A new concept of olfactory biosensor based on interdigitated microelectrodes and immobilized yeasts expressing the human receptor OR17-40

This work shows the feasibility of an olfactory biosensor based on the immobilization of Saccharomyces cerevisiae yeast cells genetically modified to express the human olfactory receptor OR17-40 onto interdigitated microconductometric electrodes. This olfactory biosensor has been applied to the detection of its specific odorant (helional) with a high sensitivity (threshold 10⁻¹⁴ M). In contrast, no significant response was observed using a non-specific odorant (heptanal), which suggests a good selectivity. Thus, this work may represent a first step towards a new kind of bioelectronic noses based on whole yeast cells and allowing a real time monitoring of olfactory receptor activation. Presented at the joint biannual meeting of the SFB-GEIMM-GRIP, Anglet, France, 14–19 October, 2006.     

Micromethods for the spectrophotometric determination of bacterial protease activities

Microassays for the spectrophotometric determination of bacterial proteases were developed using congo red elastin, a substrate specific for elastolytic activity, and hide powder azure, a substrate sensitive to more general proteolytic activity. The small reaction volume (0.1 ml) allows incubation, filtration and quantitation to be carried out in 96 well microassay plates. Using a simple spin filtration device constructed from microassay plates a large number (768) of microassays can be filtered simultaneously. The microassays are particularly useful for screening large numbers of bacterial colonies for proteolytic mutants since they allow the rapid and efficient handling of multiple samples. These assays also permit the qualitative estimation of enzyme levels.     

Fluorescent diphenylphosphonate-based probes for detection of serine protease activity during inflammation

Activity-based probes are small molecules that covalently bind to the active site of a protease in an activity-dependent manner. We synthesized and characterized two fluorescent activity-based probes that target serine proteases with trypsin-like or elastase-like activity. We assessed the selectivity and potency of these probes against recombinant enzymes and demonstrated that while they are efficacious at labeling active proteases in complex protein mixtures in vitro, they are less valuable for in vivo studies. We used these probes to evaluate serine protease activity in two mouse models of acute inflammation, including pancreatitis and colitis. As anticipated, the activity of trypsin-like proteases was increased during pancreatitis. Levels of elastase-like proteases were low in pancreatic lysates and colonic luminal fluids, whether healthy or inflamed. Exogenously added recombinant neutrophil elastase was inhibited upon incubation with these samples, an effect that was augmented in inflamed samples compared to controls. These data suggest that endogenous inhibitors and elastase-degrading proteases are upregulated during inflammation.     

Simple high-performance liquid chromatographic determination of the protease inhibitor indinavir in human plasma

Indinavir is a member of a class of protease inhibitors that actively prevent the acquired immunodeficiency syndrome virion from maturing. A high-performance liquid chromatographic (HPLC) assay was developed and validated for the determination of indinavir in human plasma. Indinavir and the internal standard were isolated from the plasma by ether extraction. The residue after evaporation of ether was reconstituted with buffer and injected onto a C₄ reversed-phase column eluted isocratically with a mobile phase consisting of 35:65 (v/v) of acetonitrile and buffer. A wavelength of 210 nm was found to be optimum for detection. The calibration range of this assay was from 10 to 5000 ng/ml and coefficients of variation for the assay ranged from 4.6% to 11.0% for three different drug concentrations and the limit of quantitation was 10 ng/ml. During the validation, short-term stability of the drug in plasma, stability during heat deactivation and on repeated freezing and thawing of plasma was evaluated. The overall recovery of indinavir by the ether extraction method was 91.4%. This HPLC assay was found to be a simple and reproducible method for monitoring indinavir levels in human plasma obtained during clinical trials of the drug.     

Comparative study on the proteases from fish pyloric caeca and the use for production of gelatin hydrolysate with antioxidative activity

