Search

We propose methods to quantitatively calculate the fluctuation rate of red blood cells with nanometric axial and millisecond temporal sensitivity at the single-cell level by using time-lapse holographic cell imaging. For this quantitative analysis, cell membrane fluctuations (CMFs) were measured for RBCs stored at different storage times. Measurements were taken over the whole membrane for both the ring and dimple sections separately. The measurements show that healthy RBCs that maintain their discocyte shape become stiffer with storage time. The correlation analysis demonstrates a significant negative correlation between CMFs and the sphericity coefficient, which characterizes the morphological type of erythrocyte. In addition, we show the correlation results between CMFs and other morphological properties such as projected surface area, surface area, mean corpuscular volume, and mean corpuscular hemoglobin., Competing Interests: The authors declare that there are no conflicts of interest related to this article.

In vitro differentiating adipocytes are sensitive to liquid manipulations and have the tendency to float. Assessing adipocyte differentiation using current microscopy techniques involves cell staining and washing, while using flow cytometry involves cell retrieval in suspension. These methods induce biases, are difficult to reproduce, and involve tedious optimizations. In this study, we present digital holographic microscopy (DHM) as a label-free, nonperturbing means to quantify lipid droplets in differentiating adipocytes in a robust medium- to high-throughput manner. Taking advantage of the high refractive index of lipid droplets, DHM can assess the production of intracellular lipid droplets by differences in phase shift in a quantitative manner. Adipocytic differentiation, combined with other morphological features including cell confluence and cell death, was tracked over 6 days in live OP9 mesenchymal stromal cells. We compared DHM with other currently available methods of lipid droplet quantification and demonstrated its robustness with modulators of adipocytic differentiation in a dose-responsive manner. This study suggests DHM as a novel marker-free nonperturbing method to study lipid droplet accumulation and may be envisioned for drug screens and mechanistic studies on adipocytic differentiation., (Copyright © 2018 Campos et al. Published by The American Society for Biochemistry and Molecular Biology, Inc.)

Aberrant activation of innate immune pathways is associated with a variety of diseases. Progress in understanding the molecular mechanisms of innate immune pathways has led to the promise of targeted therapeutic approaches, but the development of drugs that act specifically on molecules of interest remains challenging. Here we report the discovery and characterization of highly potent and selective small-molecule antagonists of the stimulator of interferon genes (STING) protein, which is a central signalling component of the intracellular DNA sensing pathway 1,2 . Mechanistically, the identified compounds covalently target the predicted transmembrane cysteine residue 91 and thereby block the activation-induced palmitoylation of STING. Using these inhibitors, we show that the palmitoylation of STING is essential for its assembly into multimeric complexes at the Golgi apparatus and, in turn, for the recruitment of downstream signalling factors. The identified compounds and their derivatives reduce STING-mediated inflammatory cytokine production in both human and mouse cells. Furthermore, we show that these small-molecule antagonists attenuate pathological features of autoinflammatory disease in mice. In summary, our work uncovers a mechanism by which STING can be inhibited pharmacologically and demonstrates the potential of therapies that target STING for the treatment of autoinflammatory disease.

The protein ASC is a central component of most inflammasome complexes, forming functional oligomeric filaments that activate large amounts of pro-caspase-1 for further IL-1β processing and the induction of Gasdermin D-dependent cell death. The central role of inflammasomes in the innate immune response pose them as new molecular targets for therapy of diverse acute, chronic and inherited autoinflammatory pathologies. In recent years, an increasing number of molecules were proposed to modulate inflammasome signalling by interacting with different components of inflammasome complexes. However, the difficult in vitro reconstitution of the inflammasome has limited the development of specific on-target biochemical assays for compound activity confirmation and for drug discovery in high throughput screening setups. Here we describe a homogeneous, pH-based ASC oligomerization assay that employs fluorescence anisotropy (FA) to monitor the in vitro filament formation of the PYD domain of human ASC. The absence of additional solubility tags as well as of proteolytic enzymes to initiate the filament reaction makes this assay suitable for testing the direct effect of small molecules on filament formation in high throughput format. The ability of the assay to detect modulators of filament formation was confirmed by using a non-filament forming PYD mutant. The high and reproducible Z'-factor of 0.7 allowed to screen 10,100 compounds by high-throughput screening (HTS) aiming to identify inhibitors of ASC filament. While none of these molecules was able to inhibit ASC filament formation in vitro , the assay is directly amenable to screen other compound classes or validate candidate molecules from other screens., Competing Interests: Conflict of interest: The authors declare no conflict of interest.

