Fluorescence linked immunosorbant assays using microtiter plates.

Fluorescence methods are widely used in the detection of antibodies and other binding events. However, as a general screening and detection tool in microtiter plates, enzyme linked immunosorbant (ELISA) methods predominate. In this paper we explore all parameters for effective use of fluorescence as a plate based detection method, including which microtiter plates can be used, the most effective means of immobilization, and the use of different fluorescent dyes or fluorescent proteins. These studies indicate that fluorescent immunosorbant assays (FLISA) can be used as effectively as enzymatic method in microtiter plate based screening methods, including the screening of phage antibody selections.

Using T7 phage display to select GFP-based binders.

Filamentous phage do not display cytoplasmic proteins very effectively. As T7 is a cytoplasmic phage, released by cell lysis, it has been prospected as being more efficient for the display of such proteins. Here we investigate this proposition, using a family of GFP-based cytoplasmic proteins that are poorly expressed by traditional phage display. Using two single-molecule detection techniques, fluorescence correlation spectroscopy and anti-bunching, we show that the number of displayed fluorescent proteins ranges from one to three. The GFP derivatives displayed on T7 contain binding loops able to recognize specific targets. By mixing these in a large background of non-binders, these derivatives were used to optimize selection conditions. Using the optimal selection conditions determined in these experiments, we then demonstrated the selection of specific binders from a library of GFP clones containing heavy chain CDR3 antibody binding loops derived from normal donors inserted at a single site. The selected GFP-based binders were successfully used to detect binding without the use of secondary reagents in flow cytometry, fluorescence-linked immunosorbant assays and immunoblotting. These results demonstrate that specific GFP-based affinity reagents, selected from T7-based libraries, can be used in applications in which only the intrinsic fluorescence is used for detection.

Eliminating helper phage from phage display.

Phage display technology involves the display of proteins or peptides, as coat protein fusions, on the surface of a phage or phagemid particles. Using standard technology, helper phage are essential for the replication and assembly of phagemid particles, during library production and biopanning. We have eliminated the need to add helper phage by using 'bacterial packaging cell lines' that provide the same functions. These cell lines contain M13-based helper plasmids that express phage packaging proteins which assemble phagemid particles as efficiently as helper phage, but without helper phage contamination. This results in genetically pure phagemid particle preparations. Furthermore, by using constructs differing in the form of gene 3 that they contain, we have shown that the display, from a single library, can be modulated between monovalent (phagemid-like) and multivalent display (phage-like) without any further engineering. These packaging cells eliminate the use of helper phage from phagemid-based selection protocols; reducing the amount of technical preparation, facilitating automation, optimizing selections by matching display levels to diversity, and effectively using the packaged phagemid particles as means to transfer genetic information at an efficiency approaching 100%.

Blood phagocyte activation during open heart surgery with cardiopulmonary bypass.

Open heart surgery with a cardiopulmonary bypass (CPB) is associated with a systemic inflammatory response which significantly contributes to adverse postoperative complications. The purpose of this study was to characterize the activation of blood phagocytes during open heart surgery with CPB. Blood samples were collected during and up to 24 h after surgery. The production of reactive oxygen species (ROS) in whole blood, the expression of surface molecules by blood phagocytes and complement activity in the plasma were determined. A cDNA microarray analysis of leukocyte RNA profile of genes was performed related to the inflammatory response. Activation of the complement was already observed at the beginning of CPB. This was followed by an increase in the neutrophil number and in both spontaneous and opsonized zymosan-activated ROS production after the onset of reperfusion. The activation of blood phagocytes was affirmed by changes in surface receptors involved in the adhesion and migration of leukocytes (CD11b, CD62L and CD31). Gene arrays also confirmed the activation of leukocytes 4 h after reperfusion. In conclusion, open heart surgery with a cardiopulmonary bypass was found to be associated with a rapid and pronounced activation of blood phagocytes and complement activation which was partly independent at the onset of CPB.

