Concentration-dependent Way Research Articles

Photostabilization Effect of Quercetin on the UV Filter Combination, Butyl Methoxydibenzoylmethane–Octyl Methoxycinnamate

The aim of the study was to investigate the effect of the natural antioxidant quercetin on the photostability of the most widely used combination of UVA (320-400 nm) and UVB (290-320 nm) filters, respectively butyl methoxydibenzoylmethane (BMDBM) and octyl methoxycinnamate (OMC). In order to reproduce the conditions prevalent in commercial sunscreen products, the stabilizing efficacy of quercetin was evaluated in model creams containing BMDBM (3%, wt/wt) together with OMC (4%, wt/wt) and exposed to a solar simulator at an irradiance corresponding to natural sunlight. Quercetin was found to enhance the photostability of the two UV filters in a concentration-dependent way. Addition of quercetin to the sunscreen formulation significantly reduced the photodegradation of BMDBM and OMC from 40.3 +/- 2.4 to 27.7 +/- 2.6% and from 51.3 +/- 2.1 to 42.2 +/- 2.0%, respectively. Moreover, comparative photodegradation studies demonstrated that quercetin was much more effective and at a lower concentration than commonly used stabilizer (octocrylene) and antioxidants (vitamin E, butylated hydroxyanisole). In vitro determination of the UVB and UVA protection parameters showed that the quercetin-based formulation fulfilled the official requirements on sunscreen products. Because of its photostabilizing and multiple antioxidant properties, quercetin represents a useful additive for the formulation of effective broad-spectrum sunscreens containing BMDBM and OMC.

The effect of immobilization of thrombin inhibitors onto self-assembled monolayers on the adsorption and activity of thrombin

Thrombus formation is the major problem associated with biomaterials for blood contact medical devices. The immobilization of inhibitors to thrombin, a serine protease that plays a central role on the coagulation system, on the surface of biomaterials should be a good strategy to avoid blood clotting and increase their hemocompatibility. The aim of this work is the design of a nanostructured surface with capacity to adsorb and inactivate thrombin. The pentapeptide sequence d-Phenylalanine–Proline–Arginine–Proline–Glycine (fPRPG), that was described as a thrombin inhibitor, was immobilized onto tetra(ethylene glycol) terminated self-assembled monolayers (EG4-SAMs). Surface containing different amounts of fPRPG were prepared using different concentrations of N,N′-Carbonyldiimidazole (CDI) during immobilization. The efficiency of fPRPG immobilization was followed using ellipsometry, contact angle measurements, Infrared reflection absorption spectroscopy (IRRAS) and X-ray photoelectron spectroscopy (XPS). Thrombin adsorption was quantified using radiolabelled thrombin and its activity in solution and after adsorption on the developed surfaces was assessed using a chromogenic assay. It was found that, although the immobilization of fPRPG on to EG4-SAMs does not increase its selectivity to thrombin, the activity of the adsorbed thrombin was inhibited in a peptide concentration dependent way. We concluded that SAMs with fPRPG immobilized in high amounts can be used as thrombin-inhibitor surfaces, which is a good step on the development of new surfaces for blood contact devices.

Interactions at Human ether-à-go-go–Related Gene Channels

Several noncardiovascular drugs have the potential to induce Torsades de Pointes cardiac arrhythmias via blockade of the rapid component of the cardiac delayed rectifier K(+) current (I(Kr)), which is encoded by human ether-à-go-go-related gene (hERG). The aim of the present study was to characterize possible interactions between terfenadine, binding to a site located inside the central cavity, and the following substances with various binding sites: dofetilide, fluvoxamine, chlorobutanol, and a hERG-specific toxin isolated from scorpion venom (CnErg1). The whole-cell configuration of the patch-clamp technique was employed on hERG channels stably expressed in human embryonic kidney 293 cells. Terfenadine does not interact with dofetilide or fluvoxamine at hERG channels. Slight subadditive inhibitory effects on hERG peak tail currents were observed when terfenadine and CnErg1 were administered in combination. Terfenadine and chlorobutanol synergistically inhibit hERG peak tail currents and enhance each other's inhibitory effect in a concentration-dependent way. In conclusion, terfenadine interacts with CnErg1 and chlorobutanol, but not with dofetilide or fluvoxamine, at hERG channels. It is shown that interactions between chlorobutanol and a hERG channel blocker binding inside the central cavity (terfenadine) produce synergistic effects on hERG currents.

