Actein alleviates 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin‐mediated cellular dysfunction in osteoblastic MC3T3‐E1 cells

The environmental pollutant 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD) is known to affect bone metabolism. We evaluated the protective effects of the triterpene glycoside actein from the herb black cohosh against TCDD‐induced toxicity in MC3T3‐E1 osteoblastic cells. We found that TCDD significantly reduced cell viability and increased apoptosis and autophagy in MC3T3‐E1 osteoblastic cells (P < .05). In addition, TCDD treatment resulted in a significant increase in intracellular calcium concentration, mitochondrial membrane potential collapse, reactive oxygen species (ROS) production, and cardiolipin peroxidation, whereas pretreatment with actein significantly mitigated these effects (P < .05). The effects of TCDD on extracellular signal‐related kinase (ERK), aryl hydrocarbon receptor, aryl hydrocarbon receptor repressor, and cytochrome P450 1A1 levels in MC3T3‐E1 cells were significantly inhibited by actein. The levels of superoxide dismutase, ERK1, and nuclear factor kappa B mRNA were also effectively restored by pretreatment with actein. Furthermore, actein treatment resulted in a significant increase in alkaline phosphatase (ALP) activity and collagen content, as well as in the expression of genes associated with osteoblastic differentiation (ALP, type I collagen, osteoprotegerin, bone sialoprotein, and osterix). This study demonstrates the underlying molecular mechanisms of cytoprotection exerted by actein against TCDD‐induced oxidative stress and osteoblast damage.     

Neutrophils contribute to intracerebral haemorrhages after treatment with recombinant tissue plasminogen activator following cerebral ischaemia

Background and purpose:

Polymorphonuclear neutrophils (PMNs) contribute to the vascular damage caused by transient cerebral ischaemia. Here we have evaluated the role of PMNs in intracerebral haemorrhage (ICH) induced in a model of thrombolysis with recombinant tissue plasminogen activator (t-PA) during the acute phase of cerebral ischaemia.

Experimental approach:

The middle cerebral artery (MCA) of male spontaneously hypertensive rats was occluded for 1 h followed by reperfusion and, 5 h later, infusion of thrombolytic products (generated in vitro by t-PA on autologous clots). Effects of pretreatment (before the MCA occlusion) with vinblastine (4 days before; 0.5 mg·kg⁻¹), monoclonal anti-neutrophil antibody (mAbRP3; 12 h, 0.3 mg·kg⁻¹) or saline on ICH, neutrophil infiltration, MCA vascular reactivity and brain infarct volume were assessed, 24 h after the beginning of reperfusion.

Key results:

Depletion of circulating neutrophils significantly reduced t-PA-induced ICH (vinblastine, 4.6 ± 1.0; mAbRP3, 5.2 ± 1.0 vs. saline, 10.8 ± 2.7 haemorrhages; P < 0.05). This depletion was associated with a decrease in cerebral infiltration by neutrophils and a decrease of endothelium-dependent, vascular dysfunction in isolated MCA, induced by the ischaemia/reperfusion and t-PA treatment. Brain infarct volume was significantly decreased after vinblastine treatment (159 ± 13 mm³ vs. 243 ± 16 mm³ with saline; P < 0.01) but not after depletion with mAbRP3 (221 ± 22 mm³).

Conclusions and implications:

Our results showed that pharmacological depletion of PMNs prevented t-PA-induced ICH, in parallel with a decrease in cerebral infiltration by PMNs and a decreased endothelial dysfunction in cerebral blood vessels.     

