Biomedical subjects

H Liu

Publications and source records attributed to H Liu.

At least 811 records · Page 45Linked to original sources

PubMed1997

Correlation between direct binding ability of synthetic T. gondii SAG1 peptides to HLA-A2 measured by a sensor for surface plasmon resonance and antigenicity of the peptides for T. gondii-infected cell-specific CTL.

Toxoplasma gondii (T. gondii) -infected B lymphoma cells present T. gondii antigens in the context of major histocompatibility complex molecules to T. gondii-specific CD8+ cytotoxic T cells (CTL). HLA-A2 molecules of T. gondii-infected human cells have been shown to be used in presenting T. gondii antigens to CD8+ CTL. SAG1, one of the major antigenic molecules of T. gondii, is an antigen for T. gondii-specific CTL, and represents a possible basis for vaccines. The direct binding of nonamer SAG1 peptides to HLA-A2 was assayed here using an automated biosensor system with a sensor for surface plasmon resonance detection. The antigenicity of synthetic SAG1 peptides to T. gondii-specific CD8+ CTL also was assayed. The present study found a high correlation between the binding ability of synthetic SAG1 peptides to HLA-A2 and the antigenicity of peptides to T. gondii-infected cell-specific CD8+ CTL.

Animals↗

PubMed1997

Temporal separation in the specification of primary and secondary motoneurons in zebrafish.

In zebrafish there are two populations of motoneurons, primary and secondary, that are temporally separate in their development. To determine if midline cells play a role in the specification of these neurons, we analyzed both secondary and primary motoneurons in mutants lacking floor plate, notochord, or both floor plate and notochord. Our data show that the specification of secondary motoneurons, those most similar to motoneurons in birds and mammals, depends on the presence of either a differentiated floor plate or notochord. In the absence of both of these structures, secondary motoneurons fail to form. In contrast, primary motoneurons, early developing motoneurons found in fish and amphibians, can develop in the absence of both floor plate and notochord. A spatial correspondence is found between secondary motoneurons and sonic hedgehog-expressing floor plate and notochord. In contrast, primary motoneuronal specification depends on the presence of sonic hedgehog in gastrula axial mesoderm, the tissue that will give rise to the notochord. These results suggest that both primary and secondary motoneurons are specified by signals from midline tissues, but at very different stages of embryonic development.

Animals↗

PubMed1997

Immobilization of Polymer-Stabilized Noble Metal Colloids and Their Catalytic Properties for Hydrogenation of Olefins

Polymer-stabilized noble metal colloids were efficiently immobilized on silica by the addition of organic acids under mild conditions. The function of organic acids in the immobilization was studied by infrared spectroscopy. Transmission electron micrographs indicate that the immobilized colloids have a controlled particle size and size distribution. They serve as catalysts in the hydrogenation of cyclohexene and cyclopentadiene, the results of which show that this new type of immobilized colloid has high selectivity and good stability.

Journal Article↗

PubMed1997

Regulation of platelet-activating factor (PAF) biosynthesis via coenzyme A-independent transacylase in the macrophage cell line IC-21 stimulated with lipopolysaccharide.

The regulation of PAF synthesis by the macrophage cell line IC-21 challenged with bacterial endotoxin was investigated. The LPS-induced increase in cellular PAF levels was rapid, sustained and attained maximal levels within 30 min following LPS stimulation. PAF accumulation was accompanied by the activation of the CoA-independent transacylase and acetyl-CoA: lyso-PAF acetyltransferase, whereas the release of free [3H]arachidonic acid in prelabeled cells reflecting the activation of phospholipase A2, occurred primarily within the initial 1-5 min of treatment with LPS. Cell lysates from LPS-stimulated macrophages exhibited a markedly increased enzymatic activity that was capable of both acylation of 1-[3H]alkyl-2-lyso-GPC (lyso-PAF) and deacylation of 1-[3H]alkyl-2-acyl-GPC generating [3H]lyso-PAF via CoA-independent transacylation of exogenous lysoplasmenylethanolamine compared with extracts from resting macrophages. Pretreatment of the cells with LPS for 5 and 30 min enhanced significantly the transfer of [14C]arachidonic acid from 1-[3H]alkyl-2-[14C]arachidonoyl-GPC into plasmenylethanolamine in prelabeled cell homogenates following the addition of exogenous lysoplasmenylethanolamine. Taken together, these data suggest that the CoA-independent transacylase, but not phospholipase A2, is a key enzyme responsible for the prolonged generation of lyso-PAF and that the increased capability of CoA-independent transacylation followed by CoA-dependent acetylation of lyso-PAF can sustain the biosynthesis of PAF in LPS-stimulated IC-21 macrophages.

