Concentration-dependent Way Research Articles

Search for serum protein-binding disaccharides and disaccharide-binding serum proteins by affinity capillary electrophoresis.

The potential use of affinity capillary electrophoresis in a microscale search for mutually interacting substances in biological fluid is demonstrated. Some disaccharides, especially gentiobiose (Gen), derivatized with 1-phenyl-3-methyl-5-pyrazolone, caused peak retardation when electrophoresed in a neutral running buffer, containing human serum. Gen, the most significantly retarded disaccharide, was converted to its negatively charged bis-mercaptoethanesulfonate derivative (MerESGen), and a serum sample was analyzed in a neutral buffer containing the derivatized disaccharide. Two peaks, belonging to the beta-globulin fraction, were found to be remarkably retarded in the buffer containing MerES-Gen in a concentration-dependent way. These findings prove an interaction between disaccharides and serum proteins.

Metmyoglobin and methemoglobin catalyze the isomerization of peroxynitrite to nitrate.

Hemoproteins, in particular, myoglobin and hemoglobin, are among the major targets of peroxynitrite in vivo. The oxygenated forms of these proteins are oxidized by peroxynitrite to their corresponding iron(iii) forms (metMb and metHb). This reaction has previously been shown to proceed via the corresponding oxoiron(iv) forms of the proteins. In this paper, we have conclusively shown that metMb and metHb catalyze the isomerization of peroxynitrite to nitrate. The catalytic rate constants were determined by stopped-flow spectroscopy in the presence and absence of 1.2 mM CO(2) at 20 and 37 degrees C. The values obtained for metMb and metHb, with no added CO(2) at pH 7.0 and 20 degrees C, are (7.7 +/- 0.1) x 10(4) and (3.9 +/- 0.2) x 10(4) M(-1) s(-1), respectively. The pH-dependence of the catalytic rate constants indicates that HOONO is the species that reacts with the iron(iii) center of the proteins. In the presence of 1.2 mM CO(2), metMb and metHb also accelerate the decay of peroxynitrite in a concentration-dependent way. However, experiments carried out at pH 8.3 in the presence of 10 mM CO(2) suggest that ONOOCO(2)(-), the species generated from the reaction of ONOO(-) with CO(2), does not react with the iron(iii) center of Mb and Hb. Finally, we showed that different forms of Mb and Hb protect free tyrosine from peroxynitrite-mediated nitration. The order of efficiency is metMbCN < apoMb < metHb < metMb < ferrylMb < oxyHb < deoxyHb < oxyMb. Taken together, our data show that myoglobin is always a better scavenger than hemoglobin. Moreover, the globin offers very little protection, as the heme-free (apoMb) and heme-blocked (metMbCN) forms only partly prevent nitration of free tyrosine.

Effects of carbonate and phosphate concentrations on hydrogen production using anaerobic sewage sludge microflora

Anaerobic sewage sludge acclimated with sucrose in a completely stirred tank reactor operating at 35°C was used as the seed (dominated by Clostridia species) in a batch experiment to investigate the concentration effects of carbonate and phosphate on biological hydrogen production. The Taguchi orthogonal array was used in the experimental design and a confirmation experiment was conducted. Experimental results indicated that the hydrogen production ability of the anaerobic microflora in sewage sludge was affected by NH 4HCO 3, Na 2HPO 4 and Na 2CO 3 with Na 2HPO 4 being the most significant supplement. Na 2HPO 4 affected the hydrogen production in a concentration-dependent way with 600 mg/l the optimal concentration. The hydrogen production rate was 1.9 times enhanced, compared with an acidogenic nutrient formulation (Proc. Soc. Civ. Engrs. 325 (1982) 61), when a proper carbonate and phosphate concentration formulation was used. The enhancement was due to a shortening of the microflora lag-time. Based on the enhancement abilities on hydrogen content and hydrogen production rate, adding phosphate instead of carbonate as a buffering capacity supplement might be a useful strategy for optimal hydrogen production operations with anaerobic sewage cultures.

