Guqi Wang

Antioxidative function of L‐FABP in L‐FABP stably transfected Chang liver cells

Liver fatty acid binding protein (L‐FABP) contains amino acids that are known to possess antioxidant function. In this study, we tested the hypothesis that L‐FABP may serve as an effective endogenous cytoprotectant against oxidative stress. Chang liver cells were selected as the experimental model because of their undetectable L‐FABP mRNA level. Full‐length L‐FABP cDNA was subcloned into the mammalian expression vector pcDNA3.1 (pcDNA‐FABP). Chang cells were stably transfected with pc‐DNA‐FABP or vector (pcDNA3.1) alone. Oxidative stress was induced by incubating cells with 400 μmol/L H2O2 or by subjecting cells to hypoxia/reoxygenation. Total cellular reactive oxygen species (ROS) was determined using the fluorescent probe DCF. Cellular damage induced by hypoxia/reoxygenation was assayed by lactate dehydrogenase (LDH) release. Expression of L‐FABP was documented by regular reverse transcription polymerase chain reaction (RT‐PCR), real‐time RT‐PCR, and Western blot. The pcDNA‐FABP–transfected cells expressed full‐length L‐FABP mRNA, which was absent from vector‐transfected control cells. Western blot showed expression of 14‐kd L‐FABP protein in pcDNA‐FABP–transfected cells, but not in vector‐transfected cells. Transfected cells showed decreased DCF fluorescence intensity under oxidative stress (H2O2 and hypoxia/reoxygenation) conditions versus control in inverse proportion to the level of L‐FABP expression. Lower LDH release was observed in the higher L‐FABP–expressed cells in hypoxia/reoxygenation experiments. In conclusion, we successfully transfected and cloned a Chang liver cell line that expressed the L‐FABP gene. The L‐FABP–expressing cell line had a reduced intracellular ROS level versus control. This finding implies that L‐FABP has a significant role in oxidative stress. (HEPATOLOGY 2005;42:871–879.)

Molecular mechanism of recombinant liver fatty acid binding protein's antioxidant activity.

Hepatocytes expressing liver fatty acid binding protein (L-FABP) are known to be more resistant to oxidative stress than those devoid of this protein. The mechanism for the observed antioxidant activity is not known. We examined the antioxidant mechanism of a recombinant rat L-FABP in the presence of a hydrophilic (AAPH) or lipophilic (AMVN) free radical generator. Recombinant L-FABP amino acid sequence and its amino acid oxidative products following oxidation were identified by MALDI quadrupole time-of-flight MS after being digested by endoproteinase Glu-C. L-FABP was observed to have better antioxidative activity when free radicals were generated by the hydrophilic generator than by the lipophilic generator. Oxidative modification of L-FABP included up to five methionine oxidative peptide products with a total of ∼80 Da mass shift compared with native L-FABP. Protection against lipid peroxidation of L-FABP after binding with palmitate or α-bromo-palmitate by the AAPH or AMVN free radical generators indicated that ligand binding can partially block antioxidant activity. We conclude that the mechanism of L-FABPs antioxidant activity is through inactivation of the free radicals by L-FABPs methionine and cysteine amino acids. Moreover, exposure of the L-FABP binding site further promotes its antioxidant activity. In this manner, L-FABP serves as a hepatocellular antioxidant.

Cell lysis with dimethyl sulphoxide produces stable homogeneous solutions in the dichlorofluorescein oxidative stress assay

The oxidation of 2′,7′-dichlorodihydrofluorescein (2′,7′-dichlorofluorescin, DCFH) to a fluorescent product, 2′,7′-dichlorofluorescein (DCF), is commonly used to quantitatively measure oxidative stress in cells using a fluorescence microplate reader. However, many cell lines tend to grow non-uniformly in the wells. This non-uniform distribution results in a high degree of variability in the fluorescence signal and decreases the precision of the method. Also, samples treated in large culture plates, dishes or flasks cannot be assayed directly in fluorescence microplate readers. This study reports an improved DCF assay method that lyses cells with DMSO/PBS (90% dimethyl sulphoxide/10% phosphate buffered saline). Oxidative stress was induced with either hydrogen peroxide or an hypoxia-reoxygenation treatment. Cell lysis with DMSO/PBS resulted in highly stable fluorescence signals in comparison to Triton X-100/PBS lysed cells. The precision of DCF fluorescence measurements of DMSO/PBS lysed cells was much better than for attached cells measured directly in 96-well plates. While DCF fluorescence in PBS was strongly quenched by albumin, no quenching occurred in DMSO/PBS. In conclusion this study describes a more convenient and accurate method for measuring cellular oxidative stress that also makes it possible to assay cells treated in large culture plates.

