A sensitive and specific two-site, sandwich-amplified enzyme immunoassay for neuropeptide Y

The development of a new enzyme immunoassay for neuropeptide Y (NPY) is reported. Two monoclonal antibodies directed against distinct epitopes of NPY are used, one as a capture antibody (NPY02) and the other one as an indicator antibody (NPY05), this latter antibody being labeled with alkaline phosphatase. The assay calibration curve was performed over concentrations of 1 to 250 pM in a NPY-free plasma. The intra-assay coefficient of variation (CV) ranged from 0.025 to 11.9%, whereas the interassay CV was comprised between 5 and 12%. The limit of detection of this assay was 1 pM (100 amol/well). Neuropeptide Y levels are related to sampling conditions; basal concentrations of NPY with low SEM are found when less than 1.2 ml of blood is taken in EDTA tubes, the sample is centrifuged at 4°C, and immediately frozen. Unanesthetized spontaneously hypertensive rats exhibited higher NPY plasma concentrations than normotensive Wistar-Kyoto controls (53 ± 7 pM and 25 ± 2 pM, respectively, mean ± SEM, p < 0.01). Plasma NPY levels are similar in 16- and 36-week-old animals. In conclusion, this technique makes it possible to assay a large number of samples within 24 h without requiring radioactivity.     

Phenolic compounds inhibit the aldose reductase enzyme from the sheep kidney

Aldose reductase (AR) is the key enzyme for the polyol pathway and responsible for sorbitol accumulation during the hyperglycemia. The present article focuses on the role of phenol, pyrogallol, hydroquinone, resorcinol, catechol, and phloroglucinol in in vitro inhibition of AR. For this purpose, AR was purified from the sheep kidney with 5.33 EU mg^?1 specific activity and 0.64% yield using several chromatographic methods. Various concentrations of the compounds were tested on in vitro AR activity. IC₅₀ values were found for phenol, pyrogallol, hydroquinone, resorcinol, catechol, and phloroglucinol as 6.5, 1.13, 5.45, 2.21, 1.8, and 2.09 mM, respectively, and their K_i constant was calculated as 3.45 ± 0.92, 0.96 ± 0.28, 3.07 ± 0.46, 1.59 ± 0.43, 2.5 ± 0.35, and 2.54 ± 0.45 mM, respectively. Pyrogallol showed better inhibitory effect compared to the other compounds. The inhibition mechanisms of all compounds were noncompetitive. In the presents study, in vitro AR inhibition was examined by the phenolic compounds.     

Metal-Induced reversible structural interconversion of human mitochondrial NAD(P)+-dependent malic enzyme

Human mitochondrial NAD(P)+-dependent malic enzyme was strongly inhibited by Lu3+. The X-ray crystal structures indicated a structural change between the metal-free and Lu3+-containing enzymes (Yang Z, Batra R, Floyd DL, Hung HC, Chang GG, Tong L. Biochem Biophys Res Commun 2000;274:440-444). We characterized the reversible slow-binding mechanism and the structural interconversion between Mn2+- and Lu3+-containing human mitochondrial malic enzymes. When Lu3+ was added, the activity of the human enzyme showed a downward curve over time, similar to that of the pigeon enzyme. The rate of the transformation (k(obs)) from the initial rate to the steady-state rate increased hyperbolically with the concentration of Lu3+, suggesting the involvement of an isomerization step. Lu3+ had a much higher affinity for the isomerized form (K*(i,Lu (app)) = 4.8 microM) than that of the native form (K(i,Lu (app)) = 148 microM). When an excess of Mn2+ was added to the Lu3+-inhibited enzyme, assays of the kinetic activity showed an upward trend, indicating reactivation. This result also indicated that the reactivation was a slow process. Fluorescence quenching experiments confirmed that the Lu3+-induced isomerization was completely reversible. The dynamic quenching constants for the metal-free, Mn2+-containing, and Lu3+-containing enzyme were 3.08, 3.07, and 3.8 M(-1), respectively. When the Lu3+-containing enzyme was treated with excess Mn2+, the dynamic quenching constant returned to the original value (3.09 M(-1)). These results indicated that binding of Mn2+ did not induce any conformational change in the enzyme. The open form transformed to the closed form only after substrate binding. Lu3+, on the other hand, transformed the open form into a catalytically inactive form. Excess Mn2+ could replace Lu3+ in the metal binding site and convert the inactive form back into the open form. This reversible process was slow in both directions because of the same but opposite structural change involved.     

