Addition Of Phloretin Research Articles

Role of phloretin as a sensitizer to TRAIL-induced apoptosis in colon cancer.

Phloretin is one of the apple polyphenols with anticancer activities. Since tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) serves important roles in inducing apoptosis, the present study examined the effect of phloretin on TRAIL-induced apoptosis in colon cancer cells. Treatment with both phloretin and TRAIL markedly suppressed the survival of cancer cells from several colon cancer cell lines compared with that of cells treated with either TRAIL or phloretin. Additionally, decreased numbers of colonies were observed following addition of phloretin and TRAIL. Furthermore, TRAIL- and phloretin-treated HT-29-Luc cells exhibited decreased luciferase activity. Increased apoptosis was observed in phloretin- and TRAIL-treated HT-29-Luc colon cancer cells, accompanying elevated levels of cleaved poly(ADP-ribose) polymerase, and caspase-3, −8 and −9. The expression levels of MCL1 apoptosis regulator BCL2 family member (Mcl-1) were decreased following addition of phloretin in colon cancer cells. In addition, overexpression of Mcl-1 in phloretin- and TRAIL-treated HT-29-Luc cells resulted in increased cell survival. Treatment of HT-29-Luc cells with a combination of cycloheximide (CHX) and phloretin led to a more prominent decrease in Mcl-1 expression compared with that in cells treated with CHX alone, while Mcl-1 expression was recovered by treatment with MG132. Binding of ubiquitin with Mcl-1 was verified using immunoprecipitation. Intraperitoneal injection of both TRAIL and phloretin into tumor xenografts was associated with a decreased tumor volume compared with that following injection with either TRAIL or phloretin. Overall, the present results suggest a synergistic effect of phloretin on TRAIL-induced apoptosis in colon cancer cells.

Interaction between phloretin and insulin: a spectroscopic study

Diabetes is among the top ten deadly diseases in the world. It occurs either when the pancreas does not produce enough insulin (INS) or when the body cannot effectively use the insulin it produces. Phloretin (PHL) has a biological effect that can treat diabetes. A spectroscopic study was carried out to explore the interaction between phloretin and insulin. UV/Vis spectroscopy, fluorescence spectroscopy, and circular dichroism spectropolarimeter were used in the study. UV/Vis spectra showed that the interaction between PHL and INS produced strong absorption at a wavelength of 282 nm. The fluorescence analysis results showed that the excitation and emission occurred at 280-nm and 305-nm wavelengths, respectively. Temperature changes did not affect INS emissions. However, the interaction of PHL–INS caused a redshift at 305 to 317 nm. Temperature affected the binding constant (Ka) and the binding site (n). Ka decreased with increasing temperature and increased the binding site. The thermodynamic parameters such as enthalpy (ΔH0) and entropy (ΔS0) each had a value of − 16,514 kJ/mol and 22.65 J/mol·K. PHL and INS interaction formed hydrogen bonds and hydrophobic interaction. The free energy (ΔG0) recorded was negative. PHL and INS interactions took place spontaneously. The quenching effect was dynamic and static. KD values were greater than KS. The higher the temperature, the less was KD and KS. The appearance of two negative signals on circular dichroism (CD) spectropolarimeter implies that phloretin could induce regional configuration changes in insulin. The addition of PHL has revealed that the proportion of α-helix in the insulin stabilizes its structure. Phloretin’s stabilization and enhancement of the α-helix structural configuration in insulin indicate that phloretin can improve insulin resistance.

