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Biomedical subjects

N Li

Publications and source records attributed to N Li.

At least 163 records · Page 9Linked to original sources
PubMed2000

Ca(2+)-binding proteins in the retina: from discovery to etiology of human disease(1).

Examination of the role of Ca(2+)-binding proteins (CaBPs) in mammalian retinal neurons has yielded new insights into the function of these proteins in normal and pathological states. In the last 8 years, studies on guanylate cyclase (GC) regulation by three GC-activating proteins (GCAP1-3) led to several breakthroughs, among them the recent biochemical analysis of GCAP1(Y99) mutants associated with autosomal dominant cone dystrophy. Perturbation of Ca(2+) homeostasis controlled by mutant GCAP1 in photoreceptor cells may result ultimately in degeneration of these cells. Here, detailed analysis of biochemical properties of GCAP1(P50L), which causes a milder form of autosomal dominant cone dystrophy than constitutive active Y99C mutation, showed that the P50L mutation resulted in a decrease of Ca(2+)-binding, without changes in the GC activity profile of the mutant GCAP1. In contrast to this biochemically well-defined regulatory mechanism that involves GCAPs, understanding of other processes in the retina that are regulated by Ca(2+) is at a rudimentary stage. Recently, we have identified five homologous genes encoding CaBPs that are expressed in the mammalian retina. Several members of this subfamily are also present in other tissues. In contrast to GCAPs, the function of this subfamily of calmodulin (CaM)-like CaBPs is poorly understood. CaBPs are closely related to CaM and in biochemical assays CaBPs substitute for CaM in stimulation of CaM-dependent kinase II, and calcineurin, a protein phosphatase. These results suggest that CaM-like CaBPs have evolved into diverse subfamilies that control fundamental processes in cells where they are expressed.

Amino Acid Sequence↗
PubMed2000

Expression of vascular endothelial growth factor isoforms and platelet-derived endothelial cell growth factor in bladder cancer.

We analyzed the expression of vascular endothelial growth factor (VEGF) messenger ribonucleic acid (mRNA) isoforms and platelet-derived endothelial cell growth factor (PDECGF) mRNA in bladder cancer. We also attempted to determine if correlation exists between their expression level and conventional clinical variables in patients with bladder cancer. Tissues obtained from 60 patients with bladder carcinoma were used for analysis. Expression levels of VEGF isoforms and PDECGF were examined using reverse transcription-polymerase chain reaction (RT-PCR). Correlations between the expression levels of each VEGF isoform and PDECGF and histopathologic findings were evaluated. Four VEGF isoforms corresponding to VEGF121, 165, 189, and 206 were detected in bladder cancer tissue by RT-PCR. Gene expression of all VEGF isoforms as a ratio of the target to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) showed no correlation with pathologic stage of bladder cancer. However, with regard to relative expression levels of VEGF isoform, which is the ratio to the sum of total VEGF isoforms, the levels of VEGF206 and VEGF189 in tumor samples of grade pT2 or higher were significantly lower than those in tumors of grade pT1 or lower (P<.05). In contrast, the levels of VEGF121 in >/=pT2 tumors tended to be higher than those in </=pT1 tumors (P=.056). The expression level of PDECGF as a ratio to GAPDH in pT2</= tumors was significantly higher than that in either pTa or pT1 tumors (P<.05). Moreover, a higher expression level of PDECGF was observed in G3 tumors than in G1 tumors (P<.05). The results indicated that gene expression of VEGF isoforms do not play a significant role in tumor progression or invasion; however, the distribution of VEGF isoforms may play a role in tumor progression of bladder cancer. A high expression level of PDECGF correlated significantly with the tumor progression of bladder cancer.

Journal Article↗
PubMed2000

ATM is required for IkappaB kinase (IKKk) activation in response to DNA double strand breaks.

Following challenge with proinflammatory stimuli or generation of DNA double strand breaks (DSBs), transcription factor NF-kappaB translocates from the cytoplasm to the nucleus to activate expression of target genes. In addition, NF-kappaB plays a key role in protecting cells from proapoptotic stimuli, including DSBs. Patients suffering from the genetic disorder ataxia-telangiectasia, caused by mutations in the ATM gene, are highly sensitive to inducers of DSBs, such as ionizing radiation. Similar hypersensitivity is displayed by cell lines derived from ataxia-telangiectasia patients or Atm knockout mice. The ATM protein, a member of the phosphatidylinositol 3-kinase (PI3K)-like family, is a multifunctional protein kinase whose activity is stimulated by DSBs. As both ATM and NF-kappaB deficiencies result in increased sensitivity to DSBs, we examined the role of ATM in NF-kappaB activation. We report that ATM is essential for NF-kappaB activation in response to DSBs but not proinflammatory stimuli, and this activity is mediated via the IkappaB kinase complex. DNA-dependent protein kinase, another member of the PI3K-like family, PI3K itself, and c-Abl, a nuclear tyrosine kinase, are not required for this response.

