Effects of platelet-activating factor, tumor necrosis factor, and interleukin-1alpha on the expression of apolipoprotein M in HepG2 cells

Apolipoprotein M (apoM) is a recently discovered human apolipoprotein predominantly present in high-density lipoprotein (HDL) in plasma, exclusively expressed in liver and in kidney. The function of apoM is yet unknown. The human apoM gene is located in the major histocompatibility complex class III region on chromosome 6. Because many genes located in this region are related to the immune response, we have investigated whether apoM might also be involved in the host inflammatory response. In this study we examined effects of the platelet-activating factor (PAF), tumor necrosis factor (TNF-alpha), and interleukin-1alpha (IL-1alpha) on apoM expression in a hepatoblastoma cell line, HepG2 cells. PAF significantly enhanced the apoM mRNA levels and the secretion of apoM in HepG2 cell cultures. The enhancement of apoM secretion is seen at a low concentration of PAF (2 ng/ml), whereas a high concentration of PAF increases both the apoM mRNA levels and apoM secretion. Neither TNF-alpha nor IL-1alpha influenced apoM mRNA level and secretion. Furthermore, Lexipafant, a PAF-receptor (PAF-R) antagonist significantly suppressed the mRNA level and the secretion of apoM in HepG2 cells in a dose-dependent manner. Neither PAF nor Lexipafant influenced the mRNA levels and the secretion of apoA-I, apoB and apoE in HepG2 cells, indicating that the effects of PAF or Lexipafant on the apoM production on hepatic cells are selective for apoM. The cellular mechanism of the effects of PAF or Lexipafant on apoM metabolism requires further investigations.     

Androgen receptor enhances myogenin expression and accelerates differentiation

Animal and clinical studies indicated that the androgen-AR signaling pathway is required for appropriate development of sexually dimorphic skeletal muscles and increases lean muscle mass, muscle strength, and muscle protein synthesis. However, the detailed mechanisms by which the androgen-AR signaling pathway regulates skeletal muscle development need further study at the molecular level. C2C12 myoblast cells stably transfected with the Flag-tagged AR were used to analyze the role of androgen-AR signaling pathway in skeletal muscle development. The results indicate that the androgen-AR signaling pathway may suppress skeletal myoblast cell growth and accelerate myoblast cell differentiation via enhanced myogenin expression. This is a first report showing the role of androgen-AR signaling pathway in regulation of myoblast cell growth and myogenic regulatory factors.     

Three-dimensional cellular deformation analysis with a two-photon magnetic manipulator workstation

The ability to apply quantifiable mechanical stresses at the microscopic scale is critical for studying cellular responses to mechanical forces. This necessitates the use of force transducers that can apply precisely controlled forces to cells while monitoring the responses noninvasively. This paper describes the development of a micromanipulation workstation integrating two-photon, three-dimensional imaging with a high-force, uniform-gradient magnetic manipulator. The uniform-gradient magnetic field applies nearly uniform forces to a large cell population, permitting statistical quantification of select molecular responses to mechanical stresses. The magnetic transducer design is capable of exerting over 200 pN of force on 4.5-microm-diameter paramagnetic particles and over 800 pN on 5.0-microm ferromagnetic particles. These forces vary within +/-10% over an area 500 x 500 microm2. The compatibility with the use of high numerical aperture (approximately 1.0) objectives is an integral part of the workstation design allowing submicron-resolution, three-dimensional, two-photon imaging. Three-dimensional analyses of cellular deformation under localized mechanical strain are reported. These measurements indicate that the response of cells to large focal stresses may contain three-dimensional global deformations and show the suitability of this workstation to further studying cellular response to mechanical stresses.     

Studies of the mechanism of phenol hydroxylase: effect of mutation of proline 364 to serine

An active site residue in phenol hydroxylase (PHHY), Pro364, was mutated to serine to investigate its role in enzymatic catalysis. In the presence of phenol, the reaction between the reduced flavin of P364S and oxygen is very fast, but only 13% of the flavin is utilized to hydroxylate the substrate, compared to nearly 100% for the wild-type enzyme. The oxidative half-reaction of PHHY using m-cresol as a substrate is similarly affected by the mutation. Pro364 was suggested to be important in stabilizing the transition state of the oxygen transfer step by forming a hydrogen bond between its carbonyl oxygen and the C4a-hydroperoxyflavin [Ridder, L., Mullholland, A. J., Rietjens, I. M. C. M., and Vervoort, J. (2000) J. Am. Chem. Soc. 122, 8728-8738]. The P364S mutation may weaken this interaction by increasing the flexibility of the peptide chain; hence, the transition state would be destabilized to result in a decreased level of hydroxylation of phenol. However, when the oxidative half-reaction was studied using resorcinol as a substrate, the P364S mutant form was not significantly different from the wild-type enzyme. The rate constants for all the reaction steps as well as the hydroxylation efficiency (coupling between NADPH oxidation and resorcinol consumption) are comparable to those of the wild-type enzyme. It is suggested that the function of Pro364 in catalysis, stabilization of the transition state, is not as important in the reaction with resorcinol, possibly because the position of hydroxylation is different with resorcinol than with phenol and m-cresol.     

