scholarly journals Dinitrophenyl-pepstatins as active-site-directed localization reagents for cathepsin D.

1983 ◽  
Vol 211 (1) ◽  
pp. 139-147 ◽  
Author(s):  
I T W Matthews ◽  
R S Decker ◽  
W Hornebeck ◽  
C G Knight
Keyword(s):  
Fluorescence Microscopy ◽  
Active Site ◽  
Cathepsin D ◽  
Enzyme Inhibitor ◽  
Ph Value ◽  
Specific Binding ◽  
Tight Binding ◽  
Water Soluble ◽  
Synovial Cells ◽  
Active Conformation

1. N-Pepstatinyl-N'-dinitrophenyl-1,6-diaminohexane, a potential active-site-directed localization reagent for cathepsin D, was found to bind non-specifically to immuno-precipitates containing cathepsin D. 2. Three new water-soluble localization reagents were synthesized, by using NN'-bis-(3-aminopropyl)piperazine, 3-oxa-1,5-diamino-pentane or 3,6-dioxa-1,8-diamino-octane, as spacer arms between the pepstatin and dinitrophenyl moieties. 3. The hydrophilic dinitrophenyl-pepstatins were all tight-binding inhibitors of cathepsin D at pH 3.5, but showed little or no binding to immuno-precipitates containing the inactive enzyme at pH 7.4. 4. Gel-chromatographic experiments showed that, at pH 5.0, all the dinitrophenyl-pepstatins were bifunctional reagents able to bind cathepsin D and anti-dinitrophenyl antibody at the same time. Enzyme-inhibitor-antibody complexes were not formed at pH 7.4, thus confirming that the reagents were active-site-directed. 5. Cultured human synovial cells were fixed and incubated with the dinitrophenyl-pepstatins at pH 5.0 or pH 7.4. After washing briefly, the cells were incubated at the appropriate pH value with anti-dinitrophenyl antibody labelled with fluorescein. When examined by fluorescence microscopy the cells stained at pH 5.0 showed fluorescent perinuclear granules, which were not seen in the cells treated at pH 7.4. The distribution of cathepsin D, determined by indirect immuno-fluorescence at pH 7.4, closely resembled that revealed by the dinitrophenyl-pepstatins at pH 5.0. 7. NN'-(3-Pepstatinylaminopropyl-3′-dinitrophenylaminopropyl)piperazine gave the most intense lysosomal staining and showed no non-specific binding. We conclude that this reagent is suitable for the subcellular localization of the active conformation of cathepsin D.

scholarly journals Bimane-labelled pepstatin, a fluorescent probe for the subcellular location of cathepsin D

1981 ◽  
Vol 199 (3) ◽  
pp. 611-617 ◽  
Author(s):  
I T W Matthews ◽  
R S Decker ◽  
C G Knight
Keyword(s):  
Fluorescent Probe ◽  
Cathepsin D ◽  
Tight Binding ◽  
Subcellular Location ◽  
Active Conformation ◽  
Apparent Dissociation Constant ◽  
Titration Curves ◽  
Kd Values ◽  
Pepstatin A ◽  
Dissociation Constant Kd

1. Pepstatinyl-cystamine was synthesized. The disulphide bond was cleaved and the pepstatin-bound thiol was made to react with monobromobimane. The fluorescent N-pepstatinyl-S-bimanyl-2-aminoethanethiol was purified. 2. Human cathepsin D showed tight binding of the bimane-labelled pepstatin at pH 3.5. The titration curves were used to determine the apparent dissociation constant, KD; values of approx. 1 x 10(-10) M were obtained. 3. Gel-chromatographic experiments showed that, like that of pepstatin, the binding of N-pepstatinyl-S-bimanyl-1-aminoethanethiol to cathepsin D was strongly pH-dependent. Binding was seen at pH 5.0, but could not be demonstrated at pH 7.4. 4. Cultured human synovial cells were fixed and incubated with the fluorescent inhibitor at pH 5.0 or pH 7.4. When examined by fluorescence microscopy the cells stained at pH 5.0 showed a punctate perinuclear distribution of bimane fluorescence. By contrast, the cells stained at pH 7.4 showed no fluorescence. 5. The distribution of cathepsin D, determined by indirect immunofluorescence at pH 7.4, closely resembled that of the fluorescent inhibitor seen at pH 5.0. 6. We conclude that N-pepstatinyl-S-bimanyl-2-aminoethanethiol is a fluorescent probe selective for the active conformation of cathepsin D.

