Vanadate-Stimulated Nadh Oxidation Requires Polymeric Vanadate, Phosphate and Superoxide

NADH oxidation. catalyzed by the microsomal enzyme system is stimulated on addition of polymeric vanitdate Maximum stimulation by polymeric vanadnte was obtained in the presence of phosphate buffer. The small stimulation obtained by metavanadat (500μM) increased on aciditication followed by neutralization. or on adding a trace amount of polymeric vanadate (1 μM).     

Enzymatic isolation and structural characterisation of polymeric suberin of cork from Quercus suber L

An enzymatic method has been used to isolate, for the first time, polymeric suberin from the bark of Quercus suber L. or cork. This was achieved by solvent extraction (dichloromethane, ethanol and water), followed by a step-by-step enzymatic treatment with cellulase, hemicellulase and pectinase, and a final extraction with dioxane/water. The progress of suberin isolation was monitored by Fourier transform infrared spectroscopy using a photoacoustic cell (FTIR-PAS). The material obtained (polymeric suberin (PS)) was characterised by solid-state and liquid-state nuclear magnetic resonance, FTIR-PAS and vapour pressure osmometry, and compared with the suberin fraction obtained by alkaline depolymerisation (depolymerised suberin (DS)). The results showed that PS is an aliphatic polyester of saturated and unsaturated fatty acids, with an average molecular weight (M(w)) of 2050 g mol(-1). Although this fraction represents only 10% of the whole suberin of cork, its polymeric nature gives valuable information about the native form of the polymer. DS was found to have an average M(w) of 750 g mol(-1) and to comprise a significant amount of acidic and alcoholic short aliphatic chains.     

Neuropeptide Y stimulates prostacyclin production in porcine vascular endothelial cells

We investigated the effects of neuropeptide Y on the prostacyclin production of cultured porcine aortic endothelial cells by measuring the stable metabolite of prostacyclin, 6-keto-prostaglandin F1 alpha, by radioimmunoassay. Neuropeptide Y induced dose- and time-dependent stimulation of prostacyclin production by cultured porcine aortic endothelial cells. The lowest stimulatory concentration of neuropeptide Y was 10(-8) M and maximal response, a 2.8 fold rise, was obtained with 10(-6) M. The stimulation lasted at least 24 h. The effect was associated with the stimulation of arachidonic acid release. Our data suggest that neuropeptide Y may inhibit the development of atherosclerosis by stimulating prostacyclin synthesis.     

Suppressive effects of co-stimulatory molecule expressions on mouse splenocytes by anti-allergic agents in vitro

The influence of anti-allergic drugs, epinastine hydrochloride (EP) and disodium cromoglycate (DSCG), on the co-stimulatory molecule expression was examined using in vitro cell culture technique. Spleen cells obtained from BALB/c mice 10 days after immunization with haemocyanin absorbed to aluminium hydroxide were cultured in the presence of 100.0 microg/ml haemocyanin and various concentrations of the agents. Low concentrations (<1.5 x 10(-4)M) of EP and DSCG did not influence spleen cell blastic activity induced by antigenic stimulation, whereas these agents caused significant inhibition of spleen cell activation when 2 x 10(-4) M of the agents were added to cell cultures. EP and DSCG also did not affect blastic activity of sensitized splenic T cells by anti-CD3 monoclonal antibody stimulation even when these cells were cultured in the presence of 2 x 10(-4) M of the agents. We next examined the influence of EP and DSCG on the expression of co-stimulatory molecules on spleen cells in response to antigenic stimulation. Sensitized spleen cells were cultured in the presence of 2 x 10(-4)M of the agents and the expression of molecules were examined by flow cytometer 24h later. EP and DSCG suppressed the expression of costimulatory molecules, CD40 and CD80, but not CD86, on splenic B cells which were enhanced by antigenic stimulation in vitro.     