Cyclooctadepsipeptides—an anthelmintically active class of compounds exhibiting a novel mode of action

There are three major classes of anthelmintics for veterinary use: the benzimidazoles/prebenzimidazoles, the tetrahydropyrimidines/imidazothiazoles, and the macrocyclic lactones. In nematodes, there are five targets for the existing anthelmintics: the nicotinergic acetylcholine receptor which is the target of tetrahydropyrimidines/imidazothiazoles and indirectly that of the acetylcholineesterase inhibitors; the GABA receptor which is the target of piperazine, the glutamate-gated chloride channel as the target of the macrocyclic lactones, and ß-tubulin as the target of prebenzimidazoles/benzimidazoles. All these anthelmintics are now in serious danger because of the worldwide spread of resistant nematodes in sheep, cattle, horses and pigs. The class of cyclooctadepsipeptides has entered the scene of anthelmintic research in the early 1990s. PF1022A, the first anthelmintically active member, is a natural compound from the fungus Mycelia sterilia that belongs to the microflora of the leaves of the Camellia japonica. PF1022A contains 4 N-Methyl- l-leucines, 2 d-lactic acids and 2- d-phenyllactic acids arranged as a cyclic octadepsipeptide with an alternating l- d- l-configuration. Emodepside is a semisynthetic derivative of PF1022A with a morpholine ring at each of the two d-phenyllactic acids in para position. The anthelmintic activity is directed against gastrointestinal nematodes in chicken, mice, rats, meriones, dogs, cats, sheep, cattle and horses. Moreover, emodepside is active against Trichinella spiralis larvae in muscles, microfilariae and preadult filariae and Dictyocaulus viviparus. PF1022A and emodepside are fully effective against benzimidazole-, levamisole or ivermectin-resistant nematodes in sheep and cattle. In Ascaris suum both cyclooctadepsipeptides lead to paralysis indicating a neuropharmacological action of these compounds. Using a PF1022A-ligand immunoscreening of a cDNA library from Haemonchus contortus a cDNA clone of 3569 base pairs could be identified. This clone codes for a novel 110 kDa heptahelical transmembrane receptor, named HC110R. Database- and phylogenetic analysis reveals that this receptor is a homolog to B0457.1 from Caenorhabditis elegans and has significant similarity to latrophilins from human, cattle and rat. HC110R is located in the plasma membrane and in lysosomes and endosomes. α-Latrotoxin, the poison of the black widow spider, binds at a 54 kDa aminoterminal fragment of HC110R. After binding a Ca 2+-influx into HEK293 cells is induced which can be blocked by EGTA, Cd 2+ or nifedipin. PF1022A or emodepside also bind to this 54 kDa aminoterminal region of HC110R and interact with the functional responses of α-latrotoxin. In C. elegans antibodies against the C-or N-terminus of HC110R bind to the B0457.1 protein located in the pharynx. Electrophysiological studies reveal that emodepside inhibits pharyngeal pumping of the nematodes in a concentration dependent way with an IC 50 value of about 4 nM. Thus, it is tempting to speculate that emodepside exerts its action on nematodes via a latrophilin-like receptor which might have an important regulatory function on pharyngeal pumping.

The inhibitory effect of aluminium on the (Na +/K +)ATPase activity of rat brain cortex synaptosomes

The effect of AlCl 3 on the (Na +/K +)ATPase activity of freeze–thawed synaptosomes, isolated from rat brain cortex, has been studied. The AlCl 3 action on the enzyme hydrolytic activity was examined using in vitro and in vivo approaches. Following exposure to AlCl 3 using both in vitro (synaptosomes incubated in the presence of AlCl 3 for 5 min) and in vivo (synaptosomes isolated from rats that received 0.03 g AlCl 3/day for 4 months) approaches, the (Na +/K +)ATPase activity was inhibited in a concentration-dependent way. The maximal inhibitory effect (∼60%) was observed in the presence of a AlCl 3 concentration >75 μM and at non-limiting ATP concentrations. Conversely, AlCl 3 did not inhibit the enzyme activity when UTP was used as substrate instead of ATP. Analysis of the substrate dependence of membrane-bound (Na +/K +)ATPase by a computer simulation model suggests that the AlCl 3-induced inhibitory effect is characterised by a reduction of the rate-limiting step velocity of the reaction cycle. Moreover, it seems that aluminium can induce impairment of the interprotomeric interaction within the oligomeric ensemble of membrane-bound (Na +/K +)ATPase. In fact, this effect was accompanied by a slight, but significant, decrease of readily accessible SH groups, which are involved in the maintenance of the membrane-bound (Na +/K +)ATPase oligomeric structure. In conclusion, during exposure to aluminium, reduction of the activation of membrane-bound (Na +/K +)ATPase by high ATP concentrations occurs, which results in a partial inhibition of the enzyme.

