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PubMed2005

Structural dissection and high-throughput screening of mannosylglycerate synthase.

The enzymatic transfer of activated mannose yields mannosides in glycoconjugates and oligo- and polysaccharides. Yet, despite its biological necessity, the mechanism by which glycosyltransferases recognize mannose and catalyze its transfer to acceptor molecules is poorly understood. Here, we report broad high-throughput screening and kinetic analyses of both natural and synthetic substrates of Rhodothermus marinus mannosylglycerate synthase (MGS), which catalyzes the formation of the stress protectant 2-O-alpha-D-mannosyl glycerate. The sequence of MGS indicates that it is at the cusp of inverting and retaining transferases. The structures of apo MGS and complexes with donor and acceptor molecules, including GDP-mannose, combined with mutagenesis of the binding and catalytic sites, unveil the mannosyl transfer center. Nucleotide specificity is as important in GDP-D-mannose recognition as the nature of the donor sugar.

Crystallography↗

PubMed2006

High throughput SNP and expression analyses of candidate genes for non-syndromic oral clefts.

BACKGROUND: Recent work suggests that multiple genes and several environmental risk factors influence risk for non-syndromic oral clefts, one of the most common birth defects in humans. Advances in high-throughput genotyping technology now make it possible to test multiple markers in many candidate genes simultaneously. METHODS: We present findings from family based association tests of single nucleotide polymorphism (SNP) markers in 64 candidate genes genotyped using the BeadArray approach in 58 case-parent trios from Maryland (USA) to illustrate how multiple markers in multiple genes can be analysed. To assess whether these genes were expressed in human craniofacial structures relevant to palate and lip development, we also analysed data from the Craniofacial and Oral Gene Expression Network (COGENE) consortium, and searched public databases for expression profiles of these genes. RESULTS: Thirteen candidate genes showed significant evidence of linkage in the presence of disequilibrium, and ten of these were found to be expressed in relevant embryonic tissues: SP100, MLPH, HDAC4, LEF1, C6orf105, CD44, ALX4, ZNF202, CRHR1, and MAPT. Three other genes showing statistical evidence (ADH1C, SCN3B, and IMP5) were not expressed in the embryonic tissues examined here. CONCLUSIONS: This approach demonstrates how statistical evidence on large numbers of SNP markers typed in case-parent trios can be combined with expression data to identify candidate genes for complex disorders. Many of the genes reported here have not been previously studied as candidates for oral clefts and warrant further investigation.

Chromosome Mapping↗

PubMed2006

Identification and validation of differences in protein levels in normal, premalignant, and malignant lung cells and tissues using high-throughput Western Array and immunohistochemistry.

The identification of proteins, which exhibit different levels in normal, premalignant, and malignant lung cells, could improve early diagnosis and intervention. We compared the levels of proteins in normal human bronchial epithelial (NHBE) and tumorigenic HBE cells (1170-I) by high-throughput immunoblotting (PowerBlot Western Array) using 800 monoclonal antibodies. This analysis revealed that 87 proteins increased by >2-fold, and 45 proteins decreased by >2-fold, in 1170-I compared with NHBE cells. These proteins are involved in DNA synthesis and repair, cell cycle regulation, RNA transcription and degradation, translation, differentiation, angiogenesis, apoptosis, cell adhesion, cytoskeleton and cell motility, and the phosphatidylinositol 3-kinase signaling pathway. Conventional Western blotting using lysates of normal, immortalized, transformed, and tumorigenic HBEs and non-small cell lung cancer cell lines confirmed some of these changes. The expression of several of these proteins has been then analyzed by immunohistochemistry in tissue microarrays containing 323 samples, including normal bronchial epithelium, hyperplasia, squamous metaplasia, dysplasias, squamous cell carcinomas, atypical adenomatous hyperplasia, and adenocarcinomas from 144 patients. The results of the immunohistochemical studies correlated with the Western blotting findings and showed gradual increases (caspase-8, signal transducers and activators of transcription 5, and p70s6K) or decrease (E-cadherin) in levels with tumor progression. These results indicate that the changes in proteins detected in this study may occur early in lung carcinogenesis and persist in lung cancer. In addition, some of the proteins detected by this approach may be novel biomarkers for early detection of lung cancer and novel targets for chemoprevention or therapy.