173 Characterization of urea transport mechanisms in the intestinal tract of growing pigs

Abstract Previous studies have indicated that pigs are capable of nitrogen salvage via urea recycling, which involves the movement of urea into the gastrointestinal tract and incorporation of nitrogen into endogenous or microbially produced amino acids. Aquaporins (AQP) and urea transporter B (UT-B) have been shown to contribute to urea transport in ruminants; however, it is unclear whether the same processes contribute to urea movement in the intestinal tract of the pig. The objective of this study was to characterize the presence and relative contribution of known urea transporters to urea flux in the growing pig. A total of 9 barrows of 50.8±0.9 kg BW were euthanized and samples of intestinal tissue were obtained from the duodenum, jejunum, ileum, cecum, and colon. All tissue samples were analyzed for mRNA abundance of UT-B and AQP-3, 7, and 10 via qPCR. Immediately after tissue collection, samples from jejunum and cecum were placed in Ussing chambers for analysis of serosal-to-mucosal urea flux using 14C-urea (49.95 kBq). Serosal-to-mucosal urea flux was measured across intestinal tissue samples with no inhibition or with addition of phloretin (1 mM) to inhibit UT-B-mediated transport, NiCl2 (1 mM) to inhibit AQP-mediated transport, or both inhibitors. UT-B was most highly expressed in the cecum (P < 0.05), while AQP-3, 7, and 10 were most highly expressed in the jejunum (P < 0.05). Serosal-to-mucosal urea flux occurred in both the jejunum and the cecum and was higher in the cecum (42.7 vs. 67.8±5.01 µmol/cm2/h; P < 0.05), confirming the capacity for urea recycling into the gut in pigs; however, neither flux rate was influenced by urea transporter inhibitors (P > 0.05). The results of this study indicate that while known urea transporters are present in the gastrointestinal tract of pigs, they do not play a significant role in urea transport.

Aquaglyceroporins but not orthodox aquaporins are involved in the cryotolerance of pig spermatozoa

BackgroundAquaporins (AQPs) are a family of transmembrane water channels that includes orthodox AQPs, aquaglyceroporins (GLPs) and superAQPs. AQP3, AQP7, AQP9 and AQP11 have been identified in boar sperm, and they are crucial for sperm maturation and osmoregulation. Water exchange is an important event in cryopreservation, which is the most efficient method for long-term storage of sperm. However, the freeze-thaw process leads to sperm damage and a loss of fertilizing potential. Assuming that the quality of frozen-thawed sperm partially depends on the regulation of osmolality variations during this process, AQPs might play a crucial role in boar semen freezability. In this context, the aim of this study was to unravel the functional relevance of the different groups of AQPs for boar sperm cryotolerance through three different inhibitors.ResultsInhibition of different groups of AQPs was found to have different effects on boar sperm cryotolerance. Whereas the use of 1,3-propanediol (PDO), an inhibitor of orthodox AQPs and GLPs, decreased total motility (P < 0.05), it increased post-thaw sperm viability, lowered membrane lipid disorder and increased mitochondrial membrane potential (MMP) (P < 0.05). When acetazolamide (AC) was used as an inhibitor of orthodox AQPs, the effects on post-thaw sperm quality were restricted to a mild increase in MMP in the presence of the intermediate concentration at 30 min post-thaw and an increase in superoxide levels (P < 0.05). Finally, the addition of phloretin (PHL), a GLP inhibitor, had detrimental effects on post-thaw total and progressive sperm motilities, viability and lipid membrane disorder (P < 0.05).ConclusionsThe effects of the different inhibitors suggest that GLPs rather than orthodox AQPs are relevant for boar sperm freezability. Moreover, the positive effect of PDO on sperm quality suggests a cryoprotective role for this molecule.

Examining urea flux across the intestine of the spiny dogfish, Squalus acanthias

Recent examination of urea flux in the intestine of the spiny dogfish shark, Squalus acanthias, has shown that feeding significantly enhances urea uptake across the intestine, and this was significantly inhibited following mucosal addition of phloretin. The present study examined potential mechanisms of urea uptake across the dogfish intestine in starved and fed dogfish. Unidirectional flux chambers were used to examine the kinetics of urea uptake, and to determine the influence of sodium, ouabain, competitive urea analogues, and phloretin on urea uptake across the gut of fed dogfish. Intestinal epithelial preparations from starved and fed dogfish were mounted in Ussing chambers to examine the effect of phloretin on bidirectional solute transport across the intestine. In the unidirectional studies, the maximum uptake rate of urea was found to be 35.3±6.9μmol.cm-2.h-1 and Km was found to be 291.8±9.6mM in fed fish, and there was a mild inhibition of urea uptake following mucosal addition of competitive agonists. Addition of phloretin, Na-free Ringers and ouabain to the mucosal side of intestinal epithelia also led to a significant reduction in urea uptake in fed fish. In the Ussing chamber studies there was a net influx of urea in fed fish and a small insignificant efflux in starved fish. Addition of phloretin blocked urea uptake in fed fish when added to the mucosal side. Furthermore, phloretin had no effect on ion transport across the intestinal epithelia with the exception of the divalent cations, magnesium and calcium.

