Tyrosine kinases and calcium dependent activation of endothelial cell phospholipase D by diperoxovanadate

Reactive oxygen species (ROS) mediated modulation of signal transduction pathways represent an important mechanism of cell injury and barrier dysfunction leading to the development of vascular disorders. Towards understanding the role of ROS in vascular dysfunction, we investigated the effect of diperoxovanadate (DPV), derived from mixing hydrogen peroxide and vanadate, on the activation of phospholipase D (PLD) in bovine pulmonary artery endothelial cells (BPAECs). Addition of DPV to BPAECs in the presence of .05% butanol resulted in an accumulation of [P-32] phosphatidylbutanol (PBt) in a dose- and time-dependent manner. DPV also caused an increase in tyrosine phosphorylation of several protein bands (Mr 20-200 kD), as determined by Western blot analysis with antiphosphotyrosine antibodies. The DPV-induced [P-32] PBt-accumulation was inhibited by putative tyrosine kinase inhibitors such as genistein, herbimycin, tyrphostin and by chelation of Ca2+ with either EGTA or BAPTA, however, pretreatment of BPAECs with the inhibitor PKC bisindolylmaleimide showed minimal inhibition. Also down-regulation of PKC alpha and epsilon, the major isotypes of PKC in BPAECs, by TPA (100 nM, 18 h) did not attenuate the DPV-induced PLD activation. The effects of putative tyrosine kinase and PKC inhibitors were specific as determined by comparing [P-32] PBt formation between DPV and TPA. In addition to tyrosine kinase inhibitors, antioxidants such as N-acetylcysteine and pyrrolidine dithiocarbamate also attenuated DPV-induced protein tyrosine phosphorylation and PLD stimulation. These results suggest that oxidation, prevented by reduction with thiol compounds, is involved in DPV-dependent protein tyrosine phosphorylation and PLD activation.

Fluorescence-polarization studies on binding of 4-methylumbelliferyl beta-D-galactopyranoside to Ricinus communis (castor-bean) agglutinin

The binding of Ricinus communis (castor-bean) agglutinin 1 to saccharides was studied by equilibrium dialysis and fluorescence polarization by using the fluorescently labelled sugar 4-methylumbelliferyl beta-D-galactopyranoside. No appreciable change in ligand fluorescence of 4-methylumbelliferyl beta-D-galactopyranoside was considerably polarized on its binding to the lectin. The association constants obtained by Scatchard analysis of equilibrium-dialysis and fluorescence-polarization data do not differ much from each other, and at 25 degrees C, Ka = 2.4 (+/- 0.2) X 10(4)M-1. These values agree reasonably well with that reported in the literature for Ricinus agglutinin 1. The number of binding sites obtained by the different experimental procedures is 1.94 +/- 0.1 per molecule of 120 000 daltons and is equal to the reported value of 2. The consistency in the values of Ka and number of binding sites indicate the absence of additional subsites on Ricinus agglutinin 1 for its specific sugars. In addition, the excellent agreement between the binding parameters obtained by equilibrium dialysis and fluorescence polarization indicate the potential of ligand-fluorescence-polarization measurements in the investigation of lectin-sugar interactions.

Fluorescence polarization as a tool to study lectin-sugar interaction. An investigation of the binding of 4-methylumbelliferyl beta-D-galactopyranoside to Abrus precatorious agglutinin

Polarization of ligand fluorescence was used to study the binding of 4-methylumbelliferyl beta-D-galactopyranoside (MeUmb-Galp) to Abrus precatorious agglutinin. The binding of the fluorescent sugar to the lectin led to considerable polarization of the MeUmb-Galp fluorescence, which was also quenched by about 30% on binding to the lectin. The binding of the fluorescent sugar was carbohydrate-specific, as evidenced by inhibition of both fluorescence polarization and quenching when lectin was preincubated with lactose. The association constant as determined by fluorescence polarization is 1.42 x 10(4) M-1 at 25 degrees C and is in excellent agreement with those determined by fluorescence quenching (Ka = 1.51 x 10(4) M-1) and equilibrium dialysis (Ka = 1.62 x 10(4) M-1) at 25 degrees C. The numbers of binding sites as determined by fluorescence polarization, quenching and equilibrium dialysis agree very well with one another, n being equal to 2.0 +/- 0.05. The consistency between the association constant value determined by fluorescence polarization, quenching and equilibrium dialysis shows the validity of this approach to study lectin-sugar interaction.