N-acetylcysteine reduces chemokine release via inhibition of p38 MAPK in human airway smooth muscle cells.

Reactive oxygen species are involved in the activation of several mitogen-activated protein kinases (MAPKs), key-players in the production of several cytokines. Therefore the current study investigated whether N-acetylcysteine (NAC), an antioxidative agent, inhibits the interleukin (IL)-1beta-induced expression and production of eotaxin and monocyte chemotactic protein (MCP)-1 in human airway smooth muscle cells (HASMC). NAC (10 mM) decreased the expression of eotaxin and MCP-1, by 46 +/- 11% (n=7) and 87 +/- 4% (n=6), respectively; the eotaxin release was inhibited by 75 +/- 5% (n=7), whereas the MCP-1 release was decreased by 69 +/- 41% (n=10). NAC (1 mM) also decreased the IL-1beta-induced activation of p38 MAPK. Compared with unstimulated cells, a four-fold increase in 8-isoprostane production in IL-1beta-stimulated HASMC was observed, which could be inhibited by NAC in a concentration-dependent way, with a maximum inhibition of 39 +/- 12%, with 1 mM NAC. The present study demonstrated that N-acetylcysteine inhibits the interleukin-1beta-induced eotaxin and monocyte chemotactic protein 1 expression and production due to a decreased activation of p38 mitogen-activated protein kinase. This study has also shown that N-acetylcysteine decreases the interleukin-1beta-induced production of reactive oxygen species, as suggested by a reduction in the 8-isoprostane production.

Selective inhibition of gamma-aminobutyric acid type A receptors in human IMR-32 cells by low concentrations of toluene.

Effects of the neurotoxic organic solvent toluene on human neuronal nicotinic acetylcholine (nACh) and gamma-aminobutyric acid type A (GABA(A)) neurotransmitter receptors were investigated in whole-cell voltage-clamped IMR-32 neuroblastoma cells. Ion currents evoked by near maximum effective concentrations of 1 mM acetylcholine (ACh) and 1 mM gamma-aminobutyric acid (GABA) are inhibited by toluene in a concentration-dependent way. Concentration-effect curves of toluene yield IC(50) values of 276+/-26 and 39+/-6 microM and slope factors of 1.4+/-0.2 and 0.8+/-0.1 for inhibition of the ACh- and GABA-induced ion currents, respectively. The results demonstrate the selective inhibition of human GABA(A) receptors by toluene at concentrations comparable with brain concentrations associated with occupational exposure.

Losartan’s molecular basis of interaction with membranes and AT 1 receptor

Physicochemical methods were used to study the thermal and dynamic changes caused by losartan in the membrane bilayers. In addition, molecular modeling was implemented to explore its topography both in membranes and AT 1 receptor. Its incorporation resulted in the modification of thermal profile of dipalmitoyl phosphatidylcholine (DPPC) bilayers in a concentration dependent way up to 20 mol% as it is depicted from the combination of differential scanning calorimetry (DSC) and MAS data. In particular, the presence of losartan caused lowering of the phase transition temperature and abolishment of the pretransition. T 1 experiments revealed the location of the drug into the membrane bilayers. The use of a combination of biophysical methods along with docking experiments brought out a possible two-step mechanism which involves incorporation of losartan at the interface of membrane bilayers and diffusion in the upper parts of AT 1 receptor helices IV–VII.

Angiotensin II phosphorylation of extracellular signal-regulated kinases in rat anterior pituitary cells.