Probing Amphotericin B Single Channel Activity by Membrane Dipole Modifiers

The effects of dipole modifiers and their structural analogs on the single channel activity of amphotericin B in sterol-containing planar phosphocholine membranes are studied. It is shown that the addition of phloretin in solutions bathing membranes containing cholesterol or ergosterol decreases the conductance of single amphotericin B channels. Quercetin decreases the channel conductance in cholesterol-containing bilayers while it does not affect the channel conductance in ergosterol-containing membranes. It is demonstrated that the insertion of styryl dyes, such as RH 421, RH 237 or RH 160, in bilayers with either cholesterol or ergosterol leads to the increase of the current amplitude of amphotericin B pores. Introduction of 5α-androstan-3β-ol into a membrane-forming solution increases the amphotericin B channel conductance in a concentration-dependent manner. All the effects are likely to be attributed to the influence of the membrane dipole potential on the conductance of single amphotericin B channels. However, specific interactions of some dipole modifiers with polyene-sterol complexes might also contribute to the activity of single amphotericin B pores. It has been shown that the channel dwell time increases with increasing sterol concentration, and it is higher for cholesterol-containing membranes than for bilayers including ergosterol, 6-ketocholestanol, 7-ketocholestanol or 5α-androstan-3β-ol. These findings suggest that the processes of association/dissociation of channel forming molecules depend on the membrane fluidity.

Dual Effects of Phloretin and Phloridzin on the Glycation Induced by Methylglyoxal in Model Systems

In the present study, the dual effects of phloretin and phloridzin on methylglyoxal (MGO)-induced glycation were investigated in three N(α)-acetyl amino acid (arginine, cysteine, and lysine) models and three N-terminal polypeptide (PP01, PP02, and PP03 containing arginine, cysteine, and lysine, respectively) models. In both N(α)-acetyl amino acids and N-terminal polypeptides models, the arginine residue was confirmed as the major target for modification induced by MGO. Meanwhile, MGO modification was significantly inhibited by the addition of phloretin or phloridzin via their MGO-trapping abilities, with phloretin being more effective. Interestingly, the cysteine residue was intact when solely incubated with MGO, whereas the consumption of N(α)-acetylcysteine and PP02 was promoted by the addition of phloretin. Additional adducts, [N(α)-acetylcysteine + 2MGO + phloretin-H(2)O] and [2N(α)-acetylcysteine + 2MGO + phloretin-2H(2)O] were formed in the model composed of N(α)-acetylcysteine, MGO, and phloretin. Another adduct, [PP02 + 2MGO + phloretin-H(2)O] was observed in the model composed of PP02, MGO, and phloretin. The generation of adducts indicates that phloretin could directly participate in the modification of the cysteine residue in the presence of MGO. When creatine kinase (model protein) was exposed to MGO, the addition of phloridzin did not show a significant effect on retaining the activity of creatine kinase impaired by MGO, whereas the addition of phloretin completely inactivated creatine kinase. Results of the mass spectrometric analysis of intact creatine kinase in different models demonstrated that phloretin could directly participate in the reaction between creatine kinase and MGO, which would lead to the inactivation of creatine kinase. Furthermore, the addition of N(α)-acetylcysteine was found to maintain the activity of creatine kinase incubated with phloretin and MGO. These results showed that phloretin and phloridzin could inhibit the modification of the arginine residue by MGO and that phloretin could directly participate in the reaction between the thiol group and MGO.