Acetyltransferases↗

PubMed1997

Novel green fluorescent protein (GFP) baculovirus expression vectors.

Baculovirus expression vectors were constructed, which contained gfp as a reporter gene. Substitutions in the amino acid sequences were carried out to produce two mutant forms of GFP. One of these mutants produces blue color when excited by ultraviolet (UV) light. The other mutant produces a yellow color that can be visualized in regular daylight. The gene of interest cloned in-frame with the gfp open reading frame allows visualization of the produced GFP-fusion protein using UV light. The presence of an in-frame 6 x His tag between the gfp cDNA and the multiple cloning site allows purification of the fusion protein on a Ni-NTA (nickel-nitrilo-triacetic acid) agarose matrix.

Amino Acid Sequence↗

PubMed1997

NMDA-receptor regulation of substance P release from primary afferent nociceptors.

Severe or prolonged tissue or nerve injury can induce hyperexcitability of dorsal horn neurons of the spinal cord, resulting in persistent pain, an exacerbated response to noxious stimuli (hyperalgesia), and a lowered pain threshold (allodynia). These changes are mediated by NMDA (N-methyl-D-aspartate)-type glutamate receptors in the spinal cord. Here we report that activation of the NMDA receptor causes release of substance P, a peptide neurotransmitter made by small-diameter, primary, sensory 'pain' fibres. Injection of NMDA in the cerebrospinal fluid of the rat spinal cord mimicked the changes that occur with persistent injury, and produced not only pain, but also a large-scale internalization of the substance P receptor into dorsal horn neurons, as well as structural changes in their dendrites. Both the pain and the morphological changes produced by NMDA were significantly reduced by substance P-receptor antagonists or by elimination of substance P-containing primary afferent fibres with the neurotoxin capsaicin. We suggest that presynaptic NMDA receptors located on the terminals of small-diameter pain fibres facilitate and prolong the transmission of nociceptive messages, through the release of substance P and glutamate. Therapies directed at the presynaptic NMDA receptor could therefore ameliorate injury-evoked persistent pain states.

2-Amino-5-phosphonovalerate↗

PubMed1997

Chemical modification and site-directed mutagenesis of the single cysteine in motif 3 of class II Escherichia coli prolyl-tRNA synthetase.

Class II prolyl-tRNA synthetase (ProRS) from Escherichia coli contains all three of the conserved consensus motifs characteristic of class II aminoacyl-tRNA synthetases. In this study, chemical modification and site-directed mutagenesis of the single cysteine located at position 443 in motif 3 of Escherichia coli ProRS is carried out. We show that chemical modification of C443 blocks the ability of the enzyme to form the activated aminoacyl-adenylate, a prerequisite for tRNA(Pro) aminoacylation. Nearly complete protection from inactivation is achieved by preincubating the enzyme with ATP or ATP and proline, but not proline alone or tRNA(Pro). Mutagenesis of C443 to amino acids Ala, Gly, and Ser resulted in significant decreases (16-225-fold) in k(cat)/K(M)(Pro) as measured by the ATP-PP(i) exchange reaction. The Ala and Gly mutations have a relatively small effect (4-7-fold) on the overall aminoacylation reaction, while the activity of the C443S mutant in this same assay is substantially reduced (80-fold). A sequence comparison of the motif 3 region of class II synthetases shows that C443 aligns with residues that have been implicated in amino acid binding specificity. The results of our study suggest that while the thiol located at position 443 of Escherichia coli ProRS is not essential for catalysis, this residue is likely to be in a buried region that forms the prolyl-adenylate substrate binding pocket.