Neuroprotective effects of erythropoietin on 6-hydroxydopamine-treated ventral mesencephalic dopamine-rich cultures

Parkinson's disease (PD) is a neurodegenerative disorder with motor symptoms caused by the loss of dopaminergic (DA) cells and consequently dopamine release in the nigrostriatal system. In vivo and in vitro 6-hydroxydopamine (6-OHDA) PD models are widely used to study the effect of striatal dopamine depletion as well as novel neuroprotective or restorative therapeutic strategies for PD. In the present study, we investigated in vitro the toxicity of 6-OHDA on DA neurons derived from E14 rat ventral mesencephalon (VM) and the neuroprotective efficiency of erythropoietin (Epo) on VM-derived cell cultures against 6-OHDA toxicity. Using E14 VM-derived DA-rich primary cultures, we could demonstrate that 6-OHDA toxicity works in a time-and concentration-dependent way, and leads to cell death not only in DA cells but also in non-DA cells in direct relation to concentration and incubation times. In addition, we found that 6-OHDA toxicity induces caspase-3 activation and an increment of intracellular reactive oxygen species (ROS) in VM-derived cultures. When 6-OHDA-treated VMs were cultured in the presence of the anti-apoptotic protein erythropoietin (Epo), the total neuronal population, including the DA neurons, was protected. However, untreated VM cultures exposed to Epo showed an increase in the total neuronal population, but not an additional increase in DA neuron cell number. These findings suggest that 6-OHDA toxicity is time and concentration-dependent and does not exclusively affect DA neurons. In high concentration and long incubation times, 6-OHDA influences the survival of other neuronal and non-neuronal cell populations derived from the VM cultures. 6-OHDA toxicity induces caspase-3 activation, indicating cell death via the apoptotic pathway which could be restricted or even prevented by pre-exposure to Epo, known to interact via the apoptotic pathway. Our results support and expand on previous findings showing that Epo is an interesting candidate molecule to mediate neuroprotective effects on DA neurons in PD. Furthermore, it could be used in promoting the survival of DA neurons after transplantation in clinical trials.

Tissue-specific inhibition and recovery of esterase activities in Lumbricus terrestris experimentally exposed to chlorpyrifos

Exposure and effect assessment of organophosphate (OP) pesticides generally involves the use of cholinesterase (ChE) inhibition. In earthworm, this enzyme activity is often measured in homogenates from the whole organism. Here we examine the tissue-specific response of ChE and carboxylesterase (CE) activities in Lumbricus terrestris experimentally exposed to chlorpyrifos-spiked field soils. Esterases were measured in different gut segments and in the seminal vesicles of earthworms following acute exposure (2 d) to the OP and during 35 d of a recovery period. We found that inhibition of both esterase activities was dependent on the tissue. Cholinesterase activity decreased in the pharynx, crop, foregut and seminal vesicles in a concentration-dependent way, whereas CE activity (4-nitrophenyl valerate) was strongly inhibited in these tissues. Gizzard CE activity was not inhibited by the OP, even an increase of enzyme activity was evident during the recovery period. These results suggest that both esterases should be determined jointly in selected tissues of earthworms. Moreover, the high levels of gut CE activity and its inhibition and recovery dynamic following OP exposure suggest that this esterase could play an important role as an enzymatic barrier against OP uptake from the ingested contaminated soil.

Musca domestica Larva Lectin Induces Apoptosis in BEL-7402 Cells through a Mitochondria-Mediated Reactive Oxygen Species Way

A lectin from Musca domestica larva was purified by affinity chromatography on a glactose-Sepharose 4B column. Musca domestica larva lectin (MLL) inhibited the growth of BEL-7402 cells in a time and concentration-dependent way. The results of Hoechst 33258 staining indicated that MLL induce BEL-7402 cells apoptosis based on the typical apoptotic morphological changes. Subsequently, we found that MLL treatment abrogated glutathione antioxidant system and induced mitochondria to generate reactive oxygen species (ROS) accumulation, resulting in reduction of mitochondrial transmembrane potential. The induction of cell apoptosis was caused by the upregulation of Bax, the downregulation of Bcl-2, the cytochrome c release and the activation of the caspases pathways.