Dipole potential as a driving force for the membrane insertion of polyacrylic acid in slightly acidic milieu

In this work, we report on the interaction of polyacrylic acid with phosphatidylcholine bilayers and monolayers in slightly acidic medium. We found that adsorption of polyacrylic acid on liposomes composed of egg lecithin at pH 4.2 results in the formation of small pores permeable for low molecular weight solutes. However, the pores were impermeable for trypsin indicating that no solubilization of liposomes occurred. The pores were permeable for both positively charged trypsin substrate N-benzoyl-l-arginine ethyl ester and negatively charged pH-indicator pyranine. Two lines of evidence were obtained confirming the involvement of the membrane dipole potential in the insertion of polyacrylic acid into lipid bilayer. (i) Addition of phloretin, a molecule which is known to decrease dipole potential of lipid bilayer, reduced the rate of a polyacrylic acid induced leakage of pyranine from liposomes. (ii) Direct measurements of air/lipid monolayer/water interface surface potential using Kelvin probe showed that adsorption of polyacrylic acid at pH 4.2 induced a decrease in both boundary and dipole potential by 37 and 62mV for ester lipid dioleoylphosphatidylcholine (DOPC). Replacement of DOPC by ether lipid 1,2-di-O-oleyl-sn-glycero-3-phosphocholine (DiOOPC) which is known to form monolayers and bilayers with only minor dipole component of membrane potential showed that addition of PAA produced similar response in the boundary potential (by 50mV) but negligible response in dipole potential of monolayer. These observations agree with our assumption that dipole potential is an important driving force for the insertion of polyacids into biological membranes.

Feeding Oscillating Dietary Crude Protein Concentrations Increases Nitrogen Utilization in Growing Lambs and This Response Is Partly Attributable to Increased Urea Transfer to the Rumen1–3

Our objective was to determine the effects of feeding oscillating compared with static dietary crude protein (CP) concentrations on nitrogen (N) retention and urea flux across ruminal epithelia. Twenty-seven Suffolk wether lambs (n = 9) were assigned to a medium-CP diet [MEDIUM; 127 g CP·kg dry matter (DM)-1] or to diets with oscillating CP content (OSC) fed in 2 different sequences, i.e. 2 d of low CP (103 g CP·kg DM-1) followed by 2 d of high CP (161 g CP·kg DM-1; OSC-HIGH) or vice versa (OSC-LOW). Diet adaptation was for 24 d, followed by 8 d of total urine and feces collection. On d 33, lambs were slaughtered 4 h after the morning feeding, such that those receiving OSC-LOW and OSC-HIGH diets were slaughtered on d 3 of receiving the low- or high-CP diets, respectively. Ruminal epithelia were collected and mounted in Ussing chambers and the serosal-to-mucosal urea flux (Jsm-urea) was measured using 14C-urea. Ruminal NH3-N concentration was lower (P = 0.001) in lambs fed OSC-LOW compared with those fed OSC-HIGH. Although N intake was similar, retained N (P = 0.001) and microbial N supply (P = 0.001) were greater in lambs fed OSC compared with those fed MEDIUM. The total Jsm-urea was higher (P = 0.001) in lambs fed OSC-LOW compared with those fed OSC-HIGH. Across diets, the addition of phloretin [a known specific inhibitor of facilitative urea transporter (UT)-B] reduced Jsm-urea by 19.5–22.3% (P = 0.001); however, phloretin-insensitive Jsm-urea was the predominant route for transepithelial urea transfer. Taken together, these data indicate that feeding oscillating dietary CP concentrations improves N retention partly by increasing urea recycling to the rumen when animals are fed low-CP diets, but the greater rates of urea transfer cannot be attributable to upregulation of UT-B.

Effect of Dipole Modifying Agents on the Surfactin Induced Conductance of Planar Lipid Bilayers

