Functional reconstitution of an eicosanoid-modulated Cl− channel from bovine tracheal smooth muscle

2002 ◽  
Vol 282 (3) ◽  
pp. C567-C577 ◽  
Author(s):  
Dany Salvail ◽  
Martin Cloutier ◽  
Eric Rousseau
Keyword(s):  
Inhibitory Concentration ◽  
Biochemical Characterization ◽  
Anion Exchange Chromatography ◽  
Biochemical Properties ◽  
Exchange Chromatography ◽  
Dependent Manner ◽  
Open Probability ◽  
Concentration Dependent Manner ◽  
Lipid Environment ◽  
Channel Control

We describe the biochemical properties of an eicosanoid-modulated Cl−channel and assess the mechanisms by which the epoxyeicosatrienoic acids (EETs) alter both its unitary conductance and its open probability ( P o). After a purification protocol involving wheat-germ agglutinin affinity and anion-exchange chromatography, the proteins were sequentially inserted into liposomes, which were then fused into PLBs. Functional and biochemical characterization tests confirm that the Cl− channel is a 55-kDa glycosylated monomer with voltage- and Ca2+concentration-independent activity. 5,6- and 8,9-EET decreased the conductance of the native channel (control conductance: 70 ± 5 pS in asymmetrical 50 mM trans/250 mM cis CsCl) in a concentration-dependent manner, with respective 50% inhibitory concentration values of 0.31 and 0.42 μM. These regioisomers similarly decreased the conductance of the purified channel (control conductance value: 75 ± 5 pS in asymmetrical 50 mM trans/250 mM cis CsCl), which had been stripped of its native proteic and lipidic environment. On the other hand, 5,6- and 8,9-EETs decreased the P o of the native channel with respective 50% inhibitory concentration values of 0.27 and 0.30 μM but failed to alter the P o of the purified protein. Thus we suggest that the effects of these EETs on channel conductance likely result from direct interactions of EET− anions with the channel pore, whereas the alteration of P o requires a lipid environment of specific composition that is lost on solubilization and purification of the protein.

scholarly journals Peptidoglycan O Acetylation and Autolysin Profile of Enterococcus faecalis in the Viable but Nonculturable State

2006 ◽  
Vol 188 (3) ◽  
pp. 902-908 ◽  
Author(s):  
John M. Pfeffer ◽  
Hendrik Strating ◽  
Joel T. Weadge ◽  
Anthony J. Clarke
Keyword(s):  
High Performance ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Hydroxyl Group ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Viable But Nonculturable ◽  
Egg White Lysozyme

ABSTRACT The O acetylation of peptidoglycan occurs specifically at the C-6 hydroxyl group of muramoyl residues. Using a combination of high-performance liquid chromatography-based organic acid analysis and carbohydrate analysis by high-pH anion-exchange chromatography, we determined that strains of Entercoccus durans, E. faecalis, E. faecium, and E. hirae produce O-acetylated peptidoglycan. The levels of O acetylation ranged from 19% to 72% relative to the muramic acid content, and they were found to vary with the growth phase of the culture. Increases of 10 to 40% in O acetylation were observed with cultures entering the stationary phase. Cells of E. faecalis in the viable but nonculturable (VBNC) state had the highest levels of peptidoglycan O acetylation. The presence of this modification to peptidoglycan was shown to inhibit the action of hen egg white lysozyme in a concentration-dependent manner. Zymography using sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels containing either O-acetylated or chemically de-O-acetylated peptidoglycan was used to monitor the production of specific autolysins in E. faecalis. Differences in the expression of specific autolysins were observed with the age of the culture, and VBNC E. faecalis produced the highest levels of these enzymes. This technique also permitted classification of the enterococcal autolysins into enzymes that preferentially hydrolyze either O-acetylated or non-O-acetylated peptidoglycan and enzymes that show no apparent preference for either substrate type.

scholarly journals Structural Characterization and Antioxidative Activity of Low-Molecular-Weights Beta-1,3-Glucan from the Residue of ExtractedGanoderma lucidumFruiting Bodies

