Biomedical subjects

X Liu

Publications and source records attributed to X Liu.

At least 973 records · Page 54Linked to original sources

PubMed1998

Determination of polyamines in human prostate by high-performance liquid chromatography with fluorescence detection.

A high-performance liquid chromatographic method for the determination of polyamines in human prostate has been developed. This method is based on pre-column derivatization with dansyl chloride (Dns-Cl). The derivatives were separated on a muBondapak C18 column (250x4.6 mm I.D.; 10 microm), and eluted with methanol and distilled water using a one-step linear gradient. The column eluate was monitored by fluorescence detection (excitation, 370 nm; emission, 506 nm). The within-assay precision of the study (C.V.) was as follows: putrescine (PUT) 2.88%, spermidine (SPD) 2.94% and spermine (SP) 1.17%. The between-assay precision (C.V.) was: PUT 2.66%, SPD 3.06%, SP 2.79%. The recovery was greater than 97%. The detection limit for PUT, SPD and SP were 0.05, 0.08 and 0.06 nmol/ml, respectively. In contrast to other studies, sample or polyamine derivatives did not require extraction with an organic solvent such as ethanol, evaporation under vacuum or other condensation procedures. This is a simple, rapid and sensitive method that can be applied to the determination of polyamines in nearly all biological tissues and body fluids, such as urine and serum.

Chromatography, High Pressure Liquid↗

PubMed1998

Identification of amino acid residues of Gsalpha critical to repression of adipogenesis.

Gsalpha regulates the differentiation of 3T3-L1 mouse embryonic fibroblasts to adipocytes, a process termed adipogenesis. Through the expression of chimera created by substituting regions of Gsalpha with corresponding regions of the G protein Gialpha2, the domain of Gsalpha involved in repression of adipogenesis was localized to sequence 146-235 of the molecule (Wang, H-y., Johnson, G. L., Liu, X. , Malbon, C. C. (1996) J. Biol. Chem. 271, 22022-22029). As a prelude to alanine-scanning mutagenesis, chimeras in Gsalpha constructed from trisection of the sequence 125-213 of Gialpha2 were expressed stably, and clones were evaluated for the ability of the chimera to repress adipogenesis in response to the inducers, dexamethasone and methylisobutylxanthine, in combination. The chimera containing sequence 150-177 of Gialpha2 repressed adipogenesis, whereas the chimeras with either sequence 125-149 or 178-213 of Gialpha2 failed to repress induction of adipogenesis. Alanine-scanning mutagenesis of these two critical domains was performed first in clusters and then confirmed by analysis of single mutations. Six residues unique to Gsalpha were identified as critical to repression of adipogenesis, Asn167, Cys200, Leu203, Ser205, Val214, and Lys216. Leu203 and Ser205 are required in tandem, as mutagenesis to alanine of either one alone was without effect on repressor activity. The remaining four residues are required for repressor activity; mutation of any one of these abolishes the ability of Gsalpha to repress adipogenesis, although not affecting the ability of the mutant form of Gsalpha to regulate adenylylcyclase. Using conserved landmarks found in the crystal structures of Gialpha1 and Gsalpha, the Leu203 and Ser205 cluster appears to be exposed, closely aligned and located in switch I region. Asn167, Val214, and Lys216 project to regions on Gsalpha that are exposed in the GTPgammaS-liganded state of the alpha subunit. We speculate that these residues constitute an important contact domain between Gsalpha and the effector controlling adipogenesis, which is yet to be identified.

3T3 Cells↗

PubMed1998

Structure of the complex of bovine pancreatic phospholipase A2 with a transition-state analogue.