Lanthanide ions inhibit the activity of dihydrofolate reductase from chicken liver

The influences of mono-, bi- and trivalent metal ions (as chloride salts) on the activity of dihydrofolate reductase (DHFR) from chicken liver have been studied to elucidate the mechanism of ion-activation of this enzyme. The results show that monovalent ions (Na⁺ and K⁺) activate DHFR at low concentration reaching a maximum activation of about 2.5 fold at 0.4–0.5 M and declining at higher concentrations. Ca²⁺ shows similar activation but at lower concentration, reaching a maximum at 0.1 M; activity declines with further increases in concentration. At very high concentration (>0.4 M), Ca²⁺ is inhibitory. The trivalent lanthanide ions, however, show a dramatic inhibition of activity of DHFR even at very low concentration. The activity of DHFR declines to 50% of that of the control at 0.02 mM EuCl₃. Intrinsic fluorescence measurements show that the ion-dependent activation in the presence of mono- and bivalent metal ions is due to the conformational changes in the protein. Energy transfer phenomenon suggests that the specific interaction of Eu³⁺ with Trp24 located in a loop at the active site of DHFR is responsible for the strong inhibition. The possible mechanism for the ion-inhibition is proposed and discussed.     

Production of monoclonal antibody to Clostridium difficile toxin A which neutralises enterotoxicity but not haemagglutination activity

Nine monoclonal antibodies (mAb) to Clostridium difficile toxin A were produced. The isotype of one mAb (37B5) was IgG2b, kappa, and that of the other eight mAbs was IgM, kappa. Immunoblot analysis after non-denatured PAGE showed that with the exception of one mAb (112G6) all mAbs gave a positive reaction with the 540 kDa band of toxin A. Immunoblot analysis showed that four mAbs (2E15, 3B4, 37B5 and 49C4) gave a positive reaction with the 240 kDa major band of toxin A. In neutralisation tests with these mAbs for enterotoxicity, mouse lethality, haemagglutination activity and cytotoxicity, 37B5 neutralised enterotoxicity in a rabbit ileal loop response test but did not neutralise any other biological activities. None of the other eight mAbs showed any neutralising activities at all.     

Blasticidin S-producing Streptomyces morookaensis possesses an enzyme activity which hydrolyzes puromycin

Abstract Puromycin was inactivated without the presence of acetyl coenzyme A when incubated with extracts of blasticidin S-producing Streptomyces morookaensis . The two derivatives from puromycin, contained in the reaction mixture, were detected by thin-layer chromatography, purified by high performance liquid chromatography and analyzed for determination of the chemical structures by ¹H-nuclear magnetic resonance and positive-ion fast atom bombardment mass spectrometries. The analytical data revealed that puromycin was inactivated by the hydrolysis of amide linkage between the aminonucleoside and 0 -methyl-l-tyrosine moieties, suggesting that S. morookaensis possesses an enzyme activity which hydrolyzes puromycin.     