2012 ◽  
Vol 2012 ◽  
pp. 1-8 ◽  
Author(s):  
Pai-Feng Kao ◽  
Shwu-Huey Wang ◽  
Wei-Ting Hung ◽  
Yu-Han Liao ◽  
Chun-Mao Lin ◽  
...  
Keyword(s):  
Cell Death ◽  
High Performance ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Water Soluble ◽  
Dependent Manner ◽  
Ros Production ◽  
Fruiting Bodies ◽  
Concentration Dependent Manner

The major cell wall constituent ofGanoderma lucidum(G. lucidum) isβ-1,3-glucan. This study examined the polysaccharide from the residues of alkaline-extracted fruiting bodies using high-performance anion-exchange chromatography (HPAEC), and it employed nuclear magnetic resonance (NMR) and mass spectrometry (MS) to confirm the structures. We have successfully isolated low-molecular-weightβ-1,3-glucan (LMG), in high yields, from the waste residue of extracted fruiting bodies ofG. lucidum. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay evaluated the capability of LMG to suppress H2O2-induced cell death in RAW264.7 cells, identifying that LMG protected cells from H2O2-induced damage. LMG treatment decreased H2O2-induced intracellular reactive oxygen species (ROS) production. LMG also influenced sphingomyelinase (SMase) activity, stimulated by cell death to induce ceramide formation, and then increase cell ROS production. Estimation of the activities of neutral and acid SMasesin vitroshowed that LMG suppressed the activities of both neutral and acid SMases in a concentration-dependent manner. These results suggest that LMG, a water-solubleβ-1,3-glucan recycled from extracted residue ofG. lucidum, possesses antioxidant capability against H2O2-induced cell death by attenuating intracellular ROS and inhibiting SMase activity.

Purification, Biochemical Characterization and Decolorization Efficiency of Laccases from Peach and Cherry Cultures of Pleutorus eryngii: A Comparative Study

2020 ◽  
Vol 27 (7) ◽  
pp. 623-634 ◽  
Author(s):  
Merve Akpinar ◽  
Raziye Ozturk Urek
Keyword(s):  
Biochemical Characterization ◽  
Gel Filtration ◽  
Industrial Effluents ◽  
Anion Exchange Chromatography ◽  
Biochemical Properties ◽  
Exchange Chromatography ◽  
Kinetic Characteristics ◽  
Chemical Agents ◽  
Decolorization Efficiency

Background:: Laccases (Lacs) are used potentially in industrial and biotechnological applications such as decolorization of dyes, degradation of industrial effluents, delignification, etc. thanks to their large varieties of substrate specificities and excellent catalytic efficiencies. The efficient utilizations of Lacs in these applications mostly depend on the identifying their biochemical properties. Objective: The goal of this research is to investigate the purification, biochemical characterization and decolorization efficiencies of Lacs. Methods: Pleurotus eryngii was incubated on peach (PC) and cherry (CC) wastes under optimized solid state fermentation conditions. Then, the enzymes extracts were purified by ammonium sulfate precipitation, anion exchange chromatography, gel filtration, respectively. Lacs fractions were subjected to electrophoretic analyses as well as their structural and kinetic characteristics. Also, the effects of selected chemical agents on purified Lacs activities and determination of decolorization efficiencies were studied. Results: As the results of purification processes of Lacs from both cultures, 3.94-fold purification was obtained for PC, while it was 5.34 for CC. The electrophoretic results of purified Lacs illustrated the single bands of protein (30±1 kDa) in accordance with the results after gel filtration. The Km values of Lacs from PC and CC were respectively detected as 1.1381 and 0.329 mM for ABTS. The selected agents partially/completely inhibited Lac activities. The highest decolorization efficiencies of purified Lacs from PC and CC were separately obtained as 53 and 11.8%. Conclusion: The results clearly indicated that the performances of Lacs from both cultures in decolorization application are different from each other depending their activities, biochemical and kinetic characteristics.