We studied the effects of ANG II on extracellular signal-regulated kinase (ERK)1/2 phosphorylation in rat pituitary cells. ANG II increased ERK phosphorylation in a time- and concentration-dependent way. Maximum effect was obtained at 5 min at a concentration of 10-100 nM. The effect of 100 nM ANG II was blocked by the AT1 antagonist DUP-753, by the phospholipase C (PLC) inhibitor U-73122, and by the MAPK kinase (MEK) antagonist PD-98059. The ANG II-induced increase in phosphorylated (p)ERK was insensitive to pertussis toxin blockade and PKC depletion or inhibition. The effect was also abrogated by chelating intracellular calcium with BAPTA-AM or TMB-8 by depleting intracellular calcium stores with a 30-min pretreatment with EGTA and by pretreatment with herbimycin A and PP1, two c-Src tyrosine kinase inhibitors. It was attenuated by AG-1478, an inhibitor of epidermal growth factor receptor (EGFR) activation. Therefore, in the rat pituitary, the increase of pERK is a Gq- and PLC-dependent process, which involves an increase in intracellular calcium and activation of a c-Src tyrosine kinase, transactivation of the EGFR, and the activation of MEK. Finally, the response of ERK activation by ANG II is altered in hyperplastic pituitary cells, in which calcium mobilization evoked by ANG II is also modified.

Inhibition of various glutathione S-transferase isoenzymes by RRR-α-tocopherol

The activity of human cytosolic glutathione S-transferases (GSTs) can positively or negatively be changed by various compounds. It is for instance known that RRR-α-tocopherol inhibits GST P1-1 [Haaften van R.I.M. et al. (2001) Alpha-tocopherol inhibits human glutathione S-transferase pi. BBRC 280, 631–633]. The effect of RRR-α-tocopherol on the other isoenzymes of GST in purified forms of the isoenzymes and in human liver cytosol (GST M and GST A) and lysate of human erythrocytes (GST P) is studied. It is found that all isoenzymes (purified enzymes and enzymes present in homogenates) are inhibited, in a concentration-dependent way, by RRR-α-tocopherol. GST P is in both cases inhibited with the highest potency compared to the other isoenzymes. It also appeared that the purified GST P1-1 isoenzyme is non-competitively inhibited by RRR-α-tocopherol. The IC 50 values of RRR-α-tocopherol for the purified isoenzymes of GST are much lower compared to the IC 50 values for human lysate and human liver cytosol. This is probably due to binding of RRR-α-tocopherol to proteins, e.g. albumin and hemoglobin, with higher affinity than to GST; so more RRR–α-tocopherol is needed to inhibit the enzyme. However, the inhibition of GSTs by RRR–α-tocopherol can still be of physiological relevance, because due to dermal application of cosmetic products very high concentrations vitamin E can be reached in the skin, where GST P1-1 is present. RRR-α-tocopherol might also be a good lead compound for the development of a new class of inhibitors of GST that can be used as adjuvant in cancer therapy.

Smooth muscle relaxant effect of kaurenoic acid, a diterpene from Copaifera langsdorffii on rat uterus in vitro.

The effect of kaurenoic acid, a diterpene isolated from the oleo-resin of the popular medicinal plant Copaifera langsdorffii (Leguminaceae), was analysed on rat uterine muscle responsiveness to various drugs in vitro. Cumulative concentration-response curves to acetylcholine and oxytocin were obtained before and after incubation of uterine segments with up to 160 microm of kaurenoic acid. The maximal contractile response (E(max)) evoked by these agonists was inhibited by kaurenoic acid in a concentration-related manner; at 160 microm, kaurenoic acid depressed the E(max) of oxytocin and acetylcholine by 83% and 91%, respectively. The relaxation caused by kaurenoic acid on oxytocin-induced contraction was unaffected in the presence of tetraethyl ammonium, a compound that blocks the calcium activated potassium channels. It was partially reversed by glibenclamide (10(-5) m), an ATP-sensitive potassium channel blocker. Also, kaurenoic acid at 160 microm concentration was found to inhibit significantly the CaCl(2)-evoked contractile responses in a medium of high potassium and zero calcium. Furthermore, kaurenoic acid was found to relax the sustained tonic contraction induced by acetylcholine, oxytocin, BaCl(2) and KCl in a concentration-dependent way. However, KCl-induced tonic contraction was only weakly inhibited by kaurenoic acid. These data indicate that the diterpene, kaurenoic acid, exerts a uterine relaxant effect acting principally through calcium blockade and in part, by the opening of ATP-sensitive potassium channels.

