Biomedical subjects

Q Liu

Publications and source records attributed to Q Liu.

At least 19 recordsLinked to original sources

PubMed2001

Mouse phosphoglycerate mutase M and B isozymes: cDNA cloning, enzyme activity assay and mapping.

Two mouse cDNAs encoding the non-muscle-specific or brain isoform (type B, Pgam1) and the muscle-specific isoform (type M, Pgam2) of phosphoglycerate mutase (PGAM) were isolated and characterized. Pgam1 contains a 765 bp open reading frame (ORF) coding for a 254-residue protein while Pgam2 contains a 762 bp ORF coding for a 253-residue protein. The deduced proteins of mouse Pgam1 and Pgam2 are highly similar to those of human and rat (> or = 93% similarity). Northern blot analysis showed that the expression patterns of Pgam1 and Pgam2 were distinct. Pgam1 was expressed as a 2.1-kb transcript highly in brain and kidney and moderately in liver, thyroid, stomach and heart, whereas Pgam2 was expressed as a 1.0-kb transcript highly in muscle, testis and moderately in heart and lung, but was not detectable in the other six tissues examined. Transfecting the cDNA fragments containing the entire ORFs of these two cDNAs into COS7 cells for transient expression, respectively, the enzyme activities of mouse Pgam1 and Pgam2 were detected to be 2.2-2.5 times of those of COS7 cells and COS7 cells transfected with vector, proving the validity of mouse Pgam1 and Pgam2 cDNAs we report here. Pgam1 and Pgam2 were assigned to 116.16 cR from D19Mit52 and 29.57 cR from D11Mit129, respectively, by radiation hybrid method. The partial genomic sequence of Pgam2 was determined, which showed that mouse Pgam2 consisted at least three exons and two introns. In addition, a pseudogene of Pgam1, Pgam1-ps1, was identified from mouse genomic sequence.

Amino Acid Sequence↗

PubMed2001

Engineering of a mammalian cell line for reduction of lactate formation and high monoclonal antibody production.

Lactate and ammonia are the two major waste products formed during mammalian cell growth. Accumulation of these side products can have a negative effect on cell growth, and has drawn recent attention because of their inhibitory effects on the specific product synthesis rate. Our aim is to reduce lactate formation in the cell culture by genetically manipulating of the pathway of lactate synthesis with an aim to achieve high monoclonal antibody production. We have partially disrupted the LDH-A gene by homologous recombination in hybridoma cells (ATCC-CRL-1606). The cells that received the newly introduced DNA were selected by G418, and an LDH-deficient cell was identified by a screening method based on medium color changing in 96-well plates. A variant cell, LDH-neo21, was identified through this screening method and was characterized. The specific productivity of lactate by LDH-neo21 cells was 50% lower than that of parental cells. Intracellular LDH enzyme activity was significantly reduced. The cell growth was improved both in terms of cell density and cell viability. Total cell density potentially reached 5 x 10(6) cells/mL while the parental hybridoma cells had a cell density of 3.5 x 10(6) cells/mL, which represented a 30% increase. The antibody production of LDH-neo21 cells was threefold greater than that of parental cells during 5-day batch culture. Polymerase chain reaction (PCR) results showed that at least one copy of the LDH-A gene was disrupted in the LDH-neo21 cells. The variant of the hybridoma cell exhibited a significant advantage of reduced lactate formation in the cell culture with a high concentration of glucose, which led to a higher production of monoclonal antibody. 2001 John Wiley & Sons, Inc.

Ammonia↗

PubMed2001

Regulation of an endogenous locus using a panel of designed zinc finger proteins targeted to accessible chromatin regions. Activation of vascular endothelial growth factor A.

We have mapped conserved regions of enhanced DNase I accessibility within the endogenous chromosomal locus of vascular endothelial growth factor A (VEGF-A). Synthetic zinc finger protein (ZFP) transcription factors were designed to target DNA sequences contained within the DNase I-hypersensitive regions. These ZFPs, when fused to either VP16 or p65 transcriptional activation domains, were able to activate expression of the VEGF-A gene as assayed by mRNA accumulation and VEGF-A protein secretion through a range exceeding that induced by hypoxic stress. Importantly, multiple splice variants of VEGF-A mRNA with defined physiological functions were induced by a single engineered ZFP transcription factor. We present evidence for an enhanced activation of VEGF-A gene transcription by ZFP transcription factors fused to VP16 and p65 targeted to two distinct chromosomal sites >500 base pairs upstream or downstream of the transcription start site. Our strategy provides a novel approach for dissecting the requirements for gene regulation at a distance without altering the DNA sequence of the endogenous target locus.

Cell Line, Transformed↗

PubMed2001

Synthesis and biological evaluation of novel pyrazoles and indazoles as activators of the nitric oxide receptor, soluble guanylate cyclase.

Database searching and compound screening identified 1-benzyl-3-(3-dimethylaminopropyloxy)indazole (benzydamine, 3) as a potent activator of the nitric oxide receptor, soluble guanylate cyclase. A comprehensive structure-activity relationship study surrounding 3 clearly showed that the indazole C-3 dimethylaminopropyloxy substituent was critical for enzyme activity. However replacement of the indazole ring of 3 by appropriately substituted pyrazoles maintained enzyme activity. Compounds were evaluated for inhibition of platelet aggregation and showed a general lipophilicity requirement. Aryl-substituted pyrazoles 32, 34, and 43 demonstrated potent activation of soluble guanylate cyclase and potent inhibition of platelet aggregation. Pharmacokinetic studies in rats showed that compound 32 exhibits modest oral bioavailability (12%). Furthermore 32 has an excellent selectivity profile notably showing no significant inhibition of phosphodiesterases or nitric oxide synthases.

Animals↗

PubMed2001

Bacterial expression of an immunologically reactive PCV2 ORF2 fusion protein.

The entire coding region of open reading frame 2 (ORF2) of porcine circovirus 2 (PCV2) was linked to the 3'-end of the maltose-binding protein (MBP)-His(8)-tag gene. The fusion protein was expressed as soluble form after induction by isopropylthio-beta-d-galactoside. MBP-His(8)-ORF2 was purified to homogeneity by immobilized metal affinity chromatography based on the interaction of the polyhistidine-tag with metal ions. Expression could represent 1% of the total protein in Escherichia coli, allowing approximately 1 mg of highly purified protein to be obtained per liter of bacterial culture. The fusion protein was recognized in Western blot by anti-PCV2 polyclonal antibody and swine sera with PCV2 infection. In addition, rabbit polyclonal antibody raised against purified MBP-His(8)-ORF2 fusion protein reacted with the ORF2 protein in immunoprecipitation. The availability of this fusion protein should permit a thorough study of prevalence of PCV2 infection in large-scale serological studies of field samples.

ATP-Binding Cassette Transporters↗

PubMed2001

Orphan G-protein-coupled receptors and natural ligand discovery.

The superfamily of seven-transmembrane-domain G-protein-coupled receptors (GPCRs) is the largest and most diverse group of transmembrane proteins involved in signal transduction. Each of the approximately 1000 family members found in vertebrates responds to stimuli as diverse as hormones, neurotransmitters, odorants and light, which selectively activate intracellular signaling events mediated by heterotrimeric G proteins. Because GPCRs are centrally positioned in the plasma membrane to initiate a cascade of cellular responses by diverse extracellular mediators, it is not surprising that modulation of GPCR function has been successful in the development of many marketed therapeutic agents. It has become clear that GPCRs for which a natural activating ligand has not yet been identified (orphan GPCRs) might provide a path to discovering new cellular substances that are important in human physiology. The process of 'de-orphanizing' these novel proteins has accelerated significantly and opened up new avenues for research in human physiology and pharmacology.

Animals↗

PubMed2001

DNA prime/canarypox boost-based immunotherapy of chronic hepatitis B virus infection in a chimpanzee.

There are about 200 million chronic hepatitis B virus (HBV) carriers at high risk of development of cirrhosis and hepatocellular carcinoma. Termination of the carrier state may avert these risks. We have investigated immunotherapy for chronic HBV infection in a chimpanzee HBV carrier using recombinant DNA-based immunization followed by a recombinant canarypox booster. One week after the booster, HBV DNA declined greater than 400-fold and remained undetectable by the quantitative polymerase chain reaction (PCR) assay for 186 weeks. Plasma levels of hepatitis B surface antigen (HBsAg) declined for only a short time. The decline in HBV DNA correlated with a boost in gamma interferon production without a corresponding boost in cytotoxic T lymphocyte levels, and decline in the transcriptional template or covalently closed circular DNA level. Confirmation of these findings requires further studies in chimpanzees and/or in humans.

Alanine Transaminase↗

PubMed2001

N-(8-Quinolyl)pyridine-2-carboxamide.

The title compound, C15H11N3O, is basically planar except that the pyridine ring is slightly titled, the dihedral angle between the pyridyl and quinolyl rings being 3.55 (5) degrees. The crystal grows in two directions and the crystal packing is stabilized by pi-pi stacking interactions.

Crystallography, X-Ray↗

PubMed2001

On sample size and inference for two-stage adaptive designs.

Proschan and Hunsberger (1995, Biometrics 51, 1315-1324) proposed a two-stage adaptive design that maintains the Type I error rate. For practical applications, a two-stage adaptive design is also required to achieve a desired statistical power while limiting the maximum overall sample size. In our proposal, a two-stage adaptive design is comprised of a main stage and an extension stage, where the main stage has sufficient power to reject the null under the anticipated effect size and the extension stage allows increasing the sample size in case the true effect size is smaller than anticipated. For statistical inference, methods for obtaining the overall adjusted p-value, point estimate and confidence intervals are developed. An exact two-stage test procedure is also outlined for robust inference.

Biometry↗

PubMed2001

Expression of the cysteinyl leukotriene 1 receptor in normal human lung and peripheral blood leukocytes.

The cysteinyl leukotrienes (CysLTs) are important mediators of human asthma. Pharmacologic and clinical studies show that the CysLTs exert most of their bronchoconstrictive and proinflammatory effects through activation of a putative, 7-transmembrane domain, G-protein-coupled receptor, the CysLT1 receptor. The initial molecular characterization of the CysLT1 receptor showed by in situ hybridization, the presence of CysLT1 receptor messenger RNA (mRNA) in human lung smooth-muscle cells and lung macrophages. We confirmed the results of these in situ hybridization analyses for the CysLT1 receptor, and produced the first immunohistochemical characterization of the CysLT1 receptor protein in human lung. The identification of the CysLT1 receptor in the lung is consistent with the antibronchoconstrictive and antiinflammatory actions of CysLT1 receptor antagonists. We also report the expression of CysLT1 receptor mRNA and protein in most peripheral blood eosinophils and pregranulocytic CD34+ cells, and in subsets of monocytes and B lymphocytes.

Blood↗

PubMed2001

Plasma levels of SDF-1 and expression of SDF-1 receptor on CD34+ cells in mobilized peripheral blood of non-Hodgkin's lymphoma patients.

CXCR4 is the receptor for the chemokine stromal derived factor-1 (SDF-1), is expressed on CD34+ cells, and has been implicated in the process of CD34+ cell migration and homing. We studied the mobilization of CD34/CXCR4 cells and the plasma levels of SDF-1 and flt3-ligand (flt3-L) in 36 non-Hodgkin's lymphoma patients receiving cyclophosphamide (Cy) plus G-CSF (arm A), Cy plus GM-CSF (arm B), or Cy plus GM-CSF followed by G-CSF (arm C) for peripheral blood stem cell (PBSC) mobilization and autotransplantation. We observed lower plasma levels of SDF-1 in PBSCs compared to premobilization bone marrow samples. The mean plasma SDF-1 levels were similar in PBSC collections in the three arms of the study. In contrast, SDF-1 levels in the apheresis collections of the "good mobilizers" (patients who collected a minimum of 2 x 10(6) CD34+ cells/kg in one to four PBSC collections) were significantly lower than the apheresis collections of the "poor mobilizers" (> or = 0.4 x 10(6) CD34+ cells/kg in the first two PBSC collections; 288 +/- 82 pg/ml versus 583 +/- 217 pg/ml; p = 0.0009). The mean percentage of CD34+ cells expressing CXCR4 in the apheresis collections was decreased in the PBSC collections compared with premobilization values from 28% to 19.4%. Furthermore, the percentage of CD34+ cells expressing CXCR4 in the good mobilizers was significantly lower compared with the poor mobilizers (14.7 +/- 2.1% versus 33.6 +/- 2.1%; p = 0.002). The good mobilizers had also significantly lower levels of flt3-L compared with the poor mobilizers (34 +/- 4 pg/ml versus 106 +/- 11 pg/ml; p = 0.006), Finally, the levels of flt3-L strongly correlated with SDF-1 levels (r = 0.8; p < 0.0001). We conclude: A) low plasma levels of SDF-1 and low expression of CXCR4 characterize patients with good mobilization outcome, and B) the levels of SDF-1 correlate with flt3-L, suggesting an association of these cytokines in mobilization of CD34+ cells.

Antigens, CD34↗

PubMed2001

Expression of adhesion molecules on CD34(+) cells in peripheral blood of non-hodgkin's lymphoma patients mobilized with different growth factors.

Adhesion molecules on CD34(+) cells were implicated in the process of peripheral blood stem cell (PBSC) mobilization and homing. We studied the mobilization of CD34(+)Thy1(+) cells, CD34(+) very late-acting antigen (VLA)4(+) cells, and CD34(+)L-selectin(+) cells in non-Hodgkin's lymphoma patients mobilized with cyclophosphamide plus G-CSF, GM-CSF, or GM-CSF followed by G-CSF. The mean percentage of CD34(+) cells in the bone marrow (BM) expressing Thy1 was 23.6% +/- 11% and 17.8% +/- 8% in the PB before mobilization, and was markedly decreased to 4.5% +/- 3.3% in the apheresis collections. Similarly, the mean percentage of CD34(+) cells expressing L-selectin was 35.8% +/- 4.3% in the BM, 21.6% +/- 4.1% in the PB before mobilization and was markedly decreased to 9.1% +/- 2.5% in the apheresis collections. Patients in the three arms of the study had a similar pattern of CD34(+)Thy1(+) and CD34(+)L-selectin(+) cell mobilization. Also, a similar pattern of coexpression of CD34(+)Thy1(+) and CD34(+)L-selectin(+) cells was observed when the patients were regrouped as "good mobilizers" (> or =2 x 10(6) CD34(+)CD45(dim) cells/kg, in four collections) and "poor mobilizers" (<0.4 x 10(6) CD34(+)CD45(dim) cells/kg, in two collections). The mean percentage of CD34(+) cells expressing VLA-4 in the BM and PB was relatively high (73.4% +/- 12% and 65.4% +/- 6.6%, respectively) and dropped considerably in the PBSC collections to 43.5% +/- 7.1% with a similar pattern observed for patients in arms A, B, and C. However, when the patients were regrouped as "good mobilizers" and "poor mobilizers," a higher percentage of CD34(+) cells expressing VLA-4 was observed in the PBSC of the pooled "good mobilizers" (50.5% +/- 9% versus 36.3% +/- 6.4%; p = 0.01). We conclude that release of CD34(+) cells to the PB involves a general downregulation of Thy1, L-selectin and VLA-4 on CD34(+) cells, irrespective of the growth factor used for mobilization. However, good mobilizers had a relatively higher percentage of CD34(+) cells expressing the VLA-4 antigen.

Adult↗

PubMed2001

[The construction of rapid amplification of cDNA ends cDNA libraries from human fetal bone and joint].

OBJECTIVE: To construct rapid amplification cDNA ends(RACE) cDNA libraries from human fetal bone and joint and provide resources for isolation of bone- and joint-specific development-related genes. METHODS: Total RNA of bone and joint were extracted with the modified single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. The double-stranded end-blunted cDNA were synthesized using TaKaRa's cDNA synthesis kit and ligated to cassette adaptors. All of the cDNA molecules were amplified by a pair of common primers. RESULTS: A protocol for RACE cDNA library construction from bone and joint was established and two RACE cDNA libraries from human fetal bone and joint were successfully constructed. CONCLUSION: The protocol of RACE cDNA library construction from limited materials proved to be simple and efficient and the library was suitable for RACE to isolate tissue-specific genes.

Bone and Bones↗

PubMed2000

Locomotor activity and occupancy of brain cannabinoid CB1 receptors by the antagonist/inverse agonist AM281.

The goals of this study were to examine the relationship between intravenous doses of the cannabinoid CB1 receptor antagonist AM281 (N-(morpholin-4-yl)-5-(4-iodophenyl)-1-(2, 4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide) and the degree of occupancy of this receptor, and to relate occupancy to the ability of this compound to antagonize the sedative effects of the cannabinoid receptor agonist WIN 55,212-2. Occupancy was determined by measuring the ability of intravenous doses of AM281 to inhibit in vivo binding of [(131)I]AM281 in brain areas, and locomotor activity was assessed by measuring the rate of beam crossings in a photocell apparatus. As previously documented, WIN 55,212-2 (1 mg/kg, i.v.) significantly reduced locomotor activity at early times after administration. Co-injection of AM281 (0.3 mg/kg i/v) and WIN 55, 212-2 restored the rate of beam crossings to that seen on injection of vehicle. In addition, AM281 (0.3 mg/kg i/v) approximately doubled locomotor activity between 60-120 min when injected alone. The IC(50) value for displacement of [(131)I]AM281 by AM281 was 0.45 mg/kg. These observations confirm earlier indications that AM281 is a CB1 receptor antagonist or inverse agonist and suggest the existence of an endogenous cannabinoid tone that moderates exploratory locomotor activity.

Animals↗

PubMed2000

Definition of the anterior choroidal artery territory in rats using intraluminal occluding technique.

This manuscript delineates the territory of the anterior choroidal artery (AChA) in rats, as defined by the induction of an AChA infarction. By advancing a 0.24-mm surgical suture up the internal carotid artery (ICA) to a point 0.5-2 mm proximal to the middle cerebral artery (MCA) origin, the AChA could be occluded and a reliable AChA distribution infarction was produced in 62% (23/37) of animals. The infarct volume, as defined by TTC staining, was 55+/-7 mm(3). Maps of the infarction, generated by measuring the entire area of overlapping coronal slices, demonstrated that the internal capsule was always damaged. Other areas that might be affected included the hippocampus, thalamus, amygdaloid complex, piriform cortex, dorsal caudatoputamen, and lateral ventricular wall. Positioning the coated suture proximal to the AChA produced a much smaller infarct involving the medial and lateral hypothalamus, preoptic region, optic chiasm, and marginal region of the internal capsule near to the lateral hypothalamus exempt from AChA territory damage. A causative relationship between AChA occlusion and a deep cerebral infarct centered on the internal capsule was further established by: (1) identifying the AChA on the non-ischemic side with colored silicone perfusion, and subsequent similar delineation on the ischemic side, and (2) delineating infarction in the silicone perfused AChA region using hematoxylin and eosin staining and the TUNEL method. The AChA usually originated from the ICA (91% of cases), 1.75+/-0.12 mm proximal to the MCA bifurcation. Approximately 27% of the AChAs had periamygdaloid branch(es) on its initial segment.

Animals↗

PubMed2000

The effect of preoperative transcatheter hepatic arterial chemoembolization on disease-free survival after hepatectomy for hepatocellular carcinoma.

BACKGROUND: Although surgical resection has produced better results than other therapies for local control of hepatocellular carcinoma (HCC), the long term results have not been satisfactory because of a low disease-free survival rate. The effects of preoperative transcatheter arterial chemoembolization (TACE) on improving disease-free survival of HCC after hepatectomy has remained controversial. METHODS: The current study was retrospective survey of 1725 patients who had hepatectomy for HCC between January 1990 and December 1995 and had follow-up data for 1457 cases. The follow-up rate was 84.5%. The significant prognostic factors were analyzed using a Cox proportional hazards survival model, and the disease-free survival was calculated by Kaplan-Meier estimation. Among 1457 cases, 120 patients underwent preoperative TACE and were divided into a one time TACE group, over two times TACE group, an effective group, and ineffective group. RESULTS: Multivariate analyses revealed significant prognostic factors as follows: preoperative TACE number, preoperative TACE effect, preoperative lesion number, intraoperative tumor thrombus, tumor size, tumor gross type, daughter nodules, vascular invasion, and postoperative alpha-fetoprotein value. Kaplan-Meier estimation showed that the 5-year disease-free survival rates of the over two times group, the one time group, and no TACE group were 51.0%, 35.5%, and 21.4%, respectively, and that the mean disease-free survival times of the three groups were 66.4, 22.5 and 12.5 months, respectively. The effective group had a 5-year disease-free survival rate of 56.8%, with a mean time of 90.1 months. CONCLUSIONS: Effective preoperative TACE may be one of the best methods, which can be clinically performed at present, for resectable HCCs including small HCCs for improving disease-free survival after hepatectomy. According to tumor size, range, location, hepatic function, and TACE effect, TACE can be performed 2-4 times preoperatively within 6 months.

Adolescent↗

PubMed2000

Mitochondrial Hsp70 Ssc1: role in protein folding.

Ssc1, the major Hsp70 of the mitochondrial matrix, is involved in the translocation of proteins from the cytosol into the matrix and their subsequent folding. To better understand the physiological mechanism of action of this Hsp70, we have undertaken a biochemical analysis of Ssc1 and two mutant proteins, Ssc1--2 and Ssc1--201. ssc1--2 is a temperature-sensitive mutant defective in both translocation and folding; ssc1--201 contains a second mutation in this ssc1 gene that suppresses the temperature-sensitive growth defect of ssc1--2, correcting the translocation but not the folding defect. We found that although Ssc1 was competent to facilitate the refolding of denatured luciferase in vitro, both Ssc1--2 and Ssc1--201 showed significant defects, consistent with the data obtained with isolated mitochondria. Purified Ssc1--2 had a lowered affinity for a peptide substrate compared with wild-type Ssc1 but only in the ADP-bound state. This peptide binding defect was reversed in the suppressor protein Ssc1--201. However, a defect in the ability of Hsp40 to stimulate the ATPase activity of Ssc1--2 was not corrected in Ssc1--201. Thus, the inability of these two mutant proteins to efficiently facilitate luciferase refolding correlates with their defect in stimulation of ATPase activity by Hsp40s, indicating that this interaction is critical for protein folding in mitochondria.

Adenosine Diphosphate↗

PubMed2000

Synthesis of Monodisperse Al-Substituted Hematite Particles from Highly Condensed Metal Hydroxide Gels.

Monodispersed aluminum-substituted hematite particles were synthesized from concentrated mixed solutions of FeCl(3) and AlCl(3) using the gel/sol process. With increasing aluminum content (Al/(Fe+Al)) in the starting solutions, hematite particles became more and more elongated. The product particles exhibited ellipsoidal, rod- and peanut-like shapes, depending on Al concentration. Higher Al content (>20 mol%) in the system greatly suppressed the formation of hematite. Atomic emission spectrometry analysis and lattice parameter measurement confirmed the substitution of Al in hematite particles. Copyright 2000 Academic Press.

Journal Article↗