[Liver transplantation for hepatitis B]. / Transplantace jater pro hepatitidu B.

Up to now the outcomes of liver transplantation in patients with chronic viral hepatitis B have not been very good because the recurrence of viral hepatitis in the graft has been high and resulted in a high early graft failure of liver transplant recipients. However, the administration of a combined therapy with lamivudine and hyperimmune anti-HBs globulin has led to a marked improvement in transplantation results and an increase in the number of liver transplantations for this indication. Four men (aged 47 to 55 years) underwent liver transplantation for cirrhosis, caused by chronic viral hepatitis B, at our centre. All were HBsAg carriers. They were our first patients who received therapy with the combined immunoprophylactic regimen of lamivudine and hyperimmune anti-HBs globulin. HBV DNA negativity was achieved in all patients prior to transplantation; three of them were pretreated with lamivudine. At 4 to 17 months of follow-up, sustained suppression of HBV replication (HBV DNA negativity) was maintained in all four patients. No complications associated with this treatment were observed and no emergence of resistant mutants was detected. The combined therapy for chronic viral hepatitis B administered to liver transplant recipients at our centre showed very good outcomes. However, the development of resistant mutants during this therapy poses a problem, which may hopefully be overcome with the use of new antivirotics, such as adefovir or tenofovir.

[Hemophagocytic syndrome (case report and literature review)]. / Hemofagocytární syndrom (Kazuistika a review literatury).

The haemophagocytic syndrome (HPS) is clinically characterized by fever, pancytopenia and hepatosplenomegaly. Usually it takes an acute course with a high mortality. The pathogenetic basis is inadequate activation of the immune system--in particular Th1-lymphocytes with subsequent overproduction of cytokines and extreme activation of macrophages with haemophagocytosis. The activated cells infiltrate organs, cause tissue damage and clinical manifestations of the syndrome. From the etiological aspect two forms exist: primary (familial) with autosomal recessive inheritance and the secondary form which forms a heterogeneous sub-group, caused as a rule by infection and/or a tumour. The prognosis seems somewhat more favourable in secondary forms. In treatment which is essentially the same for both forms, chemotherapy combined with immunosuppression proves useful, in more aggressive forms chemotherapy as used in the treatment of non-Hodgkin lymphomas. The only curative method is transplantation of haematopoietic stem cells which is also the treatment of first choice in the familial form of haemophagocytosis. In the submitted paper the authors present a review of contemporary knowledge on this treacherous and relatively rare entity. The haemopgagocytic syndrome should be always taken into account in the differential diagnosis of fever with an obscure etiology. The authors assume that the haemophagocytic syndrome is rarely considered in practice and therefore is usually inadequately diagnosed and thus not treated in time. In the conclusion the authors describe the case-records of a 26-year-old female patient with haemophagocytic syndrome which developed during pregnancy.

Mass spectral analysis of a protein complex using single-chain antibodies selected on a peptide target: applications to functional genomics.

Genome projects are identifying an ever-increasing number of genes, accelerating the need for reagents to study the expression of these genes and elucidate the function and cellular location of the gene products. Our goal was to develop a strategy to allow human single-chain variable fragment (scFv) antibodies to be used for these endeavors. A library containing 7x10(9) individual variants was displayed by bacteriophage and selected against a biotinylated peptide corresponding to the C-terminal 15 amino acid residues of Ku86, one component of a heterodimer involved in double-stranded DNA break repair. Four unique scFv antibodies were recovered that not only recognized the selected peptide, but also the intact protein. Three of the scFv antibodies were expressed in soluble form and recognized Ku86 by Western analysis. The affinity of one of the scFv antibodies for Ku86 was 16 nM as measured by BIAcore analysis. scFv immunoprecipitation of Ku86 also isolated the other component of the heterodimer, Ku70, as determined by Western analysis and mass spectrometry. These results demonstrate the utility of scFv antibodies as invaluable reagents for functional genomics.