Development of a Nitric Oxide-Releasing Analogue of the Muscle Relaxant Guaifenesin for Skeletal Muscle Satellite Cell Myogenesis

Nitric oxide (NO) mediates activation of satellite precursor cells to enter the cell cycle. This provides new precursor cells for skeletal muscle growth and muscle repair from injury or disease. Targeting a new drug that specifically delivers NO to muscle has the potential to promote normal function and treat neuromuscular disease, and would also help to avoid side effects of NO from other treatment modalities. In this research, we examined the effectiveness of the NO donor, iosorbide dinitrate (ISDN), and a muscle relaxant, methocarbamol, in promoting satellite cell activation assayed by muscle cell DNA synthesis in normal adult mice. The work led to the development of guaifenesin dinitrate (GDN) as a new NO donor for delivering nitric oxide to muscle. The results revealed that there was a strong increase in muscle satellite cell activation and proliferation, demonstrated by a significant 38% rise in DNA synthesis after a single transdermal treatment with the new compound for 24 h. Western blot and immunohistochemistry analyses showed that the markers of satellite cell myogenesis, expression of myf5, myogenin, and follistatin, were increased after 24 h oral administration of the compound in adult mice. This research extends our understanding of the outcomes of NO-based treatments aimed at promoting muscle regeneration in normal tissue. The potential use of such treatment for conditions such as muscle atrophy in disuse and aging, and for the promotion of muscle tissue repair as required after injury or in neuromuscular diseases such as muscular dystrophy, is highlighted.

Infection of myocytes with chlamydiae.

Chlamydial infection has been associated with myocarditis in animals and humans. However, the mechanism resulting in myocarditis following infection is not known. Here, evidence is presented that both Chlamydia trachomatis and Chlamydia pneumoniae can infect and replicate in myocytes isolated from neonate rats. The infected myocytes contained chlamydial inclusions, indicative of chlamydial growth, and infectious particles were recovered from the infected myocytes. It was also found that chlamydial infection at a late stage induced significant damage to the infected myocytes, as evidenced by an increased lactate dehydrogenase release, reactive oxygen species production and a reduced ATP level. However, no nuclear apoptosis was detected in the infected myocytes. Collectively, these observations have demonstrated that Chlamydia spp. are able to both infect and damage myocytes, suggesting a potential role of chlamydial infection in myocarditis.

Enhanced expression of cytosolic fatty acid binding protein and fatty acid uptake during liver regeneration in rats.

Background/Aims: Cytoplasmic liver fatty acid binding protein (L-FABP) has been suggested to be associated with cellular mitotic activity but the changes in L-FABP mRNA and protein levels during liver regeneration following partial hepatectomy (PHx) are not clear. Methods: In the present study, we determined the time course of L-FABP mRNA expression and L-FABP levels following 70% PHx using Northern and Western blot, respectively. To elucidate one of the roles for L-FABP in PHx, [3H]-palmitic acid clearance in hepatocytes isolated from 24 h post-PHx and control animals was assessed. Results: L-FABP mRNA increased at 30 min, peaked at approximately 1 h (163 ± 17%; mean ± SE, n= 5), and returned to control levels 6 h post-PHx. L-FABP level also increased at 1 h but peaked at 24-h (219 ± 41%; mean ± SE, n= 5). Hepatocyte [3H]-palmitic acid clearance increased by 29% at 24-h post-PHx, suggesting an increased intracellular transport (or binding) function by L-FABP. Pre-treatment with dexamethasone statistically reduced L-FABP levels (29%) and suppressed the regenerative process (mitotic activity). Conclusions: L-FABP mRNA increased sharply in response to PHx but the increase was short lived, while L-FABP level increased at a later stage. Both L-FABP content and fatty acid uptake increased significantly during liver regeneration induced by PHx in rats. It is likely that L-FABP is one of the factors responsible for hepatic regeneration. (Mol Cell Biochem 262: 41–49, 2004)

Binding of [3H]palmitate to BSA

Determination of the BSA-palmitate high-affinity binding constant ( K a) traditionally relied on the heptane-water partitioning technique. We used this technique to calculate K a for the BSA-[3H]palmitate complex, to determine if K a was independent of protein concentration, and to determine if the unbound [3H]palmitate concentration is constant at different BSA concentrations using constant BSA-to-palmitate molar ratios (range 1:1 to 1:4). After extensive extraction of non-[3H]palmitate radiolabeled substances, the heptane-to-buffer partition ratio, in the absence of BSA, was 702 ± 19 (mean ± SD, n = 6). This value was much lower than the predicted value of 1,376 and was highly dependent on which phase (organic or aqueous) initially contained the [3H]palmitic acid. The data were consistent with the notion of self-association of [3H]palmitate in the aqueous phase. K afor the BSA-[3H]palmitate complex was determined to be similar (2.2 ± 0.1) × 108M-1 (mean ± SD, P > 0.05) at all BSA concentrations studied. At each BSA-to-palmitate molar ratio, the equilibrium unbound ligand concentration was constant only at low BSA concentrations (<10 μM) and at low BSA-to-palmitate molar ratios (i.e., 1:1 and 1:2). At higher BSA concentrations and molar ratios, the unbound ligand concentration increased with an increase in protein concentration. Hepatocyte uptake using the manufacturer-supplied radiolabeled product was significantly higher than with the purified product, suggesting that a non-[3H]palmitate radiolabel is also a substrate for the uptake process.

