Thrombin induces the release of the Y-box protein dbpB from mRNA: A mechanism of transcriptional activation

We have recently demonstrated that thrombin induces expression of the platelet-derived growth factor B-chain gene in endothelial cells (EC) through activation of the Y-box binding protein DNA-binding protein B (dbpB). We now present evidence that dbpB is activated by a novel mechanism: proteolytic cleavage leading to release from mRNA, nuclear translocation, and induction of thrombin-responsive genes. Cytosolic, full-length dbpB (50 kDa) was rapidly cleaved to a 30-kDa species upon thrombin stimulation of EC. This truncated, “active” dbpB exhibited nuclear localization and binding affinity for the thrombin response element sequence, which is distinct from the Y-box sequence. Oligo(dT) affinity chromatography revealed that cytosolic dbpB from control EC, but not active dbpB from thrombin-treated EC, was bound to mRNA. Latent dbpB immunoprecipitated from cytosolic extracts of control EC was activated by ribonuclease treatment. Furthermore, when EC cytosolic extracts were subjected to Nycodenz gradient centrifugation, latent dbpB fractionated with mRNA, whereas active dbpB fractionated with free proteins. The cytosolic retention domain of dbpB, which we localized to the region 247–267, was proteolytically cleaved during its activation. In contrast to full-length dbpB, truncated dbpB stimulated platelet-derived growth factor B-chain and tissue factor promoter activity by over 5-fold when transiently cotransfected with reporter constructs. These results suggest a novel mode of transcription factor activation in which an agonist causes release from mRNA of a latent transcription factor leading to its transport to the nucleus and its regulation of target gene expression.

Mechanism of oligonucleotide release from cationic liposomes.

We propose a mechanism for oligonucleotide (ODN) release from cationic lipid complexes in cells that accounts for various observations on cationic lipid-nucleic acid-cell interactions. Fluorescent confocal microscopy of cells treated with rhodamine-labeled cationic liposome/ fluorescein-labeled ODN (F-ODN) complexes show the F-ODN separates from the lipid after internalization and enters the nucleus leaving the fluorescent lipid in cytoplasmic structures. ODN displacement from the complex was studied by fluorescent resonance energy transfer. Anionic liposome compositions (e.g., phosphatidylserine) that mimic the cytoplasmic facing monolayer of the cell membrane released ODN from the complex at about a 1:1 (-/+) charge ratio. Release was independent of ionic strength and pH. Physical separation of the F-ODN from monovalent and multivalent cationic lipids was confirmed by gel electrophoresis. Fluid but not solid phase anionic liposomes are required, whereas the physical state of the cationic lipids does not effect the release. Water soluble molecules with a high negative linear charge density, dextran sulfate, or heparin also release ODN. However, ATP, spermidine, spermine, tRNA, DNA, polyglutamic acid, polylysine, bovine serum albumin, or histone did not release ODN, even at 100-fold charge excess (-/+). Based upon these results, we propose that the complex, after internalization by endocytosis, induces flip-flop of anionic lipids from the cytoplasmic facing monolayer. Anionic lipids laterally diffuse into the complex and form a charged neutralized ion-pair with the cationic lipids. This leads to displacement of the ODN from the cationic lipid and its release into the cytoplasm.

Autocrine signaling through ATP release represents a novel mechanism for cell volume regulation.

Recovery of cell volume in response to osmotic stress is mediated in part by increases in the Cl- permeability of the plasma membrane. These studies evaluate the hypothesis that ATP release and autocrine stimulation of purinergic (P2) receptors couple increases in cell volume to opening of Cl- channels. In HTC rat hepatoma cells, swelling induced by hypotonic exposure increased membrane Cl- current density to 44.8 +/- 7.1 pA/pF at -80 mV. Both the rate of volume recovery and the increase in Cl- permeability were inhibited in the presence of the ATP hydrolase apyrase (3 units/ml) or by exposure to the P2 receptor blockers suramin and Reactive Blue 2 (10-100 microM). Cell swelling also stimulated release of ATP. Hypotonic exposure increased the concentration of ATP in the effluent of perfused cells by 170 +/- 36 nM in the presence of a nucleotidase inhibitor (P < 0.01). In whole-cell recordings with ATP as the charge carrier, cell swelling increased membrane current density approximately 30-fold to 16.5 +/- 10.4 pA/pF. These findings indicate that increases in cell volume lead to efflux of ATP through opening of a conductive pathway consistent with a channel, and that extracellular ATP is required for recovery from swelling. ATP may function as an autocrine factor that couples increases in cell volume to opening of Cl- channels through stimulation of P2 receptors.