Crystal and molecular structure of the ammonium salt of the dinucleoside monophosphate d(CpG)

The crystal and molecular structure of the ammonium salt of deoxycytidylyl-(3'-5')-deoxyguanosine has been determined from 0.85 A resolution single crystal X-ray diffraction data. The crystals obtained by acetone diffusion technique at -20 degrees C, are orthorhombic, P212121, a = 12.880(2), b = 17444(2) and c = 27.642(2) A. The structure was solved by high resolution Patterson and Fourier methods and refined to R = 0.136. There are two d(CpG) molecules in the asymmetric unit forming a mini left handed Z-DNA helix. This is in contrast to the earlier reported forms of d(CpG) where the molecules form self base paired duplexes. There are two ammonium ions in the asymmetric unit. The major groove NH+4 ion interacts with N7 of guanines through water bridges besides making H-bonded interactions directly with the phosphate oxygen atoms. A second NH+4 ion is found in the minor groove interacting directly with the phosphate oxygen atoms. Symmetry related molecules pack in such a way that the cytosine base stacks on cytosine and guanine base on guanine. Our structure demonstrates that alternating d(CpG) sequences have the ability to adopt the left handed Z-DNA structure even at the dimer level i.e., in a sequence which is only two base pairs long.

Synthesis and characterization of mono- and bis-D-pi-A cryptand derivatives for second-order nonlinear optics and its modulation with different metal ion inputs

Two new classes of mono- and bis-D-pi-A cryptand derivatives with a flexible and a rigid cryptand core have been synthesized. The linear and nonlinear optical properties of these molecules are probed. The three dimensional cavity of the cryptand moiety has been utilized to modulate the SHG intensity to different extents in solution with metal ion inputs such as Ni-II,Cu-II,Zn-II, and Cd-II. We also report that decomplexation events can be used to reversibly modulate their NLO responses.

Room temperature synthesis of Mn2+ doped ZnS d-dots and observation of tunable dual emission: Effects of doping concentration, temperature, and ultraviolet light illumination

Mn2+ doped (0-50.0 molar %) ZnS d-dots have been synthesized in water medium by using an environment friendly low cost chemical technique. Tunable dual emission in UV and yellow-orange regions is achieved by tailoring the Mn2+ doping concentration in the host ZnS nanocrystal. The optimum doping concentration for achieving efficient photoluminescence (PL) emission is determined to be similar to 1.10 (at. %) corresponding to 40.0 (molar %) of Mn2+ doping concentration used during synthesis. The mechanism of charge transfer from the host to the dopant leading to the intensity modulated tunable (594-610 nm) yellow-orange PL emission is straightforwardly understood as no capping agent is used. The temperature dependent PL emission measurements are carried out, viz., in 1.10 at. % Mn2+ doped sample and the experimental results are explained by using a theoretical PL emission model. It is found that the ratio of non-radiative to radiative recombination rates is temperature dependent and this phenomenon has not been reported, so far, in Mn2+ doped ZnS system. The colour tuning of the emitted light from the samples are evident from the calculated chromaticity coordinates. UV light irradiation for 150 min in 40.0 (molar %) Mn2+ doped sample shows an enhancement of 33% in PL emission intensity. (C) 2013 American Institute of Physics. http://dx.doi.org/10.1063/1.4795779]

Ultrafast photoinduced enhancement of nonlinear optical response in 15-atom gold clusters on indium tin oxide conducting film

We show that the third order optical nonlinearity of 15-atom gold clusters is significantly enhanced when in contact with indium tin oxide (ITO) conducting film. Open and close aperture z-scan experiments together with non-degenerate pump-probe differential transmission experiments were done using 80 fs laser pulses centered at 395 nm and 790 nm on gold clusters encased inside cyclodextrin cavities. We show that two photon absorption coefficient is enhanced by an order of magnitude as compared to that when the clusters are on pristine glass plate. The enhancement for the nonlinear optical refraction coefficient is similar to 3 times. The photo-induced excited state absorption using pump-probe experiments at pump wavelength of 395 nm and probe at 790 nm also show an enhancement by an order of magnitude. These results attributed to the excited state energy transfer in the coupled gold cluster-ITO system are different from the enhancement seen so far in charge donor-acceptor complexes and nanoparticle-conjugate polymer composites.