Hyperforin and its analogues inhibit CYP3A4 enzyme activity

Literature indicates that herb-drug interaction of St. John's wort is largely due to increased metabolism of the co-administered drugs that are the substrates of cytochrome P450 (CYP) 3A4 enzyme, alteration of the activity and/or expression of the enzyme. The major St. John's wort constituents, acylphloroglucinols, were evaluated for their effects on CYP3A4 enzyme activity to investigate their roles in herb-drug interaction. Hyperforin and four oxidized analogues were isolated from the plant and fully characterized by mass spectral and NMR analysis. These acylphloroglucinols inhibited activity of CYP3A4 enzyme potently in the fluorometric assay using the recombinant enzyme. Furoadhyperforin (IC(50) 0.072 microM) was found to be the most potent inhibitor of CYP3A4 enzyme activity, followed by furohyperforin isomer 1 (IC(50) 0.079 microM), furohyperforin isomer 2 (IC(50) 0.23 microM), hyperforin (IC(50) 0.63 microM) and furohyperforin (IC(50) 1.3 microM). As the acylphloroglucinols are potent inhibitors of the CYP3A4 enzyme, their modulation of the enzyme activity is unlikely to be involved in increased drug metabolism by St. John's wort.     

The influence of metal ions on malic enzyme activity and lipid synthesis in Aspergillus niger

In the presence of copper significant induction of citric acid overflow was observed, while concomitantly lower levels of total lipids were detected in the cells. Its effect was more obvious in a medium with magnesium as sole divalent metal ions, while in a medium with magnesium and manganese the addition of copper had a less pronounced effect. Since the malic enzyme was recognised as a supplier of reducing power in the form of reduced nicotinamide adenine dinucleotide phosphate for lipid biosynthesis, its kinetic parameters with regard to different concentrations of metal ions were investigated. Some inhibition was found with Fe(2+) and Zn(2+), while Cu(2+) ions in a concentration of 0.1 mM completely abolished malic enzyme activity. The same metal ions proportionally reduced the levels of total lipids in Aspergillus niger cells. A strong competitive inhibition of the enzyme by Cu(2+) was observed. It seemed that copper competes with Mg(2+) and Mn(2+) for the same binding site on the protein.     

Starch debranching enzyme (R-enzyme or pullulanase) from developing rice endosperm: purification, cDNA and chromosomal localization of the gene

Starch debranching enzyme (R-enzyme or pullulanase) was purified to homogeneity from developing endosperm of rice (Oryza sativa L. cv. Fujihikari) using a variety of high-performance liquid chromatography columns, and characterized. A cDNA clone encoding the full length of the rice endosperm debranching enzyme was isolated and its nucleotide sequence was determined. The cDNA contains an open reading frame of 2958 bp. The mature debranching enzyme of rice appears to be composed of 912 amino acids with a predicted relative molecular mass (M_r) of 102069 Da, similar in size to its M_r of about 100 000 Da estimated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The amino acid sequence of rice debranching enzyme is substantially similar to that of bacterial pullulanase, while it bears little similarity to that of bacterial isoamylase or to glycogen debranching enzymes from human muscle and rabbit muscle. Southern blot analyses strongly suggest that the debranching enzyme gene is present as a single copy in the rice genome. Analysis by restriction fragment length polymorphism with a probe including the 3′-untranslated region of cDNA for rice debranching enzyme confirmed that the debranching enzyme gene is located on chromosome 4.     

Silybin and dehydrosilybin inhibit cytochrome P450 1A1 catalytic activity: A study in human keratinocytes and human hepatoma cells

The flavonolignan silybin and its derivative dehydrosilybin have been proposed as candidate UV-protective agents in skin care products. This study addressed the effect of silybin and dehydrosilybin on the activity of cytochrome P450 isoform CYP1A1 in human keratinocytes (HaCaT) and human hepatoma cells (HepG2). CYP1A1 catalytic activity was assessed as O-deethylation of 7-ethoxyresorufin using fluorescence detection. Silybin and dehydrosylibin inhibited basal and dioxin-inducible CYP1A1 catalytic activity in both cell lines used. The inhibitory effect of tested compounds was more pronounced in HaCaT cells than in HepG2 cells, and dehydrosilybin was a much stronger inhibitor than silybin. Analyses on CYP1A1 human recombinant protein yielded IC₅₀ values of 22.9 ± 4.7 μmol/L and 0.43 ± 0.04 μmol/L for silybin and dehydrosilybin, respectively. Since CYP1A enzymes are some of the most prominent actors in the process of chemically induced carcinogenesis, the inhibitory activity of the flavonolignans tested against CYP1A1 favors their use as cytoprotective agents in terms of skin and hepatic metabolism. In addition, the capability of dehydrosilybin to inhibit CYP1A1 in submicromolar concentrations makes this compound a potential biological probe in CYP1A1 analyses.     