Taste transduction mechanism: similar effects of various modifications of gustatory receptors on neural responses to chemical and electrical stimulation in the frog

Responses in the frog glossopharyngeal nerve induced by electrical stimulation of the tongue were compared with those induced by chemical stimuli under various conditions. (a) Anodal stimulation induced much larger responses than cathodal stimulation, and anodal stimulation of the tongue adapted to 5 mM MgCl2 produced much larger responses than stimulation with the tongue adapted to 10 mM NaCl at equal current intensities, as chemical stimulation with MgCl2 produced much larger responses than stimulation with NaCl at equal concentration. (b) The enhansive and suppressive effects of 8-anilino-1-naphthalenesulfonate, NiCl2, and uranyl acetate on the responses to anodal current were similar to those on the responses to chemical stimulation. (c) Anodal stimulation of the tongue adapted to 50 mM CaCl2 resulted in a large response, whereas application of 1 M CaCl2 to the tongue adapted to 50 mM CaCl2 produced only a small response. This, together with theoretical considerations, suggested that the accumulation of salts on the tongue surface is not the cause of the generation of the response to anodal current. (d) Cathodal current suppressed the responses induced by 1 mM CaCl2, 0.3 M ethanol, and distilled water. (e) The addition of EGTA or Ca-channel blockers (CdCl2 and verapamil) to the perfusing solution of the lingual artery reversibly suppressed both the responses to chemical stimulus (NaCl) and to anodal current with 10 mM NaCl. (f) We assume from the results obtained that electrical current from the microvillus membrane of a taste cell to the synaptic area supplied by anodal stimulation or induced by chemical stimulation activates the voltage-dependent Ca channel at the synaptic area.     

Dual effects of muscarinic M2 receptors on the synthesis of cyclic AMP in CHO cells: background and model

It has been observed in several laboratories that muscarinic agonists have dual effects on the synthesis of cyclic AMP in cell lines expressing muscarinic M2 or M4 receptors, producing strong inhibition at low agonist concentrations and lesser inhibition or stimulation at high agonist concentrations. Data obtained on CHO cells (known to express adenylyl cyclases VI and VII) are best interpreted on the assumption that the upward phase of the concentration-response curves reflects simultaneous inhibition of adenylyl cyclase VI via the Gi proteins, with which the M2 and M4 receptors communicate with high affinity, and stimulation of adenyly cyclases VI and VII via the Gs proteins, with which the M2 and M4 receptors communicate with low affinity. A simplified model is described which permits one to predict how the shapes of the concentration-response curves will be affected by changes in the concentration of receptors, the affinities of activated receptors for Gi or Gs proteins, and other parameters.     

Serotonin-Elicited Amplification of Adenylate Cyclase Activity in Hippocampal Membranes from Adult Rat

The activity of the adenylate cyclase located in membranes prepared from hippocampus of adult rat can be stimulated by serotonin (5-HT) (Ka = 4 X 10(-7) M). The maximal effect is obtained with 10 microM 5-HT. Freezing of the tissue decreases the 5-HT stimulation; this stimulation is optimal in the presence of 82.5 mM Tris-maleate buffer (pH 7.4) and 50 microM GTP. The adenylate cyclase activity of membranes prepared from cortex, hypothalamus, and colliculi of adult rats is not significantly stimulated by 5-HT. Dopamine (DA) also stimulates adenylate cyclase located in hippocampal membranes; its effect can be blocked by haloperidol (10(-6) M), which fails to inhibit 5-HT stimulation. Moreover, p-chlorophenylalanine treatment for 2 weeks or selective lesion of 5-HT axons afferent to the hippocampus increases the Vmax of 5-HT stimulation, but fails to change that of DA stimulation. The 5-HT stimulation can be inhibited by metergoline, spiroperidol, and pizotyline (10(-6) M), but not by the same concentrations of mianserin, ketanserine, alprenolol, phenoxybenzamine, and mepyramine. The 5-HT stimulation of adenylate cyclase of hippocampal membranes can be mimicked by tryptamine, 5-methoxytryptamine, bufotenine, and to a lesser extent by LSD; N-methyltryptamine, N-methyltryptophan, and 5-hydroxytryptophan are inactive. Studies with kainic acid suggest that the 5-HT recognition site (5-HT1) linked to adenylate cyclase is located on the membrane of intrinsic hippocampal neurons.     

