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Hormone receptor-positive breast cancer (HR+ BC) cells depend on estrogen and its receptor, ER. Due to this dependence, endocrine therapy (ET) such as aromatase inhibitor (AI) treatment is now possible. However, ET resistance (ET-R) occurs frequently and is a priority in HR+ BC research. The effects of estrogen have typically been determined under a special culture condition, i.e., phenol red-free media supplemented with dextran-coated charcoal-stripped fetal bovine serum (CS-FBS). However, CS-FBS has some limitations, such as not being fully defined or ordinary. Therefore, we attempted to find new experimental conditions and related mechanisms to improve cellular estrogen responsiveness based on the standard culture medium supplemented with normal FBS and phenol red. The hypothesis of pleiotropic estrogen effects led to the discovery that T47D cells respond well to estrogen under low cell density and medium replacement. These conditions made ET less effective there. The fact that several BC cell culture supernatants reversed these findings implies that housekeeping autocrine factors regulate estrogen and ET responsiveness. Results reproduced in T47D subclone and MCF-7 cells highlight that these phenomena are general among HR+ BC cells. Our findings offer not only new insights into ET-R but also a new experimental model for future ET-R studies.

This study aims to explore the effect of Sini Decoction on Toll-like receptor 4(TLR4)/nuclear factor-κB(NF-κB) signaling pathway in the mice with allergic asthma(AA). Forty-eight SPF-grade BALB/c mice were randomly assigned into a blank control group, a model group, a dexamethasone group, and high-, medium-, and low-dose Sini Decoction groups, with 8 mice in each group. The sensitization solution made of ovalbumin and aluminum hydroxide powder was injected intraperitoneally in other groups except the blank control group which was injected with an equal volume of normal saline. The solution(or normal saline) was injected three times in total with an interval of 7 days. At the same time of sensitization, external cold stimulation and ice water were administered in a 4 ℃ climate box for 20 min every day. After modeling, the mice in each group were administrated with corresponding drugs by gavage for 3 weeks. At the end of administration, pentobarbital sodium(30 mg·kg~(-1)) was used for anesthesia, and then the samples were collected for the determination of various indexes. The phenol red test was conducted to evaluate tracheal excretion function. The histopathological changes of lung tissue were observed via hematoxylin-eosin(HE) staining. Masson staining was employed to reveal the deposition of blue collagen fibers around bronchi in lung tissue and the area occupied by blue collagen fibers was calculated. Immunofluorescence method was used to measure the expression of bronchial type Ⅰ collagen(Col-Ⅰ) and α-smooth muscle actin(α-SMA). The protein and mRNA levels of TLR4, NF-κB, cysteinyl aspartate specific proteinase-1(caspase-1), and interleukin-13(IL-13) were determined by Western blot and real-time fluorescence quantitative polymerase chain reaction(real-time PCR), respectively. Compared with the model group, Sini Decoction significantly increased the phenol red excretion from trachea, lowered the lung inflammation score, reduced subepithelial collagen deposition, and decreased Col-Ⅰ and α-SMA levels. Furthermore, the decoction down-regulated the protein and mRNA levels of TLR4, NF-κB, caspase-1, and IL-13 in mouse lung tissue. In conclusion, Sini Decoction can improve air remodeling by inhibiting the TLR4/NF-κB signaling pathway.

2-Monoacylglycerol (2-MAG) is one of the digestion products of dietary lipids. We recently demonstrated that a 2-MAG, 2-arachidonoyl glycerol (2-AG) potently stimulated cholecystokinin (CCK) secretion via cannabinoid receptor 1 (CB1) in a murine CCK-producing cell line, STC-1. CCK plays a crucial role in suppressing postprandial gastric emptying. To examine the effect of 2-AG on gastric emptying, we performed acetaminophen and phenol red recovery tests under oral or intraperitoneal administration of 2-AG in mice. Orally administered 2-AG (25 mg/kg) suppressed the gastric emptying rate in mice, as determined by the acetaminophen absorption test and phenol red recovery test. Intraperitoneal administration of a cholecystokinin A receptor antagonist (0.5 mg/kg) attenuated the gastric inhibitory emptying effect. In addition, both oral (10 mg/kg) and intraperitoneal (0.5 mg/kg) administration of a CB1 antagonist counteracted the 2-AG-induced gastric inhibitory effect. Furthermore, intraperitoneal 2-AG (25 mg/kg) suppressed gastric emptying. These results indicate that 2-AG exhibits an inhibitory effect on gastric emptying in mice, possibly mediated by stimulating both CCK secretion via CB1 expressed in CCK-producing cells and acting on CB1 expressed in the peripheral nerves. Our findings provide novel insights into the 2-MAG-sensing mechanism in enteroendocrine cells and the physiological role of 2-MAG., (© 2022 AOCS.)

<b>Background and Objective:</b> Phenol red, the pH indicator in cell culture media, influences the expression of cytochrome P450s (CYPs) in cell lines. This study aimed to examine how phenol red modified CYP induction by benzo[<i>a</i>]pyrene and dexamethasone in human hepatocarcinoma (HepG2), colorectal adenocarcinoma (Caco-2) and choriocarcinoma (BeWo) cells. <b>Materials and Methods:</b> The cells (1×10<sup>5</sup> cells/well in a 24-well plate) were incubated with benzo[<i>a</i>]pyrene (0.1, 1 and 10 μM) or dexamethasone (1, 5 and 10 μM) in either phenol red or phenol red-free media for 24 hrs. The mRNA expression of CYPs was determined by Real-Time Polymerase Chain Reaction (RT/qPCR). <b>Results:</b> Phenol red enhanced expression of benzo[<i>a</i>]pyrene-induced CYP1A2 inHepG2 and BeWo cells and suppressed benzo[<i>a</i>]pyrene-induced CYP2A6 expression in HepG2 and Caco-2 cells, benzo[<i>a</i>]pyrene induced CYP2B6 expression in HepG2 cells and benzo[<i>a</i>]pyrene- and dexamethasone-induced CYP3A4 expression in HepG2 and Caco-2 cells. The expression of CYP3A5 was affected differently in HepG2 and Caco-2 cell lines. Phenol red enhanced benzo[<i>a</i>]pyrene- and dexamethasone-induced CYP3A5 expression in Caco-2 cells but suppressed benzo[<i>a</i>]pyrene- and dexamethasone-induced CYP3A5 expression in HepG2 cells. <b>Conclusion:</b> Phenol red differentially influenced expression of benzo[<i>a</i>]pyrene- and dexamethasone-induced CYP1A2, CYP2A6, CYP2B6, CYP3A4 and CYP3A5 mRNAs in HepG2, Caco-2 and BeWo cells. Therefore, the inclusion of phenol red in cell culture media is of concern in studies of drug and xenobiotic metabolism via CYPs in human cell line models.

