Resonance raman detection and estimation in the aqueous phase using water dispersible cyclodextrin: reduced-graphene oxide sheets

Resonance Raman spectroscopy is a powerful analytical tool for detecting and identifying analytes, but the associated strong fluorescence background severely limits the use of the technique. Here, we show that by attaching beta-cyclodextrin (beta-CD) cavities to reduced graphene-oxide (rGO) sheets we obtain a water dispersible material (beta-CD: rGO) that combines the hydrophobicity associated with rGO with that of the cyclodextrin cavities and provides a versatile platform for resonance Raman detection. Planar aromatic and dye molecules that adsorb on the rGO domains and nonplanar molecules included within the tethered beta-CD cavities have their fluorescence effectively quenched. We show that it is possible using the water dispersible beta-CD: rGO sheets to record the resonance Raman spectra of adsorbed and included organic chromophores directly in aqueous media without having to extract or deposit on a substrate. This is significant, as it allows us to identify and estimate organic analytes present in water by resonance Raman spectroscopy.

Use of synthetic peptides to locate novel integrin alpha(2)beta(1)-binding motifs in human collagen III

A set of 57 synthetic peptides encompassing the entire triple-helical domain of human collagen III was used to locate binding sites for the collagen-binding integrin alpha(2)beta(1). The capacity of the peptides to support Mg2+-dependent binding of several integrin preparations was examined. Wild-type integrins (recombinant alpha(2) I-domain, alpha(2)beta(1) purified from platelet membranes, and recombinant soluble alpha(2)beta(1) expressed as an alpha(2)-Fos/beta(1)-Jun heterodimer) bound well to only three peptides, two containing GXX'GER motifs (GROGER and GMOGER, where O is hydroxyproline) and one containing two adjacent GXX'GEN motifs (GLKGEN and GLOGEN). Two mutant alpha(2) I-domains were tested: the inactive T221A mutant, which recognized no peptides, and the constitutively active E318W mutant, which bound a larger subset of peptides. Adhesion of activated human platelets to GER-containing peptides was greater than that of resting platelets, and HT1080 cells bound well to more of the peptides compared with platelets. Binding of cells and recombinant proteins was abolished by anti-alpha(2) monoclonal antibody 6F1 and by chelation of Mg2+. We describe two novel high affinity integrin-binding motifs in human collagen III (GROGER and GLOGEN) and a third motif (GLKGEN) that displays intermediate activity. Each motif was verified using shorter synthetic peptides.

Phosphorylation of CLEC-2 is dependent on lipid rafts, actin polymerization, secondary mediators, and Rac

The C-type lectin-like receptor 2 (CLEC-2) activates platelets through Src and Syk tyrosine kinases via a single cytoplasmic YxxL motif known as a hem immunoreceptor tyrosine-based activation motif (hemITAM). Here, we demonstrate using sucrose gradient ultracentrifugation and methyl-beta-cyclodextrin treatment that CLEC-2 translocates to lipid rafts upon ligand engagement and that translocation is essential for hemITAM phosphorylation and signal initiation. HemITAM phosphorylation, but not translocation, is also critically dependent on actin polymerization, Rac1 activation, and release of ADP and thromboxane A(2) (TxA(2)). The role of ADP and TxA(2) in mediating phosphorylation is dependent on ligand engagement and rac activation but is independent of platelet aggregation. In contrast, tyrosine phosphorylation of the GPVI-FcRgamma-chain ITAM, which has 2 YxxL motifs, is independent of actin polymerization and secondary mediators. These results reveal a unique series of proximal events in CLEC-2 phosphorylation involving actin polymerization, secondary mediators, and Rac activation.

An inclusion complex of β-Cyclodextrin-L-Phenylalanine : 1H NMR and molecular docking studies

An inclusion host-guest complex between β-cyclodextrin (β-CD) and L-phenylalanine (LPhe) was investigated using ¹H nuclear magnetic resonance spectroscopy and molecular docking techniques. ¹H chemical shift changes of β-CD were used to calculate the stability constant (K_stb) of the complex. On the basis of the Hildebrand-Benesi method, the K_stb of the 1:1 complex in D₂O solution at 300 K, pD 7.6 was of 25.5 M^-1, implying a fast intermolecular exchange rate process. Interestingly, docking simulation indicates the toroidal space can be occupied by L-Phe with two favorable arrangements. For the predicted model with the higher probability score, the L-Phe aromatic ring is facing to the secondary hydroxyl groups of β-CD. Results from NMR and docking simulation are in good agreement with the x-ray structures of β-CD/L-phenylalanine derivatives.