Amino Acid Sequence↗

PubMed1997

Nerve growth factor decreases soluble guanylate cyclase in rat pheochromocytoma PC12 cells.

Nitric oxide (NO) modulates neurotransmission in the central and peripheral nervous systems. NO acts, in part, by stimulating cGMP production by soluble guanylate cyclase (sGC), an obligate heterodimer composed of alpha and beta subunits. To investigate mechanisms that regulate responsiveness to NO in the nervous system, sGC regulation was examined in a rat pheochromocytoma cell line (PC12) exposed to nerve growth factor (NGF). NGF decreased sGC alpha1 and beta1 subunit mRNA and protein levels as well as NO-stimulated sGC enzyme activity. The NGF-mediated decrease in sGC subunit mRNA levels was blocked by 5'-deoxy-5'-methylthioadenosine (an inhibitor of NGF-induced tyrosine phosphorylation). NGF did not decrease sGC subunit mRNA levels in PC12 cells containing a mutant Ras protein that blocks Ras-dependent intracellular signaling. Incubation of PC12 cells with a transcription inhibitor (actinomycin D) or protein synthesis inhibitors (anisomycin or cycloheximide) attenuated the ability of NGF to decrease sGC subunit mRNA levels. Moreover, sGC subunit mRNA levels decreased more rapidly in NGF-treated cells than in actinomycin D-treated cells, suggesting that NGF decreases sGC subunit mRNA stability. Thus, NGF decreases sGC subunit mRNA levels via mechanisms that are dependent on protein tyrosine phosphorylation and Ras activation. The effect of NGF on sGC subunit mRNA stability appears to be transcription- and translation-dependent. Modulation of sGC subunit levels and enzyme activity in PC12 cells suggests that NO responsiveness may be regulated in the nervous system by NGF.

Animals↗

PubMed1997

Direct binding of G-protein betagamma complex to voltage-dependent calcium channels.

Voltage-dependent Ca2+ channels play a central role in controlling neurotransmitter release at the synapse. They can be inhibited by certain G-protein-coupled receptors, acting by a pathway intrinsic to the membrane. Here we show that this inhibition results from a direct interaction between the G-protein betagamma complex and the pore-forming alpha1 subunits of several types of these channels. The interaction is mediated by the cytoplasmic linker connecting the first and second transmembrane repeats. Within this linker, binding occurs both in the alpha1 interaction domain (AID), which also mediates the interaction between the alpha1 and beta subunits of the channel, and in a second downstream sequence. Further analysis of the binding site showed that several amino-terminal residues in the AID are critical for Gbetagamma binding, defining a site distinct from the carboxy-terminal residues shown to be essential for binding the beta-subunit of the Ca2+ channel. Mutation of an arginine residue within the N-terminal motif abolished betagamma binding and rendered the channel refractory to G-protein modulation when expressed in Xenopus oocytes, showing that the interaction is indeed responsible for G-protein-dependent modulation of Ca2+ channel activity.

Amino Acid Sequence↗

PubMed1997

Influenza neuraminidase inhibitors possessing a novel hydrophobic interaction in the enzyme active site: design, synthesis, and structural analysis of carbocyclic sialic acid analogues with potent anti-influenza activity.

The design, synthesis, and in vitro evaluation of the novel carbocycles as transition-state-based inhibitors of influenza neuraminidase (NA) are described. The double bond position in the carbocyclic analogues plays an important role in NA inhibition as demonstrated by the antiviral activity of 8 (IC50 = 6.3 microM) vs 9 (IC50 > 200 microM). Structure-activity studies of a series of carbocyclic analogues 6a-i identified the 3-pentyloxy moiety as an apparent optimal group at the C3 position with an IC50 value of 1 nM for NA inhibition. The X-ray crystallographic structure of 6h bound to NA revealed the presence of a large hydrophobic pocket in the region corresponding to the glycerol subsite of sialic acid. The high antiviral potency observed for 6h appears to be attributed to a highly favorable hydrophobic interaction in this pocket. The practical synthesis of 6 starting from (-)-quinic acid is also described.