Androstadienes↗
PubMed2000

Influences of fixatives on flow cytometric measurements of platelet P-selectin expression and fibrinogen binding.

Sample fixation is an important issue in flow cytometric platelet assays. However, previous reports were less than consistent regarding the influence of sample fixation on the assays. We evaluated the effects of formaldehyde and paraformaldehyde fixation on platelet P-selectin expression and fibrinogen binding using whole-blood flow cytometry and a Coulter EPICS XL-MCL cytometer. Fluorescent-labeled whole-blood samples were diluted with HEPES-buffered saline or fixed with formaldehyde (0.2, 0.5, and 1. 0%) or paraformaldehyde (0.5, 1.0, and 2.0%). Platelet P-selectin expression was 1.1+/-0.3% and 39.6+/-13.7% in unfixed resting and 10(-5) M ADP stimulated samples, respectively. Resting P-selectin expression was not significantly altered by 0.2 or 0.5% formaldehyde fixation, but was slightly decreased by 1.0% formaldehyde fixation or PFA fixation. Formaldehyde fixation caused small increases of P-selectin expression in ADP-stimulated samples. Compared to platelet fibrinogen binding of unfixed resting (4.5+/-2.1%) and ADP-stimulated (56.7+/-22.6%) samples, formaldehyde or paraformaldehyde fixation had no significant influence on resting samples, but mildly increased fibrinogen binding in stimulated samples. Unfixed samples were stable for 2 h. Fixed samples were generally stable for at least 6 h, but not thereafter. Thus, formaldehyde and paraformaldehyde have mild but complex influences on platelet P-selectin expression and fibrinogen binding measurements. To evaluate the stabilities of unfixed and fixed samples, samples were analyzed after different durations (0, 1, 2, 4, 6, 12, and 24 h) of storage at 4 degrees C in the dark. The results suggest that sample manipulation without fixation may be used when the samples are analyzed within 2 h, and that fixation with 0.5-1.0% formaldehyde or paraformaldehyde seems to be preferable when sample analysis is delayed. Effects of fixation should be carefully evaluated when establishing flow cytometric platelet assays in every laboratory.

Adenosine Diphosphate↗
PubMed2000

Tandem-cartridge solid-phase extraction followed by GC/MS analysis for measuring partition coefficients of association of polycyclic aromatic hydrocarbons to humic acid.

A tandem-cartridge solid-phase extraction system combining reversed-phase separation and dynamic ion exchange followed by GC/MS analysis was studied for measuring partition coefficients (Kdoc) of association of polycyclic aromatic hydrocarbons (PAHs) to humic acid. Time course batch experiments revealed that the association of PAHs to humic acid included a slow stage with equilibrium time of 5-7 days. The disequlibrium and retention of the PAH-humic acid associate during reversed-phase separation lead to systematic negative error for the measured partition coefficients; more accurate results were achieved with a three-cartridge tandem system in which the negative error was assessed and corrected via the PAH analytes measured on the second reversed-phase cartridge. The potential and advantages of use of C18 disk cartridges instead of conventional C18 cartridges to set up the tandem system for measuring the partition coefficients were also studied. With the tandem-cartridge system, the measured partition coefficients did not change with most experimental conditions, but a trend of decreasing partition coefficients with humic acid concentration was observed, implying possible nonlinear association of the PAHs and humic acid.

Environmental Pollutants↗
PubMed2000

Molecular cloning and functional analysis of the promoter region of rat nonmuscle myosin heavy chain-B gene.

Rat nonmuscle myosin heavy chain-B (r-nmMHC-B) mRNA was previously found downregulated in Rat 6 fibroblasts transformed by mutant p53(val135) [J. W. P. Yam, J. Y. Zheng, and W. L. W. Hsiao (1987) Biochem. Biophys. Res. Commun. 266, 472-480]. Overexpression of exogenous r-nmMHC-B could partially reverse the transforming phenotypes both in vitro and in vivo. The downregulation of r-nmMHC-B was also observed in Rat 6 transformed by c-H-ras and v-myc oncogenes. We cloned a 5.2-kb r-nmMHC-B promoter region. Sequence analysis of -1248 to +1 revealed no TATA box, but did show that it contained CAAT boxes, E12/E47, MyoD, MEF, E2F, CREB, and SP1 binding sites. Based on transient reporter assays, the promoter/enhancer activities were unusually extended to the entire 5.2 kb region in normal Rat 6 cultures, but markedly suppressed in p53(val135)-, and c-H-ras-transformed cells. The activity detected by the reporter assay corresponded to levels of mRNA as analyzed previously by Northern blots in each respective cell line. Thus, the switch-off of the r-nmMHC-B in the transformed cells is very likely controlled by upstream transcriptional factors, which might have been altered in the course of neoplastic transformation.