Clomiphene citrate elicits estrogen agonistic/antagonistic effects differentially via estrogen receptors .ALPHA.and .BETA.

Clomiphene citrate (CC) is known to possess dual actions as an estrogen agonist and an estrogen antagonist. To see how the dual actions of CC are exerted through estrogen receptor alpha (ER alpha) and/or ER beta we developed a cell-based transcription assay system in which 293T cells were transfected with the luciferase reporter plasmid with estrogen responsive element and either human ER alpha or ER beta expression plasmid. CC at lower doses (10(-10) M and 10(-12) M), but not higher doses (10(-6) M and 10(-8) M) elicited estrogenic activity via ER alpha. However, CC at concentrations between 10(-6) M and 10(-12) M did not elicit any estrogenic activity via ER beta. In the presence of 17beta-estradiol (E2), CC behaved either as an agonist or as an antagonist via ER alpha depending on the concentrations of E2, i.e., antagonistic when combined with the higher E2 concentrations, agonistic with the lower E2 concentrations. On the other hand, via ER beta, CC acted as an estrogen antagonist regardless of the concentration of E2 added together. In conclusion, CC acts as an estrogen agonist/antagonist via ER alpha in a coexisting estrogen concentration-dependent way whereas it acts as an estrogen antagonist via ER beta whether or not estrogen is present.

Spasmolytic and anti-inflammatory effects of Aloysia triphylla and citral, in vitro and in vivo studies

Aloysia triphylla is traditionally utilized for the treatment of menstrual colic (primary dysmenorrhea) in Mexico. Citral is the main chemical component found in Aloysia triphylla leaves extract. Primary dysmenorrhea is a very frequent gynecological disorder in menstruating women, affecting 30-60% of them. It is usually treated with non-steroidal anti-inflammatory drugs (NSAIDs); although their effect is rapid, they possess many side effects. Due to these shortcomings, Mexican folk therapy is considered as a feasible alternative. The effects of the hexane extract of Aloysia triphylla and citral on uterine contractions were evaluated in vitro as well as their anti-inflammatory properties and gastric wound capabilities were assessed in vivo. The inhibitory effects on the contractions were analyzed using isolated uterus strips from estrogen primed rats. Contractions were induced by KCl 60 mM, oxytocin 10 mIU/mL, charbacol 10 µM and PGF(2α) 5 µM. The anti-inflammatory effect was assessed on carrageenan-induced rat hind paw edema model. The inhibitory concentration-50 (IC(50)) of the hexane extract of Aloysia triphylla upon each contractile response was for KCl 44.73 ± 2.48 µg/mL, oxytocin 42.16 ± 3.81 µg/mL, charbacol 41.87 ± 1.73 µg/mL and PGF(2α) 28.70 ± 2.40 µg/mL in a concentration-dependent way. The extract of Aloysia triphylla produced a significant inhibitory effect on PGF(2α)-induced contraction compared to its inhibitory actions on the others. Citral exhibited the same inhibitory effect on the contraction induced by PGF(2α). The oral administration of the extract (100-800 mg/kg) and citral (100-800 mg/kg) showed anti-inflammatory activity; furthermore, the maximal dose utilized did not produce gastric injury. These results were compared with anti-inflammatory effects and gastric damage produced by 30 mg/kg of indomethacin p.o. The spasmolytic and anti-inflammatory effects support the traditional use of Aloysia triphylla leaves in the treatment of the primary dysmenorrhea in Mexican communities.

Testosterone and Cholesterol Vasodilation of Rat Aorta Involves L-Type Calcium Channel Inhibition

Testosterone has rapid nongenomic vasodilator effects which could be involved in protective cardiovascular actions. Several authors suggested specific mechanisms to explain this effect, but this matter was not clarified yet. We studied the actions of testosterone and cholesterol on endothelium-denuded rat aorta and their effects on the L-type Ca2+ current (ICa,L) and potassium current (IK). Testosterone (1–100 μM) totally relaxed, in a rapid and concentration-dependent way, the aortic rings contracted by KCl or by (−)-Bay K8644 (BAY). Cholesterol also fully relaxed the contractions induced by KCl. None of the potassium channel antagonists tested (glibenclamide, tetraethylammonium and 4-aminopyridine) modified significantly the relaxant effect of testosterone. The antagonist of classic testosterone receptors, flutamide, did not modify the vasorelaxant effect of testosterone. Furthermore, testosterone and cholesterol inhibited either basal and BAY-stimulated ICa,L in A7r5 cells and they have no effects on IK. In summary, our results demonstrate that cholesterol and testosterone relax rat aorta by inhibiting LTCC. This effect of testosterone is not mediated by the classic hormone receptor or by potassium channel activation. These results suggest that the vasodilator mechanism of cholesterol and testosterone is the same.

