Biomedical subjects

H Liu

Publications and source records attributed to H Liu.

At least 361 records · Page 20Linked to original sources

PubMed2000

Modulation of hippocampal excitability and seizures by galanin.

Previous studies have shown that the expression of the neuropeptide galanin in the hippocampus is altered by seizures and that exogenous administration of galanin into the hippocampus attenuates seizure severity. To address the role of endogenous galanin in modulation of hippocampal excitability and its possible role in seizure mechanisms, we studied two types of transgenic mice: mice with a targeted disruption of the galanin gene (GalKO) and mice that overexpress the galanin gene under a dopamine-beta-hydroxylase promoter (GalOE). GalKO mice showed increased propensity to develop status epilepticus after perforant path stimulation or systemic kainic acid, as well as greater severity of pentylenetetrazol-induced convulsions. By contrast, GalOE mice had increased resistance to seizure induction in all three models. Physiological tests of hippocampal excitability revealed enhanced perforant path-dentate gyrus long-term potentiation (LTP) in GalKO and reduced LTP in GalOE. GalKO showed increased duration of afterdischarge (AD) evoked from the dentate gyrus by perforant path simulation, whereas GalOE had increased threshold for AD induction. Depolarization-induced glutamate release from hippocampal slices was greater in GalKO and lower in GalOE, suggesting that alterations of physiological and seizure responses in galanin transgenic animals may be mediated through modulation of glutamate release. Our data provide further evidence that hippocampal galanin acts as an endogenous anticonvulsant and suggest that genetically induced changes in galanin expression modulate both hippocampal excitability and predisposition to epileptic seizures.

Animals↗

PubMed2000

Insulin activation of mitogen-activated protein (MAP) kinase and Akt is phosphatidylinositol 3-kinase-dependent in rat adipocytes.

To explore the mechanism of MAP kinase activation in adipocytes, we examined the possible involvement of several candidate signaling proteins. MAP kinase activity was markedly increased 2-4 min after treatment with insulin and declined to basal levels after 20 min. The insulin-dependent tyrosine phosphorylation of IRS-1 in the internal membrane and its association with phosphatidylinositol 3 (PI3) kinase preceded MAP kinase activation. There was little or no tyrosine phosphorylation of Shc or association of Grb2 with Shc or IRS-1. Specific PI3 kinase inhibitors blocked the insulin-mediated activation of MAP kinase. They also decreased the activation of MAP kinase by PMA and EGF but to a much lesser extent. Insulin induced phosphorylation of AKT on serine/threonine residues, and its effect could be blocked by PI3 kinase inhibitors. These results suggest that the insulin-dependent activation of MAP kinase in adipocytes is mediated by the IRS-1/PI3 kinase pathway but not by the Shc/Grb2/SOS pathway.

Adipocytes↗

PubMed2000

Drug release characteristics of unimolecular polymeric micelles.

Biodegradable, unimolecular polymeric micelles possess several features that are attractive for drug delivery applications: Thermodynamic stability, ability to encapsulate and solubilize a hydrophobic guest molecule, biodegradability, as well as size and surface characteristics that prevent rapid clearance by the RES. Here we investigate the potential of these unimolecular polymeric micelles to release a drug for an extended time. Lidocaine was used as a model drug for in vitro studies using a horizontal diffusion cell and cellulose membrane that prevented polymer transport from the source to the receiver compartment. The transport of free lidocaine from source to receiver under sink conditions was zero-order and complete within 8 h. The transport of lidocaine initially encapsulated in polymer was zero-order for the first 14 h, and 96% of the lidocaine was detected within 24 h.

Diffusion↗

PubMed2000

3,5-Bis(trifluoromethyl)pyrazoles: a novel class of NFAT transcription factor regulator.

A series of bis(trifluoromethyl)pyrazoles (BTPs) has been found to be a novel inhibitor of cytokine production. Identified initially as inhibitors of IL-2 synthesis, the BTPs have been optimized in this regard and even inhibit IL-2 production with a 10-fold enhancement over cyclosporine in an ex vivo assay. Additionally, the BTPs show inhibition of IL-4, IL-5, IL-8, and eotaxin production. Unlike the IL-2 inhibitors, cyclosporine and FK506, the BTPs do not directly inhibit the dephosphorylation of NFAT by calcineurin.