Acetamides↗

PubMed1997

Functional status among survivors of in-hospital cardiopulmonary resuscitation. SUPPORT Investigators Study to Understand Progress and Preferences for Outcomes and Risks of Treatment.

OBJECTIVES: To describe functional outcomes of seriously III patients who survived 2 months after in-hospital cardiopulmonary resuscitation (CPR) and to identify patient and clinical characteristics associated with worse functional status after CPR. METHODS: Multicenter prospective observational analysis of 162 seriously ill hospitalized patients who survived 2 months after CPR. Analysis of clinical characteristics associated with worse functional outcome. RESULTS: Among 162 survivors of in-hospital CPR, 56% had the same or improved function and 44% had worse function at 2 months compared with functional status before CPR. Patients with worse function deteriorated by a mean of 3.9 activities of daily living and were less likely to survive to hospital discharge (P < .001) or to 6 months after study entry (P < .001). Worse functional outcome was associated with greater age and longer hospital stay before CPR. CONCLUSIONS: More than half of CPR survivors had preserved functional status 2 months after CPR. However, patients with worse function are profoundly disabled. In anticipation of possible severe disability after CPR, preferences for care in such health states should be discussed with patients before the need for CPR, particularly among older patients and those with long hospital stays.

Age Factors↗

PubMed1997

The reaction pathway of the isomerization of D-xylose catalyzed by the enzyme D-xylose isomerase: a theoretical study.

Different pathways of the metal-induced isomerization of D-xylose to D-xylulose are investigated and compared in detail using energy minimization and molecular dynamics simulation. Two theoretical models are constructed for the reaction: in vacuum and in the enzyme D-xylose isomerase. The vacuum model is constructed based on the X-ray structure of the active site of D-xylose isomerase. It contains the atoms directly involved in the reaction and is studied using a semi-empirical molecular orbital method (PM3). The model in the enzyme includes the effects of the enzyme environment on the reaction using a combined quantum mechanical and molecular mechanical potential. For both models, the structures of the reactants, products, and intermediate complexes along the isomerization pathway are optimized. The effects of the position of the "catalytic Mg2+ ion" on the energies of the reactions are studied. The results indicate: 1) in vacuum, the isomerization reaction is favored when the catalytic metal cation is at site A, which is remote from the substrate; 2) in the enzyme, the catalytic metal cation, starting from site A, moves and stays at site B, which is close to the substrate; analysis of the charge redistribution of the active site during the catalytic process shows that the metal ion acts as a Lewis acid to polarize the substrate and catalyze the hydride shift; these results are consistent with previous experimental observations; and 3) Lys183 plays an important role in the isomerization reaction. The epsilon-NH3+ group of its side chain can provide a proton to the carboxide ion of the substrate to form a hydroxyl group after the hydride shift step. This role of Lys183 has not been suggested before. Based on our calculations, we believe that this is a reasonable mechanism and consistent with site-directed mutation experiments.

Aldose-Ketose Isomerases↗

PubMed1997

bcl-2 enhancement of malignant transformation in mouse epidermal JB6 cells.

Increased bcl-2 expression is a common feature of many types of human malignancies, which implies that bcl-2 plays an important role in tumorigenesis. To better understand the molecular mechanisms of bcl-2-induced oncogenesis, we examined the effects of bcl-2 expression on transformation of mouse epidermal JB6 cells induced by the tumor promoter 12-O-tetradecanoylphorbol-13 acetate (TPA). Promotion-sensitive JB6 clone41 cells were transfected with the bcl-2-containing expression vector pD5-neo/bcl-2, and the soft agar growth of bcl-2-transfected cells and control cells were compared. bcl-2 overexpression in JB6 clone41 cells caused a TPA-induced soft-agar growth fivefold greater than the growth of nontransfected or vector-transfected (neo control) cells. bcl-2 expression in the absence of TPA did not lead to colony formation in soft agar. Because the level of the transcription factor activator protein 1 (AP-1) has been shown to be critical for the responsiveness of JB6 cells to TPA-induced transformation, we compared c-jun and c-fos expression as well as the AP-1-binding activity and the AP-1-mediated transactivation of the reporter construct TRE-CAT between bcl-2-expressing cells and control cells. When compared with control cells, bcl-2-transfected cells expressed significantly more c-fos but not c-jun after TPA treatment. Furthermore, the levels of AP-1 and AP-1-induced transactivation of TRE-CAT were greater in bcl-2-transfected cells than in control cells after TPA treatment. These results showed that bcl-2 cooperates with a tumor promoter such as TPA in the induction of malignant transformation in mouse epidermal cells and that bcl-2 enhances soft-agar growth by stimulating signaling pathways that led to increased AP-1 expression.

