Results of the present study indicate an involvement of NOS in neuronal degeneration. However, the precise role of NOS in the response of motoneurons to axonal injury is not clear and needs to be further studied.

Animals↗
PubMed1995

Simultaneous measurements of Ca2+ in the intracellular stores and the cytosol of hepatocytes during hormone-induced Ca2+ oscillations.

Simultaneous Ca2+ measurements in the cytosol and intracellular stores (IS) of rat hepatocytes were performed using two Ca(2+)-sensitive probes (Fluo-3 and Mag-fura-2), and combined whole-cell patch clamp and fluorescence microscopy. A steady-state Ca2+ concentration of approximately 630 microM was estimated in the IS. alpha 1-Adrenergic stimulation induced periodic elevations of cytosolic Ca2+ and parallel synchronized transient declines in the IS. Subsequent application of the intracellular Ca(2+)-pump inhibitor thapsigargin resulted in a release of Ca2+ from the IS to reach a level of Ca2+ depletion much lower than the lowest transient decline observed during the oscillations.

Aniline Compounds↗
PubMed1995

Receptor endocytosis and dendrite reshaping in spinal neurons after somatosensory stimulation.

In vivo somatosensory stimuli evoked the release of substance P from primary afferent neurons that terminate in the spinal cord and stimulated endocytosis of substance P receptors in rat spinal cord neurons. The distal dendrites that showed substance P receptor internalization underwent morphological reorganization, changing from a tubular structure to one characterized by swollen varicosities connected by thin segments. This internalization and dendritic structural reorganization provided a specific image of neurons activated by substance P. Thus receptor internalization can drive reversible structural changes in central nervous system neurons in vivo. Both of these processes may be involved in neuronal plasticity.

Animals↗
PubMed1995

Morphological characterization of substance P receptor-immunoreactive neurons in the rat spinal cord and trigeminal nucleus caudalis.

Although there is considerable evidence that primary afferent-derived substance P contributes to the transmission of nociceptive messages at the spinal cord level, the population of neurons that expresses the substance P receptor, and thus are likely to respond to substance P, has not been completely characterized. To address this question, we used an antibody directed against the C-terminal portion of the rat substance P receptor to examine the cellular distribution of the receptor in spinal cord neurons. In a previous study, we reported that the substance P receptor decorates almost the entire dendritic and somatic surface of a subpopulation of spinal cord neurons. In the present study we have taken advantage of this labeling pattern to identify morphologically distinct subpopulations of substance P receptor-immunoreactive neurons throughout the rostral-caudal extent of the spinal cord. We observed a dense population of fusiform substance P receptor-immunoreactive neurons in lamina I at all segmental levels. Despite having the highest concentration of substance P terminals, the substantia gelatinosa (lamina II) contained almost no substance P receptor-immunoreactive neurons. Several distinct populations of substance P receptor-immunoreactive neurons were located in laminae III-V; many of these had a large, dorsally directed dendritic arbor that traversed the substantia gelatinosa to reach the marginal layer. Extensive labeling was also found in neurons of the intermediolateral cell column. In the ventral horn, we found that labeling was associated with clusters of motoneurons, notably those in Onuf's nucleus in the sacral spinal cord. Finally, we found no evidence that primary afferent fibers express the substance P receptor. These results indicate that relatively few, but morphologically distinct, subclasses of spinal cord neurons express the substance P receptor. The majority, but not all, of these neurons are located in regions that contain neurons that respond to noxious stimulation.

Afferent Pathways↗
PubMed1995

Identification and localization of two distinct microenvironments for the diacylglycerol component of lipophorin particles by 13C NMR.