Proteases from pyloric caeca extract of three fish species including brownstripe red snapper (Lutjanus vitta), bigeye snapper (Priacanthus tayenus) and threadfin bream (Nemipterus marginatus) were comparatively studied. The extracts from bigeye snapper and threadfin bream exhibited the highest hydrolytic activities toward casein, α-N-benzoyl-dl-arginine-p-nitroanilide and α-N-ρ-tosyl-l-arginine methyl ester at pH 8.0 and 60 °C and pH 8.5 and 55 °C, respectively. The extract of brownstripe red snapper showed the optimal pH and temperature of 8.0 and 60 °C with all substrates used except the optimal temperature was 65 °C when casein was used. All proteases were strongly inhibited by soybean trypsin inhibitor (SBTI) and N-ρ-tosyl-l-lysine chloromethylketone (TLCK) and partially inhibited by N-tosyl-l-phenylalanine chloromethylketone for all substrates tested, suggesting that trypsin-like proteases were the major enzymes. Substrate-gel activity staining of 40–60% ammonium sulfate (AS) fraction revealed that major activity bands were observed with molecular mass of 24, 22 and 20 kDa for brownstripe red snapper, bigeye snapper and threadfin bream, respectively. Those activity bands were partially inhibited by SBTI and TLCK. AS fraction was further used to produce gelatin hydrolysate from the skin of brownstripe red snapper with different degrees of hydrolysis (DH). Hydrolysate with DH of 15% exhibited the highest DPPH and ABTS radical scavenging activities and ferric reducing antioxidant power (p < 0.05). Therefore, the extract from pyloric caeca could be used to produce the gelatin hydrolysates possessing antioxidative activities.     

Conformational transition of the lid helix covering the protease active site is essential for the ATP-dependent protease activity of FtsH

When bound to ADP, ATP-dependent protease FtsH subunits adopt either an “open” or “closed” conformation. In the open state, the protease catalytic site is located in a narrow space covered by a lid-like helix. This space disappears in the closed form because the lid helix bends at Gly448. Here, we replaced Gly448 with various residues that stabilize helices. Most mutants retained low ATPase activity and bound to the substrate protein, but lost protease activity. However, a mutant proline substitution lost both activities. Our study shows that the conformational transition of the lid helix is essential for the function of FtsH.

Structured summary of protein interactions

FtsH and FtsHbind by molecular sieving (View Interaction)     

An impedimetric immunosensor based on interdigitated microelectrodes (IDmicroE) for the determination of atrazine residues in food samples

A novel impedimetric immunosensor for atrazine detection has been developed. The immunosensor is based on an array of interdigitated micro-electrodes (IDmicroE) and immunoreagents specifically developed to detect this pesticide. Immunochemical determination of atrazine is possible without the use of any label. An atrazine-haptenized protein was covalently immobilized on the surface of the interdigitated mu-electrodes area (interdigits space) previously activated with (3-glycidoxypropyl)trimethoxysilane. Before, the gold electrodes were blocked using N-acetylcysteamine to prevent non-specific adsorptions. All biofunctionalization steps were characterized by chemical affinity methods and impedance spectroscopy. Immunosensors measures are made by exposing the sensor to solutions containing a mixture of the analyte and the specific antibody. With this configuration, the immunosensor detects atrazine with a limit of detection of 0.04 microg L(-1) without the use of any label. The potential of the immunosensor to analyze pesticide residues in complex sample matrices, such as red wine, has been evaluated. The results shown that after solid-phase extraction atrazine can be determined in this type of sample with a limit of detection of 0.19 microg L(-1), far below the Maximum Residue Level (MRL) established by EC for residues of this herbicide in wine.     

The 10 C-terminal residues of HTLV-I protease are not necessary for enzymatic activity

Sequence alignment of human T-lymphotropic virus type I (HTLV-I) protease and other retroviral proteases reveals that the leukemia virus proteases contain residues at the C-terminus that are absent in the other proteases. We have prepared a mutant of HTLV-I protease that does not contain the 10 C-terminal residues and demonstrated that the catalytic efficiency of cleavage of a peptide substrate is unaffected.     

A rapid and non leaky way for preparation of the sharp intracellular recording microelectrodes

To fill microelectrodes using backfilling method needs excessive time approximately 4–6  h. It is often difficult to fill microelectrodes without damage or leakage. A main problem is bubble formation in microelectrodes which has an impact on the electrical properties of the electrode and thus it influences the quality of the recording. Based on Archimede's principle there is a force within a solution which pushes insoluble material with a lower specific gravity upward and outside of the solution. Centrifugation can increase the force to eliminate the bubbles.

We designed a microelectrode holder to protect microelectrode sensitive tips from mechanical damage due to the gravity tensions; it can help to eliminate the bubbles easily and simultaneously in 10  min or less.

The tests were performed for 2000, 4000, and 8000  rpm centrifugation each one for 3, 6 and 12  min duration respectively, it was found that the bubbles were completely eliminated at 8000  rpm for 6–12  min and there were no significant differences for resistance, and the number of leaky or damaged electrodes between the two methods.