Amphenicol and macrolide derived antibiotics inhibit paraoxonase enzyme activity in human serum and human hepatoma cells (HepG2) in vitro

Human serum paraoxonase (hPON1) was separately purified by ammonium sulfate precipitation and hydrophobic interaction chromatography. The in vitro effects of commonly used antibiotics, namely clarithromycin and chloramphenicol, on purified human serum paraoxonase enzyme activity (serum hPON1) and human hepatoma (HepG2) cell paraoxonase enzyme activity (liver hPON1) were determined. Serum hPON1 and liver hPON1 were determined using paraoxon as a substrate and IC(50) values of these drugs exhibiting inhibition effects were found from graphs of hydratase activity (%) by plotting concentration of the drugs. We determined that chloramphenicol and clarithromycin were effective inhibitors of serum hPON1.     

Synthesis,biological activity and molecular modelling studies of shikimic acid derivatives as inhibitors of the shikimate dehydrogenase enzyme of Escherichia coli

Shikimic acid (SA) pathway is the common route used by bacteria, plants, fungi, algae, and certain Apicomplexa parasites for the biosynthesis of aromatic amino acids and other secondary metabolites. As this essential pathway is absent in mammals designing inhibitors against implied enzymes may lead to the development of antimicrobial and herbicidal agents harmless to humans. Shikimate dehydrogenase (SDH) is the fourth enzyme of the SA pathway. In this contribution, a series of SA amide derivatives were synthesised and evaluated for in vitro SDH inhibition and antibacterial activity against Escherichia coli. All tested compounds showed to be mixed type inhibitors; diamide derivatives displayed more inhibitory activity than synthesised monoamides. Among the evaluated compounds, molecules called 4a and 4b were the most active derivatives with IC₅₀ 588 and 589?µM, respectively. Molecular modelling studies suggested two different binding modes of monoamide and diamide derivatives to the SDH enzyme of E. coli.     

Partial deletion of β9 loop in pancreatic lipase-related protein 2 reduces enzyme activity with a larger effect on long acyl chain substrates

Structural studies on pancreatic lipase have revealed a complex architecture of surface loops surrounding the enzyme active site and potentially involved in interactions with lipids. Two of them, the lid and β9 loop, expose a large hydrophobic surface and are considered as acyl chain binding sites based on their interaction with an alkyl phosphonate inhibitor. While the role of the lid in substrate recognition and selectivity has been extensively studied, the implication of β9 loop in acyl chain stabilization remained hypothetical. The characterization of an enzyme with a natural deletion of the lid, guinea pig pancreatic lipase-related protein 2 (GPLRP2), suggests however an essential contribution of the β9 loop in the stabilization of the acyl enzyme intermediate formed during the lipolysis reaction. A GPLRP2 mutant with a seven-residue deletion of β9 loop (GPLRP2-Δβ9) was produced and its enzyme activity was measured using various substrates (triglycerides, monoglycerides, galactolipids, phospholipids, vinyl esters) with short, medium and long acyl chains. Whatever the substrate tested, GPLRP2-Δβ9 activity is drastically reduced compared to that of wild-type GPLRP2 and this effect is more pronounced as the length of substrate acyl chain increases. Changes in relative substrate selectivity and stereoselectivity remained however weak. The deletion within β9 loop has also a negative effect on the rate of enzyme inhibition by alkyl phosphonates. All these findings indicate that the reduced enzyme turnover observed with GPLRP2-Δβ9 results from a weaker stabilization of the acyl enzyme intermediate due to a loss of hydrophobic interactions.     