Molecularly Imprinted Sensor for Ascorbic Acid Based on Gold Nanoparticles and Multiwalled Carbon Nanotubes

2020 ◽  
Vol 16 (7) ◽  
pp. 905-913
Author(s):  
Youyuan Peng ◽  
Qingshan Miao
Keyword(s):  
Carbon Nanotubes ◽  
Gold Nanoparticles ◽  
Ascorbic Acid ◽  
Multiwalled Carbon Nanotubes ◽  
Ph Value ◽  
Biological Fluids ◽  
Water Soluble ◽  
Multiwalled Carbon ◽  
Experimental Conditions ◽  
Molecularly Imprinted

Background: L-Ascorbic acid (AA) is a kind of water soluble vitamin, which is mainly present in fruits, vegetables and biological fluids. As a low cost antioxidant and effective scavenger of free radicals, AA may help to prevent diseases such as cancer and Parkinson’s disease. Owing to its role in the biological metabolism, AA has also been utilized for the therapy of mental illness, common cold and for improving the immunity. Therefore, it is very necessary and urgent to develop a simple, rapid and selective strategy for the detection of AA in various samples. Methods: The molecularly imprinted poly(o-phenylenediamine) (PoPD) film was prepared for the analysis of L-ascorbic acid (AA) on gold nanoparticles (AuNPs) - multiwalled carbon nanotubes (MWCNTs) modified glass carbon electrode (GCE) by electropolymerization of o-phenylenediamine (oPD) and AA. Experimental parameters including pH value of running buffer and scan rates were optimized. Scanning electron microscope (SEM), fourier-transform infrared (FTIR) spectra, cyclic voltammetry (CV) and differential pulse voltammetry (DPV) were utilized for the characterization of the imprinted polymer film. Results: Under the selected experimental conditions, the DPV peak currents of AA exhibit two distinct linear responses ranging from 0.01 to 2 μmol L-1 and 2 to 100 μmol L-1 towards the concentrations of AA, and the detection limit was 2 nmol L-1 (S/N=3). Conclusion: The proposed electrochemical sensor possesses excellent selectivity for AA, along with good reproducibility and stability. The results obtained from the analysis of AA in real samples demonstrated the applicability of the proposed sensor to practical analysis.

scholarly journals Effects of Sugar Cane Molasses Addition on the Fermentation Quality, Microbial Community, and Tastes of Alfalfa Silage

Animals ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 355
Author(s):  
Runbo Luo ◽  
Yangdong Zhang ◽  
Fengen Wang ◽  
Kaizhen Liu ◽  
Guoxin Huang ◽  
...  
Keyword(s):  
Sugar Cane ◽  
Ph Value ◽  
Electronic Tongue ◽  
Water Soluble ◽  
Soluble Carbohydrates ◽  
Cane Molasses ◽  
Fermentation Quality ◽  
Alfalfa Silage ◽  
Additive Control

The objective was to study the effects of sugar cane molasses addition on the fermentation quality and tastes of alfalfa silage. Fresh alfalfa was ensiled with no additive (Control), 1% molasses (M1), 2% molasses (M2), and 3% molasses (M3) for 206 days. The chemical composition and fermentation characteristics of the alfalfa silages were determined, the microbial communities were described by 16S rRNA sequencing, and the tastes were evaluated using an electronic tongue sensing system. With the amount of added molasses (M), most nutrition (dry matter and crude protein) was preserved and water-soluble carbohydrates (WSC) were sufficiently used to promote the fermentation, resulting in a pH reduction from 5.16 to 4.48. The lactic acid (LA) content and LA/acetic acid (AA) significantly increased, indicating that the fermentation had turned to homofermentation. After ensiling, Enterococcus and Lactobacillus were the dominant genus in all treatments and the undesirable microbes were inhibited, resulting in lower propionic acid (PA), butyric acid (BA), and NH3-N production. In addition, bitterness, astringency, and sourness reflected tastes of alfalfa silage, while umami and sourness changed with the amount of added molasses. Therefore, molasses additive had improved the fermentation quality and tastes of alfalfa silage, and the M3 group obtained the ideal pH value (below 4.5) and the best condition for long-term preservation.