Protective effect of metallothionein-III on DNA damage in response to reactive oxygen species

Metallothionein (MT)-III is a member of a brain-specific MT family, in contrast to MT-I and MT-II that are found in most tissues and are implicated in metal ion homeostasis and as an antioxidant. To investigate the defensive role of MT-III in terms of hydroxyl radical-induced DNA damage, we used purified human MT-III. DNA damage was detected by single-strand breaks of plasmid DNA and deoxyribose degradation. In this study, we show that MT-III is able to protect against the DNA damage induced by ferric ion-nitrilotriacetic acid and H(2)O(2), and that this protective effect is inhibited by the alkylation of the sulfhydryl groups of MT-III by treatment with EDTA and N-ethylmaleimide. MT-III was also able to efficiently remove the superoxide anion, which was generated from the xanthine/xanthine oxidase system. These results strongly suggest that MT-III is involved in the protection of reactive oxygen species-induced DNA damage, probably via direct interaction with reactive oxygen species, and that MT-III acts as a neuroprotective agent.     

Regulation of BRCC, a holoenzyme complex containing BRCA1 and BRCA2, by a signalosome-like subunit and its role in DNA repair

We have isolated a holoenzyme complex termed BRCC containing BRCA1, BRCA2, and RAD51. BRCC not only displays increased association with p53 following DNA damage but also ubiquitinates p53 in vitro. BRCC36 and BRCC45 are novel components of the complex with sequence homology to a subunit of the signalosome and proteasome complexes. Reconstitution of a recombinant four-subunit complex containing BRCA1/BARD1/BRCC45/BRCC36 revealed an enhanced E3 ligase activity compared to that of BRCA1/BARD1 heterodimer. In vivo, depletion of BRCC36 and BRCC45 by the small interfering RNAs (siRNAs) resulted in increased sensitivity to ionizing radiation and defects in G2/M checkpoint. BRCC36 shows aberrant expression in sporadic breast tumors. These findings identify BRCC as a ubiquitin E3 ligase complex that enhances cellular survival following DNA damage.     

STORM towards protein function: systematic tailored ORF-data retrieval and management

STORM (systematic tailored ORF-data retrieval and management) combines protein analyses of BLAST, Fasta, Pfam and ProtParam on a batch file of protein sequences. It subsequently summarises and organises the output in an Access database format.     

Conditional replication of a recombinant adenovirus studied using neomycin as a selective marker

An E1B-defective adenovirus, named r2/Ad carrying the neo expression cassette, was constructed by homologous recombination. The construction, selection (using neomycin as a selective marker), and propagation of the recombinant virus was performed in human embryonic kidney 293 cells (HEK 293). An in vitro study demonstrated that this recombinant virus has the ability to replicate in and lyse some p53-deficient human tumor cells such as human glioma tumor cells (U251) and human bladder cells (EJ), but not in some cells with functional p53, such as human adenocarcinoma cells (A549) and human fibroblast cells (MRC-5). Also, based on the cytopathic effect (CPE), it was demonstrated, under identical conditions, that the U251 cells were more sensitive to r2/Ad replication than the EJ cells. In this paper, we report that r2/Ad could be very useful in studying the in vitro selective replication of E1B-defective adenovirus and has great potential in cancer gene therapy.     

Functional integration of serial dilution and capillary electrophoresis on a PDMS microchip

For the quantitative analysis of an unknown sample a calibration curve should be obtained, as analytical instruments give relative, rather than absolute measurements. Therefore, researchers should make standard samples with various known concentrations, measure each standard and the unknown sample, and then determine the concentration of the unknown by comparing the measured value to those of the standards. These procedures are tedious and time-consuming. Therefore, we developed a polymer based microfluidic device from polydimethylsiloxane, which integrates serial dilution and capillary electrophoresis functions in a single device. The integrated microchip can provide a one-step analytical tool, and thus replace the complex experimental procedures. Two plastic syringes, one containing a buffer solution and the other a standard solution, were connected to two inlet holes on a microchip, and pushed by a hydrodynamic force. The standard sample is serially diluted to various concentrations through the microfluidic networks. The diluted samples are sequentially introduced through microchannels by electro-osmotic force, and their laser-induced fluorescence signals measured by capillary electrophoresis. We demonstrate the integrated microchip performance by measuring the fluorescence signals of fluorescein at various concentrations. The calibration curve obtained from the electropherograms showed the expected linearity.     

Simulation on long-term operation of an anaerobic bioreactor for Korean food wastes

A mathematical model was formulated to simulate the long-term performance of an anaerobic bioreactor designed to digest Korean food wastes. The system variables of various decomposition steps were built into the model, which predicts the temporal characters of solid waste, and volatile fatty acid (VFA) in the reactor, and gas production in response to various input loadings and temperatures. The predicted values of VFA and gas production were found to be in good agreement with experimental observations in batch and repeated-input systems. Finally, long-term reactor performance was simulated with respect to the seasonal temperature changes from 5°C in winter to 25°C in summer at different food waste input loadings. The simulation results provided us with information concerning the success or failure of a process during long-term operation.