Animals↗

PubMed2000

Characterization of recombinant wild type and site-directed mutations of apolipoprotein C-III: lipid binding, displacement of ApoE, and inhibition of lipoprotein lipase.

The physicochemical properties of recombinant wild type and three site-directed mutants of apolipoprotein C-III (apoC-III), designed by molecular modeling to alter specific amino acid residues implicated in lipid binding (L9T/T20L, F64A/W65A) or LPL inhibition (K21A), were compared. Relative lipid binding efficiencies to dimyristoylphosphatidylcholine (DMPC) were L9T/T20L > WT >K21A > F64A/W65A with an inverse correlation with size of the discoidal complexes formed. Physicochemical analysis (Trp fluorescence, circular dichroism, and GdnHCl denaturation) suggests that L9T/T20L forms tighter and more stable lipid complexes with phospholipids, while F64A/W65A associates less tightly. Lipid displacement properties were tested by gel-filtrating apoE:dipalmitoylphosphatidylcholine (DPPC) discoidal complexes mixed with the various apoC-III variants. All apoC-III proteins bound to the apoE:DPPC complexes; the amount of apoE displaced from the complex was dependent on the apoC-III lipid binding affinity. All apoC-III proteins inhibited LPL in the presence or absence of apoC-II, with F64A/W65A displaying the most inhibition, suggesting that apoC-III inhibition of LPL is independent of lipid binding and therefore of apoC-II displacement. Taken together. these data suggest that the hydrophobic residues F64 and W65 are crucial for the lipid binding properties of apoC-III and that redistribution of the N-terminal helix of apoC-III (L9T/T20L) enhances the stability of the lipid-bound protein, while LPL inhibition by apoC-III is likely to be due to protein:protein interactions.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed2000

The role of human O(6)-alkylguanine-DNA alkyltransferase in promoting 1,2-dibromoethane-induced genotoxicity in Escherichia coli.

The expression of the DNA repair protein human O(6)-alkylguanine-DNA alkyltransferase (AGT) in Escherichia coli strains GWR109 or TRG8 that lack endogenous AGT greatly increased the toxicity and mutagenicity of 1,2-dibromoethane (DBE). Pretreatment of strain TRG8 expressing human AGT, which is permeable to exogenous drugs, with the AGT inhibitor O(6)-benzylguanine (BG) abolished the lethal and mutagenic effects of DBE, indicating that an active AGT is required for promoting DBE genotoxicity. This was confirmed by the observation that E. coli expressing either the C145A AGT mutant, which is inactive due to loss of the alkyl acceptor site, or mutants Y114E and R128A, which are inactive due to alteration of the DNA binding domain, did not enhance the action of DBE. However, the AGT mutant protein P138M/V139L/P140K, which is active in repairing methylated DNA but is totally resistant to inactivation by BG due to alterations in the active site pocket, was unable to enhance the genotoxicity of DBE. Similarly, other mutants, G156P, Y158H and K165R that are strongly resistant to BG, were much less effective than wild type AGT in mediating the genotoxicity of DBE. Mutant P140A, which is moderately resistant to BG, did increase mutations in response to DBE but was less active than wild type. These results suggest that human AGT is able to interact with a DNA lesion produced by DBE but, instead of repairing it, converts it to a more genotoxic adduct. This interaction is prevented by mutations that modify the active site of AGT to exclude BG.

DNA, Recombinant↗

PubMed2000

Copper(2+) binding to the surface residue cysteine 111 of His46Arg human copper-zinc superoxide dismutase, a familial amyotrophic lateral sclerosis mutant.

Mutations in copper-zinc superoxide dismutase (CuZnSOD) cause 25% of familial amyotrophic lateral sclerosis (FALS) cases. This paper examines one such mutant, H46R, which has no superoxide dismutase activity yet presumably retains the gain-of-function activity that leads to disease. We demonstrate that Cu(2+) does not bind to the copper-specific catalytic site of H46R CuZnSOD and that Cu(2+) competes with other metals for the zinc binding site. Most importantly, Cu(2+) was found to bind strongly to a surface residue near the dimer interface of H46R CuZnSOD. Cysteine was identified as the new binding site on the basis of multiple criteria including UV-vis spectroscopy, RR spectroscopy, and chemical derivatization. Cysteine 111 was pinpointed as the position of the reactive ligand by tryptic digestion of the modified protein and by mutational analysis. This solvent-exposed residue may play a role in the toxicity of this and other FALS CuZnSOD mutations. Furthermore, we propose that the two cysteine 111 residues, found on opposing subunits of the same dimeric enzyme, may provide a docking location for initial metal insertion during biosynthesis of wild-type CuZnSOD in vivo.

