HYPOTHALAMIC COCAINE- AND AMPHETAMINE-REGULATED TRANSCRIPT AND CORTICOTROPHIN RELEASING FACTOR NEURONS ARE STIMULATED BY EXTRACELLULAR VOLUME AND OSMOTIC CHANGES

Several studies suggest that hypothalamic cocaine- and amphetamine-regulated transcript (CART) may interact with the hypothalamic-pituitary-adrenal (HPA) axis in the control of neuroendocrine function and may also participate in cardiovascular regulation. Therefore, this study aimed to evaluate, in experimental models of isotonic (I-EVE) and hypertonic (H-EVE) extracellular volume expansion and water deprivation (WD), the activation of CART- and corticotrophin releasing factor (CRF)-immunoreactive neurons, as well as the relative expression of CART and CRF mRNAs in the paraventricular (PVN) and supraoptic (SON) nuclei of the hypothalamus. Both H-EVE (0.30M NaCl, 2mL/100g of body weight, in 1 minute) and 24 hours of WD significantly increased plasma sodium concentrations, producing, respectively, either an increase or a decrease in extracellular volume. I-EVE (0.15M NaCl, 2mL/100g of body weight, in 1 minute) evoked a significant increase in the circulating volume accompanied by unaltered plasma concentrations of sodium. CART-expressing neurons of both magnocellular and parvocellular hypothalamic divisions were activated to produce Fos in response to H-EVE but not in response to I-EVE. Furthermore, increased expression of CART mRNA was found in the PVN of H-EVE but not I-EVE rats. These data show for the first time that EVE not only activates hypothalamic CRF neurons but also increases CRF mRNA expression in the PVN. In contrast, WD increases the number of CART-immunoreactive neurons activated to produce Fos in the PVN and SON but does not change the number of neurons double labeled for Fos and CRF or expression of CRF mRNA in the PVN. These findings provided new insights into the participation of CART in diverse processes within the PVN and SON, including its possible involvement in activation of the HPA axis and cardiovascular regulation in response to changes in extracellular volume and osmolality. (C) 2011 IBRO. Published by Elsevier Ltd. All rights reserved.

Release of cytokines by stimulated peripheral blood mononuclear cells in chronic periodontitis

Objective: The emergence of periodontal medicine increased interest in defining the behaviour of peripheral blood cells in periodontitis subjects in comparison with healthy group. The aim of this study was to evaluate the levels of interleukin (IL)-8, tumour necrosis factor-alpha (TNF-alpha), IL-6 and IL-10 released by Escherichia coli lipopolysaccharide (LPS)-stimulated peripheral blood mononuclear cells (PBMC) obtained from the peripheral blood of chronic periodontitis subjects. Design: PBMC samples were isolated from 19 systemically healthy donors, divided into generalized chronic periodontitis (n = 10) and healthy (n = 9) subjects. Cells were incubated for 24-48 h in 500 mu L wells containing RPM! 1640 and stimulated with 1.0 ng/mL of E. coli LPS. Supernatants were used to quantify the amounts of IL-8, TNF-alpha, IL-6 and IL-10 released using enzyme-linked immunosorbent assay (ELISA). Results: PBMC cells from periodontitis subjects released higher levels of TNF-alpha and IL-6 than those from healthy subjects (P < 0.05). Conversely, the supernatants of the stimulated PBMC cells obtained from healthy subjects presented higher amounts of IL-8 than those from periodontitis (P < 0.05). No differences were observed in the levels of IL-10 (P > 0.05) between groups. Conclusion: In conclusion, the results of the present study showed that E. coli LPS-stimulated PBMC from subjects with periodontitis present a different pattern of cytokine release when compared to PBMC from healthy subjects. This phenomenon could have implications locally, in periodontitis, as well as in systemic diseases. (C) 2010 Elsevier Ltd. All rights reserved.