Performance analysis of beacon-less IEEE 802.15.4 multi-hop networks

We develop an approximate analytical technique for evaluating the performance of multi-hop networks based on beacon-less CSMA/CA as standardised in IEEE 802.15.4, a popular standard for wireless sensor networks. The network comprises sensor nodes, which generate measurement packets, and relay nodes which only forward packets. We consider a detailed stochastic process at each node, and analyse this process taking into account the interaction with neighbouring nodes via certain unknown variables (e.g., channel sensing rates, collision probabilities, etc.). By coupling these analyses of the various nodes, we obtain fixed point equations that can be solved numerically to obtain the unknown variables, thereby yielding approximations of time average performance measures, such as packet discard probabilities and average queueing delays. Different analyses arise for networks with no hidden nodes and networks with hidden nodes. We apply this approach to the performance analysis of tree networks rooted at a data sink. Finally, we provide a validation of our analysis technique against simulations.

Adaptive Mesh Refinement for Fast Convergence of EFIE-Based 3-D Extraction

3-D full-wave method of moments (MoM) based electromagnetic analysis is a popular means toward accurate solution of Maxwell's equations. The time and memory bottlenecks associated with such a solution have been addressed over the last two decades by linear complexity fast solver algorithms. However, the accurate solution of 3-D full-wave MoM on an arbitrary mesh of a package-board structure does not guarantee accuracy, since the discretization may not be fine enough to capture spatial changes in the solution variable. At the same time, uniform over-meshing on the entire structure generates a large number of solution variables and therefore requires an unnecessarily large matrix solution. In this paper, different refinement criteria are studied in an adaptive mesh refinement platform. Consequently, the most suitable conductor mesh refinement criterion for MoM-based electromagnetic package-board extraction is identified and the advantages of this adaptive strategy are demonstrated from both accuracy and speed perspectives. The results are also compared with those of the recently reported integral equation-based h-refinement strategy. Finally, a new methodology to expedite each adaptive refinement pass is proposed.

Method for measurement of the angles of 90-, 45-, 45-deg and 60-, 30-, 90-deg prisms

A simple technique is devised to measure the angles of 90-, 45-, 45-deg and 60-, 30-, 90-deg prisms without using expensive spectrometers, autocollimators, and angle gauges. The method can be extended to unpolished and opaque prisms made of materials other then glass. (C) 1997 Society of Photo-Optical instrumentation Engineers.

Effect of substituent on the thermodynamics of D-glucopyranoside binding to concanavalin A, pea (Pisum sativum) lectin and lentil (Lens culinaris) lectin

Titration calorimetry measurements of the binding of phenyl-alpha (alpha PhOGlu), 3-methoxy (3MeOGlu), fluorodeoxy and deoxy derivatives of alpha-D-glucopyranose (Glu) to concanavalin A (conA), pea lectin and lentil lectin were performed at approx. 10 and 25 degrees C in 0.01 M dimethylglutaric acid/NaOH buffer, pH 6.9, containing 0.15 M NaCl and Mn2+ and Ca2+ ions. Apparently the 3-deoxy, 4-deoxy and 6-deoxy as well as the 4-fluorodeoxy and 6-fluorodeoxy derivatives of Glu do not bind to the lectins because no heat release was observed on the addition of aliquots of solutions of these derivatives to the lectin solutions. The binding enthalpies, delta H0b, and entropies, delta S0b, determined from the measurements were compared with the same thermodynamic binding parameters for Glu, D-mannopyranoside and methyl-alpha- D-glucopyranoside (alpha MeOGlu). The binding reactions are enthalpically driven with little change in the heat capacity on binding, and exhibit enthalpy-entropy compensation. Differences between the thermodynamic binding parameters can be rationalized in terms of the interactions apparent in the known crystal structures of the methyl-alpha-D-mannopyranoside-conA [Derewenda, Yariv, Helliwell, Kalb (Gilboa), Dodson, Papiz, Wan and Campbell (1989) EMBO J. 8, 2189-2193] and pea lectin-trimanno-pyranoside [Rini, Hardman, Einspahr, Suddath and Carber (1993) J. Biol. Chem. 268, 10126-10132] complexes. Increases in the entropy change on binding are observed for alpha MeOGlu binding to pea and lentil lectin, for alpha PhOGlu binding to conA and pea lectin, and for 3MeOGlu binding to pea lectin relative to the entropy change for Glu binding, and imply that the phenoxy and methoxy substituents provide additional hydrophobic interactions in the complex. Increases in the binding enthalpy relative to that of Glu are observed for deoxy and fluoro derivatives in the C-1 and C-2 positions and imply that these substituents weaken the interaction with the surrounding water, thereby strengthening the interaction with the binding site.