Amyotrophic Lateral Sclerosis↗

PubMed2000

Development of multichannel devices with an array of electrospray tips for high-throughput mass spectrometry.

The basic principles of multichannel devices with an array of electrospray tips for high-throughput infusion electrospray ionization mass spectrometry (ESI-MS) have been developed. The prototype plastic devices were fabricated by casting from a solvent-resistant resin. The sample wells on the device were arranged in the format of the standard 96-microtiter well plate, with each sample well connected to an independent electrospray exit port via a microchannel with imbedded electrode. A second plastic plate with distribution microchannels was employed as a cover plate and pressure distributor. Nitrogen gas was used to pressurize individual wells for transport of sample into the electrospray exit port. The design of independent microchannels and electrospray exit ports allowed very high throughput and duty cycle, as well as elimination of any potential sample carryover. The device was placed on a computer-controlled translation stage for precise positioning of the electrospray exit ports in front of the mass spectrometer sampling orifice. High-throughput ESI-MS was demonstrated by analyzing 96 peptide samples in 480 s, corresponding to a potential throughput of 720 samples/h. As a model application, the device was used for the MS determination of inhibition constants of several inhibitors of HIV-1 protease.

Angiotensins↗

PubMed2000

Association of disease with isolation and virulence of Rhodococcus equi from farm soil and foals with pneumonia.

OBJECTIVE: To determine whether isolation and virulence of Rhodococcus equi from soil and infected foals are associated with clinical disease. DESIGN: Cross-sectional and case-control study. SAMPLE POPULATION: R equi isolates from 50 foals with pneumonia and soil samples from 33 farms with and 33 farms without a history of R equi infection (affected and control, respectively). PROCEDURE: R equi was selectively isolated from soil samples. Soil and clinical isolates were evaluated for virulence-associated protein antigen plasmids (VapA-P) and resistance to the beta-lactam antibiotics penicillin G and cephalothin. Microbiologic cultures and VapA-P assays were performed at 2 independent laboratories. RESULTS: VapA-P was detected in 49 of 50 (98%) clinical isolates; there was complete agreement between laboratories. Rhodococcus equi was isolated from soil on 28 of 33 (84.8%) affected farms and 24 of 33 (72.7%) control farms, but there was poor agreement between laboratories. Virulence-associated protein antigen plasmids were detected on 14 of 66 (21.2%) farms by either laboratory, but results agreed for only 1 of the 14 VapA-P-positive farms. We did not detect significant associations between disease status and isolation of R equi from soil, detection of VapA-P in soil isolates, or resistance of soil isolates to beta-lactam antibiotics. No association between beta-lactam antibiotic resistance and presence of VapA-P was detected. CONCLUSIONS AND CLINICAL RELEVANCE: On the basis of soil microbiologic culture and VapA-P assay results, it is not possible to determine whether foals on a given farm are at increased risk of developing disease caused by R equi.

Actinomycetales Infections↗

PubMed2000

A second-generation synthesis of polypyrrolinone nonpeptidomimetics: prelude to the synthesis of polypyrrolinones on solid support.

[reaction: see text] A second-generation asymmetric synthesis of polypyrrolinones (3) has been achieved exploiting scalemic alpha-aminolactones (1) as building blocks. Imine formation between an appropriate lactone (1) and aldehyde (2), followed in turn by pyrrolinone ring construction promoted by KHMDS in the presence of 18-crown-6 and modified Swern oxidation furnished pyrrolinone aldehyde 3. This iterative, efficient three-step protocol paves the way for the synthesis of polypyrrolinones on solid support.

Aspartic Acid Endopeptidases↗

PubMed2000

Synthesis of polypyrrolinones on solid support.

[reaction: see text] An efficient, three-step iterative synthesis of polypyrrolinones has been achieved on solid support, setting the stage for the construction of a wide variety of libraries based on the pyrrolinone scaffold. Central to the approach is an effective end-game sequence featuring pyrrolinone ring construction with traceless release from the solid support.

Aldehydes↗

PubMed2000

Isolation and characterization of the human cyclin T1 promoter.