We previously reported that the innate sensing of the endosymbiont Leishmania RNA virus 1 (LRV1) within Leishmania (Viannia) guyanensis through Toll-like receptor 3, worsens the pathogenesis of parasite infection in mice. The presence of LRV1 has been associated with the failure of first-line treatment in patients infected with LRV1 containing - L. guyanensis and - L. braziliensis parasites. Here, we established a semi-automated image-based high-throughput drug screening (HTDS) protocol to measure parasiticidal activity of the Prestwick chemical library in primary murine macrophages infected with LRV1-containing L. guyanensis . The two-independent screens generated 14 hit compounds with over sixty-nine percent reduction in parasite growth compared to control, at a single dose in both screens. Our screening strategy offers great potential in the search for new drugs and accelerates the discovery rate in the field of drug repurposing against Leishmania . Moreover, this technique allows the concomitant assessment of the effect of drug toxicity on host cell number.

Background: Red blood cells collected in citrate-phosphate-dextrose can be stored for up to 42 days at 4 °C in saline-adenine-glucose-mannitol additive solution. During this controlled, but nevertheless artificial, ex vivo ageing, red blood cells accumulate lesions that can be reversible or irreversible upon transfusion. The aim of the present study is to follow several parameters reflecting cell metabolism, antioxidant defences, morphology and membrane dynamics during storage., Materials and Methods: Five erythrocyte concentrates were followed weekly during 71 days. Extracellular glucose and lactate concentrations, total antioxidant power, as well as reduced and oxidised intracellular glutathione levels were quantified. Microvesiculation, percentage of haemolysis and haematologic parameters were also evaluated. Finally, morphological changes and membrane fluctuations were recorded using label-free digital holographic microscopy., Results: The antioxidant power as well as the intracellular glutathione concentration first increased, reaching maximal values after one and two weeks, respectively. Irreversible morphological lesions appeared during week 5, where discocytes began to transform into transient echinocytes and finally spherocytes. At the same time, the microvesiculation and haemolysis started to rise exponentially. After six weeks (expiration date), intracellular glutathione was reduced by 25%, reflecting increasing oxidative stress. The membrane fluctuations showed decreased amplitudes during shape transition from discocytes to spherocytes., Discussion: Various types of lesions accumulated at different chemical and cellular levels during storage, which could impact their in vivo recovery after transfusion. A marked effect was observed after four weeks of storage, which corroborates recent clinical data. The prolonged follow-up period allowed the capture of deep storage lesions. Interestingly, and as previously described, the severity of the changes differed among donors.

Advances in treatment strategies together with an earlier diagnosis have considerably increased the average survival of cancer patients over the last four decades. Nevertheless, despite the growing number of new antineoplastic agents introduced each year, there is still no adequate therapy for problematic malignancies such as pancreatic, lung and stomach cancers. Consequently, it is important to ensure that existing drugs used to treat other types of cancers, and potentially other diseases, are not overlooked when searching for new chemotherapy regimens for these problematic cancer types. We describe a screening approach that identifies chemotherapeutics for the treatment of lung and pancreatic cancers, based on drugs already approved for other applications. Initially, the 1280 chemically and pharmacologically diverse compounds from the Prestwick Chemical Library® (PCL) were screened against A549 (lung cancer) and PANC-1 (pancreatic carcinoma) cells using the PrestoBlue fluorescent-based cell viability assay. More than 100 compounds from the PCL were identified as hits in one or both cell lines (80 of them, being drugs used to treat diseases other than cancer). Selected PCL hits were further evaluated in a dose-response manner. Promising candidates for repositioning emanating from this study include antiparasitics, cardiac glycosides, as well as the anticancer drugs vorinostat and topotecan., Competing Interests: The authors have declared that no competing interests exist.

Bacterial toxin-antitoxin systems (TASs) are thought to respond to various stresses, often inducing growth-arrested (persistent) sub-populations of cells whose housekeeping functions are inhibited. Many such TASs induce this effect through the translation-dependent RNA cleavage (RNase) activity of their toxins, which are held in check by their cognate antitoxins in the absence of stress. However, it is not always clear whether specific mRNA targets of orthologous RNase toxins are responsible for their phenotypic effect, which has made it difficult to accurately place the multitude of TASs within cellular and adaptive regulatory networks. Here, we show that the TAS HigBA of Caulobacter crescentus can promote and inhibit bacterial growth dependent on the dosage of HigB, a toxin regulated by the DNA damage (SOS) repressor LexA in addition to its antitoxin HigA, and the target selectivity of HigB's mRNA cleavage activity. HigB reduced the expression of an efflux pump that is toxic to a polarity control mutant, cripples the growth of cells lacking LexA, and targets the cell cycle circuitry. Thus, TASs can have outcome switching activity in bacterial adaptive (stress) and systemic (cell cycle) networks.