Conformationally constrained butyrophenones as new pharmacological tools to study 5-HT 2A and 5-HT 2C receptor behaviours

This study presents new pharmacological and molecular modelling studies on a recently described series of conformationally constrained butyrophenones. Alignment-free three-dimensional quantitative structure-activity relationship models developed on the basis of GRid Independent descriptors and partial least squares regression analysis, allow feasible predictions of activity of new compounds and reveal structural requirements for optimal affinity, particularly in the case of the 5-HT 2A receptor. The requirements for the 5-HT 2A affinity consist in a precise distance between hydrogen bond donor (protonated amino group) and hydrogen bond acceptor groups, as well as an optimal distance between the protonated amino group and the farthest extreme of the compounds. Another significant result has been the characterisation of two structurally similar compounds as interesting pharmacological tools (1-[(4-Oxo-4,5,6,7-tetrahydrobenzo[b]furan-5-yl)ethyl]-4-(6-fluorobenzisoxazol-3-yl)piperidine and 1-[(4-Oxo-4,5,6,7-tetrahydrobenzo[b]furan-6-yl)methyl]-4-(6-fluorobenzisoxazol-3-yl)piperidine). In spite of their structural similarity, the first compound shows clearly higher affinity for the 5-HT 2C receptor (about 100 fold) and higher Meltzer ratio (1.17 vs. 0.99) than the second. Moreover, the first compound inhibits arachidonic acid release in a biphasic concentration-dependent way in functional experiments at the 5-HT 2A receptor and it acts as inverse agonist at the 5-HT 2C receptor, behaviours that are not shown by the second compound.

Concentration-dependent actions of glial chondroitin sulfate on the neuritic growth of midbrain neurons

Astrocytes located in two distinct regions of midbrain differ in their neuritic growth support abilities. Midbrain neurons cultured onto astrocyte monolayers from the lateral (L) region develop long and branched neurites while neurons cultured onto astrocyte monolayers from the medial (M) region develop short or no neurites. The extracellular matrix of these astrocytes has an important role in promoting or inhibiting the growth of these neurons. Differences on the compartmental distribution, as well as on the concentration of GAGs of L and M astrocytes, may be related to their differential capacity of supporting neuritic growth. Indeed, enzymatic digestion of heparan sulfate (HS) and chondroitin sulfate (CS) chains also pointed to an important function for GAGs on axon navigation. In order to better characterize the role of CS on the growth of midbrain neurites, we treated L and M astrocyte monolayers with 1 mM of β-d-xyloside. Under these conditions, astrocytes oversynthesized and secreted CS protein-free chains to the culture medium. M astrocytes had a significant reduction in their neuritic growth-inhibiting ability after xyloside treatment, suggesting a promoting role for soluble CS in neuritic growth. Chondroitin 4-sulfate (CS-4) added in different concentrations to M astrocyte cultures turned this glia into a permissive substrate, acting in a linear way as far as the largest neurite was concerned. However, a U-shaped dose–effect curve on neurite growth resulted from the similar treatment of L astrocytes. These results suggest that glial CS-4 could be involved in the neurite growth modulating properties of midbrain neurons in a complex concentration-dependent way.

Effects of Bauhinia megalandra aqueous leaf extract on intestinal glucose absorption and uptake by enterocyte brush border membrane vesicles

Aqueous extract of Bauhinia megalandra leaves was able to inhibit the intestinal glucose absorption in a concentration-dependent way and additive to phlorizine. Moreover, B. megalandra leaf extract drastically reduced the 14C-glucose uptake by enterocyte brush border membrane vesicles. The B. megalandra leaf extract administrated orally, simultaneously with glucose improved the glucose tolerance with a significant reduction of the 30-min peak. The extract did not have an effect on the glucose tolerance when glucose was administrated subcutaneously.