13C nuclear magnetic resonance spectroscopy of lipoproteins, isolated from the insect Manduca sexta, has been employed to probe the microenvironment of diacylglycerol (DG), their major neutral lipid component. Natural abundance 13C NMR spectra of high density lipophorin exhibited several well-separated resonances derived from its lipid moiety, including those for the carbonyl carbon atoms of phospholipid and DG fatty acyl chains in the region of 175-180 ppm. To verify the assignment of the DG acyl chain carbonyl carbon resonances, di[1-13C]oleoylglycerol high density lipophorin was isolated after instilling a bolus of tri[1-13C]oleoylglycerol into the midgut of larvae fed a fat-free diet. 13C NMR spectra of the isolated lipoprotein revealed a specific and dramatic enrichment of resonances at 175.5 ppm. Expansion of this region revealed two resonances separated by 0.08 ppm. These were assigned as 1,2- and 1,3- isomers of DG, the latter presumably arising from spontaneous acyl chain migration of 1,2-DG following lipoprotein isolation. On the basis of compositional and structural analysis of this lipoprotein, it is postulated that these DG species are localized predominantly in the hydrophobic core of the particle. By contrast, natural abundance 13C NMR spectra of the DG-rich, low density lipophorin (LDLp) subspecies revealed two additional resonances, separated by 0.2 ppm, that were tentatively assigned as 1,2- and 1,3-DG present at the surface of the particle. The verify this assignment, experiments employing phospholipase C, to convert lipophorin surface associated phospholipid into DG, were performed.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Effects of solutes on optical properties of biological materials: models, cells, and tissues.

Perturbations of scattering background for absorbance measurements by photon diffusion techniques complicate algorithms used for determining the concentration of blood and the saturation of hemoglobin in tissues. In order to better define these perturbations, we have undertaken a study of the effect of solutes, some of physiological importance, upon the scattering of three types of model systems: a lipid vessel suspension (Intralipid), a cell suspension (Baker's yeast), and a tissue (perfused liver). A simple formula relates absorbancy change to proportional changes of the input/output separation rho and the square root of mu a and mu's in relation to a relevant model system. Thus, absorbance changes at 850 nm slopes and intercepts are measured as a function of rho; the absorption and reduced scattering coefficient, mu a and mu's, are calculated. We studied each of these cases as a function of the perturbation induced by low-molecular-weight polyhydroxy solutes, generally sugars (mannitol, fructose, sucrose, and glucose), alcohols (propanediol and methanol), and electrolytes (sodium and potassium chloride). Dilatometric studies indicate volume changes of the solvent system and afford correction factors. The slopes are approximately +/- 0.5 x 10(-4) OD/mM solute per centimeter separation of input/output per percent scatter (yeast or Intralipid) and indicate possible physiological detection of solutes in tissues in the millimolar range. The large optical effects of temperature upon the solute effect on model systems and of osmotic and perfusion pressures on the perfused liver further complicate the possibility of quantitative in vivo studies of these solutes.

Animals↗
PubMed1995

Properties of the alpha 1-beta anchoring site in voltage-dependent Ca2+ channels.

In voltage-dependent Ca2+ channels, the beta subunit interacts with the alpha 1 subunit via a cytoplasmic site. A biochemical assay has been developed to quantitatively describe the interaction between both subunits. In vitro synthesized 35S-labeled beta subunits specifically bind to a glutathione S-transferase (GST) fusion protein containing the alpha 1A interaction domain (AIDA, located between the amino-acids 383 and 400 of the cytoplasmic loop between the hydrophobic domains I and II). Kinetic analysis demonstrates that the association of 35S-labeled beta 1b subunit to the AIDA GST fusion protein occurs with a fast rate constant at 4 degrees C. The binding is almost irreversible as demonstrated by the absence of dissociation observed after an 8-h incubation with an 18-amino acid synthetic AIDA peptide. The alpha 1-beta binding site does not seem to be a target for cytoplasmic regulation. The interaction is mostly unaffected by changes in ionic strength, pH, and Ca2+ concentration or by protein kinase C phosphorylation. The specificity of subunit interaction in voltage-dependent Ca2+ channels was also followed by saturation analyses. The data obtained show that the AIDA GST fusion protein binds to a single site on the beta 1b with an apparent Kd of 5 nM. The affinities of AIDA GST fusion protein for various beta subunits was measured and demonstrate that beta subunits associate with different affinities to each alpha 1 interaction domain. The rank order of AIDA affinity for each beta subunit is as follows: beta 4 > beta 2a > beta 1b >> beta 3. The binding of the beta subunit to alpha 1 subunit can be inhibited in vitro by the AIDA synthetic peptide with an apparent Ki of 285 nM. This interaction can also be prevented in heterologous Ca2+ channels by the injection of the AIDA GST fusion protein into Xenopus oocytes. Our results demonstrate that the site of interaction between AID and beta subunit is responsible for anchoring the beta subunit to the alpha 1 subunit and thus allowing the beta subunit to modify Ca2+ channel activity.