The 1.89 A resolution structure of the complex of bovine pancreatic phospholipase A2 (PLA2) with the transition-state analogue L-1-O-octyl-2-heptylphosphonyl-sn-glycero-3-phosphoethanolamine (TSA) has been determined. The crystal of the complex is trigonal, space group P3121, a = b = 46.58 and c = 102.91 A and isomorphous to the native recombinant wild type (WT). The structure was refined to a final crystallographic R value of 18.0% including 957 protein atoms, 88 water molecules, one calcium ion and all 31 non-H atoms of the inhibitor at 1.89 A resolution. In all, 7 726 reflections [F>2sigma(F)] were used between 8.0 and 1.89 A resolution. The inhibitor is deeply locked into the active-site cleft and coordinates to the calcium ion by displacing the two water molecules in the calcium pentagonal bipyramid by the anionic O atoms of the phosphate and phosphonate group. The hydroxyl group of Tyr69 hydrogen bonds to the second anionic O atom of the phosphate group while that of the phosphonate group replaces the third water, 'catalytic' water, which forms a hydrogen bond to Ndelta1 of His48. The fourth water which also shares Ndelta1 of His48 is displaced by the steric hinderance of the inhibitor. The fifth conserved structural water is still present in the active site and forms a network of hydrogen bonds with the surrounding residues. The structure is compared to the other known TSA-PLA2 complexes.

Amino Acid Sequence↗

PubMed1998

Regulation of mammary differentiation by extracellular matrix involves protein-tyrosine phosphatases.

Extracellular matrix and growth factors cooperate to regulate signaling pathways and gene transcription in adherent cells. However, the mechanism of extracellular matrix signaling is poorly defined. In mammary gland, the expression of milk protein genes is controlled by cross-talk between signals derived from the basement membrane protein, laminin, and the lactogenic hormone, prolactin. Signals from basement membrane are transduced by beta1 integrins and are required for prolactin to activate DNA binding of the milk protein gene transcription factor, Stat5. Here we show that basement membrane is necessary for tyrosine phosphorylation of the prolactin receptor and thus directly affects cytokine signaling and differentiation at the level of the plasma membrane. Prolactin does not induce tyrosine phosphorylation of its receptor, Jak2, or Stat5 in nondifferentiated breast epithelia cultured on collagen I, and we show that this is due to a vanadate-sensitive activity that inhibits the prolactin pathway. We suggest that protein-tyrosine phosphatases are novel targets for regulation by extracellular matrix and in mammary cells represent an additional control to the requirement of integrins for milk protein production.

Animals↗

PubMed1998

[Polymorphism analysis of 4 loci of X-chromosome in a Chinese population of the Han nationality].

OBJECTIVE: To investigate the polymorphism of 4 loci of X-chromosome in the Hans. METHODS: Using PCR-SSLP, the authors analysed the polymorphism of DXS1068, DXS7132, DXS6804 and DXS6799 in the X-chromosome in 70 randomly selected female Hans. RESULTS: The number of alleles in the 4 loci were 5,5,5 and 6 respectively; there were no significant differences between the observed and estimated and genotype probabilities of the 4 loci; the estimates of heterozygosity of 4 loci were 0.6819, 0.6895, 0.7659 and 0.6483 respectively and there were no significant differences between the observed and estimated heterozygosity of the 4 loci. CONCLUSION: The distribution of alleles and genotype probabilities of 4 loci all observe the Hardy-Weinberg equilibrium.

Asian People↗

PubMed1998

Functional association between Arf and RalA in active phospholipase D complex.