The Wnt pathway, which controls crucial steps of the development and differentiation programs, has been proposed to influence lipid storage and homeostasis. In this paper, using an unbiased strategy based on high-content genome-wide RNAi screens that monitored lipid distribution and amounts, we find that Wnt3a regulates cellular cholesterol. We show that Wnt3a stimulates the production of lipid droplets and that this stimulation strictly depends on endocytosed, LDL-derived cholesterol and on functional early and late endosomes. We also show that Wnt signaling itself controls cholesterol endocytosis and flux along the endosomal pathway, which in turn modulates cellular lipid homeostasis. These results underscore the importance of endosome functions for LD formation and reveal a previously unknown regulatory mechanism of the cellular programs controlling lipid storage and endosome transport under the control of Wnt signaling., (© 2015 The Authors.)

Compounds tested during drug development may have adverse effects on the heart; therefore all new chemical entities have to undergo extensive preclinical assessment for cardiac liability. Conventional intensity-based imaging techniques are not robust enough to provide detailed information for cell structure and the captured images result in low-contrast, especially to cell with semi-transparent or transparent feature, which would affect the cell analysis. In this paper we show, for the first time, that digital holographic microscopy (DHM) integrated with information processing algorithms automatically provide dynamic quantitative phase profiles of beating cardiomyocytes. We experimentally demonstrate that relevant parameters of cardiomyocytes can be obtained by our automated algorithm based on DHM phase signal analysis and used to characterize the physiological state of resting cardiomyocytes. Our study opens the possibility of automated quantitative analysis of cardiomyocyte dynamics suitable for further drug safety testing and compounds selection as a new paradigm in drug toxicity screens.

Indoleamine 2,3-dioxygenase 1 (IDO1) is a key regulator of immune responses and therefore an important therapeutic target for the treatment of diseases that involve pathological immune escape, such as cancer. Here, we describe a robust and sensitive high-throughput screen (HTS) for IDO1 inhibitors using the Prestwick Chemical Library of 1200 FDA-approved drugs and the Maybridge HitFinder Collection of 14,000 small molecules. Of the 60 hits selected for follow-up studies, 14 displayed IC50 values below 20 μM under the secondary assay conditions, and 4 showed an activity in cellular tests. In view of the high attrition rate we used both experimental and computational techniques to identify and to characterize compounds inhibiting IDO1 through unspecific inhibition mechanisms such as chemical reactivity, redox cycling, or aggregation. One specific IDO1 inhibitor scaffold, the imidazole antifungal agents, was chosen for rational structure-based lead optimization, which led to more soluble and smaller compounds with micromolar activity., (Copyright © 2014 Elsevier Masson SAS. All rights reserved.)

The Biomolecular Screening Facility (BSF) is a multidisciplinary laboratory created in 2006 at the Ecole Polytechnique Federale de Lausanne (EPFL) to perform medium and high throughput screening in life sciences-related projects. The BSF was conceived and developed to meet the needs of a wide range of researchers, without privileging a particular biological discipline or therapeutic area. The facility has the necessary infrastructure, multidisciplinary expertise and flexibility to perform large screening programs using small interfering RNAs (siRNAs) and chemical collections in the areas of chemical biology, systems biology and drug discovery. In the framework of the National Centres of Competence in Research (NCCR) Chemical Biology, the BSF is hosting 'ACCESS', the Academic Chemical Screening Platform of Switzerland that provides the scientific community with chemical diversity, screening facilities and know-how in chemical genetics. In addition, the BSF started its own applied research axes that are driven by innovation in thematic areas related to preclinical drug discovery and discovery of bioactive probes.

Digital Holographic Microscopy (DHM) is a label-free imaging technique allowing visualization of transparent cells with classical imaging cell culture plates. The quantitative DHM phase contrast image provided is related both to the intracellular refractive index and to cell thickness. DHM is able to distinguish cellular morphological changes on two representative cell lines (HeLa and H9c2) when treated with doxorubicin and chloroquine, two cytotoxic compounds yielding distinct phenotypes. We analyzed parameters linked to cell morphology and to the intracellular content in endpoint measurements and further investigated them with timelapse recording. The results obtained by DHM were compared with other optical label-free microscopy techniques, namely Phase Contrast, Differential Interference Contrast and Transport of Intensity Equation (reconstructed from three bright-field images). For comparative purposes, images were acquired in a common 96-well plate format on the different motorized microscopes. In contrast to the other microscopies assayed, images generated with DHM can be easily quantified using a simple automatized on-the-fly analysis method for discriminating the different phenotypes generated in each cell line. The DHM technology is suitable for the development of robust and unbiased image-based assays.