A quasi-elastic light scattering study of smooth muscle myosin in the presence of ATP.

We have investigated the hydrodynamic properties of turkey gizzard smooth muscle myosin in solution using quasi-elastic light scattering (QELS). The effects of ionic strength (0.05-0.5 M KCl) and light chain phosphorylation on the conformational transition of myosin were examined in the presence of ATP at 20 degrees C. Cumulant analysis and light scattering models were used to describe the myosin system in solution. A nonlinear least squares fitting procedure was used to determine the model that best fits the data. The conformational transition of the myosin monomer from a folded form to an extended form was clearly demonstrated in a salt concentration range of 0.15-0.3 M KCl. Light chain phosphorylation regulates the transition and promotes unfolding of the myosin. These results agree with the findings obtained using sedimentation velocity and electron microscopy (Onishi and Wakabayashi, 1982; Trybus et al., 1982; Trybus and Lowey, 1984). In addition, we present evidence for polymeric myosin coexisting with the two monomeric myosin species over a salt concentration range from 0.05 to 0.5 M KCl. The size of the polymeric myosin varied with salt concentration. This observation supports the hypothesis that, in solution, a dynamic equilibrium exists between the two conformations of myosin monomer and filaments.     

Modulation of cholinergic neurotransmission in airways by enkephalin

We compared the effects of methionine enkephalin and leucine enkephalin on contractions of isolated canine tracheal smooth muscle strips induced by field electrical stimulation (ES) and exogenous acetylcholine (approximately 10(-5) M). Methionine and leucine enkephalin (10(-8) to 10(-5) M), when added at the peak of airway contractions induced by ES at 1 Hz, depressed the contractions in a concentration-dependent manner by a maximum of 95 and 99%, respectively. Acetylcholine-induced contractions of similar magnitude were depressed only 4% by methionine enkephalin and 12% by leucine enkephalin. Frequency-response curves (0.5-20 Hz) were also obtained before and after incubation of tracheal strips with 10(-5) M methionine and leucine enkephalin. Enkephalin depressed contractions induced by stimulation at 0.5 and 1 Hz by an average of 98 and 95%, respectively. The inhibitory effect of enkephalin progressively decreased at successively higher stimulus frequencies until at 20 Hz there was no significant difference between airway contractions obtained in the presence and absence of enkephalin. Naloxone (3 X 10(-5) M) antagonized the inhibitory effects of both enkephalins. We conclude that methionine and leucine enkephalins inhibit the release of acetylcholine from the postganglionic parasympathetic neurons that innervate airway smooth muscle.     

Lymphoblastoid cell adhesion mediated by a dimeric and polymeric endogenous beta-galactoside-binding lectin (galaptin).

Glutaraldehyde-polymerized human splenic galaptin, a beta-galactoside-binding lectin, was demonstrated to have enhanced hemagglutinating and asialofetuin binding activity relative to native dimeric galaptin when these lectins were present in solution. The polymerized lectin consisted primarily of 2-, 4- and 12-membered species after reductive alkylation. Both forms of galaptin bound, at 4 degrees C, to saturable B lymphoblastoid cell surface receptors. Estimates obtained by Scatchard analyses, with the binding data expressed in terms of 14.5 kDa subunit molarity, were 5 x 10(7) binding sites/cell with affinity constant Ka = 2.2 x 10(5) M for dimeric galaptin and 17 x 10(7) binding sites/cell with Ka = 3.4 x 10(5) M-1 for polymeric galaptin. Both forms of galaptin adsorbed to polystyrene with high efficiency; however, only plastic-adsorbed polymeric galaptin mediated adhesion of lymphoblastoid cells. Cell adhesion was inhibited by lactose. Plastic-adsorbed polymeric galaptin bound asialofetuin more efficiently than dimeric galaptin. Asialofetuin binding was inhibited 65% and 30-50% by lactose for plastic-adsorbed polymeric and dimeric galaptin, respectively. Native fetuin bound to the adsorbed dimeric galaptin in a lactose-insensitive manner. These data indicate that cell surface receptor-galaptin interaction is carbohydrate specific whereas polystyrene-adsorbed galaptin may demonstrate protein-protein interactions with soluble ligands.     