Functional blockade of tyrosine kinase A in the rat basal forebrain by a novel antagonistic anti-receptor monoclonal antibody.

We have exploited a new monoclonal antibody against the tyrosine kinase A (TrkA) nerve growth factor (NGF) receptor to block the NGF-TrkA interaction in the rat basal forebrain. The monoclonal antibody MNAC13 is a potent antagonist that prevents the binding of NGF to TrkA in a variety of systems. This antibody was used to study the maintenance of the cholinergic phenotype in the rat basal forebrain in vivo, by the implant of antibody-secreting cells. Basal forebrain cholinergic neurons (BFCNs) are greatly affected by the antibody treatment, both in terms of cell number and of cell soma size. When antibody-secreting cells are implanted at postnatal day 2 (P2), the effects observed at P8 are as severe as those obtained with anti-NGF antibodies and, interestingly, are observed also if anti-TrkA cells are implanted at P8, when anti-NGF antibodies, delivered by the same route, are no longer effective (). The effects induced by anti-TrkA, as those induced by anti-NGF, are reversible, but the time required for recovery and the critical period in the sensitivity of BFCNs to the functional inactivation of TrkA is twice as long than that observed when NGF is intercepted. These results demonstrate that BFCNs are more sensitive to the block of TrkA activation than they are to the block of NGF. The cloning of MNAC13 variable regions and their assembly into a functional polypeptide of reduced size (single chain Fv fragment) will allow its use in gene transfer applications.

Reexamination of the high mobility group-1 protein for self-association and characterization of hydrodynamic properties.

Previous studies of the 25 kDa high mobility group-1 (HMG-1) protein have generated conflicting results regarding whether HMG-1 exists as a monomer or is capable of oligomerizing to (functional) tetramers. To resolve this question, sedimentation velocity analysis yielded a s20,w value of 2.59S, which is consistent with a monomeric protein. Equilibrium sedimentation data were obtained for three HMG-1 concentrations at two rotor speeds. The six sets of data were fit to both an ideal single component and monomer-dimer equilibrium model, with essentially identical fits produced for both models, with the latter indicating a low extent (7%) of dimerization. Reaction of HMG-1 with glutaraldehyde produced a small population of oligomers consistent with a low level of dimers. This supported the monomer-dimer equilibrium model. Surprisingly, gel permeation chromatography yielded an apparent molecular mass of approx. 55 kDa for both HMG-1 and HMG-2. This finding is considered anomalous and presumably due to the high negative charge density in the C terminus of HMG-1. The sedimentation data also permit one to model HMG-1 as a hydrated prolate ellipsoid with a major axis/minor axis ratio of 2. 79. The collective evidence from the sedimentation and chemical cross-linking studies strongly supports a moderately asymmetric monomer in solution and unequivocally eliminates the possibility of a highly extended shape for HMG-1 or the existence of any extensive oligomerization.

Novel 3-Pyridyl ethers with subnanomolar affinity for central neuronal nicotinic acetylcholine receptors.

Recent evidence indicating the therapeutic potential of cholinergic channel modulators for the treatment of central nervous system (CNS) disorders as well as the diversity of brain neuronal nicotine acetylcholine receptors (nAChRs) have suggested an opportunity to develop subtype-selective nAChR ligands for the treatment of specific CNS disorders with reduced side effect liabilities. We report a novel series of 3-pyridyl ether compounds which possess subnanomolar affinity for brain nAChRs and differentially activate subtypes of neuronal nAChRs. The synthesis and structure-activity relationships for the leading members of the series are described, including A-85380 (4a), which possesses ca.50 pM affinity for rat brain [(3)H]-(-)-cytisine binding sites and 163% efficacy compared to nicotine to stimulate ion flux at human alpha4beta2 nAChR subtype, and A-84543 (2a), which exhibits 84-fold selectivity to stimulate ion flux at human alpha4beta2 nAchR subtype compared to human ganglionic type nAChRs. Computational studies indicate that a reasonable superposition of a low energy conformer of 4A with (S)-nicotine and (-)-epibatidine can be achieved.