A comparative study of the radiation resistance of four optically transparent polyimides

A comparative study of the high energy radiation resistance to formation of radicals in two pairs of polymers is reported. In one pair of polymers the phenyl groups containing the imide rings are separated by an ether linkage and in the other pair they are separated by an hexafluoroisopropylidine group. Two of the polymers contained aromatic amine units linked through an ether linkage and the other two polymers contained a trifluoromethyl biphenyl diamine. The polymers were shown to retain a high level of transparency in the visible region following radiolysis to doses as high as 8 Gy. ESR studies of the resistance to radical formation on radiolysis. at 77 K revealed that the polymers containing ether linkages were more stable than their fluorinated analogues, but all were less stable than Kapton (R). (C) 2001 Elsevier Science Ltd. All rights reserved.

Optically driven micromachine elements

We report on a proof of principle demonstration of an optically driven micromachine element. Optical angular momentum is transferred from a circularly polarized laser beam to a birefringent particle confined in an optical tweezers trap. The optical torque causes the particle to spin at up to 350 Hz, and this torque is harnessed to drive an optically trapped microfabricated structure. We describe a photolithographic method for producing the microstructures and show how a light driven motor could be used in a micromachine system. (C) 2001 American Institute of Physics.

Nocodazole inhibits insulin-stimulated glucose transport in 3T3-L1 adipocytes via a microtubule-independent mechanism

Insulin stimulates glucose transport in adipocytes and muscle cells by triggering redistribution of the GLUT4 glucose transporter from an intracellular perinuclear location to the cell surface. Recent reports have shown that the microtubule-depolymerizing agent nocodazole inhibits insulin-stimulated glucose transport, implicating an important role for microtubules in this process. In the present study we show that 2 mum nocodazole completely depolymerized microtubules in 3T3-L1 adipocytes, as determined morphologically and biochemically, resulting in dispersal of the perinuclear GLUT4 compartment and the Golgi apparatus. However, 2 mum nocodazole did not significantly effect either the kinetics or magnitude of insulin-stimulated glucose transport. Consistent with previous studies, higher concentrations of nocodazole (10-33 mum) significantly inhibited basal and insulin-stimulated glucose uptake in adi. pocytes. This effect was not likely the result of microtubule depolymerization because in the presence of taxol, which blocked nocodazole-induced depolymerization of microtubules as well as the dispersal of the perinuclear GLUT4 compartment, the inhibitory effect of 10-33 muM nocodazole on insulin-stimulated glucose uptake prevailed. Despite the decrease in insulin-stimulated glucose transport with 33 muM nocodazole we did not observe inhibition of insulin-stimulated GLUT4 translocation to the cell surface under these conditions. Consistent with a direct effect of nocodazole on glucose transporter function we observed a rapid inhibitory effect of nocodazole on glucose transport activity when added to either 3T3-L1 adipocytes or to Chinese hamster ovary cells at 4 degreesC. These studies reveal a new and unexpected effect of nocodazole in mammalian cells which appears to occur independently of its microtubule-depolymerizing effects.