By using the Telospot assay, 27 different extracts of cyanobacteria were evaluated for telomerase inhibition. All extracts showed varying, but significant activity. We selected Microcystis aeruguinosa PCC 7806 to identify the active compound and a bioassay guided fractionation led us to isolate mixtures of sulfoquinovosyl diacylglycerols (SQDGs), which were identified by 2D NMR and MS/MS experiments. Pure SQDG derivatives were then synthesized. The IC(50) values of pure synthetic sulfoquinovosyl dipalmitoylglycerol and the monopalmitoylated derivative against telomerase were determined to be 17 and 40 μM, respectively. A structure-activity relationship study allowed the identification of compounds with modified lipophilic acyl groups that display improved activity., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We introduce a label-free technology based on digital holographic microscopy (DHM) with applicability for screening by imaging, and we demonstrate its capability for cytotoxicity assessment using mammalian living cells. For this first high content screening compatible application, we automatized a digital holographic microscope for image acquisition of cells using commercially available 96-well plates. Data generated through both label-free DHM imaging and fluorescence-based methods were in good agreement for cell viability identification and a Z'-factor close to 0.9 was determined, validating the robustness of DHM assay for phenotypic screening. Further, an excellent correlation was obtained between experimental cytotoxicity dose-response curves and known IC50 values for different toxic compounds. For comparable results, DHM has the major advantages of being label free and close to an order of magnitude faster than automated standard fluorescence microscopy.

The study of membranes is at a turning point. New theories about membrane structure and function have recently been proposed, however, new technologies, combining chemical, physical, and biochemical approaches are necessary to test these hypotheses. In particular, the NCCR in chemical biology aims to visualize and characterize membrane microdomains and determine their function during hormone signaling.

Discovering new drugs to treat tuberculosis more efficiently and to overcome multidrug resistance is a world health priority. To find antimycobacterial scaffolds, we screened a kinase inhibitor library of more than 12,000 compounds using an integrated strategy involving whole cell-based assays with Corynebacterium glutamicum and Mycobacterium tuberculosis, and a target-based assay with the protein kinase PknA. Seventeen "hits" came from the whole cell-based screening approach, from which three displayed minimal inhibitory concentrations (MIC) against M. tuberculosis below 10μM and were non-mutagenic and non-cytotoxic. Two of these hits were specific for M. tuberculosis versus C. glutamicum and none of them was found to inhibit the essential serine/threonine protein kinases, PknA and PknB present in both bacteria. One of the most active hits, VI-18469, had a benzoquinoxaline pharmacophore while another, VI-9376, is structurally related to a new class of antimycobacterial agents, the benzothiazinones (BTZ). Like the BTZ, VI-9376 was shown to act on the essential enzyme decaprenylphosphoryl-β-D-ribose 2'-epimerase, DprE1, required for arabinan synthesis., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

DNA sequence information underpins genetic research, enabling discoveries of important biological or medical benefit. Sequencing projects have traditionally used long (400-800 base pair) reads, but the existence of reference sequences for the human and many other genomes makes it possible to develop new, fast approaches to re-sequencing, whereby shorter reads are compared to a reference to identify intraspecies genetic variation. Here we report an approach that generates several billion bases of accurate nucleotide sequence per experiment at low cost. Single molecules of DNA are attached to a flat surface, amplified in situ and used as templates for synthetic sequencing with fluorescent reversible terminator deoxyribonucleotides. Images of the surface are analysed to generate high-quality sequence. We demonstrate application of this approach to human genome sequencing on flow-sorted X chromosomes and then scale the approach to determine the genome sequence of a male Yoruba from Ibadan, Nigeria. We build an accurate consensus sequence from >30x average depth of paired 35-base reads. We characterize four million single-nucleotide polymorphisms and four hundred thousand structural variants, many of which were previously unknown. Our approach is effective for accurate, rapid and economical whole-genome re-sequencing and many other biomedical applications.