Stimulation of 90- and 70-kDa ribosomal protein S6 kinases by arginine vasopressin and lysophosphatidic acid in rat cardiomyocytes

Arginine vasopressin (AVP) and lysophosphatidic acid (LPA) have been shown to stimulate protein kinase C (PKC) and mitogen-activated protein (MAP) kinases and the proliferation of vascular smooth muscle cells. However, the actions of these two agents in cardiomyocytes are less well understood. To investigate the signal transduction pathways of AVP and LPA, freshly isolated adult rat cardiomyocytes were examined. Both AVP and LPA induced concentration- and time-dependent stimulation of the phosphotransferase activities of p90 ribosomal S6 kinases (RSK) and their upstream activators, extracellularly regulated kinases (ERK) 1 and 2. The activation of ERK1 and ERK2 by LPA was PKC- and phosphatidylinositol 3-kinase (PI 3-kinase)-dependent. However, AVP-induced activation of RSK2, a downstream substrate of ERK1 and ERK2, was PKC-dependent and PI 3-kinase-independent. AVP and LPA were also observed to increase the phosphotransferase activity of p70 ribosomal protein S6 kinase (p70 S6K) in a time- and concentration-dependent manner. The activation of p70 S6K by LPA and AVP was PI 3-kinase-dependent. PKC was necessary in AVP- but not in LPA-induced activation of p70 S6K. Since RSK and p70 S6K have been implicated in the regulation of translational control of protein synthesis, we concluded that AVP and LPA may stimulate the growth of cardiomyocytes through these two protein kinase cascades.     

Use of lipid rafting for the analysis of human basophil activation by flow cytometry

Background

Human leukocyte activation induced by specific and non-specific stimuli is characterized by the formation of lipid rafts defined as lipid-ordered domains that are more tightly packed than the surrounding non-raft phase of the bilayer. These lipid rafts are formed in parallel with profound membrane reorganization.

Objectives

Analyse the rafting and non-rafting proteins present on the activated and resting basophil membrane and study their interest for the flow cytometric analysis of basophil activation.

Methods

Human basophils obtained from samples used for diagnostic cellular tests such as basophil or lymphocyte activation tests were stimulated either by the formyl-methionyl-leucyl-phenylalanine peptide (fMLP), by an anti-IgE or by an allergen. After 40 min at 37°C, they were labelled by different antibodies conjugated to fluorescent dyes as an anti-IgE FITC, an anti-CCR3 PE, an anti-CD63, an anti-CD203c PE, an anti-11b, annexin V FITC or by cholera toxin FITC. Moreover, several experiments were analysed using an Amnis cytometer, allowing one to obtain the picture of the analysed cells.

Results

Anti-IgE or specific allergen elicits a membrane neo expression of CD63 at a high density and is poorly represented on resting basophil membrane. Upon an IgE-dependant activation some of the markers already present on resting basophil membrane, as CD203c, are up regulated and others, such as the IgE/IgE FcεRI receptor and CCR3 are down regulated and submitted to the formation of clusters demonstrated by the pictures taken with the Amnis cytometer. For non-IgE dependant activators, such as fMLP, the picture was different since IgE was not down regulated, whereas CCR3 was down regulated. As demonstrated using annexin V or the cholera toxin used for analysing apoptosis, these phenomenon were paralleled by the formation of lipid rafts, gangliosides domains, such as GM1, which is accessible from the extra cellular medium.

Conclusions

Basophil activation leads to membrane events close to the apoptosis phenomenon. The flow cytometric analysis of these membrane events may lead to protocols for allergen-induced activation and, may significantly increase cellular test sensitivity, particularly for drugs allergy diagnosis for which the usual protocols, such as those using CD63 alone, are insufficiently sensitive.

     

Size‐Dependent Isotropic/Nematic Phase Transition Behavior of Liquid Crystalline Peptide Nanowires

We present size‐tunable assemblies of peptide nanowires and study their liquid crystalline phase behavior. An aromatic peptide molecule of diphenylalanine was rapidly assembled into one‐dimensional nanowires, whose sizes were tunable according to the initial concentration of diphenylalanine in the preparing solution. The stable dispersion of peptide nanowires in an organic solvent exhibited colloidal nematic liquid crystalline phases. The liquid crystalline phase transition was governed by the repulsive electrostatic interaction among peptide nanowires as well as their shape anisotropy and polydispersity. Our work provides an interesting model system to investigate the liquid crystalline phase behaviors of a biomolecular assembly in terms of its assembled morphology and intermolecular interactions.