The role of Ca2+ in insulin-stimulated glucose transport in 3T3-L1 cells

We have examined the requirement for Ca2+ in the signaling and trafficking pathways involved in insulin-stimulated glucose uptake in 3T3-LI adipocytes. Chelation of intracellular Ca2+, using 1,2-bis (o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra (acetoxymethyl) ester (BAPTA-AM), resulted in >95% inhibition of insulin-stimulated glucose uptake. The calmodulin antagonist, W13, inhibited insulin-stimulated glucose uptake by 60%. Both BAPTA-AM and W13 inhibited Akt phosphorylation by 70-75%. However, analysis of insulin-dose response curves indicated that this inhibition was not sufficient to explain the effects of BAPTA-AM and W13 on glucose uptake. BAPTA-AM inhibited insulin-stimulated translocation of GLUT4 by 50%, as determined by plasma membrane lawn assay and subcellular fractionation. In contrast, the insulin-stimulated appearance of HA-tagged GLUT4 at the cell surface, as measured by surface binding, was blocked by BAPTA/AM.. While the ionophores A23187 or ionomycin prevented the inhibition of Akt phosphorylation and GLUT4 translocation by BAPTA-AM, they did not overcome the inhibition of glucose transport. Moreover, glucose uptake of cells pretreated with insulin followed by rapid cooling to 4 degreesC, to promote cell surface expression of GLUT4 and prevent subsequent endocytosis, was inhibited specifically by BAPTA-AM. This indicates that inhibition of glucose uptake by BAPTA-AM is independent of both trafficking and signal transduction. These data indicate that Ca2+ is involved in at least two different steps of the insulin-dependent recruitment of GLUT4 to the plasma membrane. One involves the translocation step. The second involves the fusion of GLUT4 vesicles with the plasma membrane. These data are consistent with the hypothesis that Ca2+/cahnodulin plays a fundamental role in eukaryotic vesicle docking and fusion. Finally, BAPTA-AM may inhibit the activity of the facilitative transporters by binding directly to the transporter itself.

Human immunodeficiency virus type 1 reverse transcription is stimulated by Tat from other lentiviruses

The tat gene is required by HIV-1 for efficient reverse transcription and this function of Tat can be distinguished from its role in transcription by RNA polymerase II using tat point mutations that abrogate each function independently The mechanism of Tat's role in reverse transcription, however, is not known, nor is it known whether this role is conserved among trans-activating factors in other retroviruses. Here we examine the abilities of heterologous viral trans-activating proteins from jembrana disease virus (jTat), HIV-2 (Tat2), and equine infectious anemia virus (eTat) to substitute for HIV-1 Tat (Tat1) and restore reverse transcription in HIV-1 carrying an inactivated tat gene. Natural endogenous reverse transcription assays showed that trans-activators from some retroviruses (Tat2 and jTat, but not eTat) could substitute for Tat1 in complementation of HIV-1 reverse transcription. Finally, we show that Y47 is critical for Tat1 to function in reverse transcription, but not HIV-1 gene expression. We mutated the homologous position in jTat to H62Y and found it did not improve its ability to stimulate reverse transcription, but an H62A mutation did inhibit jTat complementation. These data highlight the finding that the role of Tat in reverse transcription is not related to trans-activation and demonstrate that other tat genes conserve this function. (C) 2002 Elsevier Science (USA).

Reversible luminescence thermochromism in dipotassiumsodium tris[dicyanoargentate(I)] and the role of structural phase transitions

As a function of temperature, the layered compound K2Na[Ag(CN)213 displays dramatic variations in luminescence thermochromism with major trend changes occurring around 80 K. In order to understand these interesting optical properties, high-resolution neutron diffraction investigations were performed on a polycrystalline sample of this material in the temperature range from 1.5 to 300 K, and previous synchrotron X-ray data of Larochelle et al. (Solid State Commun. 114, 155 (2000)) were reinterpreted. The corresponding significant structural changes were found to be continuous with an anomalous increase of the monoclinic c-lattice parameter with decreasing temperature, associated with slight reorientations of two inequivalent, approximately linear N-C-Ag-C-N units. In the whole temperature range, the crystal structure is monoclinic with the space group C2/m. Based on the structural results, the major luminescence thermochromism changes around 80 K are attributed to the dominance of a back energy transfer process from low- to high-energy excitons at high temperatures. (E) 2002 Elsevier Science (USA).

Synthesis of optically complex core-shell colloidal suspensions: Pathways to multiplexed biological screening

The ability to generate enormous random libraries of DNA probes via split-and-mix synthesis on solid supports is an important biotechnological application of colloids that has not been fully utilized to date. To discriminate between colloid-based DNA probes each colloidal particle must be 'encoded' so it is distinguishable from all other particles. To this end, we have used novel particle synthesis strategies to produce large numbers of optically encoded particle suitable for DNA library synthesis. Multifluorescent particles with unique and reproducible optical signatures (i.e., fluorescence and light-scattering attributes) suitable for high-throughput flow cytometry have been produced. In the spectroscopic study presented here, we investigated the optical characteristics of multi-fluorescent particles that were synthesized by coating silica 'core' particles with up to six different fluorescent dye shells alternated with non-fluorescent silica 'spacer' shells. It was observed that the diameter of the particles increased by up to 20% as a result of the addition of twelve concentric shells and that there was a significant reduction in fluorescence emission intensities from inner shells as an increasing number of shells were deposited.