Cyclin T1 (CycT1) is a regulatory subunit of a general RNA polymerase II (RNAP II) elongation factor termed P-TEFb. The human immunodeficiency virus Tat protein directly associates with CycT1 to utilize CycT1/P-TEFb (also called TAK) for activation of RNAP II elongation of the integrated proviral genome. CycT1 mRNA and protein levels are induced in activated human peripheral blood lymphocytes and CycT1 protein levels are induced by a post-transcriptional mechanism when human U937 promonocytic cells are stimulated to differentiate into macrophage-like cells. To investigate mechanisms that regulate CycT1 RNA expression, we isolated the CycT1 promoter. Multiple transcription start sites were identified within 330 nucleotides upstream of the ATG initiation codon at +1. The CycT1 promoter lacks a TATA element and possesses high constitutive activity in plasmid transfection assays. Two distinct regions of the promoter were identified upstream of +1 that contain critical regulatory elements for CycT1 promoter function.

Base Sequence↗

PubMed2000

Genomic organization and characterization of promoter function of the human CDK9 gene.

CDK9 is the catalytic subunit of a general RNA polymerase II (RNAP II) elongation factor termed p-TEFb which is targeted by the human immunodeficiency virus (HIV) Tat protein to activate elongation of the integrated proviral genome. CDK9 mRNA and protein levels have been observed to be induced in activated peripheral blood lymphocytes, a cell type relevant to HIV infection. To investigate mechanisms that regulate CDK9 RNA expression, we isolated genomic sequences containing the human CDK9 gene and found that CDK9 coding sequences are interrupted by six introns. There is a major transcriptional start site located 79 nucleotides upstream of the ATG initiator codon at nucleotide +1. Nucleotides -352 to -1 contain all the transcriptional regulatory elements needed for full promoter activity in transient expression assays. The CDK9 promoter contains features characteristic of a housekeeping gene, including GC-rich sequences and absence of a functional TATA element. The CDK9 promoter possesses high constitutive activity and may therefore have utility in expression vectors or gene therapy vectors.

Base Sequence↗

PubMed2000

Microwave-assisted extraction of glycyrrhizic acid from licorice root.

In the present study, a microwave-assisted extraction (MAE) technique has been developed for the extraction of glycyrrhizic acid (GA) from licorice root. Various experimental conditions, such as extraction time, different ethanol and ammonia concentration, liquid/solid ratios, pre-leaching time before MAE and material size for the MAE procedure were investigated to optimize the efficiency of the extraction. Under appropriate MAE conditions, such as extraction times of 4-5min, ethanol concentrations of 50-60% (v/v), ammonia concentrations of 1-2% (v/v) and liquid/solid ratios of 10:1(ml/g), the recovery of GA from licorice root with MAE was equivalent with conventional extraction methods. Those methods include extraction at room temperature (ERT), the traditional Soxhlet extraction, heat reflux extraction and ultrasonic extraction. Due to the considerable savings in time and solvent, MAE was more effective than the conventional methods. This novel method is suitable for fast extraction of GA from licorice root.

Journal Article↗

PubMed2000

Natural resistance of human beta cells toward nitric oxide is mediated by heat shock protein 70.

Human beta cells exhibit increased resistance against nitric oxide (NO) radicals as compared with rodent islet cells. Here we tested whether endogenous heat shock protein 70 (hsp70) accounts for the resistance of human cells. Stable transfection of the human beta cell line CM with an antisense hsp70 mRNA-expressing plasmid (ashsp70) caused selective suppression (>95%) of spontaneously expressed hsp70 but not of hsc70 or GRP75 protein. ashsp70 transfection abolished the resistance of CM cells to the NO donors (Z)-1- (2-(2-aminoethyl)-N-(2-ammonioethyl)amino)diazen-1-ium -1,2-diolate and sodium nitroprusside and increased the proportions of necrotic cells 3-5-fold (p < 0.05) and of apoptotic cells about 2-fold (p < 0.01). Re-induction of hsp70 expression by heat shock re-established resistance to NO toxicity. hsp70 did not exert its protective effect at the level of membrane lipid integrity because radical induced lipid peroxidation appeared independent of hsp70 expression. However, after NO exposure only hsp70-deficient cells showed significantly decreased mitochondrial activity, by 40-80% (p < 0.01). These results suggest a key role of hsp70 in the natural resistance of human beta cells against NO induced injury, by preserving mitochondrial function. These findings provide important implications for the development of beta cell protective strategies in type 1 diabetes and islet transplantation.