Animals↗

PubMed1997

Genome analysis in Neurospora crassa; cloning of four loci arginine-1 (arg-1), methionine-6 (met-6), unknown-7 (un-7), and ribosome production-1 (rip-1) and associated chromosome walking.

We have cloned four Neurospora crassa genes by complementation analysis. Cloned genes include the arginine-1 (arg-1), methionine-6 (met-6), unknown-7 (un-7), and ribosome production-1 (rip-1) loci. Chromosome walks were initiated in ordered cosmid libraries from the cloned loci. A total of about 700 kb of the Neurospora genome is covered in these walks.

Arginine↗

PubMed1997

Genome analysis on linkage group VI of Neurospora crassa.

Two chromosome walks covering 420 and 110 kb on the left arm of linkage group VI (LGVIL) of Neurospora crassa were purscrooued with the goal of cloning carotenogenic loci. Complementation analysis with clones isolated in the 420-kb walk allowed identification of the yellow-1 (ylo-1) gene which is essential for Neurospora carotenogenesis. We have physically located a second gene, unknown-13 (un-13), between the cross-pathway control-1, (cpc-1) and ylo-1 loci. Cloning of a second potential carotenogenic locus, vivid (vvd), from our walks was attempted using screening of Northern blots with radiolabeled DNA fragments from walk clones to identify gene transcripts. The radiolabeled DNA fragments were used to clone complementary DNA isolates representing an additional four genes in the two walks.

Carotenoids↗

PubMed1997

Cyclic nucleotides regulate the activity of L-type calcium channels in smooth muscle cells from rat portal vein.

In order to advance our previous findings that the macroscopic slow Ca2+ currents of vascular smooth muscle (VSM) cells are regulated by cyclic nucleotides, the effects of cAMP and cGMP on the activity of single slow (L-type) Ca2+ channels were investigated using cell-attached patch clamp (22-25 degrees C). Freshly isolated VSM cells were obtained from adult male rat portal vein. For the single-channel recordings, the pipette was filled with a solution containing 90 mM Ba2+ and 1 microM Bay-K-8644 solution, and the bath contained 140 mM KCl to "zero" the membrane potential. Depolarizing pulses to 0 mV, from a holding potential (HP) of -80 mV, elicited inward unitary currents. The activity of these channels was completely blocked by superfusion of 10 microM nifedipine. Extracellular perfusion of the single cells with membrane-permeable cGMP and cAMP analogs (8Br-cGMP and 8Br-cAMP) at 1 mM caused a slight inhibition, but higher doses (3 mM), clearly showed an inhibitory effect on the single-channel activity. cAMP (100 microM) stimulated one out of five patches tested, and 100 microM cGMP showed no effect in three patches tested. Compared with control, both cyclic nucleotides at 3 mM decreased the ensemble-averaged currents by 26.7 +/- 4.1% and 37.3 +/- 2.1%, respectively. Unit amplitude and slope conductance were not changed. The normal conductance of the Ca2+ channel was 20.8 +/- 0.04 pS (n = 9), and the conductances in the presence of cAMP (n = 5) and cGMP (n = 6) were 19.3 +/- 0.04 and 20.5 +/- 0.05 pS, respectively. Single-channel kinetic analysis showed that cAMP did not affect the mean open-time, and cGMP slightly decreased the mean open-time. However, both cAMP and cGMP increased the mean closed-time. In addition, cAMP decreased the open probability (NPo) by a factor of 1.7, from 0.26 +/- 0.04 to 0.15 +/- 0.03 (P < 0.05, Student's t-test) and cGMP decreased NPo by a factor of 2.5, from 0.24 +/- 0.08 to 0.10 +/- 0.02 (P < 0.05). H-7, a non-specific protein kinase inhibitor, prevented the inhibitory effects of both cAMP and cGMP on the activity of single Ca2+ channels in rat portal vein cells. The results demonstrate that both cAMP and cGMP inhibit L-type Ca2+ channel activities in VSM cells from rat portal vein. This inhibition may be mediated by the cAMP and cGMP-dependent protein kinase phosphorylation of the L-type Ca2+ channels (or an associated regulatory protein).