Continuous differential impedance spectroscopy of single cells

A device for continuous differential impedance analysis of single cells held by a hydrodynamic cell trapping is presented. Measurements are accomplished by recording the current from two closely-situated electrode pairs, one empty (reference) and one containing a cell. We demonstrate time-dependent measurement of single cell impedance produced in response to dynamic chemical perturbations. First, the system is used to assay the response of HeLa cells to the effects of the surfactant Tween, which reduces the impedance of the trapped cells in a concentration dependent way and is interpreted as gradual lysis of the cell membrane. Second, the effects of the bacterial pore-forming toxin, Streptolysin-O are measured: a transient exponential decay in the impedance is recorded as the cell membrane becomes increasingly permeable. The decay time constant is inversely proportional to toxin concentration (482, 150, and 30 s for 0.1, 1, and 10 kU/ml, respectively).Electronic supplementary materialThe online version of this article (doi:10.1007/s10404-009-0534-2) contains supplementary material, which is available to authorized users.

Sulforaphane protects against cisplatin-induced nephrotoxicity

Cisplatin (cis-diamminedichloroplatinum II, CDDP) is a chemotherapeutic agent that induces nephrotoxicity associated with oxidative/nitrosative stress. Sulforaphane (SFN) is an isothiocyanate produced by the enzymatic action of myrosinase on glucorophanin, a glucosinolate contained in cruciferous vegetables. SFN is able to induce cytoprotective enzymes through the transcription factor Nrf2. The purpose of this study was to evaluate whether SFN induces a cytoprotective effect on the CDDP-induced nephrotoxicity. Preincubation of LLC-PK1 cells with 0.5–5 μM SFN by 24 h was able to prevent, in a concentration-dependent way, CDDP-induced cell death. Immunofluorescent staining confirmed the nuclear translocation of Nrf2 after treatment with SFN. In the in vivo studies, CDDP was given to Wistar rats as a sole i.p. injection at a dose of 7.5 mg/kg. SFN (500 μg/kg i.v.) was given two times (24 h before and 24 after CDDP-injection). Animals were killed three days after CDDP-injection. SFN attenuated CDDP-induced renal dysfunction, structural damage, oxidative/nitrosative stress, glutathione depletion, enhanced urinary hydrogen peroxide excretion and the decrease in antioxidant enzymes (catalase, glutathione peroxidase and glutathione-S-transferase). The renoprotective effect of SFN on CDDP-induced nephrotoxicity was associated with the attenuation in oxidative/nitrosative stress and the preservation of antioxidant enzymes.

Developmental toxicity of bifenthrin in embryo-larval stages of zebrafish

Bifenthrin (BF) is widely used as a miticide in orchards, nurseries and homes due to its great photostability and insecticidal activity. Recently, extensive research has been conducted on the toxicity of BF in in vitro and in vivo assays. However, no data is so far available regarding the developmental toxicity of BF to fish in early life stages. In this study, the developmental effects of BF were evaluated in embryo-larval zebrafish. At specified stages (24, 48, 72, and 96 hpf), spontaneous movement, survival and hatching as well as non-lethal malformation like curved body axis or edema were described in detail. No significant lethal effects of the treatment group compared to the control occurred except for the highest concentration group exposed for 96 h. The hatching process was accelerated by BF in a concentration-dependent way, correlated with increasing spontaneous movement. Developmental abnormalities were observed for the test compound with 96-h EC 50 of 256 μg L −1 for pericardial edema, and 109 μg L −1 for curved body axis. Results from locomotor assays showed that zebrafish larvae of 96 hpf exhibited impaired swimming behaviour after exposure to 50, 100, and 200 μg L −1 from 3 to 84 hpf. After being cultured in the BF-free embryo medium for one more day till 120 hpf, larvae from the 50 μg L −1group seemed to have recovered and showed no difference in the swimming behaviour compared to the control while animals of the two higher concentration groups were exhausted and swam in significantly lower speed. Furthermore, reverse transcription real-time PCR results showed that vitellogenin I expressions were significantly induced in larval zebrafish exposed to 150 μg L −1 BF for 72 h, indicating the disruption of the endocrine level. In summary, our studies showed that BF was developmentally toxic to zebrafish in early life stage after short-term exposure to sublethal concentrations and had the ability to impair the individual behaviours which are of great importance in the assessment of their ecological fitness.