Fluorescent 2'-deoxynucleotides containing a protecting group at the 3'-O-position are reversible terminators enabling array-based DNA sequencing by synthesis (SBS) approaches. Herein, we describe the synthesis of a new family of 3'-OH unprotected cleavable fluorescent 2'-deoxynucleotides and their evaluation as reversible terminators for high-throughput DNA SBS strategies. In this first version, all four modified nucleotides bearing a cleavable disulfide Alexa Fluor(R) 594 dye were assayed for their ability to act as a reversible stop for the incorporation of the next labeled base. Their use in SBS leaded to a signal-no signal output after successive addition of each labeled nucleotide during the sequencing process (binary read-out). Solid-phase immobilized synthetic DNA target sequences were used to optimize the method that has been applied to DNA polymerized colonies or clusters obtained by in situ solid-phase amplification of fragments of genomic DNA templates.

We designed a method termed Telospot to discover and characterize telomerase modulators as anticancer drugs or chemical biology tools. Telospot is based on a highly efficient human telomerase expression system and the detection of telomerase DNA reaction products in macroarray format. Telospot offers a highly scalable, cost- and time-effective alternative to presently available telomerase assays, which are limited by the requirement for PCR, telomerase purification or technologies not amenable to high throughput.

The development of phenyldithioethyloxycarbonyl (Phdec) and 2-pyridyldithioethyloxycarbonyl (Pydec) protecting groups, which are thiol-labile urethanes, is described. These new disulfide-based protecting groups were introduced onto the epsilon-amino group of L-lysine; the resulting amino acid derivatives were easily converted into N alpha-Fmoc building blocks suitable for both solid- and solution-phase peptide synthesis. Model dipeptide(Ardec)s were prepared by using classical peptide couplings followed by standard deprotection protocols. They were used to optimize the conditions for complete thiolytic removal of the Ardec groups both in aqueous and organic media. Phdec and Pydec were found to be cleaved within 15 to 30 min under mild reducing conditions: i) by treatment with dithiothreitol or beta-mercaptoethanol in Tris.HCl buffer (pH 8.5-9.0) for deprotection in water and ii) by treatment with beta-mercaptoethanol and 1,8-diazobicyclo[5.4.0]undec-7-ene (DBU) in N-methylpyrrolidinone for deprotection in an organic medium. Successful solid-phase synthesis of hexapeptides Ac-Lys-Asp-Glu-Val-Asp-Lys(Ardec)-NH2 has clearly demonstrated the full orthogonality of these new amino protecting groups with Fmoc and Boc protections. The utility of the Ardec orthogonal deprotection strategy for site-specific chemical modification of peptides bearing several amino groups was illustrated firstly by the preparation of a fluorogenic substrate for caspase-3 protease containing the cyanine dyes Cy 3.0 and Cy 5.0 as FRET donor/acceptor pair, and by solid-phase synthesis of an hexapeptide bearing a single biotin reporter group.

The tricarboxylate reagent benzene-1,3,5-triacetic acid (BTA) was used to attach 5'-aminated DNA primers and templates on an aminosilanized glass surface for subsequent generation of DNA colonies by in situ solid-phase amplification. We have characterized the derivatized surfaces for the chemical attachment of oligonucleotides and evaluate the properties relevant for the amplification process: surface density, thermal stability towards thermocycling, functionalization reproducibility and storage stability. The derivatization process, first developed for glass slides, was then adapted to microfabricated glass channels containing integrated fluidic connections. This implementation resulted in an important reduction of reaction times, consumption of reagents and process automation. Innovative analytical methods for the characterization of attached DNA were developed for assessing the surface immobilized DNA content after amplification. The results obtained showed that the BTA chemistry is compatible and suitable for forming highly dense arrays of DNA colonies with optimal surface coverage of about 10 million colonies/cm(2) from the amplification of initial single-template DNA molecules immobilized. We also demonstrate that the dsDNA colonies generated can be quantitatively processed in situ by restriction enzymes digestion. DNA colonies generated using the BTA reagent can be used for further sequence analysis in an unprecedented parallel fashion for low-cost genomic studies.