Combined blockade of thrombin anion binding exosite-1 and PAR4 produces synergistic antiplatelet effect in human platelets

2011 ◽  
Vol 105 (01) ◽  
pp. 88-95 ◽  
Author(s):  
Wei-Ya Wang ◽  
Chien-Kei Wei ◽  
Che-Ming Teng ◽  
Chin-Chung Wu
Keyword(s):  
Platelet Aggregation ◽  
Active Site ◽  
Antithrombotic Therapy ◽  
Specific Binding ◽  
Anticoagulant Activity ◽  
Human Platelets ◽  
Anion Binding ◽  
Inhibitory Effects ◽  
Clotting Time ◽  
Potential Strategy

SummaryThrombin exosite-1 mediates the specific binding of thrombin with fibrinogen and protease-activated receptor (PAR) 1. Exosite-1 inhibitors have been shown to effectively decrease the clotting activity of thrombin, while their antiplatelet effects are relatively weak. In the present study, the inhibitory effects of two exosite-1 inhibitors, hirugen and HD1, but not the exosite-2 inhibitor HD22, on thrombin-induced platelet aggregation and P-selectin expression were dramatically enhanced by a PAR4 antagonist, YD-3. In contrast, the PAR1 antagonist SCH-79797 did not affect the antiplatelet effects of exosite-1 inhibitors. The exosite-1 inhibitors and YD-3 prevented the Ca2+ spike and the prolonged Ca2+ response in thrombin-stimulated platelets, respectively; and combination of these two classes of agents led to abolishment of Ca2+ signal. Unlike exosite-1 inhibitors, the antiplatelet effects of the active site inhibitor PPACK and the bivalent inhibitor bivalirudin were not significantly enhanced by YD-3. In addition, the platelet-stimulating activity of γ-thrombin, an autolytic product of α-thrombin which lacks exosite-1, was inhibited by YD-3. These results suggest that the synergistic antiplatelet effects of exosite-1 inhibitor and PAR4 antagonist are resulted from combined blockade of PAR1 and PAR4 in platelets. In fibrinogen or plasma clotting assay, YD-3 neither prolonged the clotting time on its own nor enhanced the anticoagulant activity of exosite-1 inhibitors. Therefore, the combined blockade of exosite-1 and PAR4 may offer a potential strategy for improving the balance of benefits and risks of antithrombotic therapy.

scholarly journals The effect of bacterial inoculant supplementation on the fermentation process of crushed maize ears in laboratory silos

2005 ◽  
Vol 53 (4) ◽  
pp. 33-42
Author(s):  
Petr Doležal ◽  
Dušan Kořínek ◽  
Jan Doležal ◽  
Václav Pyrochta
Keyword(s):  
Lactic Acid ◽  
Ph Value ◽  
Lactobacillus Rhamnosus ◽  
Lactobacillus Brevis ◽  
Water Soluble ◽  
Bacterial Strains ◽  
Total Acid ◽  
Ethanol Content ◽  
Fermentation Quality ◽  
Biological Additive

In the experiment was the effect of biological additive on the fermentation quality of crushed maize ears of two hybrids by comparing with the untreated control. The bacterial inoculant „A“ contained selected bacterial strains of Lactobacillus rhamnosus (NCIMB 30121) and Enterococcus faecium (NCIMB 30122). As effective substances of bacterial water–soluble inoculant „B“ were selected bacterial strains of Lactobacillus rhamnosus (NCIMB 30121), Lactobacillus plantarum (DSM 12836), Lactobacillus brevis (DSM 12835), Lactobacillus buchneri (DSM 12856), Pediococcus acidialactici (P. pentosaceus) (DSM 12834). The addition of inoculant „A“ in our experiment conditions increased statistically significantly (P<0.01) the pH value (4.09±0.01), resp. 4.02±0.02 in second trial with Pedro hybrid. The bacterial inoculant „B“ increased significantly (P<0.01) the contents of lactic acid (50.95±0.1.87 g/kg DM), acetic acid (18.61±0.34 g/kg DM), sum of acids (69.55±1.62 g/kg DM) and decreased (P<0.01) in the first trial the ethanol content (5.41±0.45 g/kg DM). The highest DM content (P<0.01) was in all experimental inoculated silages with additive „A“ (54.26±0.86%, and 53.56±0.54%, resp.). The bacterial inoculant „A“ increased significantly (P<0.01) in comparison with control silage in the second trial the content of lactic acid (34.66Ī2.81 g/kg DM), sum of acids (44.68±3.54 g/kg DM), the total acids content (32.87±2.88 g/kg DM), and ethanol content (17.33±0.79 g/kg DM). The inoculation positive effect was demonstrated in reduction of ethanol amount and of total acid production. The pH value of inoculated silages was not significantly lower than that in the control silage.