The role of cytoskeletal proteins in the mechanism of insulin release

This thesis is concerned with the role of /3-cell cytoskeletal proteins in the mechanism of insulin release from islets of experimental animals, the Aston obese diabetic hyperglycaemic (ob/ob) mouse and their lean littermates and the cultural insulin secreting /?-cell lines, HIT-TT5 and RINm5F. Investigations were carried out into the glucose induced insulin response of the lean and obese mouse islets and HIT-TI5 cells and the D-glyceraldehyde response of RINm5F cells using a static incubation system. Colchicine was found to inhibit insulin release from both lean and obese mouse islets more significantly than cultured TTT-TI5 and RINm5F cells. (Colchicine pre-treatment also inhibited the second phase of insulin release from perifused lean mouse islets and HIT-TI5 cells). Cytocha-lasin B, used to investigate the role of the microfilamentous system in the mechanism of insulin release enhanced insulin release from both lean and obese mouse islets to a significantly greater degree than that from cultured HIT-TI5 and RINm5F cells. Pre-treatment of isolated lean and obese mouse islets and cultured /?-cells with a combination of colchicine and cytochalasin B significantly reduced the insulin response of the HIT-TI5 and RINm5F cells compared with the control values suggesting that intact microtubules are more important for the sustained release of insulin than the microfilamentous system. However, the response was not so clearly defined with the lean and obese mouse islets. Tubulin was separated from the extracts of lean mouse islets and the HIT-TI5 and RINm5F cells and actin was separated from all of the cell types including the obese mouse islets by SDS- polyacrylamide electrophoresis. A tubulin radioimmunoassay and a colchicine binding assay were developed to measure the tubulin content of lean and obese mouse islets, and the shift between the proportions of tubulin dimers and polymerized tubulin under stimulatory and non-stimulatory conditions. The assay methods developed were not prone to be accurate, sensitive and precise but gave some indication of the shift from unpolymerised to polymerised tubulin during glucose stimulated insulin release. These studies show that microtubules do play a fundamental role in the mechanism of insulin release from both islets and cultured HIT-TI5 and RINm5F cells.

Bio-Dis and the Paddle Dissolution Apparatuses Applied to the Release Characterization of Ketoprofen from Hypromellose Matrices

The purposes of this work were: (1) to comparatively evaluate the effects of hypromellose viscosity grade and content on ketoprofen release from matrix tablets, using Bio-Dis and the paddle apparatuses, (2) to investigate the influence of the pH of the dissolution medium on drug release. Furthermore, since direct compression had not shown to be appropriate to obtain the matrices under study, it was also an objective (3) to evaluate the impact of granulation on drug release process. Six formulations of ketoprofen matrix tablets were obtained by compression, with or without previous granulation, varying the content and viscosity grade of hypromellose. Dissolution tests were carried out at a fixed pH, in each experiment, with the paddle method (pH 4.5, 6.0, 6.8, or 7.2), while a pH gradient was used in Bio-Dis (pH 1.2 to 7.2). The higher the hypromellose viscosity grade and content were, the lower the amount of ketoprofen released was in both apparatuses, the content effect being more expressive. Drug dissolution enhanced with the increase of the pH of the medium due to its pH-dependent solubility. Granulation caused an increase in drug dissolution and modified the mechanism of the release process.

On the mechanisms of phenothiazine-induced mitochondrial permeability transition: Thiol oxidation, strict Ca(2+) dependence, and cyt c release

Phenothiazines (PTZ) are drugs widely used in the treatment of schizophrenia. Trifluoperazine, a piperazinic PTZ derivative, has been described as inhibitor of the mitochondrial permeability transition (MPT). We reported previously the antioxidant activity of thioridazine at relatively low concentrations associated to the inhibition of the MPT (Brit. J. Pharmacol., 2002;136:136-142). In this study, it was investigated the induction of MPT by PTZ derivatives at concentrations higher than 10 mu M focusing on the molecular mechanism involved. PTZ promoted a dose-response mitochondrial swelling accompanied by mitochondrial transmembrane potential dissipation and calcium release, being thioridazine the most potent derivative. PTZ-induced MPT was partially inhibited by CsA or Mg(2+) and completely abolished by the abstraction of calcium. The oxidation of reduced thiol group of mitochondrial membrane proteins by PTZ was upstream the VIP opening and it was not sufficient to promote the opening of PTP that only occurred when calcium was present in the mitochondrial matrix. EPR experiments using DMPO as spin trapping excluded the participation of reactive oxygen species on the PTZ-induced MPT. Since 117 give rise to cation radicals chemically by the action of peroxidases and cyanide inhibited the PTZ-induced swelling, we propose that VIZ bury in the inner mitochondrial membrane and the chemically generated 117 cation radicals modify specific thiol groups that in the presence of Ca(2+) result in MPT associated to cytochrome c release. These findings contribute for the understanding of mechanisms of MET induction and may have implications for the cell death induced by PTZ. (C) 2010 Elsevier Inc. All rights reserved.