This work presents a simple microfluidic device with an integrated thin-film heater for studies of DNA hybridization kinetics and double-stranded DNA melting temperature measurements. The heating characteristics of the device were evaluated with a novel, noninvasive indirect technique using molecular beacons as temperature probes inside reaction chambers. This is the first microfluidic device in which thermal dehybridization of surface-bound oligonucleotides was performed for measurement of double-stranded DNA melting temperatures with +/- 1 degrees C precision. Surface modification and oligonucleotide immobilization were performed by continuously flowing reagents through the microchannels. The resulting reproducibility of oligonucleotide surface densities, at 9% RSD, was better than for the same modification chemistries on glass slides in unstirred reagent solutions (RSD=20%). Moreover, the surface density of immobilized DNA probe molecules could be varied controllably by changing the concentration of the reagent solution used for immobilization. Thus, excellent control of surface characteristics was made possible, something which is often difficult to achieve with larger devices. Solid-phase hybridization reactions, a fundamental aspect of microarray technologies often taking several hours in conventional systems, were reduced to minutes in this device. It was also possible to determine forward rate constants for hybridization, k. These varied from 820,000 to 72,000 M(-1) s(-1), decreasing as surface densities increased. Surface densities could therefore be optimized to obtain rapid hybridization using such an approach. Taken together, this combined microfluidic/small-volume heating approach represents a powerful tool for surface-based DNA analysis.

G protein-coupled receptors (GPCR) represent a large family of drug targets for which there is no high resolution structural information. In order to understand the mechanisms of ligand recognition and receptor activation, there is a strong need for novel biophysical methods. In this Perspective we provide an overview of recent experimental approaches used to explore the molecular architecture and dynamics of GPCR and their interactions with ligands and G proteins using biophysical, non-crystallographic, methods.

A fluorescent unnatural amino acid was introduced biosynthetically at known sites into the G protein-coupled neurokinin (tachykinin) NK2 receptor by suppression of UAG nonsense codons with the aid of a chemically misacylated synthetic tRNA specifically designed for the incorporation of unnatural amino acids during heterologous expression in Xenopus oocytes. A systematic UAG-scanning mutagenesis in NK2 extra- or intracellular loops and proximal transmembrane domains established that readthrough at some UAG sites may represent a limitation to the range of applicability of the nonsense suppression methodology. Fluorescence-labeled NK2 mutants containing an unique fluorescent nitrobenzoxadiazoyl-diaminopropionic acid residue at known sites were shown to be functionnally active. Intermolecular distances were determined by measuring the fluorescence resonance energy transfer (FRET) between the fluorescent unnatural amino acid and a fluorescently labeled NK2 heptapeptide antagonist in a native membrane environment. These distances confirmed the seven transmembrane topology for G protein-coupled receptors and determined a structural model for NK2 ligand-receptor interactions. The peptide is inserted between the fifth and sixth transmembrane domains, thus suggesting that antagonism may be caused by preventing correct packing of the helices required for receptor function.

We have investigated the interaction of fluorescent peptide ligands with the G protein-coupled receptor NK2 using novel spectrofluorometric approaches. Several heptapeptide antagonists of structure PhCO-Xaa-Ala-D-Trp-Phe-D-Pro-Pro-Nle-NH2 were labelled on position 1 (Xaa) with the environment-sensitive nitrobenzoxadiazole (NBD) probe, differing only in the length of the spacer between the NBD group and the peptide. Upon binding of the labelled antagonist to NK2 receptors stably expressed in Chinese hamster ovary (CHO) cells, an increase in NBD fluorescence was observed when the spacer length was less than 10 A. Collisional quenching experiments using iodide and Co2+ ions were performed to define the accessibility of the NBD group on bound ligands to the solvent. By comparing ligands with spacer arms of varying lengths, we found that the binding pocket is buried at a depth of 5-10 A. In contrast, N-terminally NBD-labelled agonists, decapeptide neurokinin A (NKA) or heptapeptide Nle10-NKA[4-10], bound to the NK2 receptor were accessible to the solvent. Binding of fluorescent ligands to the NK2 receptor was accompanied by an enhancement in the fluorescence anisotropy. The changes in fluorescence properties were used to determine the kinetic parameters of antagonist binding and dissociation. These results indicate that the binding site on the NK2 receptor for the amino-terminal end of the heptapeptide antagonists is buried in the hydrophobic pocket of the receptor protein and clearly distinct from the binding site for the amino-terminal end of agonists, which is accessible to the solvent.(ABSTRACT TRUNCATED AT 250 WORDS)