Adult↗

PubMed2006

Physical map of the wheat high-grain protein content gene Gpc-B1 and development of a high-throughput molecular marker.

Grain protein content (GPC) is important for human nutrition and has a strong influence on pasta and bread quality. A quantitative trait locus, derived from a Triticum turgidum ssp. dicoccoides accession (DIC), with an average increase in GPC of 14 g kg(-1) was mapped on chromosome 6BS. Using the wheat-rice colinearity, a high-density map of the wheat region was developed and the quantitative trait locus was mapped as a simple Mendelian locus designated Gpc-B1. A physical map of approx. 250 kb of the Gpc-B1 region was developed using a tetraploid wheat bacterial artificial chromosome library. The constructed physical map included the two Gpc-B1 flanking markers and one potential candidate gene from the colinear rice region completely linked to Gpc-B1. The relationship between physical and genetic distances and the feasibility of isolating genes by positional cloning in wheat are discussed. A high-throughput codominant marker, Xuhw89, was developed. A 4-bp deletion present in the DIC allele was absent in a collection of 117 cultivated tetraploid and hexaploid wheat germplasm, suggesting that this marker will be useful to incorporate the high GPC allele from the DIC accession studied here into commercial wheat varieties.

Chromosomes, Plant↗

PubMed2005

Chiral aminonaphthol-catalyzed enantioselective carbonyl addition of diethylzinc to aromatic aldehydes high-throughput screened by CD-HPLC analysis.

Optically active aminonaphthols derivatives are obtained by condensation of 2-naphthol, substituted benzaldehyde, and (S)-methylbenzylamine under mild conditions, without side products. Their absolute configurations are determined by X-ray crystallographic analysis. The addition of diethylzinc to aromatic aldehydes is considerably accelerated by the presence of a catalytic amount of crystalline to give, after hydrolysis, the corresponding 1-phenylpropanol in good enantiomeric purity, as determined by CD-HPLC analysis as HTPS (high-throughput screening).

Aldehydes↗

PubMed2001

An improved high throughput heteroduplex mutation detection system for screening BRCA2 mutations-fluorescent mutation detection (F-MD).

We describe an improved, fast, automated method for screening large genes such as BRCA2 for germline genomic mutations. The method is based on heteroduplex analysis, and has been adapted for a high throughput application by combining the fluorescent technology of automated sequencers and robotic sample handling. This novel approach allows the entire BRCA2 gene to be screened with appropriate overlaps in four lanes of an ABI 377 gel. The method will detect all types of mutations, especially point mutations, more reliably and robustly than other commonly used conformational sensitive methods (e.g. CSGE). In addition we show that this approach, which relies on band shift detection, is able to detect single base substitutions that have hitherto only been detectable by direct sequencing methods.

BRCA2 Protein↗

PubMed2001

High throughput physicochemical profiling for drug discovery.

The drug development bottlenecks of attrition and development time are being addressed by acquiring a profile of the candidate's physicochemical and physiological properties during early discovery phases. This information assists selection and optimization of pharmaceutical properties in parallel with activity. High throughput methods to measure the properties: solubility, permeability, lipophilicity, pKa, stability and integrity are described and compared in this article. The underlying discovery requirements, needs and application strategies are discussed.

Animals↗

PubMed1998

Rapid assay processing by integration of dual-color fluorescence cross-correlation spectroscopy: high throughput screening for enzyme activity.