Activation of phospholipase D1 (PLD1) by Arf has been implicated in vesicle transport and membrane trafficking. PLD1 has also been shown to be associated with the small GTPase RalA, which functions downstream from Ras in a Ras-RalA GTPase cascade that facilitates intracellular signal transduction. Although PLD1 associates directly with RalA, RalA has no effect upon the activity of PLD1. However, PLD1 precipitated from cell lysates with immobilized glutathione S-transferase-RalA fusion protein is active. This suggests the presence of an additional activating factor in the active RalA-PLD1 complexes. Because Arf stimulates PLD1, we looked for the presence of Arf in the active RalA-PLD1 complexes isolated from v-Src- and v-Ras-transformed cell lysates. Low levels of Arf protein were detected in RalA-PLD1 complexes; however, if guanosine 5'-[gamma-thio]triphosphate was added to activate Arf and stimulate translocation to the membrane, high levels of Arf were precipitated by RalA from cell lysates. Interestingly, deletion of 11 amino-terminal amino acids unique to Ral GTPases, which abolished the ability of RalA to precipitate PLD activity, prevented the association between RalA and Arf. Brefeldin A, which inhibits Arf GDP-GTP exchange, inhibited PLD activity in v-Src- and v-Ras-transformed cells but not in the nontransformed cells, suggesting that the association of Arf with RalA is required for the increased PLD activity induced by v-Src and v-Ras. These data implicate Arf in the transduction of intracellular signals activated by v-Src and mediated by the Ras-RalA GTPase cascade. Because both Arf and PLD1 stimulate vesicle formation in the Golgi, these data raise the possibility that vesicle formation and trafficking may play a role in the transduction of intracellular signals.

3T3 Cells↗

PubMed1998

Accelerated reaction of nitric oxide with O2 within the hydrophobic interior of biological membranes.

We demonstrate herein dramatic acceleration of aqueous nitric oxide (NO) reaction with O2 within the hydrophobic region of either phospholipid or biological membranes or detergent micelles and demonstrate that the presence of a distinct hydrophobic phase is required. Per unit volume, at low amounts of hydrophobic phase, the reaction of NO with O2 within the membranes is approximately 300 times more rapid than in the surrounding aqueous medium. In tissue, even though the membrane represents only 3% of the total volume, we calculate that 90% of NO reaction with O2 will occur there. We conclude that biological membranes and other tissue hydrophobic compartments are important sites for disappearance of NO and for formation of NO-derived reactive species and that attenuation of these potentially damaging reactions is an important protective action of lipid-soluble antioxidants such as vitamin E.

Animals↗

PubMed1998

Tachycardia preconditions infarct size in dogs: role of adenosine and protein kinase C.

BACKGROUND: Myocardial ischemic preconditioning is a well-known phenomenon, however there is scant information in regard to nonischemic preconditioning. METHODS AND RESULTS: We studied in anesthetized dogs the preconditioning effect of tachycardia and the mediation of adenosine and protein kinase C in this process. In a control group the anterior descending coronary artery was occluded for 60 minutes and reperfused for 270 minutes. Heart rate was kept constant at 120 +/- 5 cycles/min and aortic pressure changes were damped. The infarct size (necrotic volume/risk region volume x 100) was 15.8 +/- 1.5%. In another group of dogs a similar protocol was followed, but five periods of tachycardia (213 +/- 12 cycles/min), 5 minutes in duration each, with 5 minutes of intervening periods at control heart rate, were induced previous to the coronary occlusion. The infarct size was reduced by 46% (P<.001) with respect to the nonpreconditioned group. This effect was not due to changes in collateral flow nor risk region size. During tachycardia, myocardial interstitial adenosine increased about twofold (P<.05); no metabolic, hemodynamic, or ECG evidences of ischemia were observed and the transmural vasodilatory reserve was preserved. The blockade of adenosine receptors with 8 phenyltheophylline, before or after the preconditioning tachycardia, reverted its protecting effect but it did not modify infarct size in nonpreconditioned dogs. No changes in cytosolic or particulate protein kinase C activity or translocation of alpha-, beta-, epsilon-, and zeta- protein kinase C isozyme by effect of tachycardia or ischemia were observed between preconditioned and nonpreconditioned dogs. CONCLUSIONS: Tachycardia, in the absence of ischemia, mimics the preconditioning effect of ischemia in the dog. This effect is mediated by adenosine but not by changes in protein kinase C activity or its translocation.

Adenosine↗

PubMed1998

Ischaemia and reperfusion injury of rat liver increases expression of glutathione S-transferase A1/A2 in zone 3 of the hepatic lobule.