Color and Luminescence stability of selected dental materials in vitro

To study luminescence, reflectance, and color stability of dental composites and ceramics. Materials and Methods: IPS e.max, IPS Classic, Gradia, and Sinfony materials were tested, both unpolished (as-cast) and polished specimens. Coffee, tea, red wine, and distilled water (control) were used as staining drinks. Disk-shaped specimens were soaked in the staining drinks for up to 5 days. Color was measured by a colorimeter. Fluorescence was recorded using a spectrofluorometer, in the front-face geometry. Time-resolved fluorescence spectra were recorded using a laser nanosecond spectrofluorometer. Results: The exposure of the examined dental materials to staining drinks caused changes in color of the composites and ceramics, with the polished specimens exhibiting significantly lower color changes as compared to unpolished specimens. Composites exhibited lower color stability as compared to ceramic materials. Water also caused perceptible color changes in most materials. The materials tested demonstrated significantly different initial luminescence intensities. Upon exposure to staining drinks, luminescence became weaker by up to 40%, dependent on the drink and the material. Time-resolved luminescence spectra exhibited some red shift of the emission band at longer times, with the lifetimes in the range of tens of nanoseconds. Conclusions: Unpolished specimens with a more developed surface have lower color stability. Specimens stored in water develop some changes in their visual appearance. The presently proposed methods are effective in evaluating the luminescence of dental materials. Luminescence needs to be tested in addition to color, as the two characteristics are uncorrelated. It is important to further improve the color and luminescence stability of dental materials.

One-step preparation of optically transparent Ni-Fe oxide film electrocatalyst for oxygen evolution reaction

Optically transparent cocatalyst film materials is very desirable for improved photoelectrochemical (PEC)oxygen evolution reaction (OER) over light harvesting photoelectrodes which require the exciting light to irradiate through the cocatalyst side, i.e., front-side illumination. In view of the reaction overpotential at electrode/electrolyte interface, the OER electrocatalysts have been extensively used as cocatalysts for PEC water oxidation on photoanode. In this work, the feasibility of a one-step fabrication of the transparent thin film catalyst for efficient electrochemical OER is investigated. The Ni-Fe bimetal oxide films, 200 nm in thickness, are used for study. Using a reactive magnetron co-sputtering technique, transparent(> 50% in wavelength range 500-2000 nm) Ni-Fe oxide films with high electrocatalytic activities were successfully prepared at room temperature. Upon optimization, the as-prepared bimetal oxide film with atomic ratio of Fe/Ni = 3:7 demonstrates the lowest overpotential for the OER in aqueous KOH solution, as low as 329 mV at current density of 2 mA cm 2, which is 135 and 108 mV lower than that of as-sputtered FeOx and NiOx thin films, respectively. It appears that this fabrication strategy is very promising to deposit optically transparent cocatalyst films on photoabsorbers for efficient PEC water splitting.