Dual-color fluorescence cross-correlation spectroscopy (dual-color FCS) has previously been shown to be a suitable tool not only for binding but also for catalytic rate studies. In this work, its application as a rapid method for high-throughput screening (HTS) and evolutionary biotechnology is described. This application is called RAPID FCS (rapid assay processing by integration of dual-color FCS) and does not depend on the characterization of diffusion parameters that is the prerequisite for conventional fluorescence correlation spectroscopy. Dual-color FCS parameters were optimized to achieve the shortest analysis times. A simulated HTS with homogeneous assays for different restriction endonucleases (EcoRI, BamHI, SspI, and HindIII) achieved precise yes-or-no decisions within analysis times of about 1 s per sample. RAPID FCS combines these short analysis times with the development of fast and flexible assays resulting in sensitive, homogeneous fluorescence-based assays, where a chemical linkage between different fluorophores is either cleaved or formed, or where differently labeled molecules interact by noncovalent binding. In principle, assay volumes can be reduced to submicroliters without decreasing the signal strength, making RAPID FCS an ideal tool for ultra-HTS when combined with nanotechnology. RAPID FCS can accurately probe 10(4) to 10(5) samples per day, and possibly more. In addition, this method has the potential to be an efficient tool for selection strategies in evolutionary biotechnology, where rare and specific binding or catalytic properties have to be screened in large numbers of samples.

Biochemistry↗

PubMed2004

Applications of fluorescent polymer superquenching to high throughput screening assays for protein kinases.

Protein kinases are involved in the regulation of cellular metabolism, growth, differentiation, and proliferation. Aberrations in their function can lead to diseases such as cancer and inflammation. Protein kinases are therefore possible targets for drug therapies. To address the need for high throughput screening of potential inhibitors, QTL has developed a homogeneous and robust kinase assay for use in multiwell plate format. The QTL Lightspeed fluorescence superquenching-based kinase assays do not require specialized equipment, nor do they involve the use of radioactive hazardous materials or antibodies. QTL Lightspeed kinase assays directly measure the enzymatic activity of the target and do not involve secondary (detector) enzyme. In this article, we compare QTL Lightspeed protein kinase assays using Protein Kinase A, Protein Kinase Balpha/Akt1, and ribosomal S6 kinase-2 as examples with other commercially available kinase kits. Our data show that QTL Lightspeed kinase assays offer significant advantages over the current commercial kits in terms of both sensitivity and performance. The QTL Lightspeed kinase assay also offers a kinetic assay mode where the substrate phosphorylation can be monitored in real-time.

Dose-Response Relationship, Drug↗

PubMed2004

Development and validation of a high-throughput radiometric cyp2c9 inhibition assay using tritiated diclofenac.

A rapid and sensitive radiometric assay for assessing the potential of drugs to inhibit cytochrome P450 (P450) 2C9 in human liver microsomes is described. In contrast to the conventional diclofenac 4'-hydroxylation assay, the new method does not require high performance liquid chromatography (HPLC) separation and mass spectrometry. The assay is based on the release of tritium as tritiated water that occurs upon CYP2C9-mediated 4'-hydroxylation of diclofenac labeled with tritium in the 4' position. The radiolabeled product is separated from the substrate using 96-well solid-phase extraction plates. The reaction is NADPH-dependent, and sensitive to CYP2C9 inhibitors and inhibitory monoclonal antibodies, but not to inhibitors of or antibodies against other P450 enzymes. Competition experiments using tritiated and unlabeled diclofenac indicated that CYP2C9-mediated diclofenac 4'-hydroxylation exhibits positive cooperativity and no significant kinetic isotope effect or NIH shift. IC(50) values for 18 structurally diverse chemical inhibitors were not significantly different from those determined in the diclofenac 4'-hydroxylation assay, using HPLC-tandem mass spectrometry. All the steps of the new assay, namely, incubation, product separation, and radioactivity counting, are performed in 96-well format and can be automated. This assay thus represents a high-throughput version of the classic diclofenac 4'-hydroxylation assay, which is one of the most widely used methods to assess the potential for CYP2C9 inhibition of new chemical entities.

Antibodies, Monoclonal↗

PubMed2001

High-throughput detection of West Nile virus RNA.