Animals↗
PubMed2000

Induction of heme oxygenase-1 expression in macrophages by diesel exhaust particle chemicals and quinones via the antioxidant-responsive element.

Diesel exhaust particles (DEP) contain organic chemicals that contribute to the adverse health effects of inhaled particulate matter. Because DEP induce oxidative stress in the lung and in macrophages, effective antioxidant defenses are required. One type of defense is through the expression of the antioxidant enzyme, heme oxygenase I (HO-1). HO-1 as well as phase II detoxifying enzymes are induced via antioxidant response elements (ARE) in their promoters of that gene. We show that a crude DEP total extract, aromatic and polar DEP fractions, a benzo(a)pyrene quinone, and a phenolic antioxidant induce HO-1 expression in RAW264.7 cells in an ARE-dependent manner. N-acetyl cysteine and the flavonoid, luteolin, inhibited HO-1 protein expression. We also demonstrate that the same stimuli induce HO-1 mRNA expression in parallel with the activation of the SX2 enhancer of that gene. Mutation of the ARE core, but not the overlapping AP-1 binding sequence, disrupted SX2 activation. Finally, we show that biological agents, such as oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine, could also induce HO-1 expression via an ARE-dependent mechanism. Prior induction of HO-1 expression, using cobalt-protoporphyrin, protected RAW264.7 cells against DEP-induced toxicity. Taken together, these data show that HO-1 plays an important role in cytoprotection against redox-active DEP chemicals, including quinones.

Acetylcysteine↗
PubMed2000

The role of a mitochondrial pathway in the induction of apoptosis by chemicals extracted from diesel exhaust particles.

We are interested in the cytotoxic and proinflammatory effects of particulate pollutants in the respiratory tract. We demonstrate that methanol extracts made from diesel exhaust particles (DEP) induce apoptosis and reactive oxygen species (ROS) in pulmonary alveolar macrophages and RAW 264.7 cells. The toxicity of these organic extracts mimics the cytotoxicity of the intact particles and could be suppressed by the synthetic sulfhydryl compounds, N-acetylcysteine and bucillamine. Because DEP-induced apoptosis follows cytochrome c release, we studied the effect of DEP chemicals on mitochondrially regulated death mechanisms. Crude DEP extracts induced ROS production and perturbed mitochondrial function before and at the onset of apoptosis. This mitochondrial perturbation follows an orderly sequence of events, which commence with a change in mitochondrial membrane potential, followed by cytochrome c release, development of membrane asymmetry (annexin V staining), and propidium iodide uptake. Structural damage to the mitochondrial inner membrane, evidenced by a decrease in cardiolipin mass, leads to O-*2 generation and uncoupling of oxidative phosphorylation (decreased intracellular ATP levels). N-acetylcysteine reversed these mitochondrial effects and ROS production. Overexpression of the mitochondrial apoptosis regulator, Bcl-2, delayed but did not suppress apoptosis. Taken together, these results suggest that DEP chemicals induce apoptosis in macrophages via a toxic effect on mitochondria.

Air Pollutants↗
PubMed2000

Cloning of DPK, a novel dendritic cell-derived protein kinase activating the ERK1/ERK2 and JNK/SAPK pathways.

Mitogen-activated protein kinase (MAPK) cascades are the major signaling systems transducing extracellular signals into intracellular responses, which mainly include the extracellular signal-regulated kinase (ERK) pathway, the c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) pathway, and the p38 pathway. From dendritic cell cDNA library, we isolated a full-length cDNA encoding a potentially novel 898-residue kinase, which was designated DPK. The protein contained a potential kinase domain at the N-terminal exhibiting homology with MEKK1-, MEKK2-, MEKK3-, MEKK4-, MEKK5-, Tpl-2-, and p21-activated kinases (PAKs), but no GTPase-binding domain which is characteristic of PAKs. Northern blotting analysis showed that DPK was ubiquitously expressed in normal tissues, with abundant expression in kidney, skeletal muscle, heart, and liver. When overexpressed in transfected NIH3T3 cells, it could activate both the ERK1/ERK2 pathway and the SAPK pathway in a dose-dependent manner, but not affect the p38 pathway. These findings suggested that DPK might be a novel candidate MAPKKK.