mTRAQ‐based quantification of potential endometrial carcinoma biomarkers from archived formalin‐fixed paraffin‐embedded tissues

Formalin‐fixed paraffin‐embedded (FFPE) tissues are the primary and preferred medium for archiving patients' samples. Here we demonstrate relative quantifications of protein biomarkers in extracts of laser microdissected epithelial cells from FFPE endometrial carcinoma tissues versus those from normal proliferative endometria by means of targeted proteomic analyses using LC–multiple reaction monitoring (MRM) MS with MRM Tags for Relative and Absolute Quantitation (mTRAQ) labeling. Comparable results of differential expressions for pyruvate kinase isoform M2 (PK‐M2) and polymeric Ig receptor were observed between analyses on laser microdissected epithelial cells from FFPE tissues and corresponding homogenates from frozen tissues of the same individuals that had previously been analyzed and reported. We also identified PK‐M2 in the normal proliferative phase of the endometrium. Other biomarkers in addition to PK‐M2 and polymeric Ig receptor were also observed but not consistently and/or were at levels below the threshold for quantification.     

The CDTA-soluble pectic substances from soybean meal are composed of rhamnogalacturonan and xylogalacturonan but not homogalacturonan

Structural characteristics of pectic substances extracted from soybean meal cell walls (water unextractable solids) with a chelating agent-containing buffer (0.05M 1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CDTA) and 0.05M NH(4)-oxalate in 0.05M NaOAc buffer) were studied. The arabinogalactans present as side chains to the rhamnogalacturonan backbone were largely removed by enzymatic hydrolysis using endo-galactanase, exo-galactanase, endo-arabinanase, and arabinofuranosidase B. The remaining pectic backbone appeared to be resistant to enzymatic degradation by pectolytic enzymes. After partial acid hydrolysis of the isolated pectic backbone, one oligomeric and two polymeric populations were obtained by size-exclusion chromatography. Monosaccharide and linkage analyses, enzymatic degradation, and NMR spectroscopy of these populations showed that the pectic substances in the original extract contain both rhamnogalacturonan and xylogalacturonan regions, while homogalacturonan is absent.     

Myosin accelerates synaptic vesicle recycling in the motor nerve endings

Neurotransmitter release and exocytosis of synaptic vesicles in the motor nerve endings of the frog cutaneous-pectoris muscle were studied using electrophysiological and optical methods under the conditions of inhibition of the myosin light-chain kinase and non-muscle myosin by the specific inhibitors ML-7 (12 μM) and (–)-blebbistatin (100 μM). At high-frequency stimulation (20 pulses/s), these inhibitors strengthened suppression of transmitter release during the first 20–25 s and slowed down the release of the fluorescent dye FM 1-43. The obtained results indicate that myosin accelerates rapid synaptic vesicle recycling upon high-frequency stimulation.     