The recent outbreaks of West Nile virus (WNV) in the northeastern United States and other regions of the world have made it essential to develop an efficient protocol for surveillance of WNV. In the present report, we describe a high-throughput procedure that combines automated RNA extraction, amplification, and detection of WNV RNA. The procedure analyzed 96 samples in approximately 4.5 h. A robotic system, the ABI Prism 6700 Automated Nucleic Acid workstation, extracted RNA and set up reactions for real-time reverse transcription (RT)-PCR in a 96-well format. The robot extracted RNA with a recovery as efficient as that of a commercial RNA extraction kit. A real-time RT-PCR assay was used to detect and quantitate WNV RNA. Using in vitro transcribed RNA, we estimated the detection limit of the real-time RT-PCR to be approximately 40 copies of RNA. A standard RT-PCR assay was optimized to a sensitivity similar to that of the real-time RT-PCR. The standard assay can be reliably used to test a small number of samples or to confirm previous test results. Using internal primers in a nested RT-PCR, we increased the sensitivity by approximately 10-fold compared to that of the standard RT-PCR. The results of the study demonstrated for the first time that the use of an automated system for the purpose of large-scale viral RNA surveillance dramatically increased the speed and efficiency of sample throughput for diagnosis.

Animals↗

PubMed2004

High-throughput 3D structural homology detection via NMR resonance assignment.

One goal of the structural genomics initiative is the identification of new protein folds. Sequence-based structural homology prediction methods are an important means for prioritizing unknown proteins for structure determination. However, an important challenge remains: two highly dissimilar sequences can have similar folds & how can we detect this rapidly, in the context of structural genomics? High-throughput NMR experiments, coupled with novel algorithms for data analysis, can address this challenge. We report an automated procedure, called HD, for detecting 3D structural homologies from sparse, unassigned protein NMR data. Our method identifies 3D models in a protein structural database whose geometries best fit the unassigned experimental NMR data. HD does not use, and is thus not limited by sequence homology. The method can also be used to confirm or refute structural predictions made by other techniques such as protein threading or homology modelling. The algorithm runs in O(pn + pn(5/2) log (cn)+p log p) time, where p is the number of proteins in the database, n is the number of residues in the target protein and c is the maximum edge weight in an integer-weighted bipartite graph. Our experiments on real NMR data from 3 different proteins against a database of 4,500 representative folds demonstrate that the method identifies closely related protein folds, including sub-domains of larger proteins, with as little as 10-30% sequence homology between the target protein (or sub-domain) and the computed model. In particular, we report no false-negatives or false-positives despite significant percentages of missing experimental data.

Algorithms↗

PubMed2000

Differential binding affinities of PCBs, HO-PCBs, and aroclors with recombinant human, rainbow trout (Onchorhynkiss mykiss), and green anole (Anolis carolinensis) estrogen receptors, using a semi-high throughput competitive binding assay.

A comparative study was undertaken to assess the ability of 44 polychlorinated biphenyls (PCBs), 9 hydroxylated PCBs (HO-PCBs), and 8 aroclors at concentrations ranging from 1 nM to 10 microM to compete with [3H]17beta-estradiol (E2) for binding to bacterially expressed fusion proteins using a semi-high throughput competitive-binding assay. The fusion proteins consisted of the D, E, and F domains of human (alpha), cloned reptilian (Anolis carolinensis) and recloned rainbow trout (Onchorhynkiss mykiss) estrogen receptors (ER) linked to the glutathione S-transferase (GST) protein. GST-hERalphadef (human), GST-aERdef (reptile) and GST-rtERdef (rainbow trout) fusion proteins exhibited high affinity for E2 with dissociation constants (Kd) of 0.4+/-0.1 nM, 0.7+/-0.2 nM, and 0.6+/-0.1 nM, respectively. Of the 44 PCBs examined, only PCBs 104, 184, and 188 effectively competed with [3H]E2 for binding to the GST-rtERdef protein with IC50 values ranging from 0.4-1.3 microM. In contrast, these same congeners only caused a 30% displacement of [3H]E2 in GST-hERalphadef and GST-aERdef proteins. Several additional congeners were found to bind to the GST-rtERdef fusion protein, although the degree of interaction varied among congeners. Among the HO-PCBs, 2',3',4',5'-tetrachloro-4-biphenylol and 2,6,2',6'-tetrachloro-4-biphenylol bound to all three fusion proteins with IC50 values ranging from 0.1-0.3 microM. Dimethyl sulphoxide (DMSO) concentrations of 20% significantly increased the ability of PCBs 104, 184, and 188 to compete with [3H]E2 for binding to the GST-ERdef fusion proteins, whereas at 20% DMSO, a significant reduction in saturable binding was observed. These results demonstrate that ERs from different species exhibit differential ligand preferences and relative binding affinities for PCBs, which can be dramatically affected by DMSO concentration.