Retrorsine, but not monocrotaline, is a mechanism-based inactivator of P450 3A4

Retrorsine (RTS) and monocrotaline (MCT) cause severe toxicities via P450-mediated metabolic activation. The screening of mechanism-based inhibitors showed RTS inactivated 3A4 in the presence of NADPH. Unlike RTS, MCT failed to inhibit P450 3A4 and other enzymes tested. Further studies showed the loss of P450 3A4 activity occurred in a time- and concentration-dependent way, which was not recovered after dialysis. Dextromethorphan, a P450 3A4 substrate, protected the enzyme from the inactivation. Exogenous nucleophile glutathione (GSH) and reactive oxygen species scavengers catalase and superoxide dismutase did not protect P450 3A4 from the inactivation. GSH trapping experiments showed both P450 3A4 and 2C19 converted RTS and MCT to the corresponding electrophilic metabolites which could be trapped by GSH to form 7-GSH-DHP conjugate. We conclude that RTS and MCT are metabolically activated by P450 3A4 and 2C19, and that RTS, but not MCT, is a mechanism-based inactivator of P450 3A4.

Ficus carica L.: Metabolic and biological screening

Ficus carica L. is one of the earliest cultivated fruit trees. In this work, metabolite profiling was performed on the leaves, pulps and peels of two Portuguese white varieties of F. carica (Pingo de Mel and Branca Tradicional). Phenolics and organic acids profiles were determined by HPLC/DAD and HPLC/UV, respectively. All samples presented a similar phenolic profile composed by 3- O- and 5- O-caffeoylquinic acids, ferulic acid, quercetin-3- O-glucoside, quercetin-3- O-rutinoside, psoralen and bergapten. 3- O-Caffeoylquinic acid and quercetin-3- O-glucoside are described for the first time in this species. Leaves’ organic acids profile presented oxalic, citric, malic, quinic, shikimic and fumaric acids, while in pulps and peels quinic acid was absent. The antioxidant potential of the different plant parts was checked. All materials exhibited activity against DPPH and nitric oxide radicals in a concentration-dependent way. However, only the leaves presented capacity to scavenge superoxide radical. Leaves were always the most effective part, which seems to be related with phenolics compounds. Additionally, acetylcholinesterase inhibitory capacity was evaluated, but no effect was observed. Antimicrobial potential was also assessed against several bacterial species, although no activity was noticed. This is the first study comparing the chemical composition and biological potential of F. carica pulps, peels and leaves.

Cysteamine prevents inhibition of adenylate kinase caused by cystine in rat brain cortex

Cystinosis is a systemic genetic disease caused by a lysosomal transport deficiency accumulating cystine in the lysosomes of almost all tissues. Although tissue damage might depend on cystine accumulation, the mechanisms of tissue damage are still obscures. Adenylate kinase, along with creatine kinase, is responsible for the enzymatic phosphotransfer network, crucial for energy homeostasis. Taking into account that cystine is known to inhibit creatine kinase activity, the two enzymes have thiol groups, and the strong interaction between the two activities, our main objective was to investigate the effect of cystine on adenylate kinase activity in the brain cortex of Wistar rats. For the in vivo studies, the animals were injected twice a day with 1.6 micromol/g body weight of cystine dimethylester and/or 0.46 micromol/g body weight of cysteamine from the 25th to the 29th postpartum day and sacrificed after 12 h. Cystine inhibited the enzyme activity in vitro in a concentration dependent way, whereas cysteamine prevented the inhibition. Adenylate kinase activity was found diminished in the brain cortex of rats loaded with cystine dimethylester and co-administration of cysteamine prevented the diminution of the enzyme activity. Considering that adenylate kinase together with creatine kinase is crucial for energy homeostasis, the release of cystine from lysosomes with consequent enzymes inhibition could impair energy homeostasis, contributing to tissue damage in patients with cystinosis.