In the new design of devices, the materials used and the design of the holder are simple and the approach is applicable to many laboratories worldwide.     

Development of a protease activity assay using heat-sensitive Tus-GFP fusion protein substrates

Proteases are implicated in various diseases and several have been identified as potential drug targets or biomarkers. As a result, protease activity assays that can be performed in high throughput are essential for the screening of inhibitors in drug discovery programs. Here we describe the development of a simple, general method for the characterization of protease activity and its use for inhibitor screening. GFP was genetically fused to a comparatively unstable Tus protein through an interdomain linker containing a specially designed protease site, which can be proteolyzed. When this Tus–GFP fusion protein substrate is proteolyzed it releases GFP, which remains in solution after a short heat denaturation and centrifugation step used to eliminate uncleaved Tus–GFP. Thus, the increase in GFP fluorescence is directly proportional to protease activity. We validated the protease activity assay with three different proteases, i.e., trypsin, caspase 3, and neutrophil elastase, and demonstrated that it can be used to determine protease activity and the effect of inhibitors with small sample volumes in just a few simple steps using a fluorescence plate reader.     

Factors affecting the protease activity of venom from jellyfish Rhopilema esculentum Kishinouye

In this paper, the effects of some chemical and physical factors such as temperature, pH values, glycerol, and divalent metal cations on the protease activity of venom from jellyfish, Rhopilema esculentum Kishinouye, were assayed. Protease activity was dependent on temperature and pH values. Zn²⁺, Mg²⁺, and Mn²⁺ in sodium phosphate buffer (0.02 M, pH 8.0) could increase protease activity. Mn²⁺ had the best effects among the three metal cations and the effect was about 20 times of that of Zn²⁺ or Mg²⁺ and its maximal protease activity was 2.3 × 10⁵ U/mL. EDTA could increase protease activity. PMSF had hardly affected protease activity. O-Phenanthroline and glycerol played an important part in inhibiting protease activity and their maximal inhibiting rates were 87.5% and 82.1%, respectively.     

Optimization of the hide powder azure assay for quantitating the protease of Pseudomonas fluorescens

The extracellular protease of Pseudomonas fluorescens NC 3 was optimally active at 40°C in a reaction mixture containing: 50 mM HEPES (N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid) buffer (pH 6.6), 0.5 mM CaCl₂, and 25 mg hide powder azure in 5 ml total volume. Divalent cation chelators, i.e., EDTA, o-phenanthroline, citrate or phosphate, inhibited the enzyme. Protease production by P. fluorescens NC 3 was initiated during late-logarithmic-growth phase in a sodium caseinate medium and reached its maximum at the onset of the stationary phase.     

Coupled-enzyme system for the determination of lipase activity

A new method for lipase activity determination is described, in which liberation of fatty acids by lipase-catalyzed hydrolysis is coupled to lipoxygenation. The second reaction forms hydroperoxy-fatty acids containing a conjugated double bond that are detected at 234 nm. The method is sensitive, cheap and easy to use when compared to a titration method.     

Requirement of a large K+-uptake capacity and of extracytoplasmic protease activity for protamine resistance of Escherichia coli

The effect of protamine on growing cells of Escherichia coli K-12 strains containing different K⁺-uptake systems was investigated. Immediately after the addition of the toxic peptide, growth ceased and all strains lost most of their K⁺. In addition, these cells released a significant amount of their ATP into the medium, and the cytoplasmic volume of these cells decreased by 70%. Whereas cells without rapid K⁺-uptake systems did not recover, cells containing either the Trk systems or the overproduced Kup system slowly reversed the effects of protamine, and growth resumed after the cells had reached their original volume. Experiments with a set of strains carrying mutations in the K⁺-uptake gene trkA showed a reasonably satisfactory correlation between inhibition of net K⁺ uptake and the lag time for resumption of growth after addition of protamine. Cells carrying mutations in three extracytoplasmic proteases were hypersusceptible to protamine, suggesting that the toxic peptide is degraded by these proteases. Data on the effect of a second addition of protamine suggest that protamine degradation activity is inducible. These data are interpreted to mean that reaccumulation of K⁺ by protamine-treated cells triggers recovery of the cells, thereby allowing induction of extracytoplasmic proteases. These, in turn, degrade protamine, leading to complete recovery of the cells and resumption of growth. Cells that cannot take up K⁺ rapidly remain metabolically compromised to such an extent that extracytoplasmic protease activity is not induced, leading to a prolonged susceptibility of the cells to the toxic peptide. Received: 27 August 1996 / Accepted: 25 November 1996     