Pharmacological evidence for beta(2)-adrenoceptor in right atria from stressed female rats

The purpose of the present study was to demonstrate a physiological response to TA2005, a potent beta(2)-adrenoceptor (beta(2)-AR) selective agonist, in right atria isolated from stressed female rats under the influence of the estrous cycle. We obtained concentration-response curves to the agonist in the presence and in the absence of selective antagonists in right atria isolated from female rats submitted to three daily foot-shock sessions (30 min duration, 120 pulses of 1.0 mA, 1.0 s, applied at random intervals of 5-25 s) and sacrificed at estrus or diestrus. Our results showed that the pD(2), values of TA2005 were not influenced by estrous cycle phase or foot-shock stress. However, in right atria from stressed rats sacrificed during diestrus, the concentration-response curve to TA2005 was biphasic, with a response being obtained at concentrations of 0.1 nM, whereas during estrus no response was observed at doses lower than 3 nM. ICI118,551, a beta(2)-AR antagonist, abolished the response to nanomolar concentrations of TA2005 in right atria from stressed rats at diestrus, with no changes in a,agonist pot values in right atria from control rats (7.47 +/- 0.09, p > 0.05) but a 3-fold decrease in pD(2), values of TA2005 in right atria from foot shock stressed rats (7.90 +/- 0.07, p less than or equal to 0.05). Concentration-response curves to TA2005 in the presence of ICI118,551 were best fitted by a one-site model equation. The beta(1)-AR antagonist, CGP20712A, shifted to the right only the second part of the concentration-response curves to the agonist, unmasking the putative Pz-AR-mediated response to the agonist in tissues isolated from stressed rats at diestrus. Under this condition, concentration-response curves to the agonist were best fitted by a two-site model equation. pot and maximum response of TA2005 interaction with beta(1)- and putative Pz-adrenoceptor components were calculated. Schild analyses gave a pK(B) value for CGP20712A that was typical for the interaction with beta(1)-AR in each experimental group. pK(B), values for ICI118,551 could not be obtained in stressed rats sacrificed at diestrus since Schild plot slopes were lower than 1.0. In right atria from control rats, ICI118,551 pK(B), values were similar to reported values for the interaction of the antagonist with beta(1)-AR. These results confirm that a heterogenous PAR population mediating the chronotropic response to catecholamines can be demonstrated in right atria from foot shock stressed female rats sacrificed at diestrus. The stress-induced response seems to be mediated by the beta(2)-AR subtype. Right atria from rats sacrificed during estrus are protected against stress-induced alterations on the homogeneity of PAR population.

Modulation of synaptic transmission in hippocampal CA1 neurons by a novel neurotoxin (beta-pompilidotoxin) derived from wasp venom

We examined the effects of beta-pompilidotoxin (beta-PMTX), a neurotoxin derived from wasp venom. on synaptic transmission in the mammalian central nervous system (CNS). Using hippocampal slice preparations of rodents, we made both extracellular and intracellular recordings from the CA1 pyramidal neurons in response to stimulation of the Schaffer collateral/commissural fibers. Application of 5-10 muM beta-PMTX enhanced excitatory postsynaptic potentials (EPSPs) but suppressed the fast component of the inhibitory postsynaptic potentials (IPSPs). In the presence of 10 muM bicuculline, beta-PMTX potentiated EPSPs that were composed of both non-NMDA and NMDA receptor-mediated potentials. Potentiation of EPSPs was originated by repetitive firings of the prosynaptic axons, causing Summation of EPSPs. In the presence of 10 muM CNQX and 50 muM APV, beta-PMTX suppressed GABA(A) receptor-mediated fast IPSPs but retained GABA(B) receptor-mediated slow IPSPs. Our results suggest that beta-PMTX facilitates excitatory synaptic transmission by a presynaptic mechanism and that it causes overexcitation followed by block of the activity of some population of interneurons which regulate the activity of GABA(A) receptors. (C) 2001 Published by Elsevier B.V. Ireland Ltd and the Japan Neuroscience Society.