     

Neurogliovascular unit after cerebral ischemia: is the vascular wall a pharmacological target

Ischemic stroke induces drastic alterations of the functions of the neurogliovascular unit with dramatic consequences on the well-being of the patients in terms of cognitive and motor handicap. Nowadays, only very few therapeutics are available as a treatment of ischemic stroke. Ischemia is a multifactorial pathology involving different cerebral cellular components such as neurons, astrocytes and vessels working as a functional unit. Recent experimental strategy investigation involving different agents with antioxidant properties (dt-BC, stobadine) or pleiotropic effects (lipopolysaccharide, LPS) has been developed to evaluate whether the vascular wall could be considered as a potential target in neuroprotection concept.     

Methylation of p16(INK4A) and p57(KIP2) are involved in the development and progression of gastric MALT lymphomas.

p16(INK4A) and p57(KIP2) are inhibitors of cyclin-dependent kinases and their inactivation by methylation has been reported as a major tumorigenic mechanism in tumors. To examine whether methylation of p16(INK4A) and p57(KIP2) is involved in the development and progression of gastric MALT lymphomas, 24 gastric low-grade lymphomas of MALT, 11 diffuse large B-cell lymphomas, and 10 each case of gastric lymphoid follicles with and without Helicobacter pylori infection were studied. H. pylori infection was positive in 85.7% of the gastric lymphomas. In the gastric lymphoid follicles positive for H. pylori, methylation of p16(INK4A) was detected in 10% of cases, while methylation of p57(KIP2) was not detected. In low-grade MALT lymphomas, p16(INK4A) and p57(KIP2) were methylated in 41.7 and 29.2% of the cases, respectively. In diffuse large B-cell lymphomas, methylation of p16(INK4A) and p57(KIP2) was found in 72.7 and 36.4% of the cases, respectively. All but one case with p16(INK4A) and p57(KIP2) methylation was H. pylori positive and most of them were stage I. Our results indicate that methylation of p16(INK4A) followed by p57(KIP2) methylation involves during the tumorigenesis of gastric MALT lymphomas associated with H. pylori infection. As methylation of these two genes was more frequent in the higher grade (P<0.05), it may contribute to the malignant progression of gastric MALT lymphomas.     

Effects of milrinone on blood flow of the Y-graft composed with the radial and the internal thoracic artery in patients with coronary artery disease.

OBJECTIVE: Milrinone has been known to dilate the internal thoracic artery (ITA) and the radial artery (RA). The effect of milrinone, however, on each graft is unclear when the left ITA (LITA) and the RA form a Y-graft. This study evaluated the changes in blood flow of a composite Y-graft in response to milrinone. METHODS: Thirty-two patients undergoing an isolated coronary artery bypass graft surgery were included in this study. A Y-graft was created with an in situ LITA and free RA graft attached to the proximal side of the LITA. Graft flow was measured by opening the graft end for 30s, and is expressed in 'ml/min'. Graft flow and hemodynamic data were recorded before and 10 min after intravenous milrinone (50 microg/kg) administration. RESULTS: Milrinone significantly increased the RA graft flow, measured while the LITA graft end was clamped, and total Y-graft flow. Respective graft flows were not increased by milrinone when both clamps were released simultaneously, in spite of a significant decrease in the resistance of both grafts. The ratio of flows through the RA and the LITA grafts was not changed by milrinone. CONCLUSION: Milrinone significantly reduced RA and LITA resistances and increased the total Y-graft flow. Milrinone might dilate each individual arterial graft to a different degree. Milrinone did not, however, change the flow ratio through the RA to LITA grafts when they were measured simultaneously. Therefore, it would not significantly divert graft flow to one side in a composite Y-graft.