We studied effects of compounds changing membrane dipole potential on membrane conductance induced by antimicrobial lipopeptide surfactin. Surfactin added on both sides of an artificial lipid bilayer from diphytanoyl phosphocholine in 1 M KCl (pH 6.5) produces an increase of the membrane conductance as a result of ion channel formation. Increasing a membrane dipole potential adding RH 421 to the bilayer bathing solution (10 µM , both sides) leads to ∼40 times increase of a steady-state conductance. At the same time, addition of phloretin (20 µM), known to decrease the dipole potential, results in decrease of the surfactin-induced conductance by ∼30 times. We note, that the effects of dipole modifiers on the surfactin-induced membrane conductance are clearly opposite to the effects observed with the same modifiers in case of syringomycin E-induced conductance of lipid bilayers [Ostroumova et al., Langmuir, 2008]. As we suggested earlier, the influence of dipole modifiers on syringomycin activity may be related to a promotion/retardation of a movement of positively charged syringomycin molecules in the direction of membrane hydrocarbon core. In contrast to syringomycin, surfactin is negatively charged. Hence, one can expect an inversion of the effect of dipole modifiers in case of surfactin. The obtained results are in agreement with the model proposed in [Ostroumova et al., Langmuir, 2008].The study was supported in part by the Russian Fund for Basic Research (09-04-48860), the State Program of Molecular and Cell Biology, the grant of St.-Petersburg Administration for young scientists, and the State contract (FAE Π-1372).

The effect of phloretin on sphingolipid-containing membranes modified by syringomycin E

The effects of dipole modifier phloretin on the activity of syringomycin (SRE) channels in the lipid bilayers containing different sphingolipids, N-stearoyl-phytosphingosine (PSP) and N-stearoyl-D-erythrosphingosine (DSP), have been compared. It is shown that the addition of phloretin up to a concentration of 10 μM into solutions bathing the bilayers containing 20 mol.% PSP causes a 170-fold increase in the SRE channel-forming activity. In the case of DSP-containing membranes, a more significant (5200-fold) increase of the equilibrium number of open SRE channels is observed. The enhancement of SRE activity is accompanied by about 2-fold increase of the gating charge of SRE channels in the membranes with PSP, while in the bilayers with DSP the gating charge increases about 4-fold. The revealed differences in the parameters of SRE channels in the membranes including phloretin and PSP or DSP are accounted for by different partition coefficients of the toxin and dipole modifier between the lipid and water phases. The data suggest heterogeneity of dipole potential of the PSP-containing membranes in the presence of phloretin. This heterogeneity is due to the possibility of formation in these membranes of rafts with the dipole potential not affected by modifier.

Role of membrane dipole potential-modifying agents and lipid rafts in channel-forming activity of syringomycin E

Comparison of effects of phloretin, an agent decreasing the dipole membrane potential, on ion channels in lipid bilayers containing different sphingolipids, N-stearoyl-phytosphingosine and N-stearoyl- D erythro -sphinganine, was performed. These sphingolipids have structural analogy with sphingolipids isolated from the wild-type (W303C) and mutant (W ∆ SYR2 α ) strains of Saccharomyces cerevisiae , respectively. Earlier, the mutant cells were shown to be more resistant to the action of the cyclic lipodepsipeptide syringomycin E (SRE) than the wild strain [1]. The authors of this study conclude that membranes of the wild-type yeast cells with hydroxylated sphingolipids contain lipid domains, rafts, which are lacking in the mutant cell membranes. The authors explain different SRE toxicity for yeast cells of the wild-type and mutant strains by the SRE affinity to rafts. We determined characteristics of the ion channels formed by SRE in bilayer lipid membranes (BLM) bathing in 0.1 M NaCl solutions (pH 6). In the absence of phloretin, current‐voltage characteristics of SRE channels, values of their gating charges (Q) as well as ratio of the SRE threshold concentrations required for observation of single channels are equal for the BLM containing 20 mol.% sphingolipids from the wild-type cells and sphingolipids similar to those isolated from the mutant cells. Addition to the membrane-bathing solutions of phloretin at the concentration of 10 µ M in the case of BLM containing 20 mol.% sphingolipids from the wild-type cells leads to an approximately 200-fold increase of the SRE channel-forming activity. In the case of the BLM containing sphingolipids similar to those isolated from mutant cells, a more prominent (4500-fold) rise of the steadystate number of the open SRE-channels is observed. It can be suggested that phloretin is unable to change the dipole potential of lipid rafts. Since SRE is mainly concentrated in rafts, addition of phloretin does not produce a considerable increase of the channel-forming activity in the membrane containing sphingolipids of the wild-type cells. In the membranes containing sphingolipids similar to those isolated from mutants, rafts should not be present; phloretin is uniformly distributed over the membrane thus decreasing its dipole potential and increasing sharply the SRE channel-forming activity. It has been shown in [2, 3] that an increase of the SRE channel-forming activity upon a decrease of the dipole potential of phospholipid bilayers is accompanied by a rise of the channel gating charge. In fact, in the membranes containing sphingolipids of the wild strains, Q = 2.3 ± 0.1, whereas in the membranes containing sphingolipids similar to those present in the mutant strain membranes, Q = 3.7 ± 0.5. As was expected, the increase of the SRE channel-forming activity is accompanied by the rise of the SRE channel gating charge. The obtained data allow the conclusion that in the bilayers containing sphingolipids the SRE channel-forming activity is determined predominantly by the dipole potential of lipid rafts. Thus, it can be claimed that heterogeneity of membranes plays an essential functional role in regulation of ion channels.