Lack of beta(2)-AR improves exercise capacity and skeletal muscle oxidative phenotype in mice

beta(2)-adrenergic receptor (beta(2)-AR) agonists have been used as ergogenics by athletes involved in training for strength and power in order to increase the muscle mass. Even though anabolic effects of beta(2)-AR activation are highly recognized, less is known about the impact of beta(2)-AR in endurance capacity. We presently used mice lacking beta(2)-AR [beta(2)-knockout (beta(2) KO)] to investigate the role of beta(2)-AR on exercise capacity and skeletal muscle metabolism and phenotype. beta(2) KO mice and their wild-type controls (WT) were studied. Exercise tolerance, skeletal muscle fiber typing, capillary-to-fiber ratio, citrate synthase activity and glycogen content were evaluated. When compared with WT, beta 2KO mice displayed increased exercise capacity (61%) associated with higher percentage of oxidative fibers (21% and 129% of increase in soleus and plantaris muscles, respectively) and capillarity (31% and 20% of increase in soleus and plantaris muscles, respectively). In addition, beta 2KO mice presented increased skeletal muscle citrate synthase activity (10%) and succinate dehydrogenase staining. Likewise, glycogen content (53%) and periodic acid-Schiff staining (glycogen staining) were also increased in beta 2KO skeletal muscle. Altogether, these data provide evidence that disruption of beta(2)AR improves oxidative metabolism in skeletal muscle of beta 2KO mice and this is associated with increased exercise capacity.

Increased vascular contractility and oxidative stress in beta(2)-adrenoceptor knockout mice: the role of NADPH oxidase

Background/Aims: beta(2)-adrenoceptor (beta(2)-AR) activation induces smooth muscle relaxation and endothelium-derived nitric oxide (NO) release. However, whether endogenous basal beta(2)-AR activity controls vascular redox status and NO bioavailability is unclear. Thus, we aimed to evaluate vascular reactivity in mice lacking functional beta(2)-AR (beta 2KO), focusing on the role of NO and superoxide anion. Methods and Results: Isolated thoracic aortas from beta 2KO and wild-type mice (WT) were studied. beta 2KO aortas exhibited an enhanced contractile response to phenylephrine compared to WT. Endothelial removal and L-NAME incubation increased phenylephrine-induced contraction, abolishing the differences between beta 2KO and WT mice. Basal NO availability was reduced in aortas from beta 2KO mice. Incubation of beta 2KO aortas with superoxide dismutase or NADPH inhibitor apocynin restored the enhanced contractile response to phenylephrine to WT levels. beta 2KO aortas exhibited oxidative stress detected by enhanced dihydroethidium fluorescence, which was normalized by apocynin. Protein expression of eNOS was reduced, while p47(phox) expression was enhanced in beta 2KO aortas. Conclusions: The present results demonstrate for the first time that enhanced NADPH-derived superoxide anion production is associated with reduced NO bioavailability in aortas of beta 2KO mice. This study extends the knowledge of the relevance of the endogenous activity of beta(2)-AR to the maintenance of the vascular physiology. Copyright (C) 2012 S. Karger AG, Basel

Human beta-defensin 2 and protease activated receptor-2 expression in patients with chronic periodontitis

Objective: Some previous studies have shown that gingipains, trypsin-like proteases produced by Porphyromonas gingivalis, up-regulate human beta defensin-2 (HBD-2) mRNA expression through protease-activated receptor-2 (PAR(2)) in gingival epithelial cells. This study aimed at investigating salivary HBD-2 levels and crevicular PAR(2) mRNA expression in human chronic periodontitis and evaluating whether periodontal treatment affected this process. Methods: Salivary and gingival crevicular fluid (GCF) samples were collected from periodontally healthy (control) and chronic periodontitis patients at baseline and 50 days after nonsurgical periodontal treatment. Salivary HBD-2, and GCF TNF-alpha levels were analysed by ELISA, and PAR(2) mRNA at the GCF was evaluated by RT-PCR. Results: P. gingivalis was significantly (p < 0.05) more prevalent in patients with chronic periodontitis when compared to controls. This prevalence decreased after periodontal therapy (p < 0.0001). The control group showed statistically significant lower levels of HBD-2, TNF-alpha, and PAR(2) expression when compared to the chronic periodontitis group. In addition, periodontal treatment significantly reduced PAR(2) expression and HBD-2 levels in chronic periodontitis patients (p < 0.001). Conclusions: Our results suggest that salivary HBD-2 levels and PAR(2) mRNA expression from GCF are higher in subjects with chronic periodontitis than in healthy subjects, and that periodontal treatment decreases both HBD-2 levels and PAR(2) expression. (C) 2012 Elsevier Ltd. All rights reserved.