Two methods for determination of transketolase activity

Two new optical methods for transketolase activity assay using only one substrate, xylulose 5-phosphate or glycol aldehyde, have been developed. For transketolase activity assay in the first method, it is necessary to add auxiliary enzyme, glyceraldehyde phosphate dehydrogenase. It is not needed in the second method. The range of transketolase concentration in the activity assay is 0.036–0.144 U/ml for the first method and 1.8–6.8 U/ml for the second one. Published in Russian in Biokhimiya, 2006, Vol. 71, No. 5, pp. 693–696.     

Regulation of neutral protease activity through the life cycle of Tetrahymena pyriformis

Substantial fluctuations in the intracellular specific activity of neutral proteases, as assayed at pH 8 with azocasein as substrate, occur during the life cycle of the protozoan Tetrahymena pyriformis. Specific activity increases during growth in 2% proteose peptone, despite slow secretion into the medium. The most rapid increase occurs during late stationary phase and appears to be a response to one or more low molecular weight (less than 10 000), heatstable, trypsin-insensitive, polar molecules secreted into the medium. In contrast, intracellular specific activity drops by a factor of 2–5 within the first 2–3 h after transfer to non-nutritive medium. The decrease in activity under these conditions results from an enhanced rate of secretion and the cessation of net synthesis. Its kinetics are unaffected by cycloheximide and concanavalin A.     

Identification and characterization of an extracellular protease activity produced by the marine Vibriosp 60

Abstract The marine fish pathogen Vibrio sp. 60 has been used as a host for heterologous expression of the Escherichia coli heat-labile enterotoxin B-subunit and derivatives carrying a C-terminal extension. In this study, a chimeric enterotoxin B-subunit with an extension corresponding to the carboxy-terminal nine amino acids -Tyr-Ala-Gly-Ala-Val-Val-Asn-Asp-Leu-cooH from the small subunit of herpes simplex virus type 1-encoded ribonucleotide reductase, is shown to be proteolytically cleaved in the extracellular medium by a single protease that is secreted by the host strain. Such protease behaves as a typical metalloprotease, being inhibited by EDTA but not by a serine protease inhibitor. Purification and amino acid composition analysis of the two proteolysis products revealed a specific cleavage of the peptide bond between amino acids glycine and alanine of the nine amino acid extension with loss of activity. The above observation is relevant for the biotechnological exploitation of Vibrio sp. 60.     

Photomicrobial sensors for selective determination of phosphate

A photomicrobial sensor consisting of immobilized Chlorella vulgaris and an oxygen electrode has been developed for selective determination of phosphate. When 40 mM phosphate was added to the sensor system, the photocurrent increased to a maximum under light irradiation with a response time of 1 min. The current increased with increasing phosphate concentration in the range 8–70 mM. Selectivity of the sensor was satisfactory. Good agreement was obtained between the phosphate concentrations in lake water determined by the photomicrobial sensor and by conventional colorimetry (correlation coefficient 0.96).     

Disposable screen-printed enzyme sensor for simultaneous determination of starch and glucose

A disposable screen-printed biosensor with dual working electrodes was first established for simultaneous determination of starch and glucose. The electrochemical behavior of the sensor was assessed using cyclic voltammetry and chronoamperometry. The linear response ranges of the sensor were up to 0.4% (w/v) starch and 20 mmol glucose 1^–1. Real samples were analysed using the proposed method and the reference method and the correlation coefficient was 0.997.     

Darunavir, a conceptually new HIV-1 protease inhibitor for the treatment of drug-resistant HIV

Our structure-based design strategies which specifically target the HIV-1 protease backbone, resulted in a number of exceedingly potent nonpeptidyl inhibitors. One of these inhibitors, darunavir (TMC114), contains a privileged, structure-based designed high-affinity P2 ligand, 3(R),3a(S),6a(R)-bis-tetrahydrofuranylurethane (bis-THF). Darunavir has recently been approved for the treatment of HIV/AIDS patients harboring multidrug-resistant HIV-1 variants that do not respond to previously existing HAART regimens.