Development of a RP-HPLC method for simultaneous determination of atenolol, metoprolol tartrate and phenol red for in-situ rat intestinal perfusion studies.

Single-pass intestinal perfusion (SPIP) method is a widely used experimental model to determine the intestinal permeability of drugs. These studies are performed in the presence of a reference standard (metoprolol, MT) and a zero permeability marker (phenol red, PR). Therefore, it is important to develop a validated method for simultaneous determination of the investigated compound along with MT and PR. The aim of this study was to develop a reversed phase high-performance liquid chromatography (RP-HPLC) method with UV-detection for the simultaneous determination of atenolol (ATN), MT, and PR in the perfusion medium used in SPIP experiments. Separation of compounds were performed using an InertSustain C18 (250 × 4.6 mm, 5 µm) HPLC column at 35 °C. The mobile phase was a mixture of acetonitrile and phosphate buffer (pH 7.0, 12.5 mM) in gradient elution, and was delivered at a flow rate of 1 mL/min. The acetonitrile ratio of the mobile phase increased linearly from 10 to 35 % over 15 min. The injection volume was 20 µL, and ATN, MT and PR were detected at 224 nm. The retention times under optimum HPLC conditions were 5.028 min, 12.401 min, and 13.507 min for ATN, MT and PR, respectively. The developed RP-HPLC method was validated for selectivity, specificity, calibration curve and range, accuracy and precision, carry-over effect, stability, reinjection reproducibility, recovery and robustness. The method was linear for ATN (0.76-50 µg/mL), MT (1.14-50 µg/mL), and PR (0.47-20 µg/mL) with determination coefficients of 0.9999, 0.9994 and 0.9998, respectively. The results obtained for all validation parameters of the developed RP-HPLC method met the required limits of the ICH M10 Guideline.

Plasma catalysis. A study of the influence of oxysulfur, SOx• species through the degradation of sulfonated dyes by non-thermal plasma.

This work studied the degradation reaction of sulfonated dyes, indigo carmine, phenol red, and their mixtures by non-thermal plasma (NTP). Interestingly, the degradation rate constant showed a faster process and lower activation energy (Ea) for the dye mixtures than for the degradation reaction of the individual dyes. This unexpected result opened up new opportunities for understanding plasma chemistry and the interaction between reactive species formed by the plasma and the target molecule. As no catalyst or chemical additive was added to the reactor, the decrease in Ea came from a self-synergistic effect (SSE), through the dye molecules fragmentation, which resulted in plasma catalysis. The hypothesis proposed in this work is that oxysulfur (SOx) species are formed by the desulfonation reaction of dyes. The sulfonic groups (SO3) present in the chemical structures of dyes can function as precursors for forming several SOx•- species. Studies based on oxygenated sulfonated species such as SO3•-, SO4•- and SO5•- have been widely applied in advanced oxidative and reductive processes due to their satisfactory efficiency and low cost. Among them, SO4•- is the key reactive species with the best performance in the degradation of pollutants due to its high oxidation potential (E° = 2.60 V). In addition, it is an alternative source of HO• in aqueous media, improving the oxidation reaction. In order to elucidate the SSE, the kinetic process was followed by UV-Vis analysis, and the reactive species, such as alkyl, hydroxyl, and oxy-sulfur radicals were identified by Electron Paramagnetic Resonance. The by-products of the NTP degradation reaction were analyzed by ultrafast liquid chromatography coupled with a mass spectrometer, and a fragmentation route was proposed.

Relieving Sore Throat Formula Exerts a Therapeutic Effect on Pharyngitis through Immunoregulation and NF-κB Pathway.