The beta 2-Adrenoceptor Agonist Formoterol Improves Structural and Functional Regenerative Capacity of Skeletal Muscles From Aged Rat at the Early Stages of Postinjury

Skeletal muscles from old rats fail to completely regenerate following injury. This study investigated whether pharmacological stimulation of beta 2-adrenoceptors in aged muscles following injury could improve their regenerative capacity, focusing on myofiber size recovery. Young and aged rats were treated with a subcutaneous injection of beta 2-adrenergic agonist formoterol (2 mu g/kg/d) up to 10 and 21 days after soleus muscle injury. Formoterol-treated muscles from old rats evaluated at 10 and 21 days postinjury showed reduced inflammation and connective tissue but a similar number of regenerating myofibers of greater caliber when compared with their injured controls. Formoterol minimized the decrease in tetanic force and increased protein synthesis and mammalian target of rapamycin phosphorylation in old muscles at 10 days postinjury. Our results suggest that formoterol improves structural and functional regenerative capacity of regenerating skeletal muscles from aged rats by increasing protein synthesis via mammalian target of rapamycin activation. Furthermore, formoterol may have therapeutic benefits in recovery following muscle damage in senescent individuals.

TGF-beta 1 modulates the homeostasis between MMPs and MMP inhibitors through p38 MAPK and ERK1/2 in highly invasive breast cancer cells

Background: Metastasis is the main factor responsible for death in breast cancer patients. Matrix metalloproteinases (MMPs) and their inhibitors, known as tissue inhibitors of MMPs (TIMPs), and the membrane-associated MMP inhibitor (RECK), are essential for the metastatic process. We have previously shown a positive correlation between MMPs and their inhibitors expression during breast cancer progression; however, the molecular mechanisms underlying this coordinate regulation remain unknown. In this report, we investigated whether TGF-beta 1 could be a common regulator for MMPs, TIMPs and RECK in human breast cancer cell models. Methods: The mRNA expression levels of TGF-beta isoforms and their receptors were analyzed by qRT-PCR in a panel of five human breast cancer cell lines displaying different degrees of invasiveness and metastatic potential. The highly invasive MDA-MB-231 cell line was treated with different concentrations of recombinant TGF-beta 1 and also with pharmacological inhibitors of p38 MAPK and ERK1/2. The migratory and invasive potential of these treated cells were examined in vitro by transwell assays. Results: In general, TGF-beta 2, T beta RI and T beta RII are over-expressed in more aggressive cells, except for T beta RI, which was also highly expressed in ZR-75-1 cells. In addition, TGF-beta 1-treated MDA-MB-231 cells presented significantly increased mRNA expression of MMP-2, MMP-9, MMP-14, TIMP-2 and RECK. TGF-beta 1 also increased TIMP-2, MMP-2 and MMP-9 protein levels but downregulated RECK expression. Furthermore, we analyzed the involvement of p38 MAPK and ERK1/2, representing two well established Smad-independent pathways, in the proposed mechanism. Inhibition of p38MAPK blocked TGF-beta 1-increased mRNA expression of all MMPs and MMP inhibitors analyzed, and prevented TGF-beta 1 upregulation of TIMP-2 and MMP-2 proteins. Moreover, ERK1/2 inhibition increased RECK and prevented the TGF-beta 1 induction of pro-MMP-9 and TIMP-2 proteins. TGF-beta 1-enhanced migration and invasion capacities were blocked by p38MAPK, ERK1/2 and MMP inhibitors. Conclusion: Altogether, our results support that TGF-beta 1 modulates the mRNA and protein levels of MMPs (MMP-2 and MMP-9) as much as their inhibitors (TIMP-2 and RECK). Therefore, this cytokine plays a crucial role in breast cancer progression by modulating key elements of ECM homeostasis control. Thus, although the complexity of this signaling network, TGF-beta 1 still remains a promising target for breast cancer treatment.

Dynamic computer simulation of electrophoretic enantiomer migration order and separation in presence of a neutral cyclodextrin

One-dimensional dynamic computer simulation was employed to investigate the separation and migration order change of ketoconazole enantiomers at low pH in presence of increasing amounts of (2-hydroxypropyl)-β-cyclodextrin (OHP-β-CD). The 1:1 interaction of ketoconazole with the neutral cyclodextrin was simulated under real experimental conditions and by varying input parameters for complex mobilities and complexation constants. Simulation results obtained with experimentally determined apparent ionic mobilities, complex mobilities, and complexation constants were found to compare well with the calculated separation selectivity and experimental data. Simulation data revealed that the migration order of the ketoconazole enantiomers at low (OHP-β-CD) concentrations (i.e. below migration order inversion) is essentially determined by the difference in complexation constants and at high (OHP-β-CD) concentrations (i.e. above migration order inversion) by the difference in complex mobilities. Furthermore, simulations with complex mobilities set to zero provided data that mimic migration order and separation with the chiral selector being immobilized. For the studied CEC configuration, no migration order inversion is predicted and separations are shown to be quicker and electrophoretic transport reduced in comparison to migration in free solution. The presented data illustrate that dynamic computer simulation is a valuable tool to study electrokinetic migration and separations of enantiomers in presence of a complexing agent.