Phenol red (PR) is a widely used marker for water flux correction in studies of in situ perfusion, in which intestinal absorption usually leads to the underestimation of results. In this paper, we propose a novel marker polyethylene glycol (PEG)-PR (i.e., PR modified by PEGylation) with less permeability and evaluate its application in an in situ perfusion model in rats. PEG-PR was synthesized by the chemical conjunction of polyethylene glycol-4k/5k (PEG-4k/5k) and PR. The synthesized PEG-PR was then characterized using 1 H-NMR, 13 C-NMR, ultraviolet (UV), X-ray diffraction (XRD), and differential scanning calorimetry (DSC) analyses. The low permeability of PEG-PR was assessed using everted gut sac (EGS) methods. The apparent permeability coefficients ( P app , 3-8 × 10 -7 cm/s) of PEG4k/5k-PR exhibited a nearly 15-fold reduction compared to that of PR. The different concentrations of PEG4k/5k-PR did not contribute to the P app value or cumulative permeable percentage (about 0.02-0.06%). Furthermore, the larger molecular weight due to PEGylation (PEG5k-PR) enhanced the nonabsorbable effect. To evaluate the potential application of the novel marker, atenolol, ketoprofen, and metoprolol, which represent various biopharmaceutics classification system (BCS) classes, were selected as model drugs for the recirculation perfusion method. The water flux corrected by PEG4k/5k-PR reflected the accuracy due to the nonabsorbable effect, while the effective intestinal membrane permeability ( P eff ) of atenolol corrected by PEG4k/5k-PR showed a statistically significant increase ( p < 0.05) in different intestinal segments. In conclusion, PEG-PR is a promising marker for the permeability estimation when using the in situ perfusion model in rats.

Abnormal keratinocyte differentiation is fundamental to pathologies such as skin cancer and mucosal inflammatory diseases. The ability to grow keratinocytes in vitro allows the study of differentiation however any translational value is limited if keratinocytes get altered by the culture method. Although serum lipids (SLPs) and phenol red (PR) are ubiquitous components of culture media their effect on differentiation is largely unknown. We show for the first time that PR and SLP themselves suppress expression of differentiation-specific keratins K1, K10 and K2 in normal human epidermal keratinocytes (NHEK) and two important cell lines, HaCaT and N/TERT-1. Removal of SLP increased expression of K1, K10 and K2 in 2D and 3D cultures, which was further enhanced in the absence of PR. The effect was reversed for K1 and K10 by adding all-trans retinoic acid (ATRA) but increased for K2 in the absence of PR. Furthermore, retinoid regulation of differentiation-specific keratins involves post-transcriptional mechanisms as we show KRT2 mRNA is stabilised whilst KRT1 and KRT10 mRNAs are destabilised in the presence of ATRA. Taken together, our results indicate that the presence of PR and SLP in cell culture media may significantly impact in vitro studies of keratinocyte differentiation.

Thermochromic materials exhibit a color change in response to a change in temperature. Creating nontoxic microcapsules containing thermochromic materials for applications in ink and film materials is historically challenging. In this study, we develop a nontoxic chlorophenol red (CPR)-water thermochromic system and its microcapsules with silicone shells via a reaction between water and octadecyltrichlorosilane (OTS) at the interface of a w/o emulsion. The obtained microcapsules exhibit a clear color change with full reversibility and are successfully used as inks by screen printing and as additives in films. Nontoxicity of both microcapsules and films is demonstrated through cell cytotoxicity assays. These features make these novel materials applicable to the next generation of intelligent sensors, coating, and food packaging materials.

The objective of this research is to examine the relationship between the color changes of phenol red and the growth of cancer cells, i.e., HeLa and DU145 cells, over a specific period of time. Normal mouse skin fibroblasts (L929 cells) were used as a reference. In this research, the color changes of phenol red due to the acidification of the cell culture medium from the growth of the cells over a period of nine hours showed potential colorimetric characteristics of cancer cells. The color changes of phenol red were observed using visible absorbance spectroscopy. The transformation of the absorbance spectra into coefficients of determination against the examined range of wavelengths created a distinctive spectral signature that signifies phenol red discoloration in cancer and normal cell culture lines., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The goal of this study was to create a mass transport model (MTM) model for gastric emptying and upper gastrointestinal (GI) appearance that can capture the in vivo concentration-time profiles of the nonabsorbable drug phenol red in solution in the stomach and upper small intestine by direct luminal measurement while simultaneously recording the contractile activity (motility) via manometry. We advanced from a one-compartmental design of the stomach to a much more appropriate, multi-compartmental 'mixing tank' gastric model that reflects drug distribution along the different regions of the stomach as a consequence of randomly dosing relative to the different contractile phases of the migrating motor complex (MMC). To capture the intraluminal phenol red concentrations in the different segments of the GI tract both in fasted and fed state conditions, it was essential to include a bypass flow compartment ('magenstrasse') to facilitate the transport of the phenol red solution directly to the duodenum (fasted state) or antrum (fed state). The fasted and fed state models were validated with external reference data from an independent aspiration study using another nonabsorbable marker (paromomycin). These results will be essential for the development and optimization of computational programs for GI simulation and absorption prediction, providing a realistic gastric physiologically-based pharmacokinetic (PBPK) model based on direct measurement of gastric concentrations of the drug in the stomach., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The Tixel is a novel device based on a thermo-mechanical ablation technology that combines a sophisticated motion and a temperature control. The fractional technology is used to transfer a very precise thermal energy to the skin thereby creating an array of microchannels, accompanying by no signs of pain or inconvenience. This study aimed to evaluate the effect of the Tixel on the skin permeability of three hydrophilic molecular models: verapamil hydrochloride, diclofenac sodium, and magnesium ascorbyl phosphate. Tixel's gold-platted stainless steel tip heated to a temperature of 400°C was applied on skin for 8ms or 9ms at a protrusion of 400μm (the distance in which the tip protrudes beyond the distance gauge). The experiments were carried out partly in vivo in humans using a fluorescent dye and a confocal microscopy and partly in vitro using porcine skin and a Franz diffusion cell system. The results obtained in this study have shown that (a) no significant collateral damage to the skin tissue and no necrosis or dermal coagulation have been noted, (b) the microchannels remained open and endured for at least 6h, and (c) the skin permeability of hydrophilic molecules, which poorly penetrate the lipophilic stratum corneum barrier, was significantly enhanced by using Tixel's pretreatment., (Copyright © 2016 Elsevier B.V. All rights reserved.)

With recent environmental and health concerns, biosurfactants have obtained increasing interest in replacing conventional surfactants for diverse applications. In agriculture, the use of surfactant in stimulating foliar uptake is mainly for wetting leaf surface, resisting deposition/evaporation, enhancing penetration across cuticular membrane (CM) and translocation. This paper aimed to address the improved foliar uptake by rhamnolipid (RL) in comparison with the currently used alkyl polyglucoside (APG). As found, compared with APG at 900mg/L (1×critical micellar concentration, CMC), RL at a much lower concentration of 50mg/L (1×CMC) showed much better wettability and surface activity, indicative of its high effectiveness as surfactants. Its performance on resistance to deposition and evaporation was at least as same as APG. Moreover, RL could significantly improve the penetration of herbicide glyphosate and other two small water-soluble molecules (phenol red and Fe(2+)) across CM at an equivalent efficiency as APG at 1×CMC. Finally, the greatly enhanced herbicidal actitivity of glyphosate on greenhouse plants confirmed that RL and APG could both enhance the foliar uptake including translocation. Overall, RL should be more applicable than APG in agriculture due to its more promising properties on health/environmental friendliness., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Five bromophenols isolated from three Rhodomelaceae algae (Laurencia nipponica, Polysiphonia morrowii, Odonthalia corymbifera) showed inhibitory effects against glucose 6-phosphate dehydrogenase (G6PD). Among them, the symmetric bromophenol dimer (5) showed the highest inhibitory activity against G6PD.

