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At least 631 records · Page 35Linked to original sources
PubMed2006

Identification of plant cytoskeleton-interacting proteins by screening for actin stress fiber association in mammalian fibroblasts.

Taking advantage of the high conservation of the cytoskeleton building blocks actin and tubulin between plant and animal kingdoms, we developed a functional genomic screen for the isolation of new plant cytoskeleton-binding proteins that uses a mammalian cell expression system. A yellow fluorescent protein (YFP)-fusion cDNA library from Arabidopsis was inserted into rat fibroblasts and screened for fluorescent chimeras localizing to cytoskeletal structures. The high-throughput screen was performed by an automated microscope. An initial set of candidate genes identified in the screen was isolated, sequenced, the full-length cDNAs were synthesized by RT-PCR and tested by biochemical approaches to verify the ability of the genes to bind actin directly. Alternatively, indirect binding via interaction with other actin-binding proteins was studied. The full-length cDNAs were transferred back to plants as YFP chimeras behind the CAMV-35S promoter. We give here two examples of new plant cytoskeletal proteins identified in the pilot screen. ERD10, a member of the dehydrin family of proteins, was localized to actin stress fibers in rat fibroblasts. Its direct binding to actin filaments was confirmed by several biochemical approaches. Touch-induced calmodulin-like protein, TCH2, was also localized to actin stress fibers in fibroblasts, but was unable to bind actin filaments directly in vitro. Nevertheless, it did bind to the IQ domains of Arabidopsis myosin VIII in a calcium-dependent manner. Further evidence for a cytoskeletal function of ERD10 was obtained in planta; GFP-ERD10 was able to protect the actin cytoskeleton from latrunculin-mediated disruption in Nicotiana benthamiana leaves.

Actins↗
PubMed2006

Experimental manipulations of microbial food web interactions in a humic lake: shifting biological drivers of bacterial community structure.

A previous multiyear study observed correlations between bacterioplankton community composition (BCC) and abundance and the dynamics of phytoplankton populations and bacterivorous grazers in a humic lake. These observations generated hypotheses about the importance of trophic interactions (both top-down and bottom-up) for structuring bacterial communities in this lake, which were tested using two multifactorial food web manipulation experiments that separately manipulated the intensity of grazing and the composition of the phytoplankton community. Our results, combined with field observations, suggest that a hierarchy of drivers structures bacterial communities in this lake. While other studies have noted links between aggregate measures of phytoplankton and bacterioplankton communities, we demonstrate here correlations between succession of phytoplankton assemblages and BCC as assessed by automated ribosomal intergenic spacer analysis (ARISA). We used a novel approach linking community ARISA data to phylogenetic assignments from sequence analysis of 16S rRNA gene clone libraries to examine the responses of specific bacterial phylotypes to the experimental manipulations. The synchronous dynamics of these populations suggests that primary producers may mediate BCC and diversity through labile organic matter production, which evolves in quality and quantity during phytoplankton succession. Superimposed on this resource-mediated control of BCC are brief periods of intense bacterivory that impact bacterial abundance and composition.

Animals↗
PubMed2001

High-density miniaturized thermal shift assays as a general strategy for drug discovery.

More general and universally applicable drug discovery assay technologies are needed in order to keep pace with the recent advances in combinatorial chemistry and genomics-based target generation. Ligand-induced conformational stabilization of proteins is a well-understood phenomenon in which substrates, inhibitors, cofactors, and even other proteins provide enhanced stability to proteins on binding. This phenomenon is based on the energetic coupling of the ligand-binding and protein-melting reactions. In an attempt to harness these biophysical properties for drug discovery, fully automated instrumentation was designed and implemented to perform miniaturized fluorescence-based thermal shift assays in a microplate format for the high throughput screening of compound libraries. Validation of this process and instrumentation was achieved by investigating ligand binding to more than 100 protein targets. The general applicability of the thermal shift screening strategy was found to be an important advantage because it circumvents the need to design and retool new assays with each new therapeutic target. Moreover, the miniaturized thermal shift assay methodology does not require any prior knowledge of a therapeutic target's function, making it ideally suited for the quantitative high throughput drug screening and evaluation of targets derived from genomics.

Estrogen Receptor alpha↗
PubMed2006

ADME related profiling in 96 and 384 well plate format--a novel and robust HT-assay for the determination of lipophilicity and serum albumin binding.