Nitric oxide donors improve prednisone effects on muscular dystrophy in the mdx mouse diaphragm

In Duchenne muscular dystrophy (DMD), palliative glucocorticoid therapy can produce myopathy or calcification. Since increased nitric oxide synthase activity in dystrophic mice promotes regeneration, the outcome of two nitric oxide (NO) donor drugs, MyoNovin (M) and isosorbide dinitrate (I), on the effectiveness of the anti-inflammatory drug prednisone (P) in alleviating progression of dystrophy was tested. Dystrophic mdx mice were treated (18 days) as controls or with an NO donor ± P. Fiber permeability and DNA synthesis were labeled by Evans blue dye (EBD) and bromodeoxyuridine uptake, respectively. P decreased body weight gain, M increased quadriceps mass, and I increased heart mass. P increased fiber permeability (%EBD+ fibers) and calcification in diaphragm. Treatment with NO donors + P (M+P, I+P) reduced %EBD+ fibers and calcification vs. P alone. %EBD+ fibers in M+P diaphragm did not differ from control. NO donor treatment reduced proliferation and the population of c-met+ cells and accelerated fiber regeneration. Concurrent with P, NO donor treatment suppressed two important detrimental effects of P in mice, possibly by accelerating regeneration, rebalancing satellite cell quiescence and activation in dystrophy, and/or increasing perfusion. Results suggest that NO donors could improve current therapy for DMD.

Effect of binding protein surface charge on palmitate uptake by hepatocyte suspensions

Studies were directed at determining whether hepatocytes, isolated from female Sprague‐Dawley rats, facilitate the uptake of protein‐bound long‐chain fatty acids. We postulated one form of facilitated uptake may occur through an ionic interaction between the protein‐ligand complex and the cell surface. These interactions are expected to supply additional ligand to the cell for uptake. The clearance rate of [3H]‐palmitate in the presence of α1‐acid‐glycoprotein (pI=2.7), albumin (pI=4.9) and lysozyme (pI=11.0) was investigated. Palmitate uptake was determined in the presence of protein concentrations that resulted in similar unbound ligand fractions (=0.03). The experimental clearance rates were compared to the theoretical predictions based upon the diffusion‐reaction model. By use of our experimentally determined equilibrium binding and dissociation rate constants for the various protein‐palmitate complexes, the diffusion‐reaction model predicted clearance rates were 4.9 μl s−1/106 cells, 4.8 μl s−1/106 cells and 5.5 μl s−1/106 cells for α1‐acid‐glycoprotein, albumin and lysozyme, respectively; whereas the measured hepatocyte palmitate clearance rates were 1.2±0.1 μl s−1/106 cells, 2.3±0.3 μl s−1/106 cells and 7.1±0.7 μl s−1/106, respectively. Hepatocyte palmitate clearance was significantly faster (P<0.01) in the presence of lysozyme than albumin which was significantly faster than α1‐acid‐glycoprotein (P<0.01). The marked difference in clearance rates could not be explained by considering differences in solution viscosity. Our results are consistent with the notion that ionic interactions between protein‐ligand complexes and the cell surface facilitate the ligand uptake by decreasing the diffusional distance of the unbound ligand and/or by facilitating the protein‐ligand dissociation rate.

Sequencing of rat liver cytosolic proteins by matrix-assisted laser desorption ionization–quadrupole time-of-flight mass spectrometry following electrophoretic separation and extraction

A new technique is described that enables the direct determination of the complete or partial amino acid sequence of cytosolic proteins separated by gel electrophoresis and allows for the further observation of disease- or drug-induced posttranslational modifications. The procedure uses a two-phase extraction strategy (ethyl acetate/ammonium bicarbonate) for the efficient separation of proteins/peptides from an electrophoretic matrix and subsequent sequence analysis by matrix-assisted laser desorption ionization-quadrupole time-of-flight mass spectrometry. The method was tested using hepatocyte cytosolic proteins and compared to a complementary approach using direct solvent extraction from in-gel digests. Although the latter procedure identified the proteins, it did not enable complete amino acid sequence determination. In contrast, high sequence coverage was obtained using the peptide extraction procedure, without any apparent dependence on protein size. The technique minimized the chemically inconsistent modifications generated from in-gel digestion, thus aiding mass spectrometric interpretation and valid protein sequence identification.