Animals↗

PubMed2000

Molecular cloning and functional characterization of a novel mammalian sphingosine kinase type 2 isoform.

Sphingosine-1-phosphate (SPP) has diverse biological functions acting inside cells as a second messenger to regulate proliferation and survival, and extracellularly, as a ligand for G protein-coupled receptors of the endothelial differentiation gene-1 subfamily. Based on sequence homology to murine and human sphingosine kinase-1 (SPHK1), which we recently cloned (Kohama, T., Oliver, A., Edsall, L. , Nagiec, M. M., Dickson, R., and Spiegel, S. (1998) J. Biol. Chem. 273, 23722-23728), we have now cloned a second type of mouse and human sphingosine kinase (mSPHK2 and hSPHK2). mSPHK2 and hSPHK2 encode proteins of 617 and 618 amino acids, respectively, both much larger than SPHK1, and though diverging considerably, both contain the conserved domains found in all SPHK1s. Northern blot analysis revealed that SPHK2 mRNA expression had a strikingly different tissue distribution from that of SPHK1 and appeared later in embryonic development. Expression of SPHK2 in HEK 293 cells resulted in elevated SPP levels. d-erythro-dihydrosphingosine was a better substrate than d-erythro-sphingosine for SPHK2. Surprisingly, d, l-threo-dihydrosphingosine was also phosphorylated by SPHK2. In contrast to the inhibitory effects on SPHK1, high salt concentrations markedly stimulated SPHK2. Triton X-100 inhibited SPHK2 and stimulated SPHK1, whereas phosphatidylserine stimulated both type 1 and type 2 SPHK. Thus, SPHK2 is another member of a growing class of sphingolipid kinases that may have novel functions.

3T3 Cells↗

PubMed2000

BCL10 gene mutation in lymphoma.

BCL10 is directly involved in t(1;14)(p22;q32) of mucosa-associated lymphoid tissue (MALT) lymphoma. Wild-type BCL10 promoted apoptosis and suppressed malignant transformation in vitro, whereas truncated mutants lost the pro-apoptotic activity and exhibited gain of function enhancement of transformation. We studied 220 lymphomas for genomic BCL10 mutation by polymerase chain reaction-single-strand conformational polymorphism and DNA sequencing. Nineteen mutations were found in 13 lymphoma specimens, as follows: 8 of 120 (6.7%) mucosa-associated lymphoid tissue (MALT) lymphomas, 4 of 42 (9.5%) follicular lymphomas, and 1 of 23 (4.3%) diffuse large B-cell lymphomas. No mutations were found in 14 mantle cell lymphomas or 21 T-cell lymphomas. High-grade MALT lymphoma tended to show a slightly higher mutation frequency (2 of 25, 8%) than low-grade MALT tumor (6 of 95, 6.3%). Among low-grade gastric MALT lymphoma, mutations were found in 3 of 11 tumors that did not respond to Helicobacter pylori eradication therapy, but none were found in 22 tumors that regressed completely after H pylori eradication. All 14 potentially pathogenic mutations were distributed in the carboxyl terminal domain of BCL10. Deletion accounted for 10 of these mutations; 10 of 14 mutations caused truncated forms of BCL10. Western blot analysis of a mutant case confirmed the presence of truncated BCL10 products of anticipated size. Our results suggest that BCL10 mutation may play a pathogenic role in B-cell lymphoma development, particularly in aggressive and antibiotic unresponsive MALT lymphomas, and may further implicate the biologic importance of the carboxyl terminal of the molecule. (Blood. 2000;95:3885-3890)

Adaptor Proteins, Signal Transducing↗

PubMed2000

Sideward flow in Au+Au collisions between 2A and 8A GeV

Using the large acceptance Time Projection Chamber of experiment E895 at Brookhaven, measurements of collective sideward flow in Au+Au collisions at beam energies of 2A, 4A, 6A, and 8A GeV are presented in the form of in-plane transverse momentum and the first Fourier coefficient of azimuthal anisotropy v(1). These measurements indicate a smooth variation of sideward flow as a function of beam energy. The data are compared with four nuclear transport models which have an orientation towards this energy range. All four exhibit some qualitative trends similar to those found in the data, although none show a consistent pattern of agreement within experimental uncertainties.

Journal Article↗