Duality in the Mastoparan Action on Glucose Transport in Rat Adipocytes

Mastoparan, a tetradecapeptide purified from wasp venom, has been shown to stimulate glucose transport in rat adipocytes although the mechanism of its action has remained undefined. Here, we characterized the action of mastoparan on glucose transport in rat adipocytes. Mastoparan at a concentration of 20 microM or more caused a dose-dependent release of lactate dehydrogenase (LDH) from the cells, which closely correlated with its stimulatory effect on glucose uptake. The mastoparan-induced glucose uptake was inhibited neither by deprivation of ATP with KCN nor by addition of phloretin, a direct inhibitor of glucose transporter, suggesting that the ability of mastoparan to stimulate glucose uptake did not derive from activation of the glucose transport system (i.e. translocation or activation of GLUT4 and/or GLUT1). On the other hand, mastoparan at a lower concentration (15 microM or below), which showed an insignificant effect on LDH release, potentiated the insulin action on glucose transport and Akt phosphorylation in the presence of adenosine deaminase. The effect of mastoparan was not additive to that of phenylisopropyladenosine and was completely abolished by pretreatment of adipocytes with pertussis toxin (1 microg/ml for 2 hours). Thus, the present study disclosed duality in the action of mastoparan on glucose uptake in rat adipocytes. At a concentration of 15 microM or less, it enhances the insulin action on glucose transport by a pertussis toxin-sensitive Gi protein-dependent mechanism. At higher concentrations, however, mastoparan increases non-specific permeability of the plasma membrane, which causes LDH release as well as glucose uptake not mediated through glucose transporter.

Dipole potential and head-group spacing are determinants for the membrane partitioning of pregnanolone.

The membrane interactions of pregnanolone, an intravenous general anesthetic steroid, were characterized using fluorescence spectroscopy and monolayer technique. di-8-ANEPPS [4-[2-[6-(dioctylamino)-2-naphthalenyl]ethenyl]-1-(3-sulfopropyl)-pyridinium], a membrane dipole potential (Psi)-sensitive probe, revealed pregnanolone to decrease Psi similarly as reported previously for other anesthetics. The decrement in Psi was approximately 16 and 10 mV in dipalmitoylphosphatidylcholine (DPPC) and DPPC/cholesterol (90:10, mol/mol) vesicles, respectively. Diphenylhexatriene anisotropy indicated pregnanolone to have a negligible effect on the acyl chain order. In contrast, substantial changes were observed for the fluorescent dye Prodan, thus suggesting pregnanolone to reside in the interfacial region of lipid bilayers. Langmuir balance studies indicated increased association of pregnanolone to DPPC monolayers containing cholesterol or 6-ketocholestanol at surface pressures pi > 20 mN/m as well as to monolayers of the unsaturated 1-palmitoyl-2-oleoylphosphatidylcholine. In the same surface pressure range, the addition of phloretin, which decreases Psi, reduced the penetration of pregnanolone into the monolayers. These results suggest that membrane partitioning of pregnanolone is influenced by the spacing of the phosphocholine head groups as well as by membrane dipole potential. The latter can be explained in terms of electrostatic dipole-dipole interactions between pregnanolone and the membrane lipids with their associated water molecules. Considering the universal nature of these interactions, they are likely to affect membrane partitioning of most, if not all, weakly amphiphilic drugs.