Kinetics of IFN-gamma, TNF-alpha, IL-10 and IL-4 production by mononuclear cells stimulated with gp43 peptides, in patients cured of paracoccidioidomycosis

We analyzed the kinetics of cytokine production by mononuclear cells from 17 patients who had been treated for paracoccidioidomycosis, using the stimulus of gp43 peptide groups (43kDa glycoprotein of Paracoccidioides brasiliensis) at 0.1 and 1µM, gp43 (1µg/ml) and crude Paracoccidioides brasiliensis antigen (PbAg; 75µg/ml). IFN-gamma production was a maximum at 144 hours in relation to the G2 and G8 peptide groups at 1µM and was greatest at 144 hours when stimulated by gp43 and by PbAg. The maximum TNF-alpha production was at 144 hours for the G2 group (0.1µM) and for gp43. IL-10 production was highest after 48 and 72 hours for G7 and G6 at 1µM, respectively. We also suggest the best time for analysis of IL4 production. These results may contribute towards future studies with gp43 peptides and encourage further investigations with the aim of understanding the influence of these peptides on the production of inflammatory and regulatory cytokines.

IFN stimulated gene expression in the liver is a better predictor of treatment response in chronic hepatitis c than the IL28b (IFN lambda 3) genotype

Background: Therapy of chronic hepatitis C (CHC) with pegIFNa/ribavirin achieves sustained virologic response (SVR) in ~55%. Pre-activation of the endogenous interferon system in the liver is associated non-response (NR). Recently, genome-wide association studies described associations of allelic variants near the IL28B (IFNλ3) gene with treatment response and with spontaneous clearance of the virus. We investigated if the IL28B genotype determines the constitutive expression of IFN stimulated genes (ISGs) in the liver of patients with CHC. Methods: We genotyped 93 patients with CHC for 3 IL28B single nucleotide polymorphisms (SNPs, rs12979860, rs8099917, rs12980275), extracted RNA from their liver biopsies and quantified the expression of IL28B and of 8 previously identified classifier genes which discriminate between SVR and NR (IFI44L, RSAD2, ISG15, IFI22, LAMP3, OAS3, LGALS3BP and HTATIP2). Decision tree ensembles in the form of a random forest classifier were used to calculate the relative predictive power of these different variables in a multivariate analysis. Results: The minor IL28B allele (bad risk for treatment response) was significantly associated with increased expression of ISGs, and, unexpectedly, with decreased expression of IL28B. Stratification of the patients into SVR and NR revealed that ISG expression was conditionally independent from the IL28B genotype, i.e. there was an increased expression of ISGs in NR compared to SVR irrespective of the IL28B genotype. The random forest feature score (RFFS) identified IFI27 (RFFS = 2.93), RSAD2 (1.88) and HTATIP2 (1.50) expression and the HCV genotype (1.62) as the strongest predictors of treatment response. ROC curves of the IL28B SNPs showed an AUC of 0.66 with an error rate (ERR) of 0.38. A classifier with the 3 best classifying genes showed an excellent test performance with an AUC of 0.94 and ERR of 0.15. The addition of IL28B genotype information did not improve the predictive power of the 3-gene classifier. Conclusions: IL28B genotype and hepatic ISG expression are conditionally independent predictors of treatment response in CHC. There is no direct link between altered IFNλ3 expression and pre-activation of the endogenous system in the liver. Hepatic ISG expression is by far the better predictor for treatment response than IL28B genotype.

Tegumental Ca-stimulated adenosine triphosphatase activity in adult Schistosoma mansoni worms

A Ca-stimulated ATPase activity (pH 9.5) associated with the tegumental membrane enriched (TME) fraction of Schistosoma mansoni adults was partially inhibited by NAP-taurine or by increasing concentrations of chlorpromazine; endogenous calmodulin was found associated with the TME fraction. A similar activity (pH 8.6) was histochemically visualized whithin the tegument of fixed worms on the cytoplasmic leaflet of both the doubel surface membrane and the basement membrane; this reaction was inhibited by 1 µM chloropromazine and it was also observed on the inner side of double membrane vesicles present in the TME fraction. No ATPase activity could be seen at alkaline pH with added Mg or Na/K ions. Without ATP, the addition of external Ca to the fixed worms induced the appearance of lead precipitates on the tegumental discoid bodies; this reaction was inhibited by molybdate and not by chlorpromazine. The intrategumentary regulation of calcium by the systems described and the possible use of phenothiazines against schistosimes are discussed.