The failure of about half of the drug candidates is associated with poor pharmacokinetic properties leading to a huge loss of time and money [1]. Early profiling of drug like properties provides important information in order to screen out insoluble, poorly absorbed and toxic compounds. Today, large compound libraries have to be screened, and of course the total number of compounds will rise over the next years leading to a growing demand for fully automated assays. A balance between quality, speed, throughput, cost and information content can be accomplished by the careful selection of assays and experimental conditions. Here we describe a novel 384 well format assay for two important ADME related descriptors (lipophilicity and serum protein binding) as input parameters for a precise prediction of fraction absorbed, blood/organ distribution coefficients and permeability, in order to maximize the information about a compound at an early stage of discovery.

Chromatography, High Pressure Liquid↗
PubMed1989

Molecular cloning and sequencing of genomic DNA encoding aminopeptidase I from Saccharomyces cerevisiae.

Yeast aminopeptidase I is a vacuolar enzyme, which catalyzes the removal of amino acids from the NH2 terminus of peptides and proteins (Frey, J., and Rohm, K-H. (1978) Biochim. Biophys. Acta 527, 31-41). A yeast genomic DNA encoding aminopeptidase I was cloned from a yeast EMBL3A library and sequenced. The DNA sequence encodes a precursor protein containing 514 amino acid residues. The "mature" protein, whose NH2-terminal sequence was confirmed by automated Edman degradation, consists, based only on the DNA sequence, of 469 amino acids. A 45-residue presequence contains positively and negatively charged as well as hydrophobic residues, and its NH2-terminal residues could be arrayed in an amphiphilic alpha-helix. This presequence differs from the signal sequences which direct proteins across bacterial plasma membranes and endoplasmic reticulum or into mitochondria. It remains to be established how this unique presequence targets aminopeptidase I to yeast vacuoles and how this sorting utilizes classical protein secretory pathways. Further, the aminopeptidase I gene, localized previously by genetic mapping to yeast chromosome XI and called the LAP4 gene (Trumbly, R. J., and Bradley, G. (1983) J. Bacteriol. 156, 36-48), was determined by DNA blot analyses to be a single copy gene located on chromosome XI.

Amino Acid Sequence↗
PubMed1996

Fragment walking for long DNA sequencing by using a library as small as 16 primers.

A DNA sequence can be rapidly and efficiently determined by digesting it into segments small enough to be sequenced at one time, then assembling them into contigs by searching for overlaps. Fragment walking achieves this without subcloning or preparing many kinds of primers; fragments obtained by digesting a template DNA are sequenced in parallel directly from the fragment mixture by using a set of 16 primers. Since the sequence adjacent to each cutting site is determined together with the fragment sequence, the contiguous fragment can be easily determined. The complete template DNA sequence can thus efficiently determined. The sequencing of pUC19 (2.7 kb) from both sides was done in one step by using only 15 fragments. The redundancy was only about 1.3, greatly reducing base-reading redundancy. Automating this strategy would increase the speed and efficiency of large-scale DNA sequencing.

Base Sequence↗
PubMedDate not supplied

A simple automated procedure for the detection and identification of peaks in gas chromatography--continuous scan mass spectrometry. Application to systematic toxicological analysis of drugs in whole human blood.

Gas chromatography-mass spectrometry (GC-MS), which combines the separation power of GC with the power of MS for the identification of unknown compounds, possesses high potential in systematic toxicological analysis (STA). Different factors, however, do not allow this potential to be fully exploited. Between them, the low selectivity of the mass spectrometer operating in continuous scan plays a critical role, in many cases precluding the possibility of selecting mass spectra in the total ion chromatogram (TIC) sufficiently clean for a positive identification by library search, even when using reverse search algorithms. Moreover, the large amount of information contained in GC-MS data file that results from the analysis of a biological extract makes the efforts of manual search almost useless and requires the availability of reliable methods for the automated detection and identification of peaks in a TIC. In this paper, a simple procedure that improves the performance of a bench-top GC-MS system in the purification of mass spectra of coeluting compounds and that can be easily combined with the automated processing of a GC-MS data file is described. It is based on the subtraction of the intensities of successive pairs of scans in the TIC, on the detection of positive and negative peaks in the transformed chromatograms, and on the search of the corresponding background-subtracted electron ionization mass spectra against reference libraries. In order to evaluate the proposed procedure, GC-MS data files obtained for the analysis of extracts of blank whole blood spiked with more than 100 drugs, poisons, and their metabolites at a concentration of 0.5 mg/L were used. Compared with the search of the raw TICs, the proposed procedure increased the number of identified substances and, in many cases, obtained higher match quality values for identification.