The phytoalexin-inducible multidrug efflux pump AcrAB contributes to virulence in the fire blight pathogen, Erwinia amylovora.

The enterobacterium Erwinia amylovora causes fire blight on members of the family Rosaceae, with economic importance on apple and pear. During pathogenesis, the bacterium is exposed to a variety of plant-borne antimicrobial compounds. In plants of Rosaceae, many constitutively synthesized isoflavonoids affecting microorganisms were identified. Bacterial multidrug efflux transporters which mediate resistance toward structurally unrelated compounds might confer tolerance to these phytoalexins. To prove this hypothesis, we cloned the acrAB locus from E. amylovora encoding a resistance nodulation division-type transport system. In Escherichia coli, AcrAB of E. amylovora conferred resistance to hydrophobic and amphiphilic toxins. An acrB-deficient E. amylovora mutant was impaired in virulence on apple rootstock MM 106. Furthermore, it was susceptible toward extracts of leaves of MM 106 as well as to the apple phytoalexins phloretin, naringenin, quercetin, and (+)-catechin. The expression of acrAB was determined using the promoterless reporter gene egfp. The acrAB operon was up-regulated in vitro by the addition of phloretin and naringenin. The promoter activity of acrR, encoding a regulatory protein involved in acrAB expression, was increased by naringenin. In planta, an induction of acrAB was proved by confocal laser scanning microscopy. Our results strongly suggest that the AcrAB transport system plays an important role as a protein complex required for virulence of E. amylovora in resistance toward apple phytoalexins and that it is required for successful colonization of a host plant.

Membrane Dipole Potential Modulates Proton Conductance through Gramicidin Channel: Movement of Negative Ionic Defects inside the Channel

The effect of membrane dipole potential on gramicidin channel activity in bilayer lipid membranes (BLMs) was studied. Remarkably, it appeared that proton conductance of gramicidin A (gA) channels responded to modulation of the dipole potential oppositely as compared with gA alkali metal cation conductance. In particular, the addition of phloretin, known to reduce the membrane dipole potential, resulted in a decrease in gA proton conductance, on one hand, and an increase in gA alkali metal conductance, on the other hand, whereas 6-ketocholestanol, the agent raising the membrane dipole potential, provoked an increase in gA proton conductance as opposed to a decrease in the alkali metal cation conductance. The peculiarity of the 6-ketocholestanol effect consisted in its dependence on the H + concentration. The experiments with the impermeant dipolar compound, phloridzin, showed that the response of proton transport through gramicidin channels to varying the membrane dipole potential did not change qualitatively if the dipole potential of only one monolayer or both monolayers of the BLM was altered. In contrast to gA proton conductance, the single-channel lifetime changed similarly with varying the membrane dipole potential, regardless of the kind of permeant cations (protons or potassium ions). The results of this study could be tentatively accounted for by an assumption that one of the rate-limiting steps of proton conduction through gramicidin channels represents, in fact, movement of negatively charged species (negative ionic defects) across a membrane.

Transport of ascorbate into protoplasts ofNicotiana tabacum Bright Yellow-2 cell line