Alpha 1-adrenergic and cholinergic agonists use separate signal transduction pathways in lacrimal gland

1992 ◽  
Vol 262 (6) ◽  
pp. G1087-G1096 ◽  
Author(s):  
R. R. Hodges ◽  
D. M. Dicker ◽  
P. E. Rose ◽  
D. A. Dartt
Keyword(s):  
Protein Secretion ◽  
Lacrimal Gland ◽  
Inositol Phosphates ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Cholinergic Agonists ◽  
Substrate Protein ◽  
Camp Levels

The cellular transduction pathways used by alpha 1-adrenergic and cholinergic agonists were compared in isolated acini from rat exorbital lacrimal glands. Peroxidase secretion was the index of protein secretion. Inositol phosphates were measured by anion exchange chromatography, intracellular free Ca2+ concentration ([Ca2+]i) by fluorescence methods using fura-2, cellular adenosine 3',5'-cyclic monophosphate (cAMP) levels by protein binding radioassay, and protein kinase C (PKC) activity by [32P]ATP incorporation into exogenous substrate. Protein secretion stimulated by simultaneous addition of the alpha 1-adrenergic agonist phenylephrine and the cholinergic agonist carbachol was additive. Carbachol (10(-3) M) significantly increased the ratios of inositol phosphates to inositol during a 1- or 20-min incubation in contrast to phenylephrine (10(-5) to 10(-2) M), which did not. Phenylephrine (10(-3) M) significantly increased the [Ca2+]i by a maximum of 15 +/- 3 nM compared with carbachol (10(-4) M), which increased [Ca2+]i to a maximum of 90 +/- 14 nM. Phenylephrine (10(-4) M) did not increase cAMP levels. Phenylephrine (10(-5) to 10(-3) M) decreased cytosolic PKC activity in a concentration-dependent manner. Carbachol (10(-3) M) transiently caused a slight decrease in cytosolic PKC activity. Our results indicate that alpha 1-adrenergic and cholinergic agonists use separate and different pathways to stimulate the lacrimal gland.

Chickpea (Cicer arietinum L.) Lectin Exhibit Inhibition of ACE-I, α-amylase and α-glucosidase Activity

2019 ◽  
Vol 26 (7) ◽  
pp. 494-501 ◽  
Author(s):  
Sameer Suresh Bhagyawant ◽  
Dakshita Tanaji Narvekar ◽  
Neha Gupta ◽  
Amita Bhadkaria ◽  
Ajay Kumar Gautam ◽  
...  
Keyword(s):  
Biological Effects ◽  
Exchange Chromatography ◽  
Health Concern ◽  
Carbohydrate Binding ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Ic50 Values ◽  
Chickpea Lectin

Background: Diabetes and hypertension are the major health concern and alleged to be of epidemic proportions. This has made it a numero uno subject at various levels of investigation. Glucosidase inhibitor provides the reasonable option in treatment of Diabetes Mellitus (DM) as it specifically targets post prandial hyperglycemia. The Angiotensin Converting Enzyme (ACE) plays an important role in hypertension. Therefore, inhibition of ACE in treatment of elevated blood pressure attracts special interest of the scientific community. Chickpea is a food legume and seeds contain carbohydrate binding protein- a lectin. Some of the biological properties of this lectin hitherto been elucidated. Methods: Purified by ion exchange chromatography, chickpea lectin was tested for its in vitro antioxidant, ACE-I inhibitory and anti-diabetic characteristic. Results: Lectin shows a characteristic improvement over the synthetic drugs like acarbose (oral anti-diabetic drug) and captopril (standard antihypertensive drug) when, their IC50 values are compared. Lectin significantly inhibited α-glucosidase and α-amylase in a concentration dependent manner with IC50 values of 85.41 ± 1.21 ҝg/ml and 65.05 ± 1.2 µg/ml compared to acarbose having IC50 70.20 ± 0.47 value of µg/ml and 50.52 ± 1.01 µg/ml respectively. β-Carotene bleaching assay showed antioxidant activity of lectin (72.3%) to be as active as Butylated Hydroxylanisole (BHA). In addition, lectin demonstrated inhibition against ACE-I with IC50 value of 57.43 ± 1.20 µg/ml compared to captopril. Conclusion: Lectin demonstrated its antioxidant character, ACE-I inhibition and significantly inhibitory for α-glucosidase and α-amylase seems to qualify as an anti-hyperglycemic therapeutic molecule. The biological effects of chickpea lectin display potential for reducing the parameters of medically debilitating conditions. These characteristics however needs to be established under in vivo systems too viz. animals through to humans.