Effect of Helicobacter priori on gastric epithelial cells proliferation, apoptosis and expression of p27^kip1 protein in vitro

Objective To explore the influence of Helicobacter pylori （Hp） on the proliferation, apoptosis and expression of p27^kip1 protein of gastric epithelial cells in vitro. Methods SGC-7901 cell proliferation was examined by flow cytometry after incubation with different concentration of NCTC11637. The effect of NCTC11637 on cell apoptosis was also evaluated by flow cytometry. And the expression of p27^kip1 of gastric epithelial cells was detected by immunohistochemical analysis and in situ hybridization, respectively. Results Cell proliferation was enhanced when co-incubated with the Hp in low concentration （3.4 ×10^4 to 1.9 ×10^5 CFU/ml） but inhibited in higher concentration （≥4.8 ×10^6 CFU/ml）. However, cell apoptosis was increase when co-incubated with the Hp in high concentration （ ≥9.6 ×10^5 CFU/ml） showing concentration dependent picture. In addition, the co-incubation of SGC-7901 with Hp led to decrease of the expression of p27^kip1 protein but not mRNA in a Hp concentration dependent way. Conclusion Hp could effect the gastric epithelial cells apoptosis and proliferation directly and influence the expression of p27^kip1 protein which might facilitate gastric carcinoma. Key words: Helicobacter pylori; Gastric carcinoma; Cell proliferation; Cell apoptosis; p27^kip1

A flow‐injection chemiluminescent method for the evaluation of the antioxidant activity of 5′‐nucleotides

In the present work, a simple chemiluminescence (CL) method coupled with flow-injection analysis for the evaluation of antioxidant activity of 5'-nucleotides (5'-AMP, 5'-CMP, 5'-GMP, 5'-UMP) was proposed. It is based on inhibition effect of the studied substances on CL emission of luminol-potassium ferricyanide-pyrogallol. Experiments were performed to evaluate the nature of the inhibition by 5'-nucleotides of the CL reaction and their antioxidant activities. Based on the experimental results, it was observed that 5'-nucleotides are available antioxidants that could effectively scavenge superoxide anion free radicals in a concentration-dependent way. This will provide a basis for further development of the use of nucleotides.

Cysteamine prevents inhibition of adenylate kinase caused by cystine in rat brain cortex

Cystinosis is a systemic genetic disease caused by a lysosomal transport deficiency accumulating cystine in the lysosomes of almost all tissues. Although tissue damage might depend on cystine accumulation, the mechanisms of tissue damage are still obscures. Adenylate kinase, along with creatine kinase, is responsible for the enzymatic phosphotransfer network, crucial for energy homeostasis. Taking into account that cystine is known to inhibit creatine kinase activity, the two enzymes have thiol groups, and the strong interaction between the two activities, our main objective was to investigate the effect of cystine on adenylate kinase activity in the brain cortex of Wistar rats. For the in vivo studies, the animals were injected twice a day with 1.6 micromol/g body weight of cystine dimethylester and/or 0.46 micromol/g body weight of cysteamine from the 25th to the 29th postpartum day and sacrificed after 12 h. Cystine inhibited the enzyme activity in vitro in a concentration dependent way, whereas cysteamine prevented the inhibition. Adenylate kinase activity was found diminished in the brain cortex of rats loaded with cystine dimethylester and co-administration of cysteamine prevented the diminution of the enzyme activity. Considering that adenylate kinase together with creatine kinase is crucial for energy homeostasis, the release of cystine from lysosomes with consequent enzymes inhibition could impair energy homeostasis, contributing to tissue damage in patients with cystinosis.