The uptake of ascorbate into protoplasts isolated from aNicotiana tabacum Bright Yellow-2 (BY-2) cell suspension culture was investigated. Addition of14C-labelled ascorbate to freshly isolated protoplasts resulted in a time- and substrate-dependent association of radioactive molecules with the protoplasts. The kinetic characterisation of this presumptive uptake revealed kinetics of Michaelis-Menten type with an apparent maximal uptake activity of 24 pmol/min·106 protoplasts and an apparent affinity constant of 139 μM. The amount of ascorbate molecules transported intoN. tabacum protoplasts decreased when nonlabelled dehydroascorbate or iso-ascorbate were added but was not affected by addition of 5,6-o-cyclohexylidene ascorbate or ascorbate-2-sulfate. These data indicate a carrier-mediated uptake of ascorbate into the protoplasts that shows a high structural specificity. To investigate which redox status of ascorbate is preferentially taken up by theN. tabacum protoplasts, transport was tested in the presence of various compounds that can affect the redox status of ascorbate. Testing uptake in the presence of a reductant, dithiothreitol, resulted in a significant and concentration-dependent inhibition of the amount of ascorbate molecules transported into the protoplasts. On the other hand, ascorbate uptake was significantly stimulated in the presence of the enzyme ascorbate oxidase. Ferricyanide did not affect ascorbate transport. Inhibition studies revealed that ascorbate uptake in the protoplasts is sensitive to addition of sulfhydryl reagents N-ethyl maleimide andp-chloro-mercuribenzenesulfonic acid and to a disruption of the proton gradient by the protonophore carbonylcyanide-3-chlorophenylhydrazone. The uptake of ascorbate was also inhibited by addition of cytochalasin B but not sensitive to addition of phloretin or sulfinpyrazone. Taken together these data indicate the presence of an ascorbate transport system in the plasma membrane ofN. tabacum protoplasts and suggest dehydroascorbate as the preferentially transported redox species. The putative presence of different carriers for reduced and oxidised ascorbate in the plasma membrane is discussed.

Thyroid hormones in tissues from fetal and adult rats.

Concentrations of T4 and T3 were recently measured in rat fetal tissues, and the reported values were found to be more than 10-fold higher than those found by us. The differences have been explained by the assumption that previous analytical procedures, neither avoid deiodination during autopsy of the animals or during extraction and purification, because phloretin [(3'),4',4,6-(tetra)trihydroxyaurone], a potent inhibitor of 5'-iodothyronine deiodinase activity in vitro, had not been used to prevent such problems. We here show that perfusion with phloretin during autopsy does not affect 5'-iodothyronine activity or T4 and T3 concentrations in liver, kidney, or brain. Evidence is also provided that the addition of phloretin during the homogenization process is superfluous, as the use of 80% ethanol and 0.02 M NaOH for this step results in undetectable deiodinase activity. Data are presented showing that during the final sample drying, no losses or degradation of T4 and T3 occur, confirming the adequacy of the individual recovery corrections using radiolabeled iodothyronines as internal tracers. We also present quantitative information on the intralaboratory variability of the T4 and T3 concentrations found in tissues from normal fetuses and their mothers as well as in adult males and nonpregnant females. Results are comparable to those obtained by others using entirely different analytical procedures.

Interaction of electric dipoles with phospholipid head groups. A deuterium and phosphorus-31 NMR study of phloretin and phloretin analogs in phosphatidylcholine membranes

Phloretin, 4-hydroxyvalerophenone, and 2-hydroxy-omega-phenylpropiophenone are lipophilic dipolar substances that modify ionic conductances of bilayer membranes. The structural changes at the level of the head groups and the hydrocarbon chains as induced by the incorporation of phloretin and its analogues were investigated with deuterium and phosphorus nuclear magnetic resonance. Membranes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) were selectively deuterated at the choline head group and at the hydrocarbon chains, and 2H and 31P NMR spectra were recorded with varying concentrations of dipolar agents. Incorporation of phloretin leaves the bilayer structure intact, induces only a small disordering of the hydrocarbon chains, and has no significant effect on the head-group dynamics. On the other hand, quite distinct structural changes are observed for the phosphocholine head group. While the -P-N+ dipole is oriented approximately parallel to the membrane surface for pure POPC bilayers, addition of phloretin, and to a lesser extent 4-hydroxyvalerophenone and 2-hydroxy-omega-phenylpropiophenone, rotates the N+ end of the -P-N+ dipole closer to the hydrocarbon layer. The resulting normal component of the -P-N+ dipole partly compensates the electric field of the dipolar agents. In addition to this structural change, phloretin also modifies the hydration layer at the lipid-water interface. Much less 2H2O is adsorbed to the membrane surface when the bilayer contains phloretin, 4-hydroxyvalerophenone, or 2-hydroxy-omega-phenylpropiophenone. Moreover, a rather large change in the residual phosphorus chemical shielding anisotropy argues in favor of hydrogen-bond formation between the phosphate segment and the phloretin hydroxyl groups.