In situ activation of the type 2 ryanodine receptor in pancreatic beta cells requires cAMP-dependent phosphorylation

Molecular mechanisms that regulate in situ activation of ryanodine receptors (RY) in different cells are poorly understood. Here we demonstrate that caffeine (10 mM) released Ca2+ from the endoplasmic reticulum (ER) in the form of small spikes in only 14% of cultured fura-2 loaded beta cells from ob/ob mice. Surprisingly, when forskolin, an activator of adenylyl cyclase was present, caffeine induced larger Ca2+ spikes in as many as 60% of the cells. Forskolin or the phosphodiesterase-resistant PKA activator Sp-cAMPS alone did not release Ca2+ from ER. 4-Chloro-3-ethylphenol (4-CEP), an agent that activates RYs in other cell systems, released Ca2+ from ER, giving rise to a slow and small increase in [Ca2+]i in beta cells. Prior exposure of cells to forskolin or caffeine (5 mM) qualitatively altered Ca2+ release by 4-CEP, giving rise to Ca2+ spikes. In glucose-stimulated beta cells forskolin induced Ca2+ spikes that were enhanced by 3,9-dimethylxanthine, an activator of RYs. Analysis of RNA from islets and insulin-secreting βTC-3-cells by RNase protection assay, using type-specific RY probes, revealed low-level expression of mRNA for the type 2 isoform of the receptor (RY2). We conclude that in situ activation of RY2 in beta cells requires cAMP-dependent phosphorylation, a process that recruits the receptor in a functionally operative form.

Identification of pentosidine as a native structure for advanced glycation end products in beta-2-microglobulin-containing amyloid fibrils in patients with dialysis-related amyloidosis.

beta-2-Microglobulin (beta-2m) is a major constituent of amyloid fibrils in patients with dialysis-related amyloidosis (DRA). Recently, we found that the pigmented and fluorescent adducts formed nonenzymatically between sugar and protein, known as advanced glycation end products (AGEs), were present in beta-2m-containing amyloid fibrils, suggesting the possible involvement of AGE-modified beta-2m in bone and joint destruction in DRA. As an extension of our search for the native structure of AGEs in beta-2m of patients with DRA, the present study focused on pentosidine, a fluorescent cross-linked glycoxidation product. Determination by both HPLC assay and competitive ELISA demonstrated a significant amount of pentosidine in amyloid-fibril beta-2m from long-term hemodialysis patients with DRA, and the acidic isoform of beta-2m in the serum and urine of hemodialysis patients. A further immunohistochemical study revealed the positive immunostaining for pentosidine and immunoreactive AGEs and beta-2m in macrophage-infiltrated amyloid deposits of long-term hemodialysis patients with DRA. These findings implicate a potential link of glycoxidation products in long-lived beta-2m-containing amyloid fibrils to the pathogenesis of DRA.

Mammalian karyopherin alpha 1 beta and alpha 2 beta heterodimers: alpha 1 or alpha 2 subunit binds nuclear localization signal and beta subunit interacts with peptide repeat-containing nucleoporins.

Although only 44% identical to human karyopherin alpha 1, human karyopherin alpha 2 (Rch1 protein) substituted for human karyopherin alpha 1 (hSRP-1/NPI-1) in recognizing a standard nuclear localization sequence and karyopherin beta-dependent targeting to the nuclear envelope of digitonin-permeabilized cells. By immunofluorescence microscopy of methanol-fixed cells, karyopherin beta was localized to the cytoplasm and the nuclear envelope and was absent from the nuclear interior. Digitonin permeabilization of buffalo rat liver cells depleted their endogenous karyopherin beta. Recombinant karyopherin beta can bind directly to the nuclear envelope of digitonin-permeabilized cells at 0 degree C (docking reaction). In contrast, recombinant karyopherin alpha 1 or alpha 2 did not bind unless karyopherin beta was present. Likewise, in an import reaction (at 20 degrees C) with all recombinant transport factors (karyopherin alpha 1 or alpha 2, karyopherin beta, Ran, and p10) import depended on karyopherin beta. Localization of the exogenously added transport factors after a 30-min import reaction showed karyopherin beta at the nuclear envelope and karyopherin alpha 1 or alpha 2, Ran, and p10 in the nuclear interior. In an overlay assay with SDS/PAGE-resolved and nitrocellulose-transferred proteins of the nuclear envelope, 35S-labeled karyopherin beta bound to at least four peptide repeat-containing nucleoporins--Nup358, Nup214, Nup153, and Nup98.