Amino Acid Sequence↗
PubMed2000

Sample preparation based on dynamic ion-exchange solid-phase extraction for GC/MS analysis of acidic herbicides in environmental waters.

The newly established enrichment technique, dynamic ion-exchange solid-phase extraction (DIE-SPE), was studied for sample preparation for GC/MS analysis of 16 acidic herbicides in environmental waters. C18 bonded silica was the solid-phase material used. The optimal sample pH was weakly acidic to neutral. However, for common tap water and surface water, which run pH 6-9, all the acidic herbicides except for Chloramben could be effectively extracted from a sample of 1,000-mL volume without pH adjustment. The humic acid could be concurrently extracted from water, but most of it was separated from the sample by using 3 mL of 10% methanol in acetone as the eluent, which would completely elute the analytes and leave a large part of the humic acid on the cartridge. The selective elution reduced the interference of humic acid and made the DIE-SPE an effective approach for the analysis of the acidic herbicides in surface water. Comparing DIE-SPE with conventional reversed-phase SPE (RP-SPE), the former gave higher recoveries for the acidic herbicides and was less affected by sample matrixes. A tandem-cartridge system combining RP- and DIE-SPE in sequence was set up for the simultaneous isolation of the acidic herbicides and removal of the interfering substances. Despite some minimal retention on the upper RP-SPE cartridge, most of the acidic herbicides could be extracted on the lower DIE-SPE cartridge with recovery over 80% except for Chloramben (50%), fenoprop (73%), MCPB (67%), and 2,4-DB (70%) when a 500-mL aqueous sample of pH 9.5 was percolated through the tandem-cartridge system. The effectiveness of the system in removing the long carbon chain fatty acids as well as the basic and neutral organic interfering substances from the sample was also demonstrated.

Acids↗
PubMed2000

A universal pattern of mortality decline in the G7 countries.

Human lifespan has increased enormously this century. But we remain uncertain about the forces that reduce mortality, and about the cost implications of ageing populations and their associated social burden. The poor understanding of the factors driving mortality decline, and the difficulty of forecasting mortality are due in part to the pronounced irregularity of annual to decadal mortality change. Here we examine mortality over five decades in the G7 countries (Canada, France, Germany, Italy, Japan, UK, US). In every country over this period, mortality at each age has declined exponentially at a roughly constant rate. This trend places a constraint on any theory of society-driven mortality decline, and provides a basis for stochastic mortality forecasting. We find that median forecasts of life expectancy are substantially larger than in existing official forecasts. In terms of the costs of ageing, we forecast values of the dependency ratio (that is, the ratio of people over 65 to working people) in 2050 that are between 6% (UK) and 40% (Japan) higher than official forecasts.

Aging↗
PubMed2000

Evolution of a glucose-regulated ADH gene in the genus Saccharomyces.

To determine when a glucose-repressed alcohol dehydrogenase isozyme and its regulatory gene, ADR1, arose during evolution, we surveyed species of the genus Saccharomyces for glucose-repressed ADH isozymes and for ADR1 homologues. Glucose-repressed ADH isozymes were present in all species of Saccharomyces sensu strictu and also in Saccharomyces kluyveri, the most distant member of the Saccharomyces clade. We cloned and characterized ADH promoters from S. bayanus, S. douglasii, and S. kluyveri. The ADH promoters from S. bayanus and S. douglasii had conserved sequences, including upstream regulatory elements, and an extended polydA tract. The expression of a reporter gene driven by the S. bayanus promoter was glucose-repressed and dependent on the major activator of transcription, ADR1, when it was introduced into S. cerevisiae. One S. kluyveri promoter was also glucose-repressed and ADR1-dependent in S. cerevisiae. The other S. kluyveri ADH promoter was expressed constitutively and was ADR1-independent. Although showing little sequence conservation with the S. cerevisiae ADH2 promoter, the glucose-repressed S. kluyveri promoter contains numerous potential binding sites for Adr1. The glucose-repressed ADH from S. kluyveri is a mitochondrial isozyme most closely related to S. cerevisiae ADHIII. ADR1 homologues from S. douglasii and S. paradoxus contain a trinucleotide repeat encoding polyAsn that is lacking in S. cerevisiae and S. bayanus. No ADR1 homologue could be detected in S. kluyveri, suggesting that the potential for Adr1 regulation may have arisen first, before ADR1 evolved.

Alcohol Dehydrogenase↗
PubMed2000

SNARE protein-dependent glutamate release from astrocytes.