Proteolytic processing of secretory proteins in Paramecium: immunological and biochemical characterization of the precursors of trichocyst matrix proteins

1992 ◽  
Vol 103 (2) ◽  
pp. 349-361 ◽  
Author(s):  
S.J. Shih ◽  
D.L. Nelson
Keyword(s):  
Cross Sections ◽  
Disulfide Bonds ◽  
Biochemical Characterization ◽  
Rabbit Antiserum ◽  
Anion Exchange Chromatography ◽  
Cell Extract ◽  
Exchange Chromatography ◽  
Proteolytic Processing ◽  
Matrix Proteins ◽  
Secretory Proteins

We used polyclonal serum raised against mature trichocyst matrix proteins to detect their unprocessed precursors, a group of proteins (45-55 kDa) present in the whole-cell extract. These precursor proteins were partially purified from the soluble fraction of wild-type cells by ammonium sulfate precipitation and anion-exchange chromatography. Using monoclonal antibodies against each of four families of mature (processed) matrix proteins, we showed that each family was derived from a separate group of precursors. Our results also suggest that in three of four precursors, those in which the mature proteins consist of disulfide-linked heterodimers, intrachain disulfide bonds form before proteolytic processing. Purified precursors eluted from preparative SDS-gels were used to raise rabbit antiserum, which after preadsorption with mature processed proteins specifically recognized precursors, as judged by ELISA and immunoblots. In cross-sections of developing trichocysts, the anti-precursor serum after preadsorption no longer stained the central, paracrystalline region, but still stained the peripheral as well as the structureless region of the secretory granule. In trichocyst-developing mutants tl (trichless) and ftA (football A), the precursors for all four groups of mature proteins were present but their processing was affected: severely blocked in tl (which has no recognizable crystalline trichocyst matrix), and partially blocked in ftA (which has some abnormal trichocyst matrices with crystalline centers). These observations constitute further evidence that proteolytic processing of precursors occurs in parallel with crystallization.

scholarly journals NAD(P)H-dependent aldose reductase from the xylose-assimilating yeast Candida tenuis: Isolation, characterization and biochemical properties of the enzyme

1997 ◽  
Vol 326 (3) ◽  
pp. 683-692 ◽  
Author(s):  
Wilfried NEUHAUSER ◽  
Dietmar HALTRICH ◽  
Klaus D. KULBE ◽  
Bernd NIDETZKY
Keyword(s):  
Aldose Reductase ◽  
Binding Constants ◽  
Catalytic Efficiency ◽  
Anion Exchange Chromatography ◽  
Competitive Inhibitor ◽  
Biochemical Properties ◽  
Product Inhibition ◽  
Exchange Chromatography ◽  
Binding Studies ◽  
Coenzyme Binding

During growth on D-xylose the yeast Candida tenuis produces one aldose reductase that is active with both NADPH and NADH as coenzyme. This enzyme has been isolated by dye ligand and anion-exchange chromatography in yields of 76%. Aldose reductase consists of a single 43 kDa polypeptide with an isoelectric point of 4.70. Initial velocity, product inhibition and binding studies are consistent with a compulsory-ordered, ternary-complex mechanism with coenzyme binding first and leaving last. The catalytic efficiency (kcat/Km) in D-xylose reduction at pH 7 is more than 60-fold higher than that in xylitol oxidation and reflects significant differences in the corresponding catalytic centre activities as well as apparent substrate-binding constants. The enzyme prefers NADP(H) approx. 2-fold to NAD(H), which is largely due to better apparent binding of the phosphorylated form of the coenzyme. NADP+ is a potent competitive inhibitor of the NADH-linked aldehyde reduction (Ki 1.5 μM), whereas NAD+ is not. Unlike mammalian aldose reductase, the enzyme from C. tenuisis not subject to oxidation-induced activation. Evidence of an essential lysine residue located in or near the coenzyme binding site has been obtained from chemical modification of aldose reductase with pyridoxal 5′-phosphate. The results are discussed in the context of a comparison of the enzymic properties of yeast and mammalian aldose reductase.

scholarly journals Isolation and Characterization of a Lectin-like Protein (SBLP) from the Dried Roots of Scutellaria baicalensis (Lamiaceae)

2018 ◽  
Vol 13 (12) ◽  
pp. 1934578X1801301
Author(s):  
Huiqin Wang ◽  
Guanzhen Gao ◽  
Lijing Ke ◽  
Jianwu Zhou ◽  
Pingfan Rao
Keyword(s):  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Scutellaria Baicalensis ◽  
Dependent Manner ◽  
Scutellaria Baicalensis Georgi ◽  
Isolation And Characterization ◽  
Pas Staining ◽  
Ph Range ◽  
Dose Dependent