Relieving Sore Throat Formula (RSTF) is a formula approved by the China Food and Drug Administration and has been used for the treatment of pharyngitis in clinic for many years. However, the potential pharmacological mechanism still remains unknown. We combined multiple methods including bioinformatics data digging, network pharmacology analysis, and pathway analysis to predict the potential target of RSTF. We verified our in silico prediction results with an in vivo/vitro antibacterial effect test, mouse phagocytic index test, proliferation, transformation, and migration of mouse spleen lymphocytes. Alteration of NF-κB pathway was determined by Western blotting, immunofluorescence, and PCR. The in vivo experiments demonstrated that the RSTF could significantly relieve the symptoms of pharyngitis. A rat saliva secretion test showed that RSTF can effectively relieve the xerostomia symptom. A phenol red excretion test showed that RSTF has an eliminating phlegm effect. A hot plate method and granuloma experiment proved that RSTF also have analgesic and anti-inflammatory effects. In silico prediction demonstrates that 70 active compounds of RSTF were filtered out through ADME screening and 84 putative targets correlated with different diseases. Pathway enrichment analysis showed that the candidate targets were mostly related to the response to bacteria and immunity signalling pathways, which are known contributors to pharyngitis. Experimental results confirmed that RSTF exerted therapeutic effects on pharyngitis mainly by antibacterial effect and downregulation of NF-κB activities. It is demonstrated both in silico and in vivo/vitro that RSTF exerted therapeutic effects on pharyngitis mainly through an antibiotic effect and downregulation of NF-κB signalling pathway.

In vitro tissue culture model validation-the influence of tissue culture components on IPL energy output.

Intense pulsed light (IPL) has been used therapeutically in a number of clinical settings and has been shown to have a photobiomodulatory effect on connective tissue cells, such as those derived from skin and tendon. In vitro cell culture models are essential tools preclinically in investigating such treatment modalities, as they help in optimising parameters for successful treatment. However, as culture system components have been reported to absorb part of the irradiated energy, which in turn has a bearing on the amount of light reaching the cells, it is important to establish specific parameters for the particular in vitro model used. This study, therefore, investigates the effect of our tissue culture system components on the IPL energy delivered. Individual wells of multi-well plates were irradiated with IPL at different device settings and under variable culture conditions (e.g. in the absence or presence of cell culture media with or without the pH indicator dye, phenol red), and the energy lost through the culture system determined. Our data demonstrated that the IPL device delivered significantly lower outputs than those published, and energy absorption by the culture equipment would further reduce fluencies delivered to the cell monolayer. Furthermore, energy absorption by media containing phenol red was marginally greater than clear media and resulted in only a small increase in temperature, which would not be harmful to cells. The use of phenol red-containing media therefore is valid and physiologically relevant when examining light-culture system interactions.

Caution for the routine use of phenol red - It is more than just a pH indicator.

Phenol red (PR) is the standard pH indicator in various cell and tissue culture media, as it provides a quick check for the health of the culture. PR has also been used in multiple protocols to detect cellular hydrogen peroxide as well as peroxidase activity from human peroxidase enzymes. The majority of promyelocytic leukemia cell lines (e.g. HL-60 cells) express myeloperoxidase (MPO), which may react with PR, especially as the latter is present in cell culture media at sufficient concentrations (~15 µM) to partake in redox reactions. Moreover, phenolic molecules are often efficient donor substrates for peroxidase enzymes. In this study, we hypothesized that MPO metabolism of PR via MPO-expressing HL-60 cells could result in PR metabolite(s) that could modulate cell viability. We used purified human MPO for UV-visible spectrophotometry, electron paramagnetic resonance (EPR) and LC-MS analyses to investigate PR peroxidation. 2-chloro-5,5-dimethyl-1,3-cyclohexanedione (monochloro-dimedone, MCD) was used to assess the effect of PR on MPO-catalyzed chlorination activity, and we assessed PR uptake by HL-60 cells using LC-MS analysis. Lastly, we investigated the impact of PR metabolism by intracellular MPO on cell viability (ATP, using CellTiter-Glo®), cytotoxicity (using trypan blue), and on reduced and oxidized glutathione (using GSH/GSSG-Glo™). Our results demonstrate that PR undergoes oxidative halogenation via MPO, resulting in its UV-vis spectral changes due to the formation of mono- and di-halogenated products. Moreover, a significant increase in MPO-catalyzed chlorination of MCD and an increase in glutathionyl radical detection (using EPR) were observed in the presence of PR. Our in-vitro studies revealed that PR is readily taken up by HL-60 cells and its metabolism by intracellular MPO leads to a significant decrease in cellular glutathione as well as a significant increase in glutathione disulphide formation. In spite of the latter, PR had no considerable effect on HL-60 cell viability. These results provide evidence that while no overt decrease in cell viability may be observed, PR does impart redox activity, which investigators should be wary of in experimental protocols.