The purpose with this study was to investigate the effect of phenol red (PR) on chondrogenic and osteogenic differentiation of human mesenchymal stem cells (hMSCs). hMSCs were differentiated into chondrogenic and osteogenic directions in DMEM with and without PR for 2, 7, 14, 21, and 28 days. Gene expression of chondrogenic and osteogenic markers were analyzed by RT-qPCR. The presence of proteoglycans was visualized histologically. Osteogenic matrix deposition and mineralization were examined measuring the alkaline phophatase activity and calcium deposition. During chondrogenic differentiation PR decreased sox9, collagen type 2, aggrecan on day 14 and 21 (P < 0.05), and proteoglycan synthesis on day 21 and 28. Collagen type 10 was decreased on day 21 (P < 0.05). During osteogenic differentiation PR increased alkaline phosphatase on day 7 while decreased on day 21 (P < 0.05). PR increased collagen type 1 on day 7, 14, and day 21 (P < 0.05). The alkaline phosphatase activity was increased after 2, 7, and 14 days (P < 0.05). The deposition of calcium was decreased on day 21 (P < 0.05). Our results indicate that PR should be removed from the culture media when differentiating hMSCs into chondrogenic and osteogenic directions due to the effects on these differentiation pathways.

Phenol red is widely used in cell culture as a pH indicator. Recently, it also has been reported to have estrogen-like bioactivity and be capable of promoting cell proliferation in different cell lines. However, the effect of phenol red on primary neuronal culture has never been investigated. By using patch clamp technique, we demonstrated that hippocampal pyramidal neurons cultured in neurobasal medium containing no phenol red had large depolarization-associated epileptiform bursting activities, which were rarely seen in neurons cultured in phenol red-containing medium. Further experiment data indicate that the suppressive effect of the phenol red on the abnormal epileptiform burst neuronal activities was U-shape dose related, with the most effective concentration at 28 µM. In addition, this concentration related inhibitory effect of phenol red on the epileptiform neuronal discharges was mimicked by 17-β-estradiol, an estrogen receptor agonist, and inhibited by ICI-182,780, an estrogen receptor antagonist. Our results suggest that estrogen receptor activation by phenol red in the culture medium prevents formation of abnormal, epileptiform burst activity. These studies highlight the importance of phenol red as estrogen receptor stimulator and cautions of careful use of phenol red in cell culture media.

Purpose: To develop a dry eye model of mouse induced by topical administration of benzalkonium chloride (BAC) and investigate the possible mechanisms., Methods: BAC at concentration of 0.2% was applied to the mouse ocular surface for 7 days. Phenol red thread tear test, tear break-up time (BUT) test, corneal inflammatory index scoring, fluorescein and rose bengal test were performed to evaluate the toxic effects of BAC on the ocular surface. Global specimens were collected on day (D) 7 and labeled with a series of antibodies including cytokeratin 10 (K10) and mucin 5AC (MUC5AC). Apoptosis of ocular surface epithelium was evaluated by in situ terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Histologic analysis and transmission electron microscopy (TEM) were performed on D7., Results: BAC at a concentration of 0.2% successfully induced a dry eye condition with decreased tear volume and BUTs, increased corneal fluorescein and rose bengal scores. The Inflammatory index was increased in accompaniment with higher tumor necrosis factor-α (TNF-α) expression and more inflammatory infiltration in the cornea. Immunolabeling revealed positive K10 expression in BAC-treated corneal epithelium and fewer MUC5AC-positive cells in the BAC-treated conjunctival fornix. TUNEL assay showed more apoptotic cells in the corneal basal epithelium. TEM showed that the size and intervals of the microvillis were both reduced in the corneal epithelium., Conclusions: Topical administration of 0.2% BAC in mouse induces changes resembling that of dry eye syndrome in humans, and thus, represents a novel model of dry eye.

Protein affinity is of importance for porphyrins in their application in photodynamic therapy (PDT). A new Phenol Red-modified porphyrin (R-TPP) was designed and synthesized to fully take advantage of the binding character of Phenol Red towards protein. Detailed comparisons of absorption spectra, fluorescence spectra, n-octanol/water partition coefficients, (1)O(2) quantum yields, as well as protein photocleaving abilities between R-TPP and its parent porphyrin Br-TPP clearly demonstrate the benefits stemming from the modification of Phenol Red. On one hand, the presence of Phenol Red moiety greatly enhances the binding affinity of R-TPP towards model proteins (bovine serum albumin and hen egg lysozyme), and therefore improves the availability of (1)O(2). On the other hand, the presence of Phenol Red moiety provides R-TPP with amphiphilic character, and therefore restricts aggregation and favors the generation of (1)O(2). As a result, R-TPP photocleaves proteins efficiently, showing promising application potential in PDT.

Amyloid fibril formation is a common event in more than twenty human diseases and in some normal physiological processes. The mechanism of this ordered aggregation process and the molecular forces driving it are therefore of great importance. One of the strategies used in this field is targeting the fibrillization process by different factors, like, short peptides, organic molecules, etc. Here, we targeted insulin fibril formation by a range of small aromatic molecules, with different numbers of aromatic rings and various substituent groups. Using Thioflavin T fluorescence assay and transmission electron microscopy, we found that all dicyclic and tricyclic compounds in our screen were efficient inhibitors of insulin fibril formation. A common notion regarding amyloid inhibitors is that two functional groups are essentials for interfering with the amyloid formation process; a recognition motif and a bulky group for inducing a steric interference. However, here, we showed that some monocyclic compounds as small as toluene were also found to inhibit fibrillization. In addition, we found that substituent of benzene ring have a great influence on the inhibitory potency. Specifically, cyano, methyl and nitro groups increased the inhibitory potency. The results introduced here may contribute to future rational design of amyloid inhibitors.

P2X7 receptors (P2X7Rs) gate the opening of large channels when activated by ATP or other ligands. P2X7Rs are expressed by astrocytes in culture and by reactive astrocytes in vivo, and astrocytes in culture have been shown to release glutamate and ATP in response to P2X7R activation. However, P2X7Rs are activated by ATP only at concentrations greater than 1 mM. The conditions under which astrocyte P2X7Rs would be activated in vivo are, thus, unclear. Here we show that astrocytes in culture exhibit basal P2X7R activity. Primary mouse astrocytes were found to take up the P2X7R permeant dyes YO-PRO-1 (YP) and propidium iodide in absence of any added ligands. By contrast, cultured rat astrocytes took up very little YP, consistent with their much lower level of P2X7R expression. The uptake by mouse astrocytes was inhibited by oxATP, suramin, KN-62 and brilliant blue G, and by siRNA knock-down of P2X7R. Astrocyte uptake of YP was also inhibited by phenol red at concentrations above 50 muM, suggesting that phenol red present in standard cell culture media may influence P2X7R channel activity. Treatment with apyrase, an enzyme that degrades extracellular ATP, partially decreased YP uptake in astrocytes. Conversely, exposure to the ectonucleotidase inhibitor ARL67156 enhanced YP uptake and astrocytes plated without contiguous neighboring astrocytes showed reduced basal YP uptake. These results suggest that the basal uptake of YP may be due to activation of P2X7R by release of ATP by astrocytes themselves into intercellular spaces., ((c) 2008 Wiley-Liss, Inc.)