Several fluorescent probes for the NK2 receptor were designed, synthesized, and pharmacologically characterized. These fluorescent ligands are analogues of the selective NK2 heptapeptide antagonist N-alpha-benzoyl-Ala-Ala-D-Trp-Phe-D-Pro-Pro-Nle-NH2 (1, GR94800). They were obtained by substitution of 2,n-diaminoalkyl amino acid (n = 3-6) for Ala1 and the subsequent coupling of the fluorophore NBD (7-nitrobenz-2-oxa-1,3-diazol-4-yl) or fluoresceinthiocarbamyl to the N-omega amino group. The fluorescent derivatives retained high binding affinities for the NK2 receptor in transfected CHO cells. In contrast, fluorescent derivatives made by replacing the N-alpha-benzoyl group of 1 by NBD or fluorescein were considerably less active. The effect on ligand potency of varying the length of the spacer arm between the peptide moiety and the fluorescent group was also studied. The most potent fluorescent antagonists were N-alpha-benzoyl-Dab(gamma-NBD)-Ala-D-Trp-Phe-D-Pro-Pro-Nle-NH2 (5B), pKi = 8.87 for NK2; N-alpha-benzoyl-Orn (delta-NBD)-Ala-D-Trp-Phe- D-Pro-Pro-Nle-NH2 (4B), pKi = 8.84; and N-alpha-benzoyl-Lys(epsilon-NBD)-Ala-D-Trp-Phe-D-Pro-Pro-Nle-NH2 (3B), pKi = 8.83. These three compounds were highly selective for NK2 over NK3 and NK1 receptors. We show that these fluorescent ligands are useful tools for the detection of NK2 receptor expression by flow cytometry. Additionally, these fluorescent probes should prove valuable for fluorescence microscopy and study of ligand-receptor interaction by spectrofluorimetry.

Phosphomannose isomerase catalyses the interconversion of fructose-6-P and mannose-6-P and has a critical role in the supply of D-mannose derivatives required for many eukaryotic glycosylation reactions. Three classes of enzymes possessing phosphomannose-isomerase activity have been identified in bacteria and lower eukaryotes. We have purified human phosphomannose isomerase to homogeneity from placental tissue. Protein sequence information obtained from internal fragments of the protein was used to design degenerate oligonucleotides which were used to amplify a fragment of a human phosphomannose-isomerase cDNA. A full-length cDNA was isolated from a human testes lambda gt11 library using this fragment as a probe. The cDNA encoded a protein with significant sequence identity to fungal and some bacterial phosphomannose isomerases but was unrelated to those from other bacteria. Based on amino acid sequence identity we propose a classification system for enzymes with phosphomannose-isomerase activity. The cDNA, under the control of the GAL1 promoter, was expressed in a Saccharomyces cerevisiae strain from which the native gene encoding phosphomannose isomerase had been deleted. The human enzyme was found to be able to functionally substitute for the yeast enzyme. Phosphomannose-isomerase mRNA was found in all human tissues tested but was more highly expressed in heart, brain and skeletal muscle. The cDNA was expressed in Escherichia coli permitting the isolation of pure recombinant protein which will be used for kinetic and structural studies.

A powerful chemical modification procedure has been developed to define determinants of DNA recognition by the p50 subunit of NF-kappa B. Differential labelling with [14C] iodoacetate has identified a conserved cysteine residue, Cys62, that was protected from modification by the presence of an oligonucleotide containing the specific recognition site of the protein. To determine the importance of this cysteine residue, each of the conserved cysteines in p50 was changed to serine and the DNA binding properties of the mutant proteins determined. Scatchard analysis indicated that the C62S mutant bound to its DNA recognition site with a 10-fold larger dissociation constant than the wild type protein, while the other two mutants bound with an intermediate affinity. Dissociation rate constant measurements correlated well with the dissociation constants for the wild type, C119S, and C273S p50 proteins, whereas the p50 C62S-DNA complex dissociated anomalously quickly. Competition analyses with oligonucleotide variants of the DNA recognition site and nonspecific E. coli DNA revealed that the C62S p50 mutant had an altered DNA binding site specificity and was impaired in its ability to discriminate between specific and non-specific DNA. Thus the sulphydryl group of Cys62 is an important determinant of DNA recognition by the p50 subunit of NF-kappa B.

The human ileum neurokinin NK2 receptor has been stably expressed in Chinese hamster ovary (CHO) cells using the dihydrofolate reductase (DHFR) expression system. Amplified cell populations expressing approximately 7 x 10(5) NK2 receptors/cell were selected in the presence of the DHFR inhibitor methotrexate. Cross-linking of [125I]NKA to NK2 receptor transfected cells revealed a specifically labeled protein of apparent molecular weight 64 kDa by SDS-polyacrylamide gel electrophoresis. This protein was deglycosylated by the enzymes N-glycosidase F and endoglycosydase F to a protein of apparent molecular weight of 39 kDa. The NK2 receptor was solubilized in an active form from CHO cell membranes using the zwitterionic detergent CHAPS. This method represents a valuable approach for the production of significant amounts of NK2 receptor protein from mammalian cells.