A novel lectin-like protein with MW 63.2 kDa, designated as SBLP, has been isolated and characterized from the dried roots of Scutellaria baicalensis Georgi (Lamiaceae). SBLP was purified by ammonium sulfate precipitation and anion exchange chromatography. It is a glycoprotein according to a PAS staining assay and consisting of protein (86.0%) and sugar (14.0%). Its N-terminal amino acid sequence was determined as GSAVGFLY by Edman degradation. SBLP showed hemagglutinating activity against human and rooster erythrocytes, which were stable below 60°C and in the pH range of 4 −10. Furthermore, SBLP was found to be stimulated by Ca2+, Na+, Ba2+, Zn2+ ions, which suggested it was a metal-dependent lectin. SBLP inhibited the growth of Fusarium oxysporum f.sp. lycopersici and Alternaria eichhorniae in the a dose-dependent manner, and suppressed the proliferation of HepG2 tumor cells with an IC50 of 1.00 μM. This is the first report of a lectin from Radix Scutellariae.

4-Aminopyridine block of the noninactivating cloned K+ channel Kv1.5 expressed in Xenopus oocytes

1995 ◽  
Vol 269 (2) ◽  
pp. H556-H564 ◽  
Author(s):  
T. Yamane ◽  
T. Furukawa ◽  
M. Hiraoka
Keyword(s):  
Xenopus Oocytes ◽  
Inhibitory Concentration ◽  
External Solution ◽  
K Channel ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
External Application ◽  
Charged Form ◽  
Cytoplasmic Side ◽  
Voltage Clamp Method

The blocking action of 4-aminopyridine (4-AP) on the cloned K+ channel Kv1.5 expressed in Xenopus oocytes was studied using the two-microelectrode voltage-clamp method. Application of 4-AP to the bath solution reversibly suppressed the expressed current in a voltage- and concentration-dependent manner decreasing with membrane depolarization and with a half-maximal inhibitory concentration of 0.14 mM (at +40 mV). Both block and unblock occurred mainly during a depolarization when channels were activated. With successive depolarizations, 4-AP decreased not only the peak amplitudes of the current in successive pulses, but also the current during a depolarization. Upon washout of 4-AP, the current recovered with successive depolarizations, whereas no recovery of the current was noted in the absence of depolarizations. The extent of block markedly increased with alkalization of the external solution and decreased with acidification. External application of 4-amino-pyridine methiodide, a charged form of a quaternary 4-AP derivative, did not affect the current, but internal application markedly suppressed the current, indicating the drug gained access to the channel from the cytoplasmic side. These data suggest that 4-AP crosses the membrane in its uncharged form and acts from inside of the cell in its charged form, resulting in block of the channels with higher affinity to the open state.

Effects of Buthus martensii Karsch Venom on Cell Proliferation and Cytotoxicity in Hela Cells

2011 ◽  
Vol 345 ◽  
pp. 393-398 ◽  
Author(s):  
Zhi Wang ◽  
Wen Hui Fu ◽  
Xiang Yang Lu ◽  
Guang Xian Cai
Keyword(s):  
Hela Cell ◽  
Hela Cells ◽  
Inhibitory Concentration ◽  
Scorpion Venom ◽  
Dependent Manner ◽  
Hela Cell Line ◽  
Rt Pcr ◽  
Concentration Dependent Manner ◽  
Significant Difference ◽  
Apoptosis And Necrosis

This paper focuses on the effect of the venom of the scorpion Buthus martensii on the proliferation of human cervical carcinoma Hela cell line and the related molecular mechanism. MTT test showed that the scorpion venom inhibited proliferation of Hela cells in time-dependent and concentration-dependent manner with 50% inhibitory concentration (IC50) of 34.5 μg/mL(48 h). By using flow cytometry, it was found that the scorpion venom could induce apoptosis and necrosis in Hela cells. RT-PCR and Western blot indicated there were obviously up-regulated in the expressions of p21 protein but the expression of p21 mRNA showed no significant difference in the Hela cell by the scorpion venom. These results suggest that the possible mechanism of the scorpion venom is to activate the expressions of p21 protein and to cause Hela cell apoptosis.

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