Effects of ischaemia-reperfusion injury (I/R) of liver on expression of rat glutathione S-transferase (rGST) isoenzymes that metabolize products of oxidative stress were examined. Rats underwent lobar liver ischaemia for 30 min followed by reperfusion. In ischaemic lobes, rGSTA1/A2 transcript levels increased significantly 12 h after I/R (2.94-fold) and protein levels increased significantly at 24 h (1.45-fold); increased transcript levels were also observed in nonischaemic lobes (1.78-fold). Superoxide dismutase prevented I/R and the increases in transcript and protein levels in ischaemic and non-ischaemic lobes. By in-situ hybridization, increases in transcript levels at 6 h were present in zones 2 and 3 of the ischaemic lobes and peaked at 12 h (2.5-fold zone 2, 4.5-fold zone 3). Significant increases in transcript levels also were observed at 24 h in zones 2 (2.0-fold) and 3 (2.9-fold) of non-ischaemic lobes. Nuclear run-off assays showed a 1.8-fold increase in rGSTA1/A2 transcription rates in ischaemic lobes at 3 h. We conclude that I/R causes increased rGSTA1/A2 expression in the zone of the hepatic lobule most susceptible to oxidative injury and that this expression may be an important defence against injury.

Animals↗

PubMed1998

The anemic Friend virus gp55 envelope protein induces erythroid differentiation in fetal liver colony-forming units-erythroid.

The gp55 envelope proteins of the spleen focus-forming virus initiate erythroleukemia in adult mice. Because the gp55 from the polycythemic strain (gp55-P), but not from the anemic strain (gp55-A), activates the erythropoietin receptor (EpoR) for proliferation of hematopoietic cell lines, the mechanism by which gp55-A initiates erythroleukemia has remained a mystery. We show here that gp55-A activates the EpoR in fetal liver cells. In contrast to previous studies using bone marrow cells from phenylhydrazine-treated, anemic mice, we find that both gp55-A and gp55-P induce erythroid differentiation from colony-forming unit-erythroid (CFU-E) progenitors in fetal liver cells. The effects on CFU-Es of both gp55-A and -P are mediated by the EpoR, because no colonies are seen upon expression of either gp55 in EpoR-/- fetal liver cells. However, only gp55-P induces erythroid bursts from burst-forming unit-erythroid progenitors and only gp55-P induces Epo independence in Epo-dependent cell lines. Using chimeric gp55 P/A proteins, we extend earlier work showing that the transmembrane sequence determines the capacity of gp55 proteins to differentially activate EpoR signaling. We discuss the possibilities for different signaling capacities of gp55-A and -P in fetal liver and bone marrow-derived erythroid progenitor cells.

Animals↗

PubMed1998

Complete suppresion of ethanol-induced formation of megamitochondria by 4-hydroxy-2,2,6,6-tetramethyl-piperidine-1-oxyl (4-OH-TEMPO).

An attempt has been made to suppress the ethanol-induced formation of megamitochondria (MG) in the rat liver by 4-hydroxy-2,2,6,6-tetramethyl-piperidine-1-oxyl (4-OH-TEMPO), a free radical scavenger, and by allopurinol (AP), a xanthine oxidase inhibitor. Changes observed in the liver of animals given ethanol (EtOH) for 1 month were remarkable decreases both in the body weight gains during the course of the experiment and in the liver weight at the time of sacrifice compared to those of the control; remarkable increases in the level of thiobarbituric acid reactive substances and lipid soluble fluorophores both in microsomes and mitochondria; decreases in the content of cytochrome a+a3 and b and lowered phosphorylating ability of mitochondria; and formation of MG in the liver. A combined treatment of animals with EtOH plus 4-OH-TEMPO completely suppressed the formation of MG in the liver induced by EtOH and distinctly improved the changes caused by EtOH, as specified above, while AP partly suppressed the MG formation. Results described herein provide additional insight into chronic hepatotoxicity of EtOH besides that previously reported. A novelty of the present work is that we were able for the first time to demonstrate reversibility of EtOH-mediated ultrastructural changes of the liver by a simple administration of aminoxyl-type free radical scavenger, 4-OH-TEMPO. Our results suggest that free radicals may be involved in the mechanism of the formation of MG induced by EtOH.