551 Shiga Toxin 1 Uptake By Intestinal Epithelial Cells Is CDC-42 Mediated and Clathrin-Independent

Shiga toxins (Stx) 1 and 2 are major contributors to foodborne disease caused by enterohemorrhagic E.coli (EHEC). Human intestinal epithelial cells (IEC) do not normally express Stx receptor (the glycosphingolipid Gb3), leaving the mechanism of Stx uptake poorly understood. Inhibition of clathrin-mediated endocytosis significantly increases Stx1 uptake in Gb3-free IEC. We thus now test the hypothesis that cell division cycle 42 (Cdc-42) dependent fluid phase endocytosis might be involved in clathrin-independent Stx1 uptake by IEC In Vitro and In Vivo. Methods: In Vivo: We compared pathologies in New Zealand white rabbits fed with either a) an experimental RDEC-H19A strain engineered to express Stx1, since it produces intestinal pathology quite similar to that noted in human EHEC or b) a control RDEC-1 E. coli strain that adheres to rabbit mucosa to provide an attaching and effacing lesion or c) PBS, to provide a negative control for effects of bacterial infection. Immunofluorescence of frozen sections from cecal tissue of rabbits studied at times up to 7 days after infection assessed a) uptake of Stx1 and b) presence of activated Cdc-42 in IEC. In Vitro: The apical uptake of the recombinant B-subunit of Stx1 labeled with Alexa 680 fluorescent dye (Stx1B) was assessed in T84 IEC that were: a) treated with agents known to inhibit clathrin-mediated endocytosis, b) depleted of Cdc42 by shRNA or c) untreated controls using biochemical and immunofluorescence methods. Results: 1) Cecal tissues from rabbits treated with either RDEC-1 or RDEC-H19A bacteria, but not those from PBS-treated animals, displayed Cdc42 activation in subsets of epithelial cells at sites near and those distant from attached bacteria. 2) T84 cells incubated with the inhibitors of clathrin mediated endocytosis chlorpromazine or low potassium increased Stx1B uptake in a time-dependent fashion. This stimulation was virtually eliminated, however, in T84 cells in which Cdc-42 was downregulated using shRNA. 3) The specific blocker of Cdc-42 function and nucleotide exchange, Pirl-1, also prevented stimulation of Stx1B uptake in concentration-dependent way in cells treated with inhibitors of clathrin-mediated endocytosis. 4) This clathrin independent Cdc-42 mediated Stx1B uptake was also dynamin dependent. Dynasore, a specific inhibitor of dynamin GTPase activity, inhibited the Stx1B uptake in cells with inactivated clathrin induced endocytic pathway. Conclusions: 1) Basal Stx1uptake by Gb3 negative T84 cells is regulated by Cdc-42. 2) Cdc-42 dependent clathrin independent pathway is likely to be involved in IEC uptake of Stx1 during EHEC infections.

Activity of sap from Croton lechleri on rat vascular and gastric smooth muscles

The effects of red sap from Croton lechleri (SdD), Euphorbiaceae, on vascular and gastric smooth muscles were investigated. SdD, from 10 to 1000 microg/ml, induced concentration-dependent vasoconstriction in rat caudal arteries, which was endothelium-independent. In arterial preparations pre-constricted by phenylephrine (0.1 microM) or KCl (30 mM), SdD also produced concentration-dependent vasoconstriction. To study the mechanisms implicated in this effect we used selective inhibitors such as prazosin (0.1 microM), an antagonist of alpha(1)-adrenoceptors, atropine (0.1 microM), an antagonist of muscarinic receptors, and ritanserin (50 nM), a 5-HT(2A) antagonist; none of these influenced vasoconstriction caused by SdD. Likewise, nifedipine (50 nM), an inhibitor of L-type calcium channels, did not modify the action of SdD. Capsaicin (100 nM), an agonist of vanilloid receptors, also did not affect vasoconstriction by SdD. We also investigated the action of SdD (10-1000 microg/ml) on rat gastric fundus; per se the sap slightly increased contractile tension. When the gastric fundus was pre-treated with SdD (100 microg/ml) the contraction induced by carbachol (1 microM) was increased, whereas that by KCl (60mM) or capsaicin (100 nM) were unchanged. The data shows that SdD increased contractile tension in a concentration-dependent way, both on vascular and gastric smooth muscles. The vasoconstriction is unrelated to alpha(1), M, 5-HT(2A) and vanilloid receptors as well as L-type calcium channels. SdD increased also contraction by carbachol on rat gastric fundus. Thus for the first time, experimental data provides evidence that sap from C. lechleri owns constricting activity on smooth muscles.