Amino Acid Sequence↗

PubMed2005

High-throughput screening of protein binding by equilibrium dialysis combined with liquid chromatography and mass spectrometry.

A new approach for screening plasma protein binding is presented. The method is based on equilibrium dialysis combined with rapid generic LC-MS bioanalysis by using a sample pooling approach enabling high-throughput screening of protein binding in the drug discovery phase. The method is evaluated by a comparison of measured unbound free fractions f(u) (%) between single and pooled compounds for a test set of structurally diverse compounds with a wide range of unbound fractions. Test compounds include 1 acidic and 10 basic drug standards along with 36 new chemical entities. A good correlation (R2>0.95) of f(u) (%) between the single and pooled compounds is found, suggesting that at least 10 compounds can be simultaneously measured with acceptable accuracy. A simplified drug-protein binding model is applied to calculate the f(u) (%) of drugs at various drug and protein concentrations and this is applied to elucidate the applicability of the sample pooling approach from a theoretical standpoint. Moreover, pH shifts in the plasma were observed after dialysis when using different types of buffers and the impact of that on the f(u) is illustrated in association with their physicochemical properties, in particular the ionization state of compounds by the profile of effective mobility as a function of pH. A new buffer is proposed being able to minimize the pH shift of plasma during the dialysis. In addition, the application of the proposed buffer does not necessarily require adjusting plasma pH before the dialysis and utilizing a CO2 incubator during the dialysis. The effect of the ionic strengths of different buffers on MS signals is investigated with regard to ion suppression. The sample pooling method not only significantly reduces the plasma volume required but also the number of bioanalysis samples as compared to the single compound measurements by a conventional approach. The new proposed approach is especially beneficial for measuring in vitro protein binding in matrices such as mouse plasma where plasma is available only in limited amounts. The current new development will facilitate the drug discovery process by more rapidly assessing the protein binding potential of drug candidates.

Animals↗

PubMed2005

A high-throughput, near-saturating screen for type III effector genes from Pseudomonas syringae.

Pseudomonas syringae strains deliver variable numbers of type III effector proteins into plant cells during infection. These proteins are required for virulence, because strains incapable of delivering them are nonpathogenic. We implemented a whole-genome, high-throughput screen for identifying P. syringae type III effector genes. The screen relied on FACS and an arabinose-inducible hrpL sigma factor to automate the identification and cloning of HrpL-regulated genes. We determined whether candidate genes encode type III effector proteins by creating and testing full-length protein fusions to a reporter called Delta79AvrRpt2 that, when fused to known type III effector proteins, is translocated and elicits a hypersensitive response in leaves of Arabidopsis thaliana expressing the RPS2 plant disease resistance protein. Delta79AvrRpt2 is thus a marker for type III secretion system-dependent translocation, the most critical criterion for defining type III effector proteins. We describe our screen and the collection of type III effector proteins from two pathovars of P. syringae. This stringent functional criteria defined 29 type III proteins from P. syringae pv. tomato, and 19 from P. syringae pv. phaseolicola race 6. Our data provide full functional annotation of the hrpL-dependent type III effector suites from two sequenced P. syringae pathovars and show that type III effector protein suites are highly variable in this pathogen, presumably reflecting the evolutionary selection imposed by the various host plants.

Arabidopsis↗

PubMed2000

Ultra-high throughput screen of two-million-member combinatorial compound collection in a miniaturized, 1536-well assay format.