Amino Acid Sequence↗
PubMed1995

PER3, a gene required for peroxisome biogenesis in Pichia pastoris, encodes a peroxisomal membrane protein involved in protein import.

PER genes are essential for the biogenesis of peroxisomes in the yeast Pichia pastoris. Here we describe the cloning of PER3 and functional characterization of its product Per3p. The PER3 sequence predicts that Per3p is a 713-amino acid (81-kDa) hydrophobic protein with at least three potential membrane-spanning domains. We show that Per3p is a membrane protein of the peroxisome. Methanol- or oleate-induced cells of per3-1, a mutant strain generated by chemical mutagenesis, lack normal peroxisomes but contain numerous abnormal vesicular structures. The vesicles contain thiolase, a PTS2 protein, but only a small portion of several other peroxisomal enzymes, including heterologously expressed luciferase, a PTS1 protein. These results suggest that the vesicles in per3-1 cells are peroxisomal remnants similar to those observed in cells of patients with the peroxisomal disorder Zellweger syndrome, and that the mutant is deficient in PTS1 but not PTS2 import. In a strain in which most of PER3 was deleted, peroxisomes as well as peroxisomal remnants appeared to be completely absent, and both PTS1- and PTS2-containing enzymes were located in the cytosol. We propose that Per3p is an essential component of the machinery required for import of all peroxisomal matrix proteins and is composed of independent domains involved in the import of specific PTS groups.

Acetyl-CoA C-Acetyltransferase↗
PubMed1995

ATP-sensitive binding of a 70-kDa cytosolic protein to the glucose transporter in rat adipocytes.

We have identified a 70-kDa cytosolic protein (GTBP70) in rat adipocytes that binds to glutathione S-transferase fusion proteins corresponding to the cytoplasmic domains of the facilitative glucose transporter isoforms Glut1, Glut2, and Glut4. GTBP70 did not bind to irrelevant fusion proteins, indicating that the binding is specific to the glucose transporter. GTBP70 binding to the glucose transporter showed little isoform specificity but was significantly subdomain-specific; it bound to the C-terminal domain and the central loop, but not to the N-terminal domain of Glut4. The GTBP70 binding to Glut4 was not affected by the presence of 2 mM EDTA, 2.4 mM Ca2+, or 150 mM K+. The binding was inhibited by ATP in a dose-dependent manner, with 50% inhibition at 10 mM ATP. This inhibition was specific to ATP, as ADP and AMP-PCP (adenosine 5'-(beta, gamma-methylenetriphosphate)) were without effect. GTBP70 did not react with antibodies against phosphotyrosine, phosphothreonine, or phosphoserine, suggesting that it is not a phosphoprotein. The binding of GTBP70 to Glut4 was not affected by the pretreatment of adipocytes with insulin. When these experiments were repeated using rat hepatocyte cytosols, no ATP-sensitive 70-kDa protein binding to the glucose transporter fusion proteins was evident, suggesting that either GTBP70 expression or its function is cell-specific. These findings strongly suggest the possibility that GTBP70 may play a key role in glucose transporter regulation in insulin target cells such as adipocytes.

Adenosine Triphosphate↗