Studies on follicular atresia: role of gonadotropins and gonadal steroids in regulating cathepsin-D activity of preovulatory follicles in the rat

Preovulatory follicular atresia was studied using pregnant mare serum gonadotropin (PMSG)-primed rats (15 IU/rat) which were deprived of hormonal support either by allowing the metabolic clearance of the PMSG or by injecting a specific PMSG antiserum (PMSG a/s). Atresia was monitored by an increase in lysosomal cathepsin-D activity and a decrease in the receptor activity of the granulosa cells (GC) isolated from the preovulatory follicles. It was shown that the increase in lysosomal activity and the decrease in receptor activity seen at 96 h after PMSG (or PMSG plus PMSG a/s) could be arrested both by follicle stimulating hormone (FSH) and luteinizing hormone (LH). Injection of cyanoketone or clomiphene citrate together with FSH/LH prevented this 'rescue' suggesting a role for estrogens in the regulation of atresia. Although the administration of estradiol-17 beta (20 micrograms/rat) together with PMSG a/s could show a 'rescue effect' in terms of reduction in cathepsin-D activity the gonadotropin receptor activities of these granulosa cells were not restored. The injection of dihydrotestosterone (DHT) to 48 h PMSG-primed rats induced atresia as noted by an increase in cathepsin-D activity. However, the exogenous administration of FSH along with DHT prevented this atretic effect suggesting that DHT is not having a direct effect on atresia. Determination of androgen: estrogen content of the granulosa cells and an analysis of the individual profile of androgen and estrogen revealed that the increase in cathepsin-D activity could be correlated only with the decrease in GC estrogen content. This along with the observation that GC showed a loss of estrogen synthesis well before the increase in cathepsin-D activity strongly points out that the lack of estrogen rather than an increase in androgen is the principle factor responsible for the atresia of preovulatory follicles in the rat.

X-ray Studies on Crystalline Complexes Involving Amino Acids and Peptides. XIII. Effect of Chirality on Molecular Aggregation: The Crystal Structures of L-Arginine D-Aspartate and L-Arginine D-Glutamate Trihydrate

The crystal structures of (1) L-arginine D-asparate, C6HIsN40~.C4H6NO4 [triclinic, P1, a=5.239(1), b=9.544(1), c=14.064(2)A, a=85"58(1), /3=88.73 (1), ~/=84.35 (1) °, Z=2] and (2) L-arginine D-glutamate trihydrate, C6H15N40~-.CsHsNO4.3H20 [monoclinic, P2~, a=9.968(2), b=4.652(1), c=19.930 (2) A, fl = 101.20 (1) °, Z = 2] have been determined using direct methods. They have been refined to R =0.042 and 0.048 for 2829 and 2035 unique reflections respectively [I>2cr(I)]. The conformations of the two arginine molecules in the aspartate complex are different from those observed so far in the crystal structures of arginine, its salts and complexes. In both complexes, the molecules are organized into double layers stacked along the longest axis. The core of each double layer consists of two parallel sheets made up of main-chain atoms, each involving both types of molecules. The hydrogen bonds within each sheet and those that interconnect the two sheets give rise to EL-, DD- and DE-type head-to-tail sequences. Adjacent double layers in (1) are held together by side-chain-side-chain interactions whereas those in (2) are interconnected through an extensive network of water molecules which interact with sidechain guanidyl and carboxylate groups. The aggregation pattern observed in the two LD complexes is fundamentally different from that found in the corresponding EL complexes.

Split-Freedom And Mvd-Intersection - A New Characterization Of Multivalued Dependencies Having Conflict-Free Covers

Sets of multivalued dependencies (MVDs) having conflict-free covers are important to the theory and design of relational databases [2,12,15,16]. Their desirable properties motivate the problem of testing a set M of MVDs for the existence of a confiict-free cover. In [8] Goodman and Tay have proposed an approach based on the possible equivalence of M to a single (acyclic) join dependency (JD). We remark that their characterization does not lend an insight into the nature of such sets of MVDs. Here, we use notions that are intrinsic to MVDs to develop a new characterization. Our approach proceeds in two stages. In the first stage, we use the notion of “split-free” sets of MVDs and obtain a characterization of sets M of MVDs having split-free covers. In the second, we use the notion of “intersection” of MVDs to arrive at a necessary and sufficient condition for a split-free set of MVDs to be conflict-free. Based on our characterizations, we also give polynomial-time algorithms for testing whether M has split-free and conflict-free covers. The highlight of our approach is the clear insight it provides into the nature of sets of MVDs having conflict-free covers. Less emphasis is given in this paper to the actual efficiency of the algorthms. Finally, as a bonus, we derive a desirable property of split-free sets of MVDs,thereby showing that they are interesting in their own right.