Dehydromonocrotaline induces cyclosporine A-insensitive mitochondrial permeability transition/cytochrome c release

Monocrotaline (MCT) is a pyrrolizidine alkaloid present in plants of the genus Crotalaria that causes cytotoxicity and genotoxicity in animals and humans. It is well established that the toxicity of MCT results from its hepatic bioactivation to dehydromonocrotaline (DHM), an alkylating agent, but the exact mechanism of action remains unknown. In a previous study, we demonstrated DHM`s inhibition of mitochondrial NADH-dehydrogenase activity at micromolar concentrations, which is an effect associated with a significant reduction in ATP synthesis. As a follow-up study, we have evaluated the ability of DHM to induce mitochondrial permeability transition (MPT) and its associated processes in isolated rat liver mitochondria. In the presence of 10 mu M Ca(2+), DHM (50-250 mu M) elicited MPT in a concentration-dependent, but cyclosporine A-independent manner, as assessed by mitochondrial swelling, which is associated with mitochondrial Ca(2+) efflux and cytochrome c release. DHM (50-250 mu M) did not cause hydrogen peroxide accumulation but did deplete endogenous glutathione and NAD(P)H, while oxidizing protein thiol groups. These results potentially indicate the involvement of mitochondria, via apoptosis, in the well-documented cytotoxicity of monocrotaline. (C) 2009 Elsevier Ltd. All rights reserved.

Effects of Mg2+ on Ca2+ release from sarcoplasmic reticulum of skeletal muscle fibres from yabby (crustacean) and rat

1. The role of myoplasmic [Mg2+] on Ca2+ release from the sarcoplasmic reticulum (SR) was examined in the two major types of crustacean muscle fibres, the tonic, long sarcomere fibres and the phasic, short sarcomere fibres of the fresh mater decapod crustacean Cherax: destructor (yabby) and in the fast-twitch rat muscle fibres using the mechanically skinned muscle fibre preparation. 2. A robust Ca2+-induced Ca2+-release (CICR) mechanism was present in both long and short sarcomere fibres and 1 mM Mg2+ exerted a strong inhibitory action on the XR Ca2+ release in both fibre types. 3. The XR displayed different properties with respect to Ca2+ loading in the long and the short sarcomere fibres and marked functional differences were identified with respect to Mg2+ inhibition between the two crustacean fibre types. Thus, in long sarcomere fibres, the submaximally loaded XR was able to release Ca2+ when [Mg2+] was lowered from 1 to 0.01 mw in the presence of 8 mM ATP(total) and in the virtual absence of Ca2+ (< 5 nM) even when the CICR was suppressed. In contrast, negligible Ca2+ was released from the submaximally loaded SR of short sarcomere yabby fibres when [Mg2+] was lowered from 1. to 0.01 mM under the same conditions as for the long sarcomere fibres. Nevertheless, the rate of XR Ca2+ release in short sarcomere fibres increased markedly when [Mg2+] was lowered in the presence of [Ca2+] approaching the normal resting levels (50-100 nM). 4. Rat fibres were able to release SR Ca2+ at a faster rate than the long sarcomere yabby fibres when [Mg2+] was lowered from 1 to 0.01 mM in the virtual absence of Ca2+ but, unlike with yabby fibres, the net rate of Ca2+ release was actually increased for conditions that were considerably less favourable to CICR. 5. In summary it is concluded that crustacean skeletal muscles have more that one functional type of Ca2+-release channels, that these channels display properties that are intermediate between those of mammalian skeletal and cardiac isoforms, that the inhibition exerted by Mg2+ at rest on the crustacean SR Ca2+-release channels must be removed during excitation-contraction coupling and that, unlike in crustacean fibres, CICR cannot play the major role in the activation of XR Ca2+-release channels in the rat skeletal muscle.

Uncommon mechanism of egg incubation in the endemic Southern hermit crab Loxopagurus loxochelis: how is this phenomenon related to egg production?

The aim of the present work was to characterize the egg production of Loxopagurus loxochelis. A total of 71 ovigerous females were obtained, 28% of which were simultaneously incubating eggs at different developmental stages. This phenomenon can be the result of incomplete fertilization, or may represent a rapid gonadal cycle by this species in this area, which is, to our understanding, the best explanation of this phenomenon. Egg volume decreased 25.6% during the incubation period. The reproductive output based on dry and wet weight was 6.8 and 19.3%, respectively. Water was the prevailing component of the eggs, representing 86.0% of the total weight at initial stage, increasing to 95.1% at the final stage. Ash content increased at the same time as a decrease in the organic content occurred, indicating the consumption of yolk and absorption of salts from the water medium. In conclusion, we hypothesized that this population has the capacity to copulate and deposit another brood even before the release of the larvae from the previous one, intensifying the reproductive effort of these hermit crabs as a strategy of adaptation in a region considered the northern limit of the geographical distribution of this species.

