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S Turconi

Publications and source records attributed to S Turconi.

9 recordsLinked to original sources
PubMed2001

Real experiences of uHTS: a prototypic 1536-well fluorescence anisotropy-based uHTS screen and application of well-level quality control procedures.

This paper describes, for the first time, a true ultra-high throughput screen (uHTS) based upon fluorescence anisotropy and performed entirely in 1536-well assay plates. The assay is based upon binding and displacement of a BODIPY-FL-labeled antibiotic to a specific binding site on 70S ribosomes from Escherichia coli (Kd approximately 15 nM). The screen was performed at uHTS rates (i.e., >100,000 assay wells/24 h) using entirely commercially available equipment. In order to examine the reproducibility of detection of test compound effects, assays were performed in duplicate. Both overall assay statistics and reproducibility for individual compound results were excellent, at least equivalent to conventional HTS assays. Interference artifacts occurred mainly as a result of autofluorescence from test compounds. Well-level quality control procedures were developed to detect, eliminate, or even correct for such effects. Moreover, development of a brighter, longer wavelength probe (based upon Cy3B) markedly reduced such interferences. Overall, the data demonstrate that fluorescence anisotropy-based uHTS is now a practical reality.

Anti-Bacterial Agents↗
PubMed2000

Expression, purification, and functional analysis of the human serine protease HtrA2.

HumHtrA2 or Omi is a recently described member of a novel family of mammalian serine proteases homologous to the Escherichia coli htrA gene product. Although the physiological function of members of this new family is unclear, the current understanding is that as well as being involved with the degradation aberrantly folded proteins during conditions of cellular stress, they may possess a chaperone-like role under normal conditions. In this report we describe the overexpression of humHtrA2 in two heterologous systems comparing the merits of each. We found that molecular analysis of processing events in Sf9 cells allowed us to revisit E. coli expression systems which were initially unsuccessful. Using E. coli we were able to produce milligram amounts of >90% pure recombinant enzyme as determined by SDS-PAGE gels. By means of fluorescently labeled substrates alpha- and beta-casein and zymography, the proteolytic activity of recombinant HumHtrA2 was also demonstrated.

Baculoviridae↗
PubMed2000

Characterization of human HtrA2, a novel serine protease involved in the mammalian cellular stress response.

Human HtrA2 is a novel member of the HtrA serine protease family and shows extensive homology to the Escherichia coli HtrA genes that are essential for bacterial survival at high temperatures. HumHtrA2 is also homologous to human HtrA1, also known as L56/HtrA, which is differentially expressed in human osteoarthritic cartilage and after SV40 transformation of human fibroblasts. HumHtrA2 is upregulated in mammalian cells in response to stress induced by both heat shock and tunicamycin treatment. Biochemical characterization of humHtrA2 shows it to be predominantly a nuclear protease which undergoes autoproteolysis. This proteolysis is abolished when the predicted active site serine residue is altered to alanine by site-directed mutagenesis. In human cell lines, it is present as two polypeptides of 38 and 40 kDa. HumHtrA2 cleaves beta-casein with an inhibitor profile similar to that previously described for E. coli HtrA, in addition to an increase in beta-casein turnover when the assay temperature is raised from 37 to 45 degrees C. The biochemical and sequence similarities between humHtrA2 and its bacterial homologues, in conjunction with its nuclear location and upregulation in response to tunicamycin and heat shock suggest that it is involved in mammalian stress response pathways.

Alanine↗
PubMed1999

A Homogenous 384-Well High Throughput Screen for Novel Tumor Necrosis Factor Receptor: Ligand Interactions Using Time Resolved Energy Transfer.

The herpes virus entry mediator (HVEM) receptor and its ligand, HVEM-L, are involved in both herpes simplex virus type-1 (HSV-1) herpes simplex virus type-2 (HSV-2) infection, and in T-cell activation such that antagonists of this interaction are expected to have utility in viral and inflammatory diseases. In this report we describe the configuration of a homogeneous 384-well assay based on time-resolved energy transfer from a europium chelate on the HVEM receptor to an allophycocyanin (APC) acceptor on the ligand. Specific time resolved emission from the acceptor is observed on receptor:ligand complex formation. The results of various direct and indirect labeling strategies are described. Several assay optimization experiments were necessary to obtain an assay that was robust to automation and file compound interference while sensitive to the effect of potential inhibitors. The signal was stable for more than 24 h at room temperature using the Eu(3+) chelates, suggesting no dissociation of the lanthanide ion. The 384-well assay was readily automated and was able to identify more than 99.5% of known positive controls in the validation studies successfully. Screening identified both a series of known potent inhibitors and several structural classes of hits that readily deconvoluted to yield single compound inhibitors with the desired functional activity in secondary biological assays. The equivalence of the data in 384- and 1536-well formats indicates that routine implementation of 1536-well chelate-based energy transfer screening appears to be primarily limited by liquid handling rather than detection issues.

Journal Article↗
PubMed1999

Single Molecule Detection Technologies in Miniaturized High Throughput Screening: Fluorescence Correlation Spectroscopy.