Concentration dependent mitochondrial effect of amiodarone

Although, the antiarrhythmic effect of amiodarone is well characterized, its effect on post-ischemic heart and cardiomyocytes, as well as the mechanism of its toxicity on extracardiac tissues is still poorly understood. In this study, we analyzed energy metabolism in situ during ischemia-reperfusion in Langendorff-perfused heart model by measuring the high-energy phosphate metabolites using 31 P NMR spectroscopy. The toxicity of amiodarone on cardiomyocytes and cell lines of extracardiac origin, as well as direct effect of the drug on mitochondrial functions in isolated mitochondria was also analyzed. Amiodarone, when was present at low concentrations and predominantly in membrane bound form, protected heart and mitochondrial energy metabolism from ischemia-reperfusion-induced damages in Langendorff-perfused heart model. Toxicity of the drug was significantly higher on hepatocytes and pancreatic cells than on cardiomyocytes. In isolated mitochondria, amiodarone did not induce reactive oxygen species formation, while it affected mitochondrial permeability transition in a concentration dependent way. Up to the concentration of 10 μM, the drug considerably inhibited Ca 2+-induced permeability transition, while at higher concentrations it induced a cyclosporin A independent permeability transition of its own. At concentrations where it inhibited the Ca 2+-induced permeability transition ( ic 50=3.9±0.8 μM), it did not affect, between 6 and 30 μM it uncoupled, while, at higher concentrations it inhibited the respiratory chain. Thus, the concentration dependent nature of amiodarone’s effect on permeability transition together with the different sensitivities of the tissues toward amiodarone can be involved in the beneficial cardiac and the simultaneous toxic extracardiac effects of the drug.

Vitamin E and thyroid disease: a potential link that kindles hope.

Vitamin E was discovered in 1922 and is the best known lipid-soluble antioxidant [1]. Natural vitamin E represents a complex of 8 tocopherol and tocotrienol isomers of which α-tocopherol (atoc) is predominant in most species and significantly more potent than any other naturally occurring tocopherol [2]. Vitamin E is considered the prototype of phenolic based chain-breaking antioxidants, its primary role consisting in preventing free radical initiated lipid peroxidation damage and thus in protecting the integrity of tissues [3]. Vitamin E is mainly distributed in adipose tissue and in the subcellular membrane fractions, the intracellular transport and release of α-tocopherol into the plasma involves via a Golgi-dependent pathway a specific protein, the α-tocopherol transfer protein [4]. Oxidative stress is defined as the overproduction of oxygen free radicals (OFR) that can no longer be compensated by the antioxidant defence system [5]. Oxidative stress appears to play a pivotal role in various diseases such as atherosclerosis, cancer and Alzheimer’s and is associated with diminution of antioxidant molecules. α-tocopherol decreases, in a specific and concentration dependent way, the proliferation of vascular smooth cells by interacting with the protein kinase C (PKC) [6]. The interaction with the cytosolic PKC results in inhibition of its membrane translocation and activation [7]. The PKC specific activity is diminished due to dephosphorylation following the activation of protein phosphatase 2A1 activity by α-tocopherol at a cellular level [8]. Thus, the diminution of a-toc in smooth muscle cells may consequently augment the PKC activity and lead to an increase in cell proliferation also by a non-antioxidant mechanism [8].

INDUCTION OF CYP1A BY THE N-IMIDAZOLE DERIVATIVE, 1-BENZYLIMIDAZOLE

Xenobiotics can induce cytochrome P4501A (CYP1A) by ligand binding to the aryl hydrocarbon receptor (AhR). Typical AhR ligands are polycyclic aromatic compounds with planar molecular conformation. The present work investigated the ability of the N-imidazole derivative, 1-benzylimidazole (BIM), to induce CYP1A in rainbow trout hepatocytes. Benzylimidazole increased hepatocellular CYP1A catalytic activity (determined as 7-ethoxyresorufin-O-deethylase [EROD] activity) and CYP1A mRNA in a concentration-dependent way. Computational studies on the molecular structure of BIM indicated that the energetically most stable BIM conformer has the imidazole ring and the phenyl ring in different planes, i.e., does not take a planar conformation. This property of BIM does not agree with the structural requirements of a typical AhR ligand. In line with this observation, we found that the AhR antagonist, alpha-naphthoflavone (alphaNF), was not able to inhibit BIM induction of EROD activity and CYP1A mRNA, although it inhibited the induction of CYP1A by the prototypic AhR ligand, beta-naphthoflavone (betaNF). The results suggest that transcriptional activation of CYP1A by the N-imidazole derivative, BIM, is not mediated through direct ligand binding to the AhR.