Effect of academic psychological stress in post-graduate students: The modulatory role of cortisol on superoxide release by neutrophils

Experimental and clinical evidence shows that neutrophils play an important role in the mechanism of tissue injury in immune complex diseases through the generation of reactive oxygen species. In this study, we examined the influence of academic psychological stress in post-graduate students on the capacity of their blood neutrophils to release superoxide when stimulated by immune complexes bound to nonphagocytosable surfaces and investigated the modulatory effect of cortisol on this immune function. The tests were performed on the day before the final examination. The state-trait anxiety inventory questionnaire was used to examine whether this stressful event caused emotional distress. In our study, the psychological stress not only increased plasma cortisol concentration, but it also provoked a reduction in superoxide release by neutrophils. This decrease in superoxide release was accompanied by diminished mRNA expression for subunit p47(phox) of the phagocyte superoxide-generating nicotinamide adenine dinucleotide phosphate-oxidase. These inhibitory effects were also observed by in vitro exposure of neutrophils from control volunteers to 10(-7) M hydrocortisone, and could be prevented by the glucocorticoid receptor antagonist RU-486. These results show that in a situation of psychological stress, the increased levels of cortisol could inhibit superoxide release by neutrophils stimulated by IgG immune complexes bound to nonphagocytosable surfaces, which could attenuate the inflammatory state.

Central but not systemic inhibition of inducible nitric oxide synthase modulates oxytocin release during endotoxemic shock

Previous studies have shown that immunological challenges as lipopolysaccharide (LPS) administration increases plasma oxytocin (OT) concentration. Nitric oxide (NO), a free radical gas directly related to the immune system has been implicated in the central modulation of neuroendocrine adaptive responses to immunological stress. This study aimed to test the hypothesis that the NO pathway participates in the control of OT release induced by LPS injection. For this purpose, adult male Wistar rats received bolus intravenous (i.v.) injection of LPS, preceded or not by iv. or intracerebroventricular (i.c.v.) injections of aminoguanidine (AG), a selective inducible nitric oxide synthase (iNOS) inhibitor. Rats were decapitated after 2, 4 and 6 h of treatment, for measurement of OT by radioimmunoassay. In a separate set of experiments, mean arterial pressure (MAP) and heart rate (HR) were measured every 15 min over 6 h, using a polygraph. These studies revealed that LPS reduced MAP and increased HR at 4 and 6 h post-injection. LPS significantly increased plasma OT concentration at 2 and 4 h post-injection. Pre-treatment with i.c.v. AG further increased plasma OT concentration and attenuated the LPS-induced decrease in MAP, however, i.v. AG failed to show similar effects. Thus, iNOS pathway may activate a central inhibitory control mechanism that attenuates OT secretion during endotoxemic shock. (C) 2009 Elsevier Inc. All rights reserved.

Compound 48/80 induces endothelium-dependent and histamine release-independent relaxation in rabbit aorta

Compound 48/80 (C48/80) is a synthetic condensation product of N-methyl-p-methoxyphenethyl am me with formaldehyde and is an experimental drug used since the 1950s to induce anaphylactic shock through histamine release. This study was carried out to further elucidate the mechanism by which this drug induces nitric oxide (NO) release. Our specific goals were: (a) to verify if C48/80`s relaxation occurs through the stimulation of histamine receptors; (b) to evaluate the endothelium-dependent relaxation induced by C48/80; (c) to identify NO as the endothelium-relaxing factor released by C48/80; (d) to identify the NO synthase (NOS) responsible for NO release; and (e) to verify if the relaxation induced by C48/80 is calcium and cyclic guanidine monophosphate (cGMP) dependent. Rabbit aorta segments, with and without endothelium, were suspended in organ chambers (25 ml) filled with Krebs solution maintained at 37 degrees C, bubbled with 95% O-2/5% CO2 (pH 7.4). Phenylephrine was used to contract the segments. Other protocol drugs included H-1- and H-2-receptor antagonists, cyclooxygenase, NOS, guanylyl cyclase and phospholipase C (PLC) inhibitors. Endothelium-dependent relaxation induced by C48/80 was also studied in calcium-free Krebs solution associated with a calcium chelator. In summary, our investigation demonstrated that the C48/80 vasodilating action: (a) does not depend on H-1 and H-2 histamine receptors; (b) is NO endothelium-dependent; (c) is dependent on the endothelial constitutive NOS (NOS-3) isoform activation; (d) is cGMP-dependent; and that NOS-3 activation by C48/80: (a) is independent of PLC up to 25 mu g/ml and (b) is partially dependent of this lipase in higher doses. (C) 2007 Elsevier Inc. All rights reserved.