Results of a complete survey of the more than 2-million-member Pharmacopeia compound collection in a 1536-well microvolume screening assay format are reported. A complete technology platform, enabling the performance of ultra-high throughput screening in a miniaturized 1536-well assay format, has been assembled and utilized. The platform consists of tools for performing microvolume assays, including assay plates, liquid handlers, optical imagers, and data management software. A fluorogenic screening assay for inhibition of a protease enzyme target was designed and developed using this platform. The assay was used to perform a survey screen of the Pharmacopeia compound collection for active inhibitors of the target enzyme. The results from the survey demonstrate the successful implementation of the ultra-high throughout platform for routine screening purposes. Performance of the assay in the miniaturized format is equivalent to that of a standard 96-well assay, showing the same dependence on kinetic parameters and ability to measure enzyme inhibition. The survey screen identified an active class of compounds within the Pharmacopeia compound collection. These results were confirmed using a standard 96-well assay.

Chemistry, Pharmaceutical↗

PubMed2006

An alternative strategy for high throughput generation and characterization of monoclonal antibodies against human plasma proteins using fractionated native proteins as immunogens.

Construction of a monoclonal antibody (mAb) bank containing a vast variety of antibodies against human tissue proteins is important for proteomic research. A novel strategy of subtractive immunization using fractionated native proteins was developed for high throughput generation of mAb against human plasma proteins. By this novel approach, the bottleneck of antigen preparation can be overcome by combining repeated immunization of animals with subtracted fractions of plasma or tissue proteins and identification of target antigen by immunoprecipitation/mass spectrum strategies. Plasma freshly collected from healthy adults was pooled and three fractions were prepared by size exclusion chromatography. Mice were immunized with the fractionated plasma proteins, and 205 strains of hybridomas secreting mAb were obtained after two-round subtractive immunizations and cell fusions. In the first round, 110 strains of hybridomas were established, in which 77 strains secreting mAb were identified against 10 human plasma high-abundant proteins. In the second round, plasma fraction I was absorbed with mAb against IgM, IgG, ceruloplasmin and haptoglobin. The absorbed fraction I was used as immunogen for the second round immunization and cell fusion. Ninety-five strains of hybridomas secreting mAb were obtained. Although the target antigens of mAb from 82 strains of hybridomas were identified as IgM, IgA, alpha2-macroglobulin and fibrinogen, about 85% antibodies obtained from this round were identified as new antibodies when compared with mAb obtained in the first round immunization with plasma fraction I. The results suggest that subtractive immunization with fractionated plasma proteins followed by identification of antigens with immunoprecipitation/mass spectrum may be an effective approach for rapid preparation of mAb against high-and medium-abundant plasma or tissue proteins.

Amino Acid Sequence↗

PubMed2001

High-throughput cytochrome P450 (CYP) inhibition screening via cassette probe-dosing strategy: III. Validation of a direct injection/on-line guard cartridge extraction-tandem mass spectrometry method for CYP2C19 inhibition evaluation.

A new direct injection/on-line guard cartridge extraction-tandem mass spectrometry (DI/GCE-MS-MS) method has been validated for high-throughput evaluation of cytochrome P450 (CYP) 2C19 inhibition potential using human hepatic microsomes and 96-well microtiter plates. Microsomal incubations were quenched with formic acid, centrifuged, and the resulting supernatants were injected for DI/GCE-MS-MS analysis. Due to the use of a C(18) guard cartridge (4x2.0 mm I.D.), this method exhibits several advantages such as little sample preparation, rapid on-line extraction, short analysis time (2.5 min), and minimal source contamination and performance deterioration. The DI/GCE-MS-MS method demonstrates an inter-day accuracy ranged from 0.3 to 2.4% with precision ranging from 2.0 to 3.0% for the quantification of 4-hydroxymephenytoin, a marker metabolite of S-mephenytoin mediated by CYP2C19, in microsomal incubations. The CYP2C19 inhibition assay has been validated using tranylcypromine as a known inhibitor of the isoform. The IC(50) value (43.5 microM) measured by the new method is in agreement with a reported literature value (approximately 30 microM).

Aryl Hydrocarbon Hydroxylases↗