Allopurinol↗

PubMed1998

Myosin VIIa as a common component of cilia and microvilli.

The distribution of myosin VIIa, which is defective or absent in Usher syndrome 1B, was studied in a variety of tissues by immunomicroscopy. The primary aim was to determine whether this putative actin-based mechanoenzyme is a common component of cilia. Previously, it has been proposed that defective ciliary function might be the basis of some forms of Usher syndrome. Myosin VIIa was detected in cilia from cochlear hair cells, olfactory neurons, kidney distal tubules, and lung bronchi. It was also found to cofractionate with the axonemal fraction of retinal photoreceptor cells. Immunolabeling appeared most concentrated in the periphery of the transition zone of the cilia. This general presence of a myosin in cilia is surprising, given that cilia are dominated by microtubules, and not actin filaments. In addition to cilia, myosin VIIa was also found in actin-rich microvilli of different types of cell. We conclude that myosin VIIa is a common component of cilia and microvilli.

Animals↗

PubMed1998

In vivo investigation of blood compatibility of titanium oxide films.

Titanium oxide films were synthesized by ionbeam-enhanced deposition. The films were prepared by depositing titanium atoms and simultaneously bombarding them with Xe+ ions at an energy of 40 keV in an O2 environment. An in vivo investigation, which entailed implanting low-temperature isotropic pyrolytic carbon (LTI carbon) cylinders, widely used to fabricate artificial heart valves, and titanium-oxide-coated LTI carbon cylinders with diameters of 5 mm and thicknesses of 0.5 mm into the ventral aorta of dogs for 14 days, showed that the amount of thrombus on the titanium-oxide-coated LTI carbon was much less than that formed on the surface of LTI carbon alone. Scanning electron microscopy (SEM) was used to observe the morphology of thrombus. On the titanium oxide films no platelet aggregation was found, almost no red blood cells were damaged, and almost no fibrin was found on the surface. However, all three characteristics were found on the surface of LTI carbon alone, proving that the blood compatibility of titanium oxide films is better than that of LTI carbon and titanium-oxide-coated LTI carbon.

Animals↗

PubMed1998

Basic FGF and FGF receptor 1 are expressed in microglia during experimental autoimmune encephalomyelitis: temporally distinct expression of midkine and pleiotrophin.

Heparin-binding growth factors have been implicated in central nervous system development, regeneration and pathology. To assess the expression pattern and possible function in multiple sclerosis, the heparin-binding growth factors pleiotrophin (PTN), midkine (MK), basic fibroblast growth factor (FGF-2) and one of its receptors (FGFR1/flg) mRNA and protein levels were examined in an experimental autoimmune encephalomyelitis (EAE) model in the Lewis rat. We assessed the time course of expression of PTN, MK and FGF-2 during EAE and determined the cellular origin of FGF-2 and FGFR1 in normal spinal cord and during inflammatory demyelination. Basal expression of PTN and MK mRNAs in normal spinal cords was significantly upregulated after induction of EAE. MK expression was upregulated two to threefold correlating with disease progression, whereas PTN expression reached peak levels threefold above basal levels during the clinical recovery period. FGF-2 mRNA expression was low in normal spinal cord and dramatically increased in correlation with progressive demyelination. FGF-2 was confined to neurons in normal tissue and shifted dramatically to microglia, paralleling their activation during EAE. Double immunohistochemistry revealed colocalization of FGF-2 to activated microglia/macrophages with strongest expression in the macrophage-rich perivascular core area and microglial expression at the edges of white and gray matter perivascular regions. FGFR1, like its ligand, was induced in activated macrophages/microglia. Growth factor expression in demyelinating diseases could serve several functions, e.g., to modulate the activity of microglia/macrophage in an autocrine fashion, to induce the expression of other factors like insulin-like growth factor 1 or plasminogen activator, which can effect regeneration or degeneration, respectively, and finally to stimulate directly localized proliferation and/or regeneration of oligodendrocytes within the lesion area.