Because an increase in the HCO(3)(-) concentration of oviductal liquid at midcycle is believed to markedly enhance fertility, we have studied active secretion of HCO(3)(-) across highly differentiated cultures of monkey oviductal epithelium. Cultured cell sheets were mounted in Ussing chambers and bathed in medium containing 25 mM HCO(3)(-). Purinergic agents potently stimulated short-circuit current (I(sc)) with an initial transient response declining within approximately 2 min to a sustained response. The potency sequence of ATP approximately UTP > ADP >> AMP suggested that the I(sc) response was mediated mainly by P2Y(2) receptors. Acetazolamide, an inhibitor of carbonic anhydrase, had little or no effect on baseline I(sc) or the transient response to ATP but abolished the sustained response to ATP. Similar results were obtained on sheets of native epithelium. In pH-stat experiments, the abluminal medium of cell cultures was bathed in HCO(3)(-)-CO(2) medium, and the pH of the unbuffered luminal medium was maintained at approximately 7.4 by addition of strong acid or base. ATP stimulated base secretion, and this was inhibited by acetazolamide. Furthermore, these changes in secretion of base were in good quantitative agreement with the I(sc) responses. When phenol red (an estrogen) was removed from the culture medium, ATP-dependent HCO(3)(-) secretion was markedly reduced but could be restored by treatment with estradiol. Estrogens also markedly increased ciliation of the cultures. These results suggest that the midcycle increase in the HCO(3)(-) concentration of oviductal liquid may be mediated by the effects of estradiol on purinergic pathways or on ATP secretion.

The study of the mechanism of amyloid fibril formation and its inhibition is of key medical importance due to the lack of amyloid assembly inhibitors that are approved for clinical use. We have previously demonstrated the potent inhibitory potential of phenolsulfonphthalein, a nontoxic compound that was approved for diagnostic use in human subjects, on aggregation of islet amyloid polypeptide (IAPP) that is associated with type 2 diabetes. Here, we extend our studies on the mechanism of action of phenolsulfonphthalein by comparing its antiamyloidogenic effect to a very similar compound that is also approved for human use, phenolphthalein. While these compounds have very similar primary chemical structures, they significantly differ in their three-dimensional conformation. Our results clearly demonstrated that these two compounds had completely different inhibitory potencies: While phenolsulfonphthalein was a very potent inhibitor of amyloid fibril formation by IAPP, phenolphthalein did not show significant antiamyloidogenic activity. This behavior was observed with a short amyloid fragment of IAPP and also with the full-length polypeptide. The NMR spectrum of IAPP 20-29 in the presence of phenolsulfonphthalein showed chemical shift deviations that were different from the unbound or phenolphthalein-bound peptide. Differential activity was also observed in the inhibition of insulin amyloid formation by these two compounds, and density-gradient experiments clearly demonstrated the different inhibitory effect of the two compounds on the formation of prefibrillar assemblies. Taken together, our studies suggest that the three-dimensional arrangement of the polyphenol phenolsulfonphthalein has a central role in its amyloid formation inhibition activity.

It has been reported that the transport function for organic anions on the kidney is maintained in multidrug resistance-associated protein 2 (Mrp2)-deficient rats. Different from Mrp2-deficient rats, Long-Evans Cinnamon (LEC) rats have impaired urinary excretion of Mrp2-substrate, phenolsulfonphthalein (PSP). PSP is transported by the potential-sensitive urate transport system in rat brush-border membranes. We analyzed the function of PSP transport system in LEC rats. Unlike Long-Evans Agouti (LEA) rats, the initial uptake of PSP and urate into the renal brush-border membrane vesicles of LEC rats were not significantly enhanced in the presence of positive intravesicular potential, suggesting that the potential-sensitive urate transport system is impaired in LEC rats. LEC rats should be useful for elucidating the potential-sensitive urate transport system in rats at the molecular level.

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.

We reported recently that roscovitine (ROSC), a selective cyclin-dependent kinase (CDK) inhibitor, arrested human MCF-7 breast cancer cells in G2 phase of the cell cycle and concomitantly induced apoptosis. On the other hand, ROSC-induced G1 arrest observed by another group has not been accompanied by apoptosis. Therefore, we decided to prove to which extent components of tissue culture media could affect the primary action of ROSC. For this purpose we compared the efficacy of the ROSC treatment on MCF-7 cells cultivated in medium with and without phenol red. The kinetics of MCF-7 cell proliferation strongly depended on the presence of phenol red that has been recognized previously as a weak estrogen. Exposure of MCF-7 cells cultivated in phenol red-deprived medium to ROSC resulted in a strong G2 arrest and apoptosis. However, the anti-proliferative and pro-apoptotic action of ROSC was strongly diminished in cells maintained in medium containing phenol red. The ratio of the G2 cell population after 12 h ROSC was reduced by approximately 20% in the latter and correlated with the lack of CDK2 inactivation. Moreover, the kinetics of ROSC-induced apoptosis was delayed in the presence of phenol red. These results clearly evidence that the efficacy of the therapy of ER-positive breast cancers by CDK inhibitors is diminished in the presence of estrogen-mimicking compounds and indicate that phytoestrogens and xenoestrogens could interfere with the therapy. Therefore, the exposure of cancer patients to the estrogen mimics should be avoided at least during chemotherapy by CDK inhibitors., ((c) 2006 Wiley-Liss, Inc.)

To enhance fungal disease resistance, wheat plants (cv. Bobwhite) were engineered to constitutively express the potent antimicrobial protein Ace-AMP1 from Allium cepa, driven by a maize ubiquitin promoter along with its first intron. The bar gene was used for selection of putative transformants on medium containing phosphinothricin (PPT). Transgene inheritance, integration and stability of expression were confirmed over two generations by PCR, Southern, northern and western blot analyses, respectively. The levels of Ace-AMP1 in different transgenic lines correlated with the transcript levels of the transgene. Up to 50% increase in resistance to Blumeria graminis f. sp. tritici was detected in detached leaf assays. In ears of transgenic wheat inoculated with Neovossia indica, Ace-AMP1 intensified expression of defense-related genes. Elevated levels of salicylic acid and of transcripts of phenylalanine ammonia lyase (PAL), glucanase (PR2) and chitinase (PR3) in the transgenic plants indicated manifestation of systemic acquired resistance (SAR).