A C-terminal region of the Saccharomyces cerevisiae transcription factor ADR1 plays an important role in the regulation of peroxisome proliferation by fatty acids.

The Saccharomyces cerevisiae transcriptional activator ADR1, which controls ADH2 gene expression, was shown to be involved in the regulation of peroxisome proliferation. To study the mode of action of ADR1, we compared strains carrying the adr1-1 mutation, high or low copy numbers of the ADR1 gene, the constitutive allele ADR1-5c, and 3'-deletions of ADR1. High ADR1 gene dosage increased the transcription of genes encoding peroxisomal proteins as compared to one copy of the ADR1 gene. Furthermore, overexpression of ADR1 under ethanol growth conditions induced the proliferation of peroxisomal structures. The organelles were observed to be localized in clusters, a typical feature of peroxisomes induced by oleic acid. In contrast, the ADR1-5c allele, which induces ADH2 expression to a level comparable to that of high ADR1 gene dosage was found to have only a small effect. An analysis of functional domains of the ADR1 protein revealed that the N-terminal 220 amino acids of ADR1 were sufficient for wild-type levels of transcription of the FOX2, FOX3, and PAS1 genes, but the entire ADR1 protein was required for complete induction of the CTA1 gene and for growth oleic acid medium. Our data suggest that a functional domain of the ADR1 protein localized between residues 643 and 1323 is required for the induction of peroxisomal structures and for the utilization of oleic acid.

The glycerol kinase (GUT1) gene of Saccharomyces cerevisiae: cloning and characterization.

The GUT1 gene of Saccharomyces cerevisiae, encoding glycerol kinase, was cloned and sequenced. The cloned genomic DNA fragment contains an open reading frame potentially coding for a protein of 709 amino acids with homology to bacterial glycerol kinases (40.8% identity over 502 amino acids, and 42.1% identity over 496 amino acids, in comparison to the smaller E. coli and B. subtilis enzymes). Disruption of GUT1 showed that the gene is required for growth on glycerol, but not on glucose or ethanol media. No glycerol kinase activity was detected in the disruption mutant. According to enzyme activity and transcript analysis, synthesis of glycerol kinase is repressed by glucose, and derepression is ADR1-dependent.

A fast new approach to pharmacophore mapping and its application to dopaminergic and benzodiazepine agonists.

In the absence of a 3D structure of the target biomolecule, to propose the 3D requirements for a small molecule to exhibit a particular bioactivity, one must supply both a bioactive conformation and a superposition rule for every active compound. Our strategy identifies both simultaneously. We first generate and optimize all low-energy conformations by any suitable method. For each conformation we then use ALADDIN to calculate the location of points to be considered as part of the superposition. These points include atoms in the molecule and projections from the molecule to hydrogen-bond donors and acceptors or charged groups in the binding site. These positions and the relative energy of each conformation are the input to our new program DISCO. It uses a clique-detection method to find superpositions that contain at least one conformation of each molecule and user-specified numbers of point types and chirality. DISCO is fast; for example, it takes about 1 min CPU to propose pharmacophores from 21 conformations of seven molecules. We typically run DISCO several times to compare alternative pharmacophore maps. For D2 dopamine agonists DISCO shows that the newer 2-aminothiazoles fit the traditional pharmacophore. Using site points correctly identifies the bioactive enantiomers of indoles to compare with catechols whereas using only ligand points leads to selecting the inactive enantiomer for the pharmacophore map. In addition, DISCO reproduces pharmacophore maps of benzodiazepines in the literature and proposes subtle improvements. Our experience suggests that clique-detection methods will find many applications in computational chemistry and computer-assisted molecular design.