Amitriptyline↗
PubMed2006

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%.

Bacteria↗
PubMed2002

Fully automated ab initio protein structure prediction using I-SITES, HMMSTR and ROSETTA.

MOTIVATION: The Monte Carlo fragment insertion method for protein tertiary structure prediction (ROSETTA) of Baker and others, has been merged with the I-SITES library of sequence structure motifs and the HMMSTR model for local structure in proteins, to form a new public server for the ab initio prediction of protein structure. The server performs several tasks in addition to tertiary structure prediction, including a database search, amino acid profile generation, fragment structure prediction, and backbone angle and secondary structure prediction. Meeting reasonable service goals required improvements in the efficiency, in particular for the ROSETTA algorithm. RESULTS: The new server was used for blind predictions of 40 protein sequences as part of the CASP4 blind structure prediction experiment. The results for 31 of those predictions are presented here. 61% of the residues overall were found in topologically correct predictions, which are defined as fragments of 30 residues or more with a root-mean-square deviation in superimposed alpha carbons of less than 6A. HMMSTR 3-state secondary structure predictions were 73% correct overall. Tertiary structure predictions did not improve the accuracy of secondary structure prediction.

Algorithms↗
PubMed2002

Analytical methods for the monitoring of solid phase organic synthesis.

Solid phase synthesis (SPS) is a powerful technique to assemble compound libraries in high-throughput parallel and combinatorial synthesis. The widespread applications of these techniques required the development of analytical methods for both structural elucidation and reaction monitoring. This review covers some recently developed techniques for on-bead analyses together with solution-state ones. Particular emphasis is devoted to software and hardware improvements for automated high-throughput analysis.

Chemistry Techniques, Analytical↗
PubMed2005

Identification of an immunogenic EWS-FLI1-derived HLA-DR-restricted T helper cell epitope.

Immunotherapy with tumor-specific peptide-loaded dendritic cells represents a promising therapeutic approach for patients with multifocal primary or early relapsed Ewing family tumors (EFT). The authors therefore screened a peptide library derived from the fusion region of the EFT-specific chimeric transcription factor EWS-FLI1 for immunogenic peptides. T-cell priming with 10 peptides was evaluated using IFN(gamma) video-assisted automated enzyme-linked immunospot technique. The authors report the identification of the first EFT-specific immunogenic T-cell epitope so far. Its identification will lead to a better understanding of EFT immunology and may improve DC-based immunotherapy.

Amino Acid Sequence↗
PubMed1996

A survey of the goat genome transcribed in the lactating mammary gland.

To fulfill its primary function, which is to synthesize milk during the course of lactation, the mammary gland requires efficient transcriptional, translational, and secretory machineries involving multiple genes among which promising candidates underlying the genetic variation of milk production have to be found. With the aim of providing a first transcriptional profile of lactating mammary tissue, a non-normalized cDNA library has been constructed from the udder of a lactating goat. After having discarded cDNA clones encoding the major milk proteins the rapid characterization of genes expressed in this tissue, by automated partial cDNA sequencing, was used to analyze a total of 435 cDNA clones. Examination of the Expressed Sequence Tags (ESTs) for similarities with sequence databases identified 234 cDNAs corresponding to 140 unique genes or proteins. Eighty-three clones, not similar to any current database entries, representing 77 novel sequences unrelated to previously described genes, were thus identified. Tissue specificity and relative abundance of 18 of these 77 unidentified clones were examined by dot blot and RT-PCR experiments. Sequence data were subsequently used to assign six genes of unknown localization in the bovine genome, to synteny groups by use of bovine-hamster cell hybrids and PCR.

Animals↗
PubMed2004

Identification of compounds that enhance the anti-lymphoma activity of rituximab using flow cytometric high-content screening.

In this report, we describe a new flow cytometry technique termed flow cytometric high-content screening (FC-HCS) which involves semi-automated processing and analysis of multiparameter flow cytometry samples. As a first test of the FC-HCS technique, we used it to screen a 2000-compound library, called the National Cancer Institute (NCI) Diversity Set, to identify agents that would enhance the anti-lymphoma activity of the therapeutic monoclonal antibody rituximab. FC-HCS identified 15 compounds from the Diversity Set that significantly enhanced the ability of rituximab to inhibit cell cycle progression and induce apoptosis in lymphoma cells. The validity of the screening results was confirmed for several compounds using additional assays of cell proliferation, apoptosis and cell growth. The FC-HCS technique was relatively simple and reliable and could process up to 1000 samples/day on a single flow cytometer. The FC-HCS technique may be useful for a variety of applications including drug discovery, immunologic monitoring of patients, functional genomics studies and tissue engineering efforts.