Hydrogen peroxide- and cell-density-regulated expression of NADH-cytochrome b5 reductase in HeLa cells.

Environmental conditions regulate the expression of different antioxidant enzymes in cell culture. We have studied the effect of cell density and hydrogen peroxide on the expression of NADH-cytochrome b5 reductase in HeLa cells. Polypeptide levels of the NADH-cytochrome b5 reductase increased about three fold in confluent HeLa cells compared to sparse cells. Addition of H2O2 to HeLa cells altered expression levels of the NADH-cytochrome b5 reducatase in a concentration-dependent way, being sparse cells more sensitive to H2O2 addition than confluent cells. The presence of pyruvate, a H2O2 scavenger, produced a significant increment (200%) in the levels of NADH-cytochrome b5 reductase in sparse cells, but less increase (25%) in confluent cells, suggesting that generation of endogenous H2O2 could repress NADH-cytochrome b5 reductase expression, particularly in sparse cultures. Accordingly, confluent HeLa cells showed significantly lower levels of reactive oxygen species than cells in sparse cultures. Addition of tert-butylhydroquinone, a compound which generates reactive oxygen species through redox cycling, also reduced expression of the NADH-cytochrome b5 reductase. Increments in several antioxidant enzymes taking place during confluency could participate in the increase of NADH-cytochrome b5 reductase expression by reducing reactive oxygen species levels in cells. Overall, our results support that acute oxidative stress caused by H2O2 inhibits the expression levels of NADH-cytochrome b5 reductase, most likely due to inhibition of SP1 transcriptional activity. On the other hand, adaptation to H2O2 involved increased expression of the cytochrome b5 reductase, supporting the existence of additional regulatory mechanisms.

Influence of isolation procedure, extracellular matrix and dexamethasone on the regulation of membrane transporters gene expression in rat hepatocytes

The influence of the isolation procedure of hepatocytes, extracellular matrix (ECM) configuration and incubation medium supplementation by dexamethasone (DEX) on the cell morphology and on the gene expression of membrane transporters was examined in rat hepatocytes. The mRNA levels were determined using oligonucleotide microarrays, in liver, in suspension and in primary culture in monolayer (CPC), and in collagen gels sandwich (SPC) in absence and presence of DEX (100 and 1000 nM). The results indicated pronounced morphological differences between CPC and SPC in response to DEX demonstrating that the hepatocytes re-formed, as in vivo, multicellular arrays with extensive bile canalicular network only in SPC in presence of DEX. The mRNA levels of membrane transporters were not affected significantly during isolation procedure. However, plating hepatocytes in CPC resulted in a decrease of major basolateral transporters mRNA level whereas mRNA levels of mdr1b and mrp3 were increased (>100-fold). Similar observations were made in SPC in the absence of DEX demonstrating that the ECM configuration alone did not play a critical role in the regulation of membrane transporters. However, adding DEX to the incubation medium in SPC resulted in an up-regulation of mdr2, oatp2 and mrp2 in a concentration-dependent way for the two latter genes, whereas mdr1b and mrp3 expression were maintained to their baseline liver levels. These data suggested therefore that the combination of ECM and DEX supplementation is essential for the formation of the bile canalicular network and is a determinant factor in the regulation of membrane transporters in cultured rat hepatocytes.

Adduct of 2-[ 18F]FDG and 2-nitroimidazole as a putative radiotracer for the detection of hypoxia with PET: synthesis, in vitro- and in vivo-characterization

A new sugar-coupled 2-nitroimidazole derivative ([ 18F] 1 ) has been prepared in good radiochemical yields starting from peracetylated 2-[ 18F]FDG obtained from an automated 2-[ 18F]FDG production module. The corresponding glucose derivative 2 has proved to be able to inhibit 2-[ 18F]FDG uptake into tumor cells in a concentration dependent way. However, [ 18F] 1 failed to show a retention in hypoxic tumor tissue thus excluding itself from further investigations.