We investigated the cellular mechanisms underlying the Ca(2+)-dependent release of glutamate from cultured astrocytes isolated from rat hippocampus. Using Ca(2+) imaging and electrophysiological techniques, we analyzed the effects of disrupting astrocytic vesicle proteins on the ability of astrocytes to release glutamate and to cause neuronal electrophysiological responses, i.e., a slow inward current (SIC) and/or an increase in the frequency of miniature synaptic currents. We found that the Ca(2+)-dependent glutamate release from astrocytes is not caused by the reverse operation of glutamate transporters, because the astrocyte-induced glutamate-mediated responses in neurons were affected neither by inhibitors of glutamate transporters (beta-threo-hydroxyaspartate, dihydrokainate, and L-trans-pyrrolidine-2,4-dicarboxylate) nor by replacement of extracellular sodium with lithium. We show that Ca(2+)-dependent glutamate release from astrocytes requires an electrochemical gradient necessary for glutamate uptake in vesicles, because bafilomycin A(1), a vacuolar-type H(+)-ATPase inhibitor, reduced glutamate release from astrocytes. Injection of astrocytes with the light chain of the neurotoxin Botulinum B that selectively cleaves the vesicle-associated SNARE protein synaptobrevin inhibited the astrocyte-induced glutamate response in neurons. Therefore, the Ca(2+)-dependent glutamate release from astrocytes is a SNARE protein-dependent process that requires the presence of functional vesicle-associated proteins, suggesting that astrocytes store glutamate in vesicles and that it is released through an exocytotic pathway.

ATP-Binding Cassette Transporters↗
PubMed2000

Formation and actions of cyclic ADP-ribose in renal microvessels.

Recent studies indicated that cyclic ADP-ribose (cADPR) serves as a second messenger for intracellular Ca(2+) mobilization in a variety of mammalian cells. However, the metabolism and actions of cADPR in the renal vasculature are poorly understood. In the present study, we characterized the enzymatic pathway of the production and metabolism of cADPR along the renal vascular tree and determined the role of cADPR in the control of intracellular [Ca(2+)] and vascular tone. The high performance liquid chromatographic analyses showed that cADPR was produced and hydrolyzed along the renal vasculature. The maximal conversion rate of nicotinamide guanine dinucleotide (NGD) into cyclic GDP-ribose (that represents ADP-ribosyl cyclase activity for cADPR formation) was 8.69 +/- 2.39 nmol/min/mg protein in bulk-dissected intrarenal preglomerular vessels (n = 7) and 4.35 +/- 0.13, 2.23 +/- 0.27, 2.40 +/- 0.19, and 0.31 +/- 0.02 nmol/min/mg protein, respectively, in microdissected arcuate arteries (n = 6), interlobular arteries (n = 6), afferent arterioles (n = 7), and vasa recta (n = 10). The activity of cADPR hydrolase was also detected in the renal vasculature. Using the fluorescence microscopic spectrometry, cADPR was found to produce a large rapid Ca(2+) release from beta-escin-permeabilized renal arterial smooth muscle cells (SMCs). In isolated, perfused, and pressurized small renal arteries, cADPR produced a concentration-dependent vasoconstriction when added into the bath solution. The vasoconstrictor effect of cADPR was completely blocked by tetracaine, a Ca(2+)-induced Ca(2+) release (CICR) inhibitor. These results suggest that an enzymatic pathway for cADPR production and metabolism is present along the renal vasculature and that cADPR may importantly contribute to the control of renal vascular tone through CICR.

Adenosine Diphosphate Ribose↗
PubMed2000

Resonance Rayleigh scattering method for the determination of trace amounts of cadmium with iodide-basic triphenylmethane dye systems.

In dilute phosphoric acid solution, cadmium (II) reacts with a large excess of I- to form [CdI4]2- which reacts further with basic triphenylmethane dyes such as crystal violet (CV), ethyl violet (EV), methyl violet (MV), brilliant green (BG) or malachite green (MG) to form an ion-association complex. This results in a significant enhancement of resonance Rayleigh scattering (RRS) intensity and the appearance of new RRS spectra. The characteristics of RRS spectra of the ion-association complexes, the influencing factors and the optimum conditions of these reactions have been investigated. The intensity of RRS is directly proportional to the concentration of cadmium from 0 to 60 ng mL(-1) for EV and MV systems, 0 to 80 ng mL(-1) for CV system, and 0 to 100 ng mL(-1) for BG and MG systems. The methods exhibit high sensitivities and the detection limits for cadmium are between 0.35 and 2.00 ng mL(-1) depending on the different reaction systems. The new RRS method was applied to the direct determination of traces of cadmium in pure zinc and synthetic water samples.

Journal Article↗