Antibodies, Monoclonal↗
PubMedDate not supplied

Solution phase synthesis of a library of tetrasubstituted pyrrole amides.

An efficient strategy for the solution-phase parallel synthesis of a library of pyrrole-amides is described. Key reactions include functional homologation of beta-ketoesters with a set of aldehydes followed by oxidation to produce a series of differently substituted 1,4-dicarbonyl compounds. Rapid cyclization using a microwave-assisted Paal-Knorr reaction provided a set of 24 pyrrole esters that were further functionalized through a trimethylaluminum-mediated aminolysis to obtain a larger library of 288 diverse pyrrole-3-amides. The tetrasubstitution allows a good exploration of the chemical space around the central pyrrole core. The last step was entirely automated with a Bohdan Myriad personal synthesizer.

Amides↗
PubMed2003

Transcriptome analysis of the acoelomate human parasite Schistosoma mansoni.

Schistosoma mansoni is the primary causative agent of schistosomiasis, which affects 200 million individuals in 74 countries. We generated 163,000 expressed-sequence tags (ESTs) from normalized cDNA libraries from six selected developmental stages of the parasite, resulting in 31,000 assembled sequences and 92% sampling of an estimated 14,000 gene complement. By analyzing automated Gene Ontology assignments, we provide a detailed view of important S. mansoni biological systems, including characterization of metazoa-specific and eukarya-conserved genes. Phylogenetic analysis suggests an early divergence from other metazoa. The data set provides insights into the molecular mechanisms of tissue organization, development, signaling, sexual dimorphism, host interactions and immune evasion and identifies novel proteins to be investigated as vaccine candidates and potential drug targets.

Animals↗
PubMed1993

DNA sequencing: modular primers assembled from a library of hexamers or pentamers.

Here we report a striking effect displayed by "modular primers," which consist of hexamer or pentamer oligonucleotide modules base-stacked to each other upon annealing to a DNA template. Such a combination of modules is found to prime DNA sequencing reactions uniquely, unlike either of the modules alone. We attribute this effect in part to the increase in the affinity of an oligonucleotide for the template in the presence of an adjacent module. All possible pentamer (or hexamer) sequences total 1024 (or 4096) samples, a manageable size for a presynthesized library. This approach can replace the synthesis of primers, which is the current bottleneck in time and cost of the primer walking sequencing, and can allow full automation of the closed cycle of walking.

Base Sequence↗
PubMed2005

Unsaturated fatty acids are inhibitors of bacterial conjugation.

This report describes a high-throughput assay to identify substances that reduce the frequency of conjugation in Gram-negative bacteria. Bacterial conjugation is largely responsible for the spread of multiple antibiotic resistances in human pathogens. Conjugation inhibitors may provide a means to control the spread of antibiotic resistance. An automated conjugation assay was developed that used plasmid R388 and a laboratory strain of Escherichia coli as a model system, and bioluminescence as a reporter for conjugation activity. Frequencies of conjugation could be measured continuously in real time by the amount of light produced, and thus the effects of inhibitory compounds could be determined quantitatively. A control assay, run in parallel, allowed elimination of compounds affecting cell growth, plasmid stability or gene expression. The automated conjugation assay was used to screen a database of more than 12,000 microbial extracts known to contain a wide variety of bioactive compounds (the NatChem library). The initial hit rate was 1.4 %. From these, 48 extracts containing active compounds and representing a variety of organisms and extraction conditions were subjected to fractionation (24 fractions per extract). The 52 most active fractions were subjected to a secondary analysis to determine the range of plasmid inhibition. Plasmids R388, R1 and RP4 were used as representatives of a variety of plasmid transfer systems. Only one fraction (of complex composition) affected transfer of all three plasmids, while four other fractions were active against two of them. Two separate compounds were identified from these fractions: linoleic acid and dehydrocrepenynic acid. Downstream analysis showed that the chemical class of unsaturated fatty acids act as true inhibitors of conjugation.

Conjugation, Genetic↗