Highly sensitive chemiluminescence enzyme immunoassay for the quantification of carcinoembryonic antigen in the presence of an enhancer and a stabilizer.

Using the advantages of phenol red, a signal enhancer, and bovine serum albumin (BSA), a stabilizer of horseradish peroxidase (HRP), added in HRP enzyme reaction of Amplex Red and H2O2, highly sensitive 1,1'-oxalyldiimidazole chemiluminescence enzyme immunoassay (ODI-CLEIA) was developed to rapidly quantify trace levels of carcinoembryonic antigen (CEA) in human serum. Phenol red acts as an enhancer in ODI-CLEIA while BSA supported rapid and stable activation of HRP. The CL emission of resorufin formed from the HRP enzyme reaction in the presence of BSA and phenol red was about 70-fold brighter than that in the absence of both materials. ODI-CLEIA in the presence of BSA (1.5 mg/ml).and phenol red (1 mM) was able to rapidly analyze CEA in human serum with the wide linear calibration curve (2.5-100 ng/ml). The limit of detection (LOD = 3σ/slope) of ODI-CLEIA was as low as 0.19 ng/ml. Additionally, it was confirmed that the accuracy, precision, and reproducibility of ODI-CLEIA in the presence of BSA and phenol red were good with the statistically acceptable error range.

Zn(2+)-cyclen-based complex enable a selective detection of single-stranded thymine-rich DNA in aqueous buffer.

It is a big challenge to develop fluorescent probes for selective detection of DNA with specific sequences in aqueous buffers. We report a new tetraphenylethene-based Zn(2+)-cyclen complex (TPECyZn), and a chemo-sensing ensemble of the Zn complex with phenol red. TPECyZn showed significant fluorescence enhancement upon binding to thymine-rich DNA in HEPES buffers. But its selectivity was not high enough to eliminate the interference from some random DNA. By constructing the chemo-sensing ensemble of TPECyZn with phenol red, the background fluorescence was eliminated due to the energy transfer from TPECyZn to phenol red. Moreover, this chemo-sensing ensemble revealed high selectivity in detecting thymine-rich single-stranded DNA over other DNA in aqueous buffer. It can detect poly deoxythymidylic acid sequence as short as 2 nt. This detection in aqueous media makes this probe feasible in real application.

Mechanism of triphenylmethane Cresol Red degradation by Trichoderma harzianum M06.

Cresol Red belongs to the triphenylmethane (TPM) class of dyes which are potentially carcinogenic or mutagenic. However, very few studies on biodegradation of Cresol Red were investigated as compared to other type dyes such as azo and anthraquinone dye. The aim of this work is to evaluate triphenylmethane dye Cresol Red degradation by fungal strain isolated from the decayed wood in Johor Bahru, Malaysia. Detailed taxonomic studies identified the organisms as Trichoderma species and designated as strain Trichoderma harzianum M06. In this study, Cresol Red was decolorized up to 88% within 30 days under agitation condition by Trichoderma harzianum M06. Data analysis revealed that a pH value of 3 yielded a highest degradation rate among pH concentrations (73%), salinity concentrations of 100 g/L (73%), and a volume of 0.1 mL of Tween 80 (79%). Induction in the enzyme activities of manganese peroxidase, lignin peroxidase, laccase, 1,2- and 2,3-dioxygenase indicates their involvement in Cresol Red removal. Various analytical studies such as Thin-Layer Chromatography (TLC), UV-Vis spectrophotometer, and Gas chromatography mass spectrometry (GC-MS) confirmed the biotransformation of Cresol Red by the fungus. Two metabolites were identified in the treated medium: 2,4-dihydroxybenzoic acid (t R 7.3 min and m/z 355) and 2-hydroxybenzoic acid (t R 8.6 min and m/z 267). Based on these products, a probable pathway has been proposed for the degradation of Cresol Red by Trichoderma harzianum M06.

Adenosine reagent-free detection by co-immobilization of adenosine deaminase and phenol red on an optical biostrip.

Adenosine detection in human serum is important because this ribonucleoside has established clinical applications, modulating many physiological processes. Furthermore, a simple and cheap detection method is useful in adenosine production processes. Adenosine can be determined enzymatically using either S-adenosyl-homocysteine hydrolase and (3) [H]-adenosine, or adenosine kinase combined with GTP and luciferase, or an amperometric biosensor carrying adenosine deaminase (ADA), purine nucleoside phosphorylase, and xanthine oxidase. We developed a simple and cheap method relying on a transparent biostrip bearing ADA and the indicator phenol red (PR), co-immobilized to polyacrylamide, itself chemically adhered to a derivatized glass strip. The ADA-catalyzed conversion of adenosine to inosine and ammonia leads to a local pH alteration, changing the absorbance maximum of PR (from 425 to 567 nm), which is measured optically. The biostrip shows an analytical range 0.05-1.5 mM adenosine and is reusable when stored at 4 °C. When the biostrip was tested with serum, spiked with adenosine (70 and 100 µM), and filtered for protein and adenosine phosphates depletion, it showed good adenosine recovery. In summary, we show the proof-of-concept that adenosine can be determined reagent-free, at moderate sensitivity on an easy to construct, cheap, and reusable biostrip, based on commercially available molecular entities.

A rapid and sensitive fluorimetric ß-galactosidase assay for coliform detection using chlorophenol red-ß-D-galactopyranoside.

We report on a new fluorimetric assay for ß-galactosidase (ß-gal) and faecal coliform bacteria that utilizes a long-wavelength dye, chlorophenol red-ß-D-galactopyranoside (CPRG), that has been widely used for colorimetric assays. The novel feature of this new assay is the unexpected development of a large fluorescence response from liberated chorophenol red (CPR) upon complexation with poly-L-arginine (pR) in solution. The binding of CPR to pR occurs through the sulphonate group of CPR, causing formation of a charge-transfer complex and up to a 70-fold increase in emission intensity. A major advantage of the assay is the ability to utilize excitation and emission wavelengths in the red end of the spectrum, which avoids common interferences obtained when using UV-absorbing dyes such as 4-methylumbelliferyl-ß-D-galactopyranoside. We provide data on the utility of CPRG as a fluorimetric reporter for both ß-gal and Escherichia coli ATCC 25922 and demonstrate optimized reaction conditions for rapid and sensitive detection of E. coli at a level of 1 colony-forming unit (cfu)/10 mL after 12 h of culture followed by a 1-h assay, which is below the regulatory limit for testing of recreational water.

Sonodynamic and sonocatalytic damage of BSA molecules by Cresol Red, Cresol Red-DA and Cresol Red-DA-Fe under ultrasonic irradiation.

The interaction of Cresol Red derivatives (Cresol Red (o-Cresolsulfonphthalein), Cresol Red-DA (3,3'-Bis [N,N-di (carboxymethyl) aminomethyl]-o-cresolsulfonphthalein) and Cresol Red-DA-Fe(III) (3,3'-Bis [N,N-di (carboxymethyl) aminomethyl]-o-cresolsulfonphthalein-Ferrous(III)) with bovine serum albumin (BSA) were studied by the combination of ultraviolet spectroscopy, circular dichroism (CD) spectroscopy, fluorescence spectroscopy and synchronous fluorescence spectroscopy. On that basis, the sonodynamic and sonocatalytic damages of Cresol Red derivatives to BSA under ultrasonic irradiation were also investigated by means of corresponding spectrum technology. Meanwhile, some influenced factors such as ultrasonic irradiation time, Cresol Red derivatives concentration and ionic strength on the damage degree of BSA molecules were also reviewed. In addition, the binding site and damage site of BSA molecules were estimated by synchronous fluorescence spectra. Finally, the results of oxidation-extraction photometry (OEP) using several reactive oxygen species (ROS) scavengers indicated that the damage of BSA molecules is mainly due to the generation of ROS. Perhaps, this paper may offer some important subjects for broadening the application of Cresol Red derivatives in sonodynamic therapy (SDT) and sonocatalytic therapy (SCT) technologies for tumor treatment.

Cytotoxicity of phenol red in toxicity assays for carbon nanoparticles.

To explore the novel properties of carbon nanoparticles (CNPs) in nanotoxicity assays, the adsorption of phenol red (a pH indicator for culture medium) by multi-walled carbon nanotubes (MWNTs) and three kinds of carbon blacks (CBs) with nanosize, and its effects on cytotoxicity were studied. Results indicated that the phenol red adsorbed and delivered into cells by CBs was responsible for the toxicity to Hela cells in the medium without serum. The cellular uptake of phenol red was verified using 125I-labeling techniques. The size-dependent cytotoxicity of CBs was found to closely correlate to adsorption of phenol red, cellular uptake of phenol red-CB complexes and the amount of phenol red delivered into the cells by CBs. Although the CBs were either nontoxic or slightly toxic, as vehicles of phenol red, they played an essential role in the cytotoxicity induced by phenol red. However, MWNTs showed an intrinsic cytotoxicity independent of phenol red. The implications associated with these findings are discussed.

Medium-mediated effects increase cell killing in a human keratinocyte cell line exposed to solar-simulated radiation.

PURPOSE: The objective of this study was to investigate whether cell culture medium is a biologically relevant exposure medium that can be employed in non-ionising photobiological investigations. METHODS: The effect of solar-simulated irradiation on cell culture medium and its ability to elicit cell death was studied. The role of reactive oxygen species (ROS), cell secreted factors, and the contribution of individual components of the medium were investigated. RESULTS: Cell death was found to be primarily mediated through the formation of ROS via riboflavin photosensitisation and degradation in the cell culture medium. Phenol red was found to significantly reduce the cell killing ability of riboflavin. Exposures in riboflavin-free medium resulted in significantly increased cell survival compared to identical exposures in riboflavin containing medium. CONCLUSIONS: This study has shown that solar radiation toxicity is augmented by cell culture medium due to the presence of riboflavin. Results suggest that exposures performed in phenol red-free medium may serve to increase phototoxic effects if riboflavin is present. Riboflavin-free media is recommended for solar radiation investigations to eliminate concerns regarding riboflavin photosensitisation and nutrient deprivation.

Self-assembling peptide nanofiber scaffolds for controlled release governed by gelator design and guest size.

The aim of this study was to develop controlled drug delivery by network scaffolds based on self-assembling peptide RADAFI and RADAFII. These two peptides self-assembled into interconnected nanofibrilar network structures with distinct physical morphologies. The hydrogels were also utilized for entrapment and release of some model guests, promising their future application as a drug delivery vehicle. Fickian diffusion controlled the release kinetics. Furthermore, the obtained release function was dependent on both rational design of the peptides used for hydrogel formation and choice of the entrapped molecules. On the basis of the striking different releases of these two peptide scaffolds, we suggested that guest size and lipophilicity influenced the release competitively. The release of RADAFI system was dominated by guest size, and the guest lipophilicity controlled the release behavior in RADAFII system. In a word, this work would potentially provide a spatially and temporally controlled delivery system for some functional drugs in the future.

Total anti-oxidant capacity of cell culture media.

1. The composition of synthetic cell culture media is important for the behaviour of cultured cells in vitro and may affect the results of many in vitro experiments. The total anti-oxidant capacity (TAC) of an extracellular medium may be an important factor in cell redox homeostasis. 2. In the present study, the TAC of cell culture media used for the cultivation of mammalian, yeast and bacterial cells (RPMI1640, Iscove's modified Dulbecco's medium, Dulbecco's modified Eagle's medium, minimum essential medium Eagle's 1959 with Earle's salts, Parker medium 199 with Hanks salts, bacterial Luria-Bertani medium, yeast extract-peptone-glucose and yeast nitrogen base media) was estimated using the 2,2'-azinobis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS(.+)) decolourization assay and the ferric ion reducing anti-oxidant power assay. 3. We found that components of the media such as cysteine, tyrosine, tryptophan and Phenol Red are important contributors to the TAC of cell culture media.

Phenol red in the culture medium strongly affects the susceptibility of human MCF-7 cells to roscovitine.

Estrogens play an important role in the growth and terminal differentiation of the mammary gland. Prolonged exposure to estrogens seems to predispose women to breast cancer. It recently became evident that not only the intrinsic hormonal status but also external factors such as the occurrence of pharmaceuticals and chemicals with hormone activity in the environment may put women at greater risk of developing breast cancer. We focused on the interference of endocrine disruptors in breast cancer therapy. We observed that phenol red added to the culture medium strongly promoted the cell proliferation and cell cycle progression of human cells expressing the estrogen receptor, and affected their susceptibility to chemotherapy.

Phenol red interacts with the protofibril-like oligomers of an amyloidogenic hexapeptide NFGAIL through both hydrophobic and aromatic contacts.

Amyloid-associated diseases affect millions of people worldwide. Phenol red exhibits modest inhibition toward fibril formation of human Islet amyloid polypeptide (hIAPP) and its toxicity, which is associated with type II diabetes mellitus. However, the molecular level mechanisms of interactions remain elusive. The binding of phenol red molecules to the protofibrils of an amyloidogenic fragment (NFGAIL) of hIAPP has been investigated by molecular dynamics simulations with explicit solvent. The phenol red molecules were observed to bind primarily along either beta-sheet stacking or beta-strand directions. Through its three aromatic rings, the phenol red molecule preferentially interacted with the hydrophobic side chains of Phe, Leu, and Ile; and the polar sulfone and hydroxyl groups were mainly exposed in solvent. Thus, phenol red improves the solubility of the early protofibrils and represses further growth. Interestingly, there was no obvious preference toward the aromatic Phe residue in comparison to the hydrophobic Leu or Ile residues. The lack of binding along the hydrogen bond direction indicates that phenol red does not directly block the beta-sheet extension. Further free energy analysis suggested that a phenol red analog may potentially improve the binding affinity.

Antagonism of ATP responses at P2X receptor subtypes by the pH indicator dye, Phenol red.

1 Many types of culture media contain a pH-sensitive dye. One commonly occurring dye, Phenol red sodium (Na(+)) salt, was tested for blocking activity at rat P2X(1-4) receptors (P2X(1-4)Rs) expressed in Xenopus oocytes. 2 Phenol red Na(+)-salt antagonised adenosine 5'-triphosphate (ATP) responses at P2X(1)R (IC(50), 3 microM) and, at higher concentrations, also blocked P2X(2)R and P2X(3)R. Phenol red Na(+)-salt, purified of lipophilic contaminants, blocked P2X(1)R and P2X(3)R by acting as an insurmountable antagonist. 3 Two lipophilic extracts of Phenol red antagonised ATP responses at P2XRs. Extract A was a potent antagonist at P2X(1)R (IC(50), 1.4 microM), whereas extract B was a potent antagonist at P2X(3)R (IC(50), 4.1 microM). A bisphenolic compound (RS151030) found in these extracts was a potent antagonist at P2X(1)R (IC(50), 0.3 microM) and at P2X(3)R (IC(50), 2.4 microM). 4 Phenolphthalein base was a potent irreversible antagonist at P2X(1)R (IC(50), 1 microM), whereas Phenolphthalein K(+)-salt was 25-fold less potent here. 5 Phenolphthalein base was a reversible antagonist of ATP responses at rat P2X(4)R (IC(50), 26 microM), whereas Phenolphthalein K(+)-salt was inactive. 6 Dimethyl sulphoxide (DMSO), used to dissolve lipophilic extracts, showed pharmacological activity by itself at rat P2X(1)R and P2X(4)R. 7 Thus, Phenol red and related compounds are antagonists at rat P2X(1)R, but are also active at other rat P2XRs. Phenolphthalein base is a newly identified, low potency antagonist of ATP responses at P2X(4)R. Culture media containing these red dyes should be used cautiously in future pharmacological studies of P2XRs. Also, wherever possible, the solvent DMSO should be used with caution.

Coordinate late expression of trefoil peptide genes (pS2/TFF1 and ITF/TFF3) in human breast, colon, and gastric tumor cells exposed to X-rays.

The trefoil factors (TFFs) are pleiotropic factors involved in organization and homeostasis of the gastrointestinal tract, estrogen responsiveness, inflammatory disorders, and carcinogenesis. In an earlier study using cDNA array technologies to identify new genes expressed in irradiated cell survivors, we isolated a cDNA clone corresponding to the reported human TFF1 gene (E. K. Balcer-Kubiczek et al., Int. J. Radiat. Biol., 75: 529-541, 1999). To determine whether expression of other TFFs is altered by ionizing radiation, we quantified changes in expression of TFF3 as well as TFF1 in RNA samples obtained from irradiated and control human tumor breast, colon, and gastric tumor cells and examined expression kinetics up to 2 weeks after irradiation. X-ray-induced TFF1 and TFF3 expression profiles were compared with those induced by hydrogen peroxide (H2O2) or 17beta-estradiol (ES). The results revealed that TFF1 and TFF3 mRNA are coinduced by X-irradiation in a subset of the lines, but substantial heterogeneity in their responses was observed in cells derived from a single cell type. TFF1 and TFF3 transcriptional response to X-irradiation differed from that to H2O2 or ES in the timing of their induction as well as tissue-type dependence, i.e., their induction pattern after X-irradiation was late and sustained, whereas their induction by H2O2 or ES was early and transient. TFF1 mRNA, protein production in the cytoplasm, and secretion in the culture supernatant were coordinately regulated after X-irradiation. There was no requirement for TP53 in this induction. These results demonstrate the existence of a novel class of radiation-responsive genes that might be involved in bystander effects.

Improvement of receptor-mediated gene delivery to HepG2 cells using an amphiphilic gelling agent.

Gene transfer was performed using asialo-oroso-mucoid-polylysine (ASOR-PL) conjugates to allow targeted expression of the gene in cells of hepatic origin. In a gel-electrophoretic analysis, the ASOR-PL conjugate produced a complete DNA retardation effect at the optimal ratio of 222:1 (ASOR-PL conjugate/pCMV beta-gal plasmid). The gene-transfer efficiency of the ASOR-PL conjugate was evaluated in HepG2 cells that express asialoglycoprotein receptor and NIH 3T3 cells that do not. The expression was assayed by 5-bromo-4-chloroindol-3-yl beta-D-galactopyranoside ('X-Gal') staining and Chlorophenol Red beta-D-galactopyranoside. When an expression vector for the tumour-suppressor gene p53, pCMVp53, complexed to ASOR-PL conjugate, was transfected into HepG2 cells, the exogenously provided p53 gene was detected in the HepG2 cells by PCR. To improve the efficiency of DNA delivery and expression of the therapeutic proteins poloxamer 407, a fusogenic peptide, influenza-virus haemagglutinin HA2 and chloroquine were individually incorporated into the system. The expression level of beta-galactosidase in HepG2 cells was increased by about four times by the presence of poloxamer 407, whereas the fusogenic peptide HA2 and chloroquine had no effects. When HepG2 cells were transfected with pCMVp53 in the presence of poloxamer 407, the mRNA of transfected p53 could be detected by reverse transcriptase PCR. The current findings open the possibility that a receptor-mediated gene-delivery system for hepatic gene therapy using ASOR-PL conjugate in combination with poloxamer 407 may be developed in the future.