scholarly journals Asymmetric reassociation of calf spleen NAD+ glycohydrolase into liposomes

1987 ◽  
Vol 246 (2) ◽  
pp. 319-324 ◽  
Author(s):  
H M Muller ◽  
F Schuber
Keyword(s):  
Outer Surface ◽  
Active Site ◽  
Gel Filtration ◽  
Water Soluble ◽  
Unilamellar Vesicles ◽  
Phosphatidylcholine Liposomes ◽  
Nad Glycohydrolase ◽  
Large Unilamellar Vesicles ◽  
Solubilized Enzyme

NAD+ glycohydrolase (NAD+ nucleosidase, EC 3.2.2.6) can be solubilized from calf spleen microsomes (microsomal fractions) by steapsin or by detergents to yield respectively a hydrophilic (i.e. water-soluble) and a hydrophobic form of the enzyme. The detergent-solubilized enzyme was successfully reassociated into phosphatidylcholine liposomes either by a cholate-dialysis or by a gel-filtration procedure. In both cases the incorporation of NAD+ glycohydrolase was found to be completely asymmetric, i.e. the active site of the enzyme was exposed only at the outer surface of the vesicles. By contrast, as judged by flotation experiments, the hydrophilic form of NAD+ glycohydrolase could not be reassociated into liposomes. These results are in agreement with the hypothesis that calf spleen NAD+ glycohydrolase is an amphipathic protein. When incorporated into large unilamellar vesicles composed of phosphatidylcholine, NAD+ glycohydrolase was not found to catalyse vectorial transfer of NAD+ by transglycosidation with nicotinamide as acceptor.

scholarly journals A specific amino acid context in EGFR and HER2 phosphorylation sites enables selective binding to the active site of Src homology phosphatase 2 (SHP2)

2020 ◽  
Vol 295 (11) ◽  
pp. 3563-3575 ◽  
Author(s):  
Zachary Hartman ◽  
Werner J. Geldenhuys ◽  
Yehenew M. Agazie
Keyword(s):  
Amino Acid ◽  
Active Site ◽  
Specific Binding ◽  
Tyrosine Phosphatase ◽  
Strong Inhibitor ◽  
Selective Binding ◽  
Specific Amino Acid ◽  
Src Homology ◽  
Her2 Signaling

The Src homology phosphatase 2 (SHP2) is a cytoplasmic enzyme that mediates signaling induced by multiple receptor tyrosine kinases, including signaling by the epidermal growth factor receptor (EGFR) family (EGFR1–4 or the human homologs HER1–4). In EGFR (HER1) and EGFR2 (HER2) signaling, SHP2 increases the half-life of activated Ras by blocking recruitment of Ras GTPase-activating protein (RasGAP) to the plasma membrane through dephosphorylation of docking sites on the receptors. However, it is unclear how SHP2 selectively recognizes RasGAP-binding sites on EGFR and HER2. In this report, we show that SHP2-targeted pTyr residues exist in a specific amino acid context that allows selective binding. More specifically, we show that acidic residues N-terminal to the substrate pTyr in EGFR and HER2 mediate specific binding by the SHP2 active site, leading to blockade of RasGAP binding and optimal signaling by the two receptors. Molecular modeling studies revealed that a peptide derived from the region of pTyr992-EGFR packs well and makes stronger interactions with the SHP2 active site than with the SHP1 active site, suggesting a built-in mechanism that enables selective substrate recognition by SHP2. A phosphorylated form of this peptide inhibits SHP2 activity in vitro and EGFR and HER2 signaling in cells, suggesting inhibition of SHP2 protein tyrosine phosphatase activity by this peptide. Although we do not expect this peptide to be a strong inhibitor by itself, we foresee that the insights into SHP2 selectivity described here will be useful in future development of active-site small molecule-based inhibitors.