Animals↗

PubMed1998

Localization of the proenzyme form of the vitellin-processing protease in Blattella germanica by affinity-purified antibodies.

During Blattella germanica embryo development, the nutritive yolk protein vitellin is processed by a cysteine protease, which is activated proteolytically from a proprotease during acidification of yolk granules. A murine polyclonal antiserum was generated with the purified proprotease as the immunogen. The antiserum was made monospecific to proprotease by subtractive affinity chromatography using proprotease-free yolk proteins as ligand. The purified antibodies were employed to investigate the temporal and spatial expression of the proprotease during vitellogenesis and embryo development. Anti-proprotease-reactive peptides appeared in extracts of fat bodies and ovarian follicles of post-mating females, but not in fat bodies of males or the fat bodies or follicles of unmated females, suggesting that the proprotease is synthesized extraovarially. Use of the antibodies was extended to monitor the kinetics of proprotease disappearance during early embryo development.

Animals↗

PubMed1998

Jak-STAT pathway is involved in the induction of TNF-beta gene during stimulation by IL-2.

IL-2 is the major regulatory cytokine of the immune system. It plays a key role in T cell survival, growth and activation. IL-2 may induce the expression of multiple genes including some cytokine genes. The induction of these genes is triggered by different signal pathways, one of them being the Jak-STAT signal pathway. The genes regulated by this pathway remain to be determined. By studying IL-2-inducible genes, we have confirmed that the TNF-beta gene is one of the immediate early genes activated by IL-2. By analysis of the DNA sequences around 180-300 bases upstream of the transcription initiation point of the mouse TNF-beta gene, we demonstrate that there is a STAT5 binding site which is essential to the inducibility of the TNF-beta gene. Furthermore, in BA/F3 cells co-transfected with the STAT5A gene and IL-2R beta gene, the activation of the TNF-beta gene promoter by IL-2 was greatly promoted, whereas the TNF-beta gene promoter became IL-2-non-inducible if the STAT5A gene was substituted with a dominant negative STAT5A, i.e. a C-terminally truncated mutant. Taken together, our results show that the Jak-STAT signal pathway is involved in induction of the TNF-beta gene in cells stimulated by IL-2.

Animals↗

PubMed1998

Cloning, expression, and purification of a catalytic fragment of Moloney murine leukemia virus reverse transcriptase: crystallization of nucleic acid complexes.

Reverse transcriptase is an essential retroviral enzyme that uses RNA- and DNA-directed DNA polymerase activities as well as an RNaseH activity to synthesize a double-stranded DNA copy of the single-stranded RNA genome. In an effort to obtain high-resolution structural information regarding the polymerase active site of reverse transcriptase, we have pursued studies on a catalytic fragment from Moloney murine leukemia virus reverse transcriptase. DNA encoding the catalytic fragment, defined originally by limited proteolytic digestion, has been cloned, and the protein has been expressed and purified from Escherichia coli. The fragment obtained by limited proteolytic digestion and the bacterially expressed fragnment retain polymerase activity. Crystallization studies involving nucleic acid complexes with a catalytic fragment from both sources are reported, including variables screened to improve crystals and cryocooling. Three crystal forms of catalytic fragment-nucleic acid complexes have been characterized, which all contain at least two protein molecules in the asymmetric unit. As isolated, the catalytic fragment is monomeric. This analysis indicates that the enzyme dimerizes in the presence of nucleic acid.

Base Sequence↗

PubMed1998

An AI approach to dynamic visual field testing.

Visual field test results are crucial to the accuracy and efficiency of diagnosing blinding diseases such as glaucoma. Herein, a method of integrating self-organizing neural networks and empirical heuristics is used to perform visual field tests via a dynamic test strategy, which can lead to a reduction in the number of trials in a perimetric test. Experiments performed using clinical test records show that we are able to reduce by 20% to 30% the number of trials per test without much adverse effect on the accuracy of the tests.

Algorithms↗