Fluorescence assay technologies used for miniaturized high throughput screening are broadly divided into two classes. Macroscopic fluorescence techniques (encompassing conventional fluorescence intensity, anisotropy [also often referred to as fluorescence polarization] and energy transfer) monitor the assay volume- and time-averaged fluorescence output from the ensemble of emitting fluorophores. In contrast, single-molecule detection (SMD) techniques and related approaches, such as fluorescence correlation spectroscopy (FCS), stochastically sample the fluorescence properties of individual constituent molecules and only then average many such detection events to define the properties of the assay system as a whole. Analysis of single molecular events is accomplished using confocal optics with an illumination/detection volume of approximately 1 fl (10(-15) L) such that the signal is insensitive to miniaturization of HTS assays to 1 µl or below. In this report we demonstrate the general applicability of one SMD technique (FCS) to assay configuration for target classes typically encountered in HTS and confirm the equivalence of the rate/equilibrium constants determined by FCS and by macroscopic techniques. Advantages and limitations of the current FCS technology, as applied here, and potential solutions, particularly involving alternative SMD detection techniques, are also discussed.

Journal Article↗
PubMed1998

Tyrosine YZ and YD of photosystem II . Comparison of optical spectra to those of tyrosine oxidised by pulsed radiolysis

We have compared the optical spectrum of tyrosine oxidised in aqueous solution by pulsed radiolysis with spectra of redox active tyrosines YZ and YD of photosystem II. This indicates a "tyrosinate" state for these tyrosines and also casts doubt on the assumption that YZ and YD optical spectra are very similar in different photosystem II preparations. It suggests that further optical spectra of YZ in more intact oxygen-evolving preparations are needed before the role of Yz in water oxidation can be clarified. Copyright 1998 Elsevier Science B.V.

Journal Article↗
PubMed1997

EPR investigation of water oxidizing photosystem II: detection of new EPR signals at cryogenic temperatures.

Experiments are described which allow the detection and characterization of new EPR signals in photosystem II (PSII). PSII has been extensively studied with the water oxidising complex (WOC) poised in the S1 and S2 states. Other stages in the cycle of water oxidation lack characteristic EPR signals for use as probes. In this study, experiments use multiple turnovers of PSII from an initial S1 state to allow new states of PSII to be studied. The first EPR signal detected, centered at g = 4.85 and termed the g = 5 signal, is suggested to be a new form of S2 probably formed by decay of S3 at cryogenic temperatures, but a novel form of oxidized non-heme iron cannot be fully excluded at present. The second signal is split around g = 2 and shows characteristics of signals formed by spin-spin interaction between two paramagnetic species. The split g = 2 signal is reversibly formed by illumination at <30 K of a sample containing the g = 5 signal. The g = 2 signal may be a form of the "S3" EPR signal previously only found in a variety of PSII preparations where oxygen evolution has been inhibited. Those "S3" signals are thought to arise from the interaction of an oxidized amino acid radical and the S2 state, i.e., S2X+. Illumination at higher temperatures or illumination at <30 K, followed by dark-adaptation at 77 K, removes the g = 5 signal and prevents subsequent detection of the g = 2 signal on illumination at <30 K. The most likely explanation of our data is that illumination at <30 K of centers containing the g = 5 species allows accumulation of an oxidized intermediate and that at higher temperatures electron transfer proceeds to re-form an EPR-silent S state equivalent to that initially trapped during sample preparation. Study of these signals should provide an important new insight into the WOC and PSII.

Electron Spin Resonance Spectroscopy↗
PubMed1997

Analysis of the interaction of water with the manganese cluster of photosystem II using isotopically labeled water.

The association of water with the Mn of the water oxidizing complex was investigated using H2(17)O- and 2H2O-reconstituted lyophilized photosystem II particles. The pulsed electron paramagnetic resonance (EPR) technique of electron spin echo envelope modulation (ESEEM) was used to investigate the interaction of the magnetic 2H and 17O nuclei with the paramagnetic S2 state of the Mn complex and other photosystem II components. ESEEM offers a much more specific and sensitive detection of this type of interaction than continuous wave (CW) EPR. Unlike earlier reports using CW EPR, these experiments did not detect any interaction of water with the multiline EPR signal from the S2 state of the Mn complex. No signals indicating specific interaction of either H or O with the multiline signal were detected. Signals due to 2H and 17O were detected only at the Larmour frequency, indicating nonspecific "distant ENDOR" effects. A weak interaction with 17O was detected both in S1, when the Mn is EPR silent, and in S2, but only on the high-field side of g = 2. This interaction may be with the Rieske iron-sulfur center in the cytochrome b6f complex. The results were the same whether the multiline signal was generated by 200 K illumination of dark-frozen samples, or by room temperature illumination in the presence of the inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). Illumination at room temperature in the presence of an electron acceptor to allow multiple turnovers of the system with cycling of the S states did not result in the appearance of any new interactions. These results appear to exclude close (less than 6 A) binding of water to the Mn center giving rise to the multiline signal, and also to exclude mechanisms in which water oxidation involves the breaking and re-formation of the mu-oxo bridges of the Mn complex. They cannot, however, exclude models in which water binding to the manganese complex and direct oxidation by the manganese complex occur in the higher S states, or are catalyzed by one bis(mu-oxo) Mn dimer while oxidizing equivalents are accumulated in the S2 state by a second bis(mu-oxo) Mn dimer.

Deuterium Oxide↗