Pre-junctional inhibitory action of prostaglandin E2 on excitatory neuro-effector transmission in the human bronchus

The effects of prostaglandin E2 (PGE2) and indomethacin on excitatory neuro-effector transmission in the human bronchus were investigated by tension recording and microelectrode methods. PGE2 (10(-10)-10(-9)M) suppressed the amplitude of twitch contractions and excitatory junction potentials (e.j.ps) evoked by field stimulation at a steady level of basal tension obtained by the combined application of indomethacin (10(-5) M) and FPL55712 (10(-6) M). In doses over 10(-8)M, PGE2 reduced the muscle tone and dose-dependently suppressed the amplitude of twitch contractions. Indomethacin (10(-5) or 5 x 10(-5) M) reduced the muscle tone and enhanced the amplitude of twitch contractions and e.j.ps evoked by field stimulation in the presence of FPL55712. PGE2 (10(-9) M) had no effect on the post-junctional response of smooth muscle cells to exogenously applied acetylcholine (ACh) (4 x 10(-7) M). However, indomethacin (10(-5) M) significantly enhanced the ACh-induced contraction of the human bronchus. These results indicate that PGE2 in low concentrations has a pre-junctional action to inhibit excitatory neuro-effector transmission in addition to a post-junctional action, presumably by suppressing transmitter release from the vagus nerve terminals in the human bronchial tissues.     

Fluorescent labeling of RAFT-generated poly(N-isopropylacrylamide) via a facile maleimide-thiol coupling reaction

We report a facile labeling technique in which the telechelic thiocarbonylthio functionality of well-defined poly(N-isopropylacrylamide) (PNIPAM) prepared by room temperature RAFT polymerization is first converted to the thiol and subsequently reacted with a maleimido-functional fluorescent dye, N-(1-pyrene)maleimide (PM). Nearly monodisperse PNIPAM (M(n) = 39 500 g/mol, M(w)/M(n) = 1.07) was synthesized using a trithiocarbonate-based CTA, 2-dodecylsulfanylthiocarbonylsulfanyl-2-methyl propionic acid (DMP), and a conventional azo-initiator, namely, 2,2'-azobis(4-methoxy-2,4-dimethylvaleronitrile) (V-70), as the primary source of radicals. The key to successful conjugation of PM to PNIPAM is the implementation of a two-step reduction process involving (1) the cleavage of the trithiocarbonate with a strong reducing agent, in this case, NaBH4, to form a mixture of polymeric thiols and disulfides and (2) the conjugation of PM to the pure polymeric thiol in the presence of tris(2-carboxyethyl)phosphine.HCl (TCEP). We show that TCEP efficiently eliminates the formation of polymeric disulfides and thus allows for the desired addition of the free polymeric thiol across the maleimide double bond. This concept is demonstrated using SEC-MALLS and UV-vis spectroscopy measurements.     

Disulfide bond polymerization of a cyclic peptide derived from the surface loop 4 of class 1 OMP of Neisseria meningitidis

Summary Two peptides derived from the surface loop 4 of class 1 Outer Membrane Protein (OMP) ofNeisseria meningitidis were synthesized on solid phase using the Boc/Bzl strategy: one containing the entire loop 4 cyclized and the other representing the polymerized cyclic loop 4. To test a more efficient cyclic peptide presentation, in the present study a strategy was developed to obtain polymers of cyclic peptides. In order to obtain the polymeric cyclic peptide, two protecting groups for cysteine were used — Acm and Mob. The Cys(Acm)-protected cyclic peptide was obtained after removing the Mob group. The polymerization reaction was carried out by simultaneous deprotection/oxidation ofS-Acm with iodine. Analysis of the polymeric cyclic peptide in Tris-tricine-SDS-PAGE showed different bands with molecular weights higher than expected for the corresponding monomeric cyclic peptide. Both peptides were used in immunization of four different mouse strains. The antisera raised against the peptides were evaluated by ELISA and Western blotting vs. OMP preparation ofN. meningitidis. The titers raised against the polymerized cyclic peptide were higher than the ones raised against the cyclic peptide. The antisera elicited did not show bactericidal activity. Nevertheless, the antisera elicited against the polymeric cyclic peptide in the CBA/J mouse strain showed opsonic activity. The antibodies raised against the polymeric cyclic peptide were successfully used as probes in Western blotting experiments to verify the display of loop 4 peptide on the surface of filamentous phage M13.     