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1997

Ethylisopropylamiloride diminishes changes in intracellular Na, Ca and pH in ischemic newborn myocardium.

Numerous studies suggest that in adult hearts myocardial ischemic injury is in part the result of proton stimulation of Na/H exchange which increases intracellular Na (Nai) and thus leads to increases in intracellular Ca concentration ( [Ca]i) due to changes in Na/Ca exchange flux. Corollary to the hypothesis, inhibition of Na/H exchange diminishes Na and Ca accumulation and improves heart function after ischemia. To test this hypothesis and its corollary in newborn hearts, NMR spectroscopy was used to measure intracellular pH (pHi), Nai, [Ca]i, and high energy phosphates in isolated, 4-7-day-old rabbit hearts, Langendorff-perfused with Krebs-Henseleit solution at pH 7.4+/-0.5 equilibrated with 95% O2/5% CO2 at 36+/-1 degrees C. Control hearts were perfused for 30 min before initiating 40 min of global ischemia followed by 40 min of reperfusion. In a second group of hearts ethylisopropylamiloride (EIPA-10 microM) was added to the perfusate 20 min before global ischemia to inhibit Na/H exchange. After 15 min ischemia, pHi in EIPA-treated hearts (6.41+/-0.04) was higher than that of the control hearts (6.20+/-0.08; P<0.05). EIPA also limited the increase in Nai and [Ca]i during ischemia and improved Nai and [Ca]i recovery during reperfusion (P<0.05). Nai (mEq/kg dry weight) rose from 18. 1+/-3.2 to 110.6+/-14.0 and recovered to 53.3+/-12.3 in the control group. The corresponding Nai values for EIPA-treated hearts were 16. 2+/-2.4, 39.6+/-9.6 and 12.6+/-3.5, respectively. In control hearts [Ca]i (nM/l) rose from 332+/-42 to 1157+/-89 and recovered to 842+/-55, whereas in EIPA-treated hearts the values were 255+/-32, 616+/-69 and 298+/-34, respectively. EIPA also preserved cellular ATP during ischemia and reperfusion and diminished inorganic phosphate during reperfusion (P<0.05). Finally, EIPA treatment improved recovery of left ventricular developed pressure (68.2+/-8.9 v 16.2+/-3.6% of control) and limited myocardial injury as indicated by decreased total creatine kinase release during reperfusion (348+/-132 v 2432+/-639 IU/g dry weight). Thus, as in adults, the results from newborn hearts are consistent with the hypothesis.

Adenosine Triphosphate↗

PubMed1997

Rotational correlation times of internuclear vectors in a DNA duplex with G-A mismatch determined in aqueous solution by complete relaxation matrix analysis of off-resonance ROESY (O-ROESY) spectra.

Complete relaxation matrix analysis of the off-resonance ROESY (O-ROESY) spectra is presented and demonstrated for a synthetic DNA duplex with two G-A mismatches, d(GCTGTC-GAAAGC)2, in solution. The internuclear distance and the rotational correlation time of the internuclear vector could be garnered simultaneously using complete relaxation matrix analysis of O-ROESY, by which spin diffusion effects could be accommodated. Correlation times in the terminal and the mismatched regions were significantly reduced compared to those in other regions, indicating the conformational flexibility of the mismatched pair. The average structure obtained by restrained molecular dynamics simulation with inclusion of variations of the rotational correlation times also indicated a general tendency of the mismatched and contiguous bases to flip to the outside of the double strand. Off-resonance ROESY combined with the complete relaxation matrix analysis method may offer an alternative way to investigate the structures and dynamics of biological macromolecules.

DNA↗