Objectives: The current study tested the hypothesis that the extracellular environment mediates mitochondrial suppression of oral epithelial cells and fibroblasts by blue light., Methods: We exposed Balb fibroblasts (Balb), normal human epidermal keratinocytes (NHEK), and oral squamous carcinoma cells (OSC2) to blue light (30-120J/cm2) in different cell-culture media and in phosphate buffered saline (PBS). Mitochondrial activity (MTT method) was used to assess cellular response 72 h post-light exposure. Cell-culture media were replaced or supplemented before or after light exposure to assess the variables of exposure time and medium degradation as mediators of blue light-induced effects., Results: Mitochondrial activity of NHEK was not suppressed by exposure to blue light regardless of extracellular conditions. The mitochondrial activity of OSC2 and Balb cells was suppressed most when cells were exposed to light in cell-culture medium (versus PBS). Blue light suppressed mitochondrial activity more when irradiated medium remained in contact with the cells at least 1h, indicating a time-dependence of the medium effects. Neither a replacement nor a supplementation of medium components reduced blue light-induced mitochondrial suppression., Significance: Our results suggest that tissue environments influence cellular responses to blue light and that these environments should be considered when assessing any biological effects of blue light during the photopolymerization of restorative resins.

A simple reversed-phase high-performance liquid chromatography (HPLC) method with ultraviolet detection at 280 nm was developed for simultaneous quantitation of furosemide and hydrochlorothiazide along with phenol red as a nonabsorbable marker for in situ permeability studies in anaesthetized rats. A jejunal segment of approximately 10 cm was isolated and cannulated in both ends for inlet and outlet solution. The perfusate was collected every 10 min, and samples were analyzed using the developed method. The mobile phase was acetonitrile-water-triethylamine-glacial acetic acid (41.5 + 57.4 + 0.1 + 0.9, adjusted to pH 5.6) at a flow rate of 1 mL/min; the run time was 9 min. The calibration graphs were linear for all 3 compounds (r > 0.999) across the concentration range of 7.93-125 microg/mL for phenol red and 6.25-100 microg/mL for hydrochlorothiazide and furosemide. The limits of quantitation were 7.2, 8.9, and 6.8 microg/mL for furosemide, hydrochlorothiazide, and phenol red, respectively. The coefficients of variation for intraassay and interassay precision were less than or equal to 7.6%, and the accuracy was between 93.2-103.4%. Using the single pass intestinal perfusion technique and the suggested HPLC method for sample analysis, mean values of 0.25 x 10(-4) (+/-0.16) cm/s and 0.22 x 10(-4) (+/-0.13) cm/s were obtained for furosemide and hydrochlorothiazide, respectively.

The purpose of this study was to elucidate the transporter-mediated secretion systems for phenolsulfonphthalein in brush-border membranes. In human and rat renal brush-border membranes, a potential-sensitive transport system has been shown to be involved in the efflux of organic anions. The uptake of phenolsulfonphthalein into rat renal brush-border membrane vesicles was stimulated by an inside-positive membrane potential. This potential-sensitive uptake of phenolsulfonphthalein was inhibited by probenecid, pyrazinoate and urate. p-Aminohippurate had no effect on the potential-sensitive uptake of phenolsulfonphthalein. Moreover, urate competitively inhibited the uptake of phenolsulfonphthalein. On the other hand, the uptake of phenolsulfonphthalein was slightly increased in the presence of an outward Cl- gradient. These results suggest that phenolsulfonphthalein has high affinity for the potential-sensitive urate transport system but has low affinity for an anion exchanger.

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.

Ten years ago, we reported that in adult human females the ovarian surface epithelium (OSE) is a source of germ cells. Recently, we also demonstrated that new primary follicles are formed by assembly of oocytes with nests of primitive granulosa cells in the ovarian cortex. The components of the new primary follicles, primitive granulosa and germ cells, differentiated sequentially from the OSE, which arises from cytokeratin positive mesenchymal progenitor cells residing in the ovarian tunica albuginea. In the present study, we investigated the possibility that the oocytes and granulosa cells may differentiate in cultures derived from adult human ovaries. Cells were scrapped from the surface of ovaries and cultured for 5 to 6 days, in the presence or absence of estrogenic stimuli [phenol red (PhR)]. The OSE cells cultured in the medium without PhR differentiated into small (15 micron) cells of granulosa phenotype, and epithelial, neural, and mesenchymal type cells. In contrast, OSE cells cultured in the presence of PhR differentiated directly into large (180 micron) cells of the oocyte phenotype. Such cells exhibited germinal vesicle breakdown, expulsion of the polar body, and surface expression of zona pellucida proteins, i.e. characteristics of secondary oocytes. These in vitro studies confirm our in vivo observations that in adult human ovaries, the OSE is a bipotent source of oocytes and granulosa cells. Development of numerous mature oocytes from adult ovarian stem cells in vitro offers new strategies for the egg preservation, IVF utilization, and treatment of female infertility. In addition, other clinical applications aiming to utilize stem cells, and basic stem cell research as well, may employ totipotent embryonic stem cells developing from fertilized oocytes.

We assayed the effects of phenol red (pr), estrogen (Es), and progesterone (Pg) in three-dimensional organotypic cultures of human uterine endocervix. Small intact fragments deposited on sponges submerged in DMEM with 10% fetal bovine serum were cultured in three different conditions: with pr (DMEM(pr+)), without pr (DMEM(pr-)), and without pr but with the addition of physiological concentrations of Es and Pg [DMEM(pr-)(Es+Pg)]. Cell viability and cellular responses were assayed after 4, 10, and 21 days using immunohistochemistry for the expression and distribution of the following markers: mucins and mucopolysaccharides (PAS staining), pan-cytokeratins (AE1/AE3), CK19, p63, Ki-67, vimentin, estrogen receptor-alpha (ER-alpha), and progesterone receptor (PR). The fragments cultivated in DMEM(pr+) showed a cuboidal, poorly differentiated epithelial phenotype and signs of stroma degeneration. In DMEM(pr-), both tissue architecture and cellular heterogeneity were much better preserved: epithelial cells showed a more columnar shape, and stroma was very well conserved, maintaining cell density. The addition of Es and Pg further improved the histology and physiology of the fragments: in DMEM(pr-)(Es+Pg), epithelial cells retained a columnar shape, secreted mucins, and formed areas of squamous hyperplasia.

The formation of amyloid fibril is associated with major human diseases, including Alzheimer's disease, prion diseases, and type 2 diabetes. Methods for efficient inhibition of amyloid fibril formation are therefore highly clinically important. A principal approach for the inhibition of amyloid formation is based on the use of modified molecular recognition elements. Here, we demonstrate efficient inhibition of amyloid formation of the type 2 diabetes-related human islet amyloid polypeptide (hIAPP) by a modified aromatic peptide fragment and a small aromatic polyphenol molecule. A molecular recognition assay using peptide array analysis suggested that molecular recognition between hIAPP and its core amyloidogenic module is mediated by aromatic rather than hydrophobic interactions. To study the possible effect of aromatic interactions on inhibition of hIAPP fibril formation, we have used peptide and small molecule inhibitors. The addition of a nonamyloidogenic peptide analogue of the core module NFGAILSS, in which phenylalanine was substituted with tyrosine (NYGAILSS), resulted in substantial inhibition of fibril formation by hIAPP. The inhibition was significantly stronger than the one achieved using a beta-sheet breaker-conjugated peptide NFGAILPP. On the basis of the molecular arrangement of the tyrosine-phenylalanine interaction, we suggest that the inhibition stems from the geometrical constrains of the heteroaromatic benzene-phenol interaction. In line with this notion, we demonstrate remarkable inhibition of hIAPP fibril formation and cytotoxicity toward pancreatic beta-cells by a small polyphenol molecule, the nontoxic phenol red compound. Taken together, our results provide further experimental support for the potential role of aromatic interactions in amyloid formation and establish a novel approach for its inhibition.

Clinical hemodialysis systems achieve high single pass extraction of small solutes that are not bound to plasma proteins. But they clear protein-bound solutes much less effectively. This study examines the extent to which clearance of a protein-bound test solute is improved by increasing the dialyzer mass transfer area coefficient (KoA) and the dialysate flow rate (Qd). A reservoir containing test solutes and artificial plasma with albumin concentration approximately 4 g/dl was dialyzed with a standard clinical dialysate delivery system. The clearance of phenol red (ClPR) was compared with the clearances of urea and creatinine at a plasma flow rate (Qp) of 200 ml/min with varying values of KoA and Qd. ClPR increased from 11 +/- 2 ml/min to 23 +/- 2 ml/min when KoA for phenol red, KoAPR, was increased from 238 to 640 ml/min and Qd was increased from 286 +/- 6 ml/min to 734 +/- 9 ml/min. Increasing either KoAPR or Qd alone had lesser effects. Clearance values for phenol red were much lower than clearance values for the unbound solutes urea and creatinine, which ranged from 150 to 200 ml/min and were less affected by varying KoA and Qd. A mathematical model was developed to predict ClPR from values of Qp, Qd, the fraction of phenol red bound to albumin (94% +/- 1%) and KoAPR. The model accurately predicts the pattern of measured results and shows further that ClPR can be made to approach Qp only by very large increases in both KoAPR and Qd.

The effect of light on proton transport across lipid membranes of small unilamellar liposomes containing incorporated major light-harvesting pigment-protein complex of Photosystem II (LHCII) has been studied with the application of pH-sensitive dyes entrapped inside vesicles. Proton permeability coefficient for LHCII-modified membranes was found to be about twice as high as in the case of the control pure lipid vesicles. Illumination of the samples with light absorbed by the LHCII-bound photosynthetic pigments considerably affects the kinetics of proton transport: it increases the rate and decreases the steady-state level of proton gradient across the membranes. The effect was interpreted in terms of heat-induced conformational changes of LHCII molecular structures that affect proton buffering capacity of this protein. Both the control and the LHCII-modified lipid membranes have been found to be practically impermeable to Ca(++) ions, as demonstrated by fluorescence of liposome-entrapped calcium-sensitive probe calcium crimson. The slight differences in the proton transport across the LHCII-containing membranes under the presence of Ca(++) suggest calcium binding to this antenna protein.

The general properties of the taurine uptake in human endometrial tumoral Ishikawa cells were similar to those usually found in other tissues. Uptake was notably affected by the oxygen pressure, being higher at the physiological pO(2) of the endometrium (40 mm Hg, equivalent to 5% O(2)) compared to that used under standard experimental culture conditions (160 mm Hg or 20% O(2)). Uptake of taurine was also density-dependent in Ishikawa cells and was significantly decreased at confluence. Uptake regulation by PKC driven phosphorylation occurs only in growing cells and not in resting cells. The taurine uptake of three Ishikawa cell lines was very different. The taurine uptake of one of the cell lines was affected by estradiol, probably through a non-genomic pathway, whereas tamoxifen had no effect in all cell lines.

The sulfonephthalein indicator, phenol red, exhibits an unusually slow rate of oxidation by laccase from Poliporus pinsitus, in spite of the fact that it is a phenol and therefore a natural substrate for this phenoloxidase enzyme. Nevertheless, after prolonged exposure to laccase (24 h) phenol red is oxidized by more than 90%. We found that phenol red, which can be oxidatively converted into a resonance-stabilized phenoxy radical, performs as a mediator in the laccase-catalyzed oxidation of a nonphenolic substrate (4-methoxybenzyl alcohol) and also of a hindered phenol (2,4,6-tri-tert-butylphenol). In particular, phenol red was found to be at least 10 times more efficient than 3-hydroxyanthranilate (a reported natural phenolic mediator of laccase) in the oxidation of 4-methoxybenzyl alcohol. Other phenols, which do not bear structural analogies to phenol red, underwent rapid degradation and did not perform as laccase mediators. On the other hand, several variously substituted sulfonephthaleins, of different pK2 values, mediated the laccase catalysis, the most efficient being dichlorophenol red, which has the lowest pK2 of the series. The mediating efficiency of phenol red and dichlorophenol red was found to be pH dependent, as was their oxidation Ep value (determined by cyclic voltammetry). We argue that the relative abundance of the phenoxy anion, which is easier to oxidize than the protonated phenol, may be one of the factors determining the efficiency of a phenolic mediator, together with its ability to form relatively stable oxidized intermediates that react with the desired substrate before being depleted in undesired routes.

Osteoblastic induction is commonly studied using the colony-forming unit-fibroblastic (CFU-f) assay, in which bone marrow stromal cells (BMC) are grown in a tissue culture environment permissive for osteoblastic differentiation (DMEM containing dexamethasone, ascorbic acid and beta-glycerophosphate). These cells form colonies, which express alkaline phosphatase, and form a collagenous matrix that becomes calcified. However, these same cells originate in the bone marrow where under normal circumstances they do not proliferate or differentiate despite being subjected to many of the same growth factors and hormones present within the tissue culture environment. We show here that phenol red, present within tissue culture medium as a pH indicator, may itself be a factor that permits osteoblastic recruitment. BMC cultured in the presence of the bone anabolic agents PGE2, PGA2, or bFGF, but in the absence of phenol red, failed to respond to these agents in terms of total or osteoblastic colony number. This effect was dose dependent, with low (2.5 mg/l) and high (15-20 mg/l) doses of phenol red being nonpermissive for the stimulatory effects of PGE2 whereas doses of 5-10 mg/l were permissive. Furthermore, the effects observed in the absence of phenol red could not be abrogated by the addition of 17beta-estradiol indicating that these effects cannot be attributed to estrogenic impurities within the phenol red preparation. This indicates that phenol red itself can affect the differentiation of BMC by a mechanism not previously described.

Epidermal growth factor (EGF) stimulates repair in the damaged intestine, but its role in the normal intestine is not clear. Because EGF receptors are found on the basolateral surface but not the luminal surface, we hypothesized that mucosal permeability regulates EGF binding. Adult male rats were divided into 3 groups, one that was fed normal chow (the control), one that was starved for 4 days, and one that was given methotrexate (MTX) intragastrically (10 mg/kg/day for 3 days). The rats were sacrificed and everted sacks of the jejunum were made and incubated in EGF solution. Western blot analysis of mucosal homogenates showed that the amount of phosphotyrosyl EGF receptor in the starved and MTX-treated groups was, respectively, about 1.5 times and 2 times that in the control group. The mucosal permeability in the starved and MTX treated groups also increased and varied directly with the amount of phosphotyrosyl EGF receptor. These results suggest that in the adult rat intestine, luminal EGF may play a role only under tissue damage, where enhanced permeability permits the EGF to pass through the mucosa and bind to its receptor on the basolateral membrane.

Estrogens play an important role in the regulation of placental function, and 17-beta-estradiol (E2) production rises eighty fold during human pregnancy. Although term placenta has been found to specifically bind estrogens, cellular localization of estrogen receptor alpha (ER-alpha) in trophoblast remains unclear. We used western blot analysis and immunohistochemistry with h-151 and ID5 monoclonal antibodies to determine the expression and cellular localization of ER-alpha protein in human placentae and cultured trophoblast cells. Western blot analysis revealed a ~65 kDa ER-alpha band in MCF-7 breast carcinoma cells (positive control). A similar band was detected in five normal term placentae exhibiting strong expression of Thy-1 differentiation protein in the villous core. However, five other term placentae, which exhibited low or no Thy-1 expression (abnormal placentae), exhibited virtually no ER-alpha expression. In normal placentae, nuclear ER-alpha expression was confined to villous cytotrophoblast cells (CT), but syncytiotrophoblast (ST) and extravillous trophoblast cells were unstained. In abnormal placentae no CT expressing ER-alpha were detected. Normal and abnormal placentae also showed ER-alpha expression in villous vascular pericytes and amniotic (but not villous) fibroblasts; no staining was detected in amniotic epithelial cells or decidual cells. All cultured trophoblast cells derived from the same normal and abnormal placentae showed distinct ER-alpha expression in western blots, and the ER-alpha expression was confined to the differentiating CT, but not to the mature ST. Trophoblast cells from six additional placentae were cultured in normal medium with phenol red (a weak estrogen) as above (PhR+), or plated in phenol red-free medium (PhR-) without or with mid-pregnancy levels of E2 (20 nM). Culture in PhR- medium without E2 caused retardation of syncytium formation and PhR-medium with E2 caused acceleration of syncytium formation compared to cultures in PhR+ medium. These data indicate that the considerable increase in estrogen production during pregnancy may play a role, via the ER-alpha, in the stimulation of CT differentiation and promote function in normal placentae. This mechanism, however, may not operate in abnormal placentae, which show a lack of ER-alpha expression.

Aims: To examine the effect on the leakage of low molecular weight cytoplasmic constituents from Staphylococcus aureus using phenolics singly and in combination, and to see if the observations could be modelled using a non-linear dose response., Methods and Results: The rate of potassium, phosphate and adenosine triphosphate leakage was examined in the presence of chlorocresol and m-cresol. Individually, leakage was observed only at long contact times or high concentrations. Combined at these ineffective concentrations, the cytoplasmic pool of all constituents studied was released within minutes. Both chlorocresol and m-cresol were shown to have non-linear dose responses. A rate model for the combinations, which takes account of these non-linear responses, accurately predicted the observations., Conclusions: Antimicrobials, which when used alone exhibit a non-linear dose response, will also give a non-linear dose response in combination. The simple linear-additive model ignores the concept of the dilution coefficient and will always describe the phenomenon of synergy for combinations where one or more of the components has a dilution coefficient greater than one. This has been borne out by examination of the purported prime lesion of chlorocresol and m-cresol, alone and in combination., Significance and Impact of the Study: Studies aimed at producing synergistic mixtures of antimicrobials, which ignore the non-linear additive effect, may waste valuable research effort looking for a physiological explanation for an apparent synergy, where none, in-fact, exists. Patents granted on the basis of analyses using the linear-additive model for combinations of compounds with non-linear dose responses may no longer be supportable.

Uptake of phosphate by the everted intestinal sacs of mice was measured at various concentrations of this anion in the incubation medium. Phenol red when added to the serosal side was able to inhibit the uptake of phosphate significantly, while uptake of glucose or proline was unaffected. When para amino hippuric acid was present along with phenol red in the serosal compartment, the inhibition exerted by the dye on phosphate uptake was significantly less. These findings suggest that entry of phenol red into gut tissue from the serosal side interferes with the mucosal uptake of phosphate by the everted intestinal sacs of mice.

Laccase production by the white-rot fungus Trametes versicolor (CBS100.29) grown in submerged cultures was studied. Addition of different insoluble lignocellulosic materials into the culture medium in order to enhance laccase production was investigated. The lignocellulosic materials were grape seeds, grape stalks and barley bran, selected because of their availability and low cost, since they are agro-industrial wastes abundant in most countries. Barley bran gave the highest activities, a maximum value of 639U/l, which was 10 times the value attained in the cultures without lignocellulosics addition. The decolourisation of a model dye, Phenol Red, by the ligninolytic fluids obtained in the above-mentioned cultures was investigated. Grape stalk and barley bran cultures showed the highest ability to decolourise the dye, attaining a percentage of decolourisation of around 60% in 72 h.

In human colorectal carcinoma Caco-2 cells, sodium butyrate (NaBT) induces the expression of the reticulocyte, 15-lipoxygenase-1 (15-LO-1) and causes these cells to undergo differentiation and apoptosis. 15-LO-1 is also expressed in human colorectal epithelium with a significant higher expression observed in colorectal tumors. In this study, we have prepared stable Caco-2 cells that expressed 15-LO-1 under control of an inducible promoter. These cells provide a model system to study regulation of 15-LO-1 activity in colorectal cells without the interfering presence of NaBT and are useful to study the biological function of 15-LO-1. The expressed 15-LO-1 was highly active as measured in cell lysates, but we were unable to detect metabolism in intact cells. The addition of calcium to the media for the Caco-2 cells was required for 15-LO-1 to translocate from the cytosol to the membrane which is frequently a requirement for lipoxygenase activity. Despite the addition of calcium and translocation, little lipoxygenase activity was detected with intact cells. However, after removal of phenol red, a common constituent of cell culture media, we were able to detect 15-LO-1 activity in the transfected Caco-2 cultured cells. Thus the presence of calcium and the absence of antioxidants present in commonly used culture media are required for expressed 15-LO-1 to be catalytically active and to permit an examination of its biological effects., (Copyright 2001 Harcourt Publishers Ltd.)

Previous studies have demonstrated that cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-mediated Cl(-)channel found in most epithelia including reproductive tract, could be regulated by various culture conditions. The present study further investigated the effect of phenol red, a pH indicator widely used in growth medium, and steroid hormones, present in the supplement fetal bovine serum (FBS), on primary cultured endometrial epithelial cells by monitoring ion channel activities using the short-circuit current technique. When compared to the results obtained with normal medium supplemented with regular FBS, the forskolin-stimulated I(SC), presumably mediated by CFTR, obtained in phenol red-free medium was significantly reduced, from 16.95+/-1.53 microA/cm(2)(control) to 9.72+/-0.89 microA/cm(2)(medium without phenol red, P< 0.05). The forskolin-activated I(SC)was further attenuated to 5.29+/-0.46 microA/cm(2)in the phenol red-free medium when supplemented with charcoal/ dextran-treated FBS where steroid hormones were removed. Our data suggest that phenol red and steroid hormones present in culture medium and FBS supplement, respectively, may somehow upregulate CFTR expression in vitro. Our study demonstrates the need for carefully choosing the culture media and supplements due to the effect of steroid hormones., (Copyright 2001 Academic Press.)

The reported estrogenic action of phenol red and/or its lipophilic contaminants has led to the widespread use of indicator-free culture medium to conduct endocrine studies in vitro. Because we have recently developed methods to measure large-magnitude estrogen effects in the tissue culture medium containing phenol red, we concluded that the indicator issue required further evaluation. To do this, we selected nine estrogen receptor positive (ER+) cell lines representing four target tissues and three species. We investigated phenol red using five different experimental protocols. First, 17beta-estradiol (E2) responsive growth of all nine ER+ cells lines was compared in the medium with and without the indicator. Second, using representative lines we asked if phenol red was mitogenic in the indicator-free medium. The dose-response effects of phenol red were compared directly to those of E2. Third, we asked if tamoxifen-inhibited growth equally in phenol red-containing and indicator-free medium. This study was based on a report indicating that antiestrogen effects should be seen only in phenol red-containing medium. Fourth, we asked if phenol red displaced the binding of 3H-E2 using ERK intact human breast cancer cells. Fifth, we compared E2 and phenol red as inducers of the progesterone receptor using a human breast cancer cell line. All the experiments presented in this report support the conclusion that the concentration of phenol red contaminants in a standard culture medium available today is not sufficient to cause estrogenic effects. In brief, our studies indicate that the real issue of how to demonstrate estrogenic effects in culture resides elsewhere than phenol red. We have found that the demonstration of sex steroid hormone-mitogenic effects in culture depends upon conditions that maximize the effects of a serum-borne inhibitor(s). When the effects of the inhibitor are optimized, the presence or absence of phenol red makes no everyday difference to the demonstration of estrogen mitogenic effects with several target cell types from diverse species.

The efflux transport of oestrone-3-sulphate, a steroid hormone sulphate, across the blood-cerebrospinal fluid barrier has been examined following its intracerebroventricular administration. [3H]Oestrone-3-sulphate was eliminated from cerebrospinal fluid (CSF) with an apparent efflux clearance of 205 microL min(-1) per rat. There was 25% of unmetabolized [3H]oestrone-3-sulphate in the plasma 5 min after intracerebroventricular administration, indicating that at least a part of [3H]oestrone-3-sulphate is transported from CSF to the circulating blood across the blood-CSF barrier. This efflux transport was inhibited by co-administration of excess oestrone-3-sulphate (25 mM 10 microL = 0.25 micromol) into rat cerebral ventricle. To characterize the oestrone-3-sulphate transport process, an in-vitro uptake experiment was performed using isolated rat choroid plexus. Oestrone-3-sulphate uptake by isolated rat choroid plexus was found to be a saturable process with a Michaelis-Menten constant (Km) of 18.1 +/- 6.3 microM, and a maximum uptake rate (Vmax) of 48.0 +/- 15.1 pmol min(-1) microL(-1) of tissue. The oestrone-3-sulphate transport process was temperature dependent and was inhibited by metabolic inhibitors such as 2,4-dinitrophenol and rotenone, suggesting an energy dependence. This uptake process was also inhibited by steroid hormone sulphates (1 mM dehydroepiandrosterone sulphate and 1 mM oestrone sulphate), bile acids (1 mM taurocholic acid and 1 mM cholic acid) and organic anions (1 mM sulphobromophthalein and 1 mM phenolsulphonphthalein), whereas 1 mM p-aminohippuric acid, 1 mM p-nitrophenol sulphate, 0.1 mM methotrexate and the cardiac glycoside, 2.5 microM digoxin, had little effect. In conclusion, these results provide evidence that oestrone-3-sulphate is transported from CSF to the circulating blood across the blood-CSF barrier via a carrier-mediated efflux transport system.

Androgens regulate the expression of both human prostatic acid phosphatase (PAcP) and prostate-specific antigen (PSA), two major prostate epithelium-specific differentiation antigens. Due to the important role of these two enzymes as prostate epithelium differentiation markers, we investigated their regulation of expression at the mRNA level in LNCaP human prostate carcinoma cells. Interestingly, phenol red, a pH indicator in the culture medium, promoted cell growth. To eliminate this non-specific effect, a phenol red-free, steroid-reduced medium was utilized. When high-density cells were grown in that medium, 5alpha-dihydrotestosterone (DHT) suppressed PAcP but stimulated PSA. However, tumor promoter phorbol ester 12-o-tetradecanoyl phorbol-13-acetate (TPA) functioned as a potent inhibitor of both PAcP and PSA expression. Prolonged treatment with DHT as well as TPA resulted in a similar down-regulation of protein kinase C and cellular PAcP activities. Thus, the levels of PAcP and PSA mRNA are differentially regulated by androgens in LNCaP cells., (Copyright 2000 Academic Press.)

Drugs for testing the function of liver and kidney (sulfobromophthalein, phenolsulfonphthalein, indigo carmine and indocyanine green) and other organic anions (rose bengal and haematin) were found to potently inhibit human liver aldehyde reductase that is involved in the detoxification of 3-deoxyglucosone and methylglyoxal, reactive intermediates, during the formation of advanced glycation end products. The inhibition patterns by the compounds were non-competitive with respect to both coenzyme (NADPH) and substrate (D-glucuronate). The kinetics of the inhibition by a mixture of the 2 inhibitors suggests that all the inhibitory compounds bind to overlapping sites on the enzyme. The binding of rose bengal, sulfobromophthalein and phenylsulfonphthalein to the free enzyme was detected by ultrafiltration assay. However, in the reverse reaction, the enzyme was inhibited competitively with respect to the alcohol substrate by rose bengal, haematin, phenolsulfonphthalein, sulfobromophthalein, indigo carmine and indocyanine green, which showed Ki values of 0.1, 1, 3, 4, 4 and 10 microM, respectively. The results suggest that these potent inhibitors bind weakly to the free enzyme and tightly to the enzyme-NADP binary complex.

Retinoids are natural differentiation-inducing compounds that are promising as anticancer agents. Cancer cell lines are valuable in the investigation of the potential of retinoids for the treatment of specific cancers. However, using different treatment conditions but the same cell lines, investigators have produced markedly contradictory results for the effectiveness of retinoids. The present study examined different factors in the treatment conditions that may have masked or interfered with the effects of retinoids and, thereby, resulted in this conflict. Our studies revealed that the effects of retinoids on cancer cell proliferation were influenced by serum, the choice of vehicle (DMSO vs ethanol) and its concentration, phenol red, the degree of cellular confluence, and the method of assessing proliferation (cell number or [3H]thymidine uptake vs the MTT assay). Optimized conditions were the use of serum-free, ethanol-free, and phenol red-free media, investigating cells in the log phase of growth, using