The activation of cAMP-dependent protein kinase is directly linked to the stimulation of bone resorption by parathyroid hormone.

The present study was performed to characterize the direct involvement of cAMP in the stimulation of bone resorption by parathyroid hormone (PTH), using Sp-cAMPS and Rp-cAMPS, which were the direct agonist and antagonist in the activation of cAMP-dependent protein kinase (PKA), respectively. Bone resorbing activity was estimated as the number of pits formed on the dentine slice and total area of pits per slice in bone marrow cells derived from 2 week-old mice. Dibutyryl cAMP (dbcAMP)(10(-4)M) and Sp-cAMPS (10(-4)M) caused the remarkable stimulation of bone resorption. Although Rp-cAMPS (10(-4)M) did not affect bone resorption by itself, it significantly inhibited dbcAMP- and Sp-cAMPS-induced stimulation of bone resorption. Moreover, Rp-cAMPS (10(-4)M) antagonized 10(-7)M human PTH-(1-34)-induced stimulation of bone resorption, although it did not affect 10(-8)M 1,25(OH)2D3-induced stimulation of bone resorption. Present study indicates the direct involvement of PKA in the stimulation of bone resorption by PTH.     

Stimulation of glycosaminoglycan sulfotransferase from chick embryo cartilage by basic proteins and polyamines

A soluble glycosaminoglycan sulfotransferase (3'-phosphoadenylylsulfate:chondroitin 4'-sulfotransferase, EC 2.8.2.5) from chick embryo cartilage has been prepared free from endogenous acceptor. The reaction with this enzyme preparation was stimulated by basic proteins and polyamines, the degree of stimulation being dependent on the chemical nature of both basic compounds and acceptor glycosaminoglycans. A maximum stimulation was obtained when protamine (basic compound) and chondroitin (acceptor) were involved in the reaction mixture at a molar ratio of protamine to repeating disaccharide units of chondroitin, 1:100. The stimulation of sulfotransferase activity by basic substances was much higher than that by Mn2+. However, increasing the Mn2+ concentration immediately reduced the stimulation by basic substances. The Km value for 3'-phosphoadenosine 5'-phosphosulfate of the sulfotransferase, when chondroitin was used as acceptor, was 1 . 10(-6) M in the presence of 25 microgram/ml protamine, compared to 2 . 10(-5) M in the absence of protamine. These observations indicate that the basic proteins and polyamines may interact with acceptor polysaccharide, thereby causing an increase in the affinity of the enzyme toward 3'-phosphoadenosine 5'-phosphosulfate.     

Effects of the cannabinoid receptor agonist CP 55,940 and the cannabinoid receptor antagonist SR 141716 on intracranial self-stimulation in Lewis rats.

Lewis rats were trained to self-stimulate the medial forebrain bundle (MFB) using a rate-frequency paradigm. They were then tested for the effects of the cannabinoid receptor agonist CP 55,940, the selective cannabinoid receptor antagonist SR 141716 and the dopamine D1 receptor antagonist SCH 23390. CP 55,940 (0, 10, 25 and 50 microg/kg i.p.) had no effect on MFB self-stimulation behaviour as assessed by the M50, the stimulation frequency at which half-maximal response rates were obtained. With SR 141716, only a very high dose (20 mg/kg i.p.) caused a significant inhibition of the rewarding efficacy of the stimulation. This was seen as an increase in the M50. All other doses of SR 141716 (0, 1, 3, 10 mg/kg i.p.) were ineffective in modulating the M50. By comparison, a relatively low dose (0.06 mg/kg i.p.) of SCH 23390 caused a large increase in M50. These results indicate a relatively modest influence, if any at all, of exogenous or endogenous cannabinoids on reward-relevant neurotransmission.