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Results for “combinatorial indexing”

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100 records · Page 6Linked to original sources
PubMed1999

Use of a cell-based, lawn format assay to rapidly screen a 442,368 bead-based peptide library.

A cell-based, lawn format assay utilizing an in situ photocleavage method has been developed that allows the rapid examination of large bead-based compound libraries as discrete molecules. The format uses frog melanophore cells in a contiguous, adherent, confluent layer in small petri dishes covered with a 0.5-1-mm layer of agarose containing 130 micron diameter TentaGel beads at a density of 2-20 beads/mm2. Employing this technique a 9-mer, 442,368-member peptide library (designed around the 13 amino acid alpha-MSH peptide sequence) made up of 12 separate pools of 36,864 peptides/pool was assayed. Initially, a fraction (approximately 10%) of each pool was scanned (approximately 3700 beads from each pool) in 60-mm petri dishes to identify the most active pools. Upon direct photocleavage of the beads with UV light (365 nm), each petri dish was photographed over a 60-min period with a CCD camera to record changes in light intensity as an index of melanosome dispersion. Active beads were those that were surrounded by a localized decrease in light transmittance indicating melanosome dispersed cells. Upon examination with a dissecting microscope, single beads centrally located to a circular array of dispersed cells were identified and removed from the agarose and sequenced by Edman degradation to determine the peptide sequence. Re-synthesized peptides were re-examined against alpha-MSH receptor to confirm and quantify the activity. Several 9-mer peptides were identified with potencies similar to the natural 13-mer peptide. This method allows for the rapid screening of large bead-based photo-cleavable peptide libraries with the advantage that each compound is screened as a discrete molecule in a well-less format.

Animals↗
PubMed2002

Current trends in lead discovery: are we looking for the appropriate properties?

The new drug discovery paradigm is based on high-throughput technologies, both with respect to synthesis and screening. The progression HTS hits --> lead series --> candidate drug --> marketed drug appears to indicate that the probability of reaching launched status is one in a million. This has shifted the focus from good quality candidate drugs to good quality leads. We examined the current trends in lead discovery by comparing MW (molecular weight), LogP (octanol/water partition coefficient, estimated by Kowwin) and LogSw (intrinsic water solubility, estimated by Wskowwin) for the following categories: 62 leads and 75 drugs; compounds in the development phase (I, II, III and launched), as indexed in MDDR; and compounds indexed in medicinal chemistry journals, categorized according to their biological activity. Comparing the distribution of the above properties, the 62 lead structures show the lowest median with respect to MW (smaller) and LogP (less hydrophobic), and the highest median with respect to LogSw (more soluble). By contrast, over 50% of the medicinal chemistry compounds with activities above 1 nanomolar have MW > 425, LogP > 4.25 and LogSw < -4.75, indicating that the reported active compounds are larger, more hydrophobic and lesssoluble when compared to time-tested quality leads. In the MDDR set, a progressive constraint to reduce MW and LogP, and to increase LogSw, can be observed when examining trends in the developmental sequence: phase I, II, III and launched drugs. These trends indicate that other properties besides binding affinity, e.g., solubility and hydrophobicity, need to be considered when choosing the appropriate leads.

Chemistry, Pharmaceutical↗
PubMed2001

Mining the chemical quarry with joint chemical probes: an application of latent semantic structure indexing (LaSSI) and TOPOSIM (Dice) to chemical database mining.

In this study we use a novel similarity search technique called latent semantic structure indexing (LaSSI) with joint chemical probes as queries to mine the MDL drug data report database. LaSSI is based on latent semantic indexing developed for searching textual databases. We use atom pair and topological torsion descriptors in our calculations. The results obtained with LaSSI are compared with another in-house similarity search technique TOPOSIM. The results from the similarity searches using joint chemical probes are significantly better than searches using single chemical probes for both LaSSI and TOPOSIM. The selected molecules are closely related in activity to their queries and are ranked among the top 300 scoring molecules of the 82 860 entries in the database. Our implementation of LaSSI is very fast and efficient in finding active compounds. The results also show that LaSSI consistently retrieves more diverse chemical structures representative of the joint chemical probes in comparison to TOPOSIM. The use of multimolecule topological probes to identify compounds complements the use of searching databases with 3D pharmacophore hypotheses.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed2004

Antifibrotic effects of a tissue inhibitor of metalloproteinase-1 antibody on established liver fibrosis in rats.

Liver fibrosis is characterized by increased synthesis, and decreased degradation, of extracellular matrix (ECM) within the injