Biomedical subjects

G Liu

Publications and source records attributed to G Liu.

At least 289 records · Page 16Linked to original sources

PubMed1999

Site-directed mutagenesis of the AcMNPV p143 gene: effects on baculovirus DNA replication.

Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) encodes a 143-kDa protein (P143) required for viral DNA synthesis and involved in host range determination. The predicted amino acid sequence of P143 contains seven motifs (I, Ia, II-VI) shared with a superfamily of helicases involved in the unwinding of duplex nucleic acids; a putative DNA binding motif; a putative nuclear localization signal (NLS); and a demonstrated host range motif. In this study, the functional significance of these conserved P143 motifs was examined by site-specific mutation resulting in amino acid substitutions of conserved residues within each of them. An in vivo complementation replication assay was developed and each mutated P143 protein expressed from a transfected plasmid was tested for its ability to complement the replication-negative ts8 baculovirus mutant for the amplification of an origin-containing plasmid. Mutations in the helicase motifs I, Ia, and II and in a potential helix-turn-helix motif abolished the ability of P143 to complement the ts8 defect in DNA replication, suggesting that these conserved amino acid residues may be essential for the replication function of the protein. In contrast, mutation of conserved amino acid residues in the helicase motifs IV, V, and VI did not affect the ability of the P143 proteins to complement the replication defect of ts8. A mutation in motif III caused a reduction in the replication function of P143. Deletion of Gly552 in the host range region eliminated the replication function of P143. Mutations within a putative NLS had no effect on the ability of P143 to support DNA replication, suggesting that these residues are nonessential and that the putative P143 NLS sequence may not be responsible for the nuclear localization of the protein. The transient complementation system used in this study provides a simple method for functional analysis of essential baculovirus genes in infected cell cultures.

Adenosine Triphosphate↗

PubMed1999

Affinity capillary electrophoresis coupled with on-line microdialysis by attachable electrode.

This paper developed a new affinity capillary electrophoresis method coupled with on-line microdialysis using attachable electrodes in order to study the interaction of biomacromolecules with their small molecule ligands in the desired physiological conditions. The sampling mechanism was discussed and the experimental conditions were optimized. The affinity constant of sulfamethoxazole (SMZ) with bovine serum albumin (BSA) was determined with homemade equipment. The method is simple and practical.

Calibration↗

PubMed1999

Stable transfection of human fetal chondrocytes with a type II procollagen minigene: expression of the mutant protein and alterations in the structure of the extracellular matrix in vitro.

OBJECTIVE: To perform stable transfections of human chondrocytes under conditions that allow the maintenance of the chondrocyte-specific phenotype, and to examine the effects of the stable transfection of a mutated type II procollagen gene (COL2A1) on the structure of the cartilaginous extracellular matrix produced in vitro. METHODS: A type II procollagen minigene that lacks exons 16-27 was stably transfected into human fetal epiphyseal chondrocytes in vitro. Expression of the minigene was detected by reverse transcriptase-polymerase chain reaction, and the encoded protein was identified by Western blot with a human type II collagen-specific antibody. The cartilaginous extracellular matrix produced by the cultured transfected chondrocytes was characterized using histochemical staining, polarized light microscopy analysis, and transmission electron microscopy. RESULTS: A shortened type II collagen encoded by the transfected minigene was biosynthesized and produced in the cultures of transfected cells. Histologic analyses demonstrated a more dense, negatively charged cartilaginous matrix in control cultures. In contrast, COL2A1 minigene-transfected cultures were more cellular, were populated with cells of irregular shape and less-chondrocytic appearance, contained abundant intracellular dense granules, and were surrounded by a less-dense matrix. Polarized light microscopy and transmission electron microscopy revealed a well-organized collagen fibrillar matrix in untransfected, control chondrocyte cultures, while the matrix in the transfected cultures was less birefringent and contained numerous truncated collagen fibrils. CONCLUSION: The findings illustrate the feasibility of gene transfer into human fetal chondrocytes under conditions that allow the preservation of their specific phenotype, and also shed light on the function of type II collagen in the maintenance of the structural integrity of articular cartilage matrix.

3T3 Cells↗

PubMed1999

Increased risk of hepatocellular carcinoma in male hepatitis B surface antigen carriers with chronic hepatitis who have detectable urinary aflatoxin metabolite M1.

We followed 145 men with chronic hepatitis B virus (HBV) hepatitis for 10 years to determine whether exposure to aflatoxin, or concomitant exposure to hepatitis C virus (HCV), or family history of hepatocellular carcinoma (HCC) increased the risk of developing HCC. We collected 8 monthly urine samples before beginning follow-up and pooled them to detect aflatoxin metabolite M1 (AFM1). AFM1 was detected in 78 (54%) of the subjects. The risk of HCC was increased 3.3-fold (with a 95% confidence interval of 1.2-8.7) in those with detectable AFM1 (above 3.6 ng/L). This relative risk was adjusted for age and for HCV status. The attributable risk from exposure to detectable AFM1 was 0.553 (0.087, 0.94). The relative risk of fatal cirrhosis for those with elevated AFM1 was 2.8 (0.6, 14.3), and the odds of having a persistently elevated alanine transaminase (ALT) were 2.5-fold greater in those with detectable AFM1 (P =.007). Concomitant infection with HCV increased the risk of HCC 5.8-fold (2. 0-17), adjusted for age and AFM1 status. A family history of HCC increased the risk of HCC 5.6-fold, adjusted for age and AFM1. Four men with detectable AFM1 and HCC all had missense mutation in codon 249 of the p53 gene in cancer tissues. This study shows that exposure to AFM1 can account for a substantial part of the risk of HCC in men with chronic HBV hepatitis and adds importantly to the evidence that HCV and family history of HCC increase the risk of HCC in men with chronic HBV hepatitis.

Adolescent↗

PubMed1999

Analysis of splice variants of the fibronectin gene in thyroid carcinomas by reverse transcription-polymerase chain reaction: increased expression of oncofetal fibronectin mRNA in papillary carcinomas is not caused by the alternation in splicing.

The expression levels of each splice variant of the fibronectin gene in the normal thyroid and in thyroid tumors were examined by reverse transcription-polymerase chain reaction (RT-PCR). In thyroid papillary carcinomas, insertion of a variant exon in the ED-A and ED-B domains, and three of five types of splice variants in the IIICS domain were observed. In spite of the marked increase in the expression of oncofetal fibronectin mRNA with the IIICS sequence in papillary and anaplastic carcinomas in the previous reports, the relative expression levels of each splice variant with or without the IIICS sequence showed no difference among all the tumor types. Therefore, the much increased expression of oncofetal fibronectin mRNA in these carcinomas is not caused by the alternation in splicing, but may be caused by an increase in promoter activity or stability of mRNA of the fibronectin gene.

Adenocarcinoma, Follicular↗

PubMed1999

The aetiology and pathogenesis of cardiopulmonary bypass-associated metabolic acidosis using polygeline pump prime.

OBJECTIVE: The pathogenesis of the metabolic acidosis of cardiopulmonary bypass (CPB) is not fully understood. New quantitative methods of acid-base balance now make it possible to describe it more clearly. Accordingly, we studied acid-base changes during CPB with polygeline pump prime and defined and quantified the factors which contribute to metabolic acidosis. DESIGN: Prospective cohort study. SETTING: Tertiary institution. PARTICIPANTS: 10 cardiac bypass graft surgery patients. INTERVENTIONS: Sampling of arterial blood at four time intervals: post-induction, on CPB during cooling and rewarming, and at skin closure. Measurement of serum Na+, K+, Mg++, Ca++, Cl-, bicarbonate, and phosphate concentrations, arterial blood gases, and serum albumin, lactate, and pyruvate concentrations at each collection point. Analysis of findings according to quantitative physicochemical principles, including calculation of the strong ion difference apparent, the strong ion difference effective, and the strong ion gap (SIG). MEASUREMENTS AND MAIN RESULTS: All patients developed a mild metabolic acidosis. The median serum standard bicarbonate concentration decreased from 25.0 mEq/l post-induction to 22.3 mEq/l at cooling and 22.2 mEq/l at rewarming (p < 0.05). The standard base excess decreased from a median of 1.55 mEq/l prior to CPB, to -2.50 mEq/l at cooling, -1.65 mEq/l at rewarming and, -0.85 mEq/l at skin closure (p < 0.001). This mild metabolic acidosis occurred despite a decrease in the median serum lactate concentration from 3.20 mEq/l post-induction to 1.83, 1.80, and 1.58 mEq/l at the three other time points. The increase in the median serum chloride concentration from 104.9 mEq/l post induction to 111.0, 111.1, and 110.0 mEq/l at the subsequent time points (p < 0.0001) was the main cause of the acidosis. There was also a significant increase in the SIG of 3.8 mEq/l at cooling and rewarming (p < 0.0001), suggesting a role for other unmeasured anions (polygeline) in the genesis of this acidosis. CONCLUSIONS: Using quantitative biophysical methods, it can be demonstrated that, in patients receiving a pump prime rich in chloride and polygeline, the metabolic acidosis of CPB is mostly due to iatrogenic increases in serum chloride concentration and unmeasured strong anions (SIG). Its development is partially attenuated by iatrogenic hypoalbuminaemia. Changes in lactate concentrations did not play a role in the development of metabolic acidosis in our patients.

Acid-Base Equilibrium↗

PubMed1999

Early but not delayed continuous arteriovenous hemofiltration improves cardiovascular function in sepsis in dogs.

OBJECTIVE: Continuous arteriovenous hemofiltration (CAVH) has been advocated as treatment to remove inflammatory mediators and thereby to improve hemodynamic parameters in sepsis. However, the results obtained with CAVH have been inconsistent. In a canine model of bacteremic Pseudomonas aeruginosa pneumonia, we tested the hypothesis that the time course of the institution of CAVH may be important in obtaining a beneficial treatment effect. METHODS: Two protocols were performed in phenobarbital-anesthetized dogs. In the early hemofiltration study (EHS), CAVH for 3 h was initiated 2 h post-pneumonia before mean arterial pressure (MAP) fell. In the late hemofiltration study (LHS), CAVH for 3 h was initiated at 5 h post-pneumonia when a decrease in MAP had already occurred. Hemodynamic measurements included cardiac output (CO), stroke volume (SV), and stroke work (SW). Myocardial depressant activity [filterable cardiodepressant substance (FCS)] found in plasma was assessed by bioassay at each measurement interval. RESULTS: In EHS, after 5 h of sepsis, SW, CO, and SV in the hemofiltered pneumonia group were higher as compared with the nonhemofiltered pneumonia group. In contrast, in LHS, no differences in hemodynamic parameters were found between the two pneumonia groups. In both EHS and LHS, plasma FCS activity was decreased to similar extents by CAVH. CONCLUSION: These results suggest the time course of institution of CAVH may be important in obtaining a beneficial treatment effect in sepsis.

Animals↗

PubMed1999

Polymorphism at an Sp1 binding site of COL1A1 and bone mineral density in premenopausal female twins and elderly fracture patients.

Polymorphism at an Sp1 binding site in the COL1A1 gene has been reported to be associated with bone mineral density (BMD) and osteoporotic vertebral fracture. We therefore examined for associations and linkage of the Sp1 polymorphism in the COL1A1 gene and BMD at the lumbar spine and femoral neck in 38 monozygotic (MZ) and 40 dizygotic (DZ) twin pairs of white adult women. All twins were premenopausal with an age range of 21-49 years. Sp1 genotypes of 56 patients with idiopathic osteoporotic vertebral fracture were examined for a preponderance of either genotype relative to our normal healthy twin subjects. In the twin sample no significant association was found between Sp1 genotypes and BMD at the spine and femoral neck. No linkage of Sp1 genotype and BMD at the spine or femoral neck was observed in DZ twins discordant for genotype. Frequencies of Sp1 genotypes were similar in our healthy (twin) and fracture population samples. In conclusion, in our American sample of premenopausal twins we found no association or linkage of the Sp1 polymorphism at the COL1A1 gene and BMD at the lumbar spine and femoral neck, and no over-representation of any Sp1 genotype was observed in our sample of patients with osteoporotic vertebral fracture. Taken together these results indicate that the Sp1 polymorphism is not related to BMD in our American sample, and contrasts with the findings in a British population.

Adult↗

PubMed1999

Bicuculline sensitive depressor response to ethanol infusion into the lateral hypothalamus.

Decreased GABA function in the hypothalamus increases mean arterial pressure (MAP) and heart rate (HR). Since ethanol acts on GABA-A receptors, blocking GABA-A receptors can prevent a decrease of MAP and HR by ethanol in the lateral hypothalamus (LH). Ethanol at 5-30 mM, with or without 25 ng/microl bicuculline, was infused into the LH, and the activity of the site was validated with 100 nmoles of serotonin. Male rats were anesthetized with pentobarbital, and the femoral artery was catheterized to measure MAP and HR. Microinfusion was performed with a 28-gauge cannula placed into the LH. Serotonin increased MAP and HR within 15 sec. Ethanol decreased the MAP by -21.15 +/- 3.92 mmHg and HR by -53.61 +/- 14.95 BPM, at 15 min, which recovered by 15 min after the infusion was terminated. These maximum decreases were produced by 20 mM ethanol giving a U-shaped dose response. The aCSF vehicle had no effect. Bicuculline prevented ethanol-induced changes and had no effect when administered alone. Both serotonin and ethanol have direct effects on LH neurons with cardiovascular function. Ethanol produces this effect through GABA-A receptors.

Animals↗

PubMed1999

Variability of neurotransmitter concentration and nonsaturation of postsynaptic AMPA receptors at synapses in hippocampal cultures and slices.

To understand the elementary unit of synaptic communication between CNS neurons, one must know what causes the variability of quantal postsynaptic currents and whether unitary packets of transmitter saturate postsynaptic receptors. We studied single excitatory synapses between hippocampal neurons in culture. Focal glutamate application at individual postsynaptic sites evoked currents (I(glu)) with little variability compared with quantal excitatory postsynaptic currents (EPSCs). The maximal I(glu) was >2-fold larger than the median EPSC. Thus, variations in [glu]cleft are the main source of variability in EPSC size, and glutamate receptors are generally far from saturation during quantal transmission. This conclusion was verified by molecular antagonism experiments in hippocampal cultures and slices. The general lack of glutamate receptor saturation leaves room for increases in [glu]cleft as a mechanism for synaptic plasticity.

Animals↗

PubMed1999

Discovery of a series of pyrrolidine-based endothelin receptor antagonists with enhanced ET(A) receptor selectivity.

Endothelins, ET-1, ET-2, and ET-3 are potent vasoconstricting and mitogenic 21-amino acid bicyclic peptides, which exert their effects upon binding to the ET(A) and ET(B) receptors. The ET(A) receptor mediates vasoconstriction and smooth muscle cell proliferation, and the ET(B) receptor mediates different effects in different tissues, including nitric oxide release from endothelial cells, and vasoconstriction in certain vascular cell types. Selective antagonists of endothelin receptor subtypes may prove useful in determining the role of endothelin in various tissue types and disease states, and hence as therapeutic agents for such diseases. The pyrrolidine carboxylic acid A-127722 has been disclosed as a potent and ET(A)-selective antagonist, and is currently undergoing clinical trials. In our efforts to find antagonists with altered selectivity (ET(A)-selective, ET(B)-selective, or nonselective), we investigated the SAR of the 2-substituent on the pyrrolidine. Compounds with alkyl groups at the 2-position possessed ET(A) selectivity improved over A-127722 (1400-fold selective), with the best of these compounds showing nearly 19,000-fold selectivity.

Animals↗

PubMed1999

Markers of splanchnic perfusion and intestinal translocation of endotoxins during cardiopulmonary bypass: effects of dopamine and milrinone.

OBJECTIVES: To investigate markers of splanchnic perfusion and the extent of endotoxemia during cardiopulmonary bypass (CPB) and to compare the effects of dopamine and milrinone on both splanchnic perfusion and endotoxemia. DESIGN: Prospective, randomized, blinded study. SETTING: University teaching hospital. PARTICIPANTS: Twenty-four patients scheduled for elective coronary artery bypass graft surgery (CABG). INTERVENTIONS: Patients were allocated to receive placebo (eight patients), dopamine (eight patients), or milrinone (eight patients) during CPB, and at seven times intraoperatively assays were performed of arterial and hepatic venous endotoxin levels, as well as measurements and/or calculations of intramucosal gastric pH (pHi), arterial and hepatic venous lactate-pyruvate ratio (lac/pyr), and hepatic venous oxygen saturation (S(HV)O2). MEASUREMENTS AND MAIN RESULTS: Both splanchnic and systemic endotoxin levels increased significantly, and this was unaffected by either dopamine or milrinone. Gastric pHi did not change, and there were only modest increases in lac/pyr, which remained within the normal range of less than 10 in both splanchnic and systemic blood. In the placebo group, S(HV)O2 decreased at the onset of CPB and also significantly decreased during rewarming and at the end of CPB and surgery. In the dopamine-treated patients, S(HV)O2 was greater compared with placebo and milrinone during both hypothermic and rewarming phases. CONCLUSION: Endotoxemia occurs during routine CPB. Neither pHi nor lac/pyr values showed adverse change, but hepatic venous oximetry may be a more sensitive indicator of splanchnic dysoxia in that S(HV)O2 was reduced during rewarming. Whether dopamine or milrinone confer protection against splanchnic ischemia remains uncertain.

Adult↗

PubMed1999

A novel method to determine the localization of high and low-affinity GABA transporters to the luminal and antiluminal membranes of brain capillary endothelial cells.

Some evidence has shown that the luminal plasma membrane and the antiluminal plasma membrane of the blood-brain barrier (BBB) are functionally distinct. This polarity permits brain capillary endothelial cells (BCECs) to actively regulate the internal milieu of the brain. Especially, polarity also exists concerning the transport of some endogenous substances such as amino acids and glucose. It is important to determine the luminal and antiluminal membrane localization of some transporters of endogenous substances, which will contribute to a better understanding of the transport mechanisms of the endogenous substances at the BBB. The present paper describes a novel procedure for combining isolated brain capillaries with primary cultured BCECs to determine the subcellular localization of some transporters, which is relatively simpler than the methods described previously. This method was used for the first time to determine successfully the luminal and antiluminal distribution of high and low-affinity GABA transporters. The low-affinity GABA transporter is probably localized to the luminal membrane of BCECs, and the high-affinity GABA transporter may be localized to the antiluminal membrane.

Animals↗

PubMed1999

Regulation of paracellular absorption of cimetidine and 5-aminosalicylate in rat intestine.

PURPOSE: Isolating the relative contributions of parallel transcellular and paracellular transport to the intestinal absorption of small hydrophilic molecules has proven experimentally challenging. In this report, lumenal appearance of drug metabolite is utilized as a tool to assess the contribution of paracellular transport to the absorption of cimetidine and 5-aminosalicylate (5ASA) in rat small intestine. METHODS: Steady-state intestinal absorption and elimination of cimetidine and 5ASA were studied in single-pass intestinal perfusions in rats. RESULTS: Both drugs were metabolized in intestinal epithelia with subsequent metabolite secretion into the intestinal lumen. Jejunal cimetidine absorption decreased with increasing perfusion concentration while the ratio of lumenal metabolite to lumenal drug loss increased. Cimetidine uptake at perfusion concentrations above 0.4 mM resulted in over 80% drug elimination into the jejunal lumen. Inhibition of intracellular metabolism of cimetidine by methimazole did not alter epithelial uptake but totally abolished transepithelial cimetidine flux indicating an elevation of intracellular cimetidine. Similarly, co-perfusion of 5ASA with cimetidine and methimazole totally abolished 5ASA absorption but increased lumenal levels of N-acetyl 5ASA indicating an increase in intracellular uptake of 5ASA. CONCLUSIONS: Cimetidine and 5ASA absorption across rat jejunal epithelia are exclusively paracellular. Elevation of intracellular cimetidine, inferred from mass balance considerations, restricts paracellular transport of both drugs.

Anesthesia↗

PubMed1999

Systematic study of the substituted active C-terminus of hirudin.

Hirudin, a potent clinical thrombin inhibitor from Hirudo medicinalis, consists of 65 amino acids in a single chain. In this paper, we systematically synthesize a series of C-terminal (desulfo hirudin45-65) peptides substituted by 20 natural L-amino acids via the Multipin method. The resulting peptide library is subsequently screened using an alpha-thrombin-mediated fibrinogen clotting assay and alpha-thrombin-induced amidolytic hydrolysis assay.

Amides↗

PubMed1999

The cytoplasmic domain of FtsK protein is required for resolution of chromosome dimers.

Chromosome dimers, formed by homologous recombination between sister chromosomes, normally require cell division to be resolved into monomers by site-specific recombination at the dif locus of Escherichia coli. We report here that it is not in fact cell division per se that is required for dimer resolution but the action of the cytoplasmic domain of FtsK, which is a bifunctional protein required both for cell division and for chromosome partition.

Binding Sites↗

PubMed1999

Advances in the signal transduction of ceramide and related sphingolipids.

Recently, the sphingolipid metabolites ceramide, sphingosine, ceramide 1-P, and sphingosine 1-P have been implicated as second messengers involved in many different cellular functions. Publications on this topic are appearing at a rapidly increasing rate and new developments in this field are also appearing rapidly. It is thus important to summarize the results obtained from many different laboratories and from different fields of research to obtain a clearer picture of the importance of sphingolipid metabolites. This article reviews the studies from the last few years and includes the effects of a variety of extracellular agents on sphingolipid signal transduction pathways in different tissues and cells and on the mechanisms of regulation. Sphingomyelin exists in a number of functionally distinct pools and is composed of distinct molecular species. Sphingomyelin metabolites may be formed by many different pathways. For example, the generation of ceramide from sphingomyelin can be catalyzed by at least five different sphingomyelinases. A large variety of stimuli can induce the generation of ceramide, leading to activation or inhibition of various cellular events such as proliferation, differentiation, apoptosis, and inflammation. The effect of ceramide on these physiological processes is due to its many different downstream targets. It can activate ceramide-activated protein kinases and ceramide-activated protein phosphatases. It also activates or inhibits PKCs, PLD, PLA2, PC-PLC, nitric oxide synthase, and the ERK and SAPK/JNK signaling cascades. Ceramide activates or inhibits transcription factors, modulates calcium homeostasis and interacts with the retinoblastoma protein to regulate cell cycle progression. Most of the work in this field has involved the study of ceramide effects, but the roles of the other three sphingomyelin metabolites is now attracting much attention. The complex interactions between signaling components and ceramide and the controls regulating these interactions are now being identified and are presented in this review.

Animals↗

PubMed1999

Measurement of the expression of oncofetal fibronectin mRNA in thyroid carcinomas by competitive reverse transcription-polymerase chain reaction.

Abundant expression of oncofetal fibronectin mRNA has been observed in thyroid papillary and anaplastic carcinomas. In this study, we measured relative expression levels of oncofetal fibronectin mRNA in thyroid cancer tissues by competitive reverse transcription polymerase chain reaction (RT-PCR) using thyroglobulin mRNA as an internal control. By this method, all papillary and anaplastic carcinomas and 3 of 6 follicular carcinomas were distinguished from benign tissues, such as normal thyroid tissues, follicular adenomas, and adenomatous goiters. Furthermore, 2 anaplastic carcinomas were clearly distinguished from differentiated carcinomas. These results suggest the possibility of establishing a more accurate preoperative or postoperative diagnosis of papillary and anaplastic carcinomas by measuring the relative expression level of oncofetal fibronectin to thyroglobulin in thyroid tumors.

Adenoma↗