Human recombinant soluble 37 kDa CD23 has been expressed in insect cells and secreted into the culture medium using the IL-2 leader sequence. The 37 kDa CD23 was purified 600-fold to homogeneity by monoclonal antibody affinity chromatography and gel filtration. The pure protein is monomeric, glycosylated, depleted of one N terminal amino acid and contains four disulphide bonds. It degrades into smaller fragments of 33, 29 and 25 kDa if purified in the absence of protease inhibitors. The same pattern of proteolytic fragments is observed when the pure preparation is incubated at room temperature for 3 weeks. Physical characterization of the 37 kDa CD23 by circular dichroism indicates that the protein contains mainly beta sheet and 20% of alpha helical structures. Specific binding of IgE to natural CD23 (low affinity IgE receptor) was inhibited by purified recombinant 37 kDa CD23. Moreover, purified recombinant 37kDa CD23 and interleukin-1 promoted the survival of germinal centre B cells.

Human interleukin-5 is a homodimer; each subunit contains two cysteine residues that form two inter-subunit disulfide bonds. The topology of the disulfides in recombinant human interleukin-5 produced in Escherichia coli was studied by proteolytic digestion and peptide mapping. Disulfide linked peptides containing cysteine 42 linked to cysteine 84 were isolated. This indicated that cysteines 42 and 84 of one subunit were linked in an antiparallel manner to cysteines 84 and 42 of the other subunit.

The partial amino acid sequence of p140 calf thymus DNA topoisomerase II was determined by analysis of cyanogen bromide peptides. Five peptides were aligned and shared extensive homology with sequences derived from cDNA clones for the human topoisomerase II isoenzyme forms. Less homology was seen with the Drosophila, yeast and bacterial type II enzymes. Calf and human enzymes shared epitopes allowing isolation of a cDNA clone to human topoisomerase II isoenzyme alpha. Our results indicate that calf thymus p140 topoisomerase II is an active N-terminal proteolytic fragment of the native p180 enzyme and demonstrate that mammalian type II enzymes exhibit close sequence similarity.

The urine of some febrile patients has been shown to contain a tumor necrosis factor-alpha-inhibiting activity (TNF-alpha INH) when tested in a cytotoxicity assay using the TNF-susceptible cell line L-929. The inhibitor was purified to homogeneity using a simple three-step procedure which included a TNF-alpha affinity column, cation exchange and reverse-phase chromatography. The NH2-terminal amino acid sequence of the inhibitor showed no sequence similarity with proteins in the data bases used. Using gel filtration, it was shown that TNF-alpha and the inhibitor form a stable complex which eluted with a molecular weight of about 75,000. This value corresponds to the sum of the inhibitor (approximately 30,000) and TNF-alpha (approximately 45,000-50,000) molecular weight. The TNF-alpha INH blocked prostaglandin E2 production by dermal fibroblasts in a dose-dependent manner, providing evidence for antiinflammatory activity. TNF-alpha INH also blocked class I antigen expression in a dose-dependent manner as measured using the human Colo 205 tumor cell line. Furthermore, TNF-alpha INH affected TNF-alpha synergism with IFN-gamma-induced HLA-DR antigen expression but had no effect on IFN-gamma activity. The data presented demonstrate that TNF-alpha bioactivity can be regulated at the protein level.

An aminopeptidase specific for methionine (peptidase M) has been purified from wild-type and mutant Salmonella typhimurium strains. Recombinant peptidase M was also purified from Escherichia coli. These preparations were characterized with respect to their physicochemical properties using analytical ultracentrifugation, SDS/PAGE, isoelectric focusing, titration curve analysis, amino acid analysis, N-and C-terminal sequencing and various spectroscopic methods. Peptidase M activity is stimulated by Co2+, in agreement with previous studies using crude extracts of Salmonella. The purified preparations did not contain significant amounts of any metal. Enzymically important metal is loosely associated and lost during enzyme purification. Peptidase M was shown to contain seven free sulphydryl residues none of which are involved in either intra-or inter-molecular disulphide bonds. Most appear solvent-accessible as evidenced by their reactivity under native conditions. Limited modification of the sulphydryl residues with either iodoacetamide or 5,5'-dithiobis(2-nitrobenzoic acid) led to inactivation. Several cysteines were shown to be labelled to various degrees by peptide mapping of inactivated S-[14C]carboxymethylated protein. Whether cysteine modification affects enzymic activity directly (blocking an active site) or indirectly (by causing conformational change) remains to be established.