An Improved Method for the Quantification of Thiamine (Vitamin B1) in Cocoa-Containing Beverage Powders

2017 ◽  
Vol 100 (5) ◽  
pp. 1511-1515
Author(s):  
Frédéric Martin ◽  
Liliane Meyer ◽  
Konstantinos Zelianos ◽  
Esther Campos Gimenez
Keyword(s):  
Ph Value ◽  
Solid Phase ◽  
Vitamin B1 ◽  
Water Soluble ◽  
Local Market ◽  
Improved Method ◽  
Test Portion ◽  
Beverage Samples ◽  
Water Soluble Vitamin ◽  
Thiamine Content

Abstract The purpose of this work was to understand low recoveries of thiamine (vitamin B1) when extracted from cocoa-containing beverage powders fortified with water-soluble vitamin B1, and to develop and validate a new procedure to improve these results. Based on the literature, previous trials have focused on two main factors: pH value prior to paper filtration and the need for solid-phase extraction (SPE) clean up. We demonstrate that by following European Standard EN 14122, recovery of thiaminein cocoa-containing beverage powders is low and dependent on the test portion (86 and 72% for 0.5 and 1.5 g test portions, respectively). Our improved method resolved this problem by keeping the pH low (around 1) prior to paper filtration, leading to a 96.3% recovery and high precision (RSDr of 3.5%). The use of strong cation-exchange SPE cartridges for cleanup prior to the thiamine oxidation reaction proved to be essential. A comparison between our improved method and EN 14122 on nine cocoa-containing beverage samples available on local market from different manufacturers showed a systematic increase in thiamine content (up to 70%) when the improved methodwas applied. The highest difference was observed forthe sample that contained the highest amount of cocoa. However, for beverage powders that contained bothcocoa and milk, no difference was observed.

SYNTHESIS AND OPTICAL PROPERTIES OF WATER SOLUBLE ZnSe NANOCRYSTALS

2001 ◽  
Vol 15 (28n30) ◽  
pp. 3881-3884 ◽  
Author(s):  
N. Murase ◽  
M. Y. Gao ◽  
N. Gaponik ◽  
T. Yazawa ◽  
J. Feldmann
Keyword(s):  
Ph Value ◽  
Water Soluble ◽  
Final Solution ◽  
Blue Fluorescence ◽  
Wet Chemistry ◽  
Preparation Conditions ◽  
Transmission Electron ◽  
Mean Size ◽  
Surface Capping ◽  
Znse Nanocrystals

ZnSe nanocrystals are prepared in water by a wet chemistry method. By selecting an appropriate pH value and surface-capping agents, a whitish blue fluorescence peaking at 470 nm is observed under ZV irradiation. The intensity of this fluorescence increases dramatically under reflux and saturates after ~ 40 hrs. The final mean size of the ZnSe nanocrystals measured by transmission electron microscopy is aboyt 2 nm in diameter. The quantum efficiency of the fluorescence form the final solution is estimated to be ~1%, although the preparation conditions have not yet been completely optimized. These properties are discussed in comparison with those of similarly prepared CdTe and differently prepared ZnSe nanocrystals.

Modified Mechanism and Properties of Water-Soluble Sodium Silicate for Oil and Gas Field

2015 ◽  
Vol 814 ◽  
pp. 220-229
Author(s):  
Hong Xu Zhang ◽  
Yu Jie Zhao ◽  
Jia Zhuang ◽  
Hai Yang Qin ◽  
Han Ling Zhang
Keyword(s):  
Sodium Silicate ◽  
Oil And Gas ◽  
Ph Value ◽  
Water Soluble ◽  
Drilling Fluids ◽  
Gas Field ◽  
Structure Of Water ◽  
Oil And Gas Field ◽  
Inherent Nature ◽  
Poor Stability

With an analysis on the structure of water-soluble sodium silicate and its polymerization, it was found that the poor stability of silicate drilling fluids lies in the relevance between the inherent nature and the pH value of sodium silicate. The modification of water-soluble sodium silicate in this paper was to improve its stability and keep the inhibitive property simultaneously. The a-olefin sulfonate (AOS) was employed as the modifer agent acted on the water-soluble sodium silicate monomer and oligomers. Furthermore, the modification mechanism was discussed through FTIR, Laser particle size, Zeta potential and SEM. A stable sodium silicate drilling fluids with better inhibitive property was obtained by the comparison of modified sodium silicate and unmodified ones.

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