Washingtonia filifera seed extracts inhibit the islet amyloid polypeptide fibrils formations and α-amylase and α-glucosidase activity

Washingtonia filifera seeds have revealed to possess antioxidant properties, butyrylcholinesterase and xanthine oxidase inhibition activities. The literature has indicated a relationship between Alzheimer’s disease (AD) and type-2 diabetes (T2D). Keeping this in mind, we have now evaluated the inhibitory properties of W. filifera seed extracts on α-amylase, α-glucosidase enzyme activity and the Islet Amyloid Polypeptide (IAPP) fibrils formation.Three extracts from seeds of W. filifera were evaluated for their enzyme inhibitory effect and IC₅₀ values were calculated for all the extracts. The inhibition mode was investigated by Lineweaver-Burk plot analysis and the inhibition of IAPP aggregate formation was monitored.W. filifera methanol seed extract appears as the most potent inhibitor of α-amylase, α-glucosidase, and for the IAPP fibril formation.Current findings indicate new potential of this extract that could be used for the identification or development of novel potential agents for T2D and AD.     

(−)‐Epigallocatechin‐3‐gallate inhibits human angiotensin‐converting enzyme activity through an autoxidation‐dependent mechanism

We investigated the molecular mechanisms involved in the angiotensin‐converting enzyme (ACE) inhibition by (?)‐epigallocatechin‐3‐gallate (EGCg), a major tea catechin. EGCg inhibited both the ACE activity in the lysate of human colorectal cancer cells and human recombinant ACE (rh‐ACE) in a dose‐dependent manner. Co‐incubation with zinc sulfate showed no influence on the rh‐ACE inhibition by EGCg, whereas it completely counteracted the inhibitory effect of ethylenediaminetetraacetic acid, a chelating‐type ACE inhibitor. Although hydrogen peroxide was produced by the autoxidation of EGCg, hydrogen peroxide itself had little effect on the ACE activity. Conversely, the co‐incubation of EGCg with borate or ascorbic acid significantly diminished the EGCg inhibition. A redox‐cycling staining experiment revealed that rh‐ACE was covalently modified by EGCg. A Lineweaver–Burk plot analysis indicated that EGCg inhibited the ACE activity in a non‐competitive manner. These results suggested that EGCg might allosterically inhibit the ACE activity through oxidative conversion into an electrophilic quinone.     

镉及其与钙组合对褐脉少花龙葵毛状根生长、抗氧化酶活性和吸收镉的影响

为了探讨利用褐脉少花龙葵毛状根来修复重金属镉(Cd)污染的可能性,采用溶液培养法研究了Cd单独及其与钙(Ca)组合对褐脉少花龙葵毛状根生长、抗氧化酶超氧化物歧化酶(SOD)和过氧化物酶(POD)活性及对Cd吸收的影响。结果表明,Cd≤50μmol/L时能促进毛状根生长,而高于100μmol/LCd则抑制毛状根生长,使其侧根根尖变褐和变短,数目减少。与对照相比,不同浓度Cd培养的毛状根可溶性蛋白含量和SOD活性先升高后逐渐下降;其丙二醛(MDA)含量显著提高;100μmol/LCd使毛状根POD活性逐渐升高,但300μmol/LCd则使毛状根POD活性逐渐降低。与对照(仅添加100μmol/L或300μmol/LCd的毛状根)相比,Cd和10～30mmol/LCaCl2组合培养使毛状根可溶性蛋白含量和MDA含量降低;但提高其SOD活性;而100μmol/LCd和10～30mmol/LCaCl2结合培养的毛状根POD活性均比对照低;而300μmol/LCd和10～30mmol/LCaCl2结合培养的毛状根POD活性则均比对照提高。原子吸收分光光度法测定结果表明,毛状根吸收和吸附的重金属Cd含量随着培养基中Cd浓度的升高而增加。但外源加入10～30mmol/LCaCl2能减少毛状根对Cd的吸收,并调节其抗氧化酶SOD和POD活性,降低其膜脂过氧化水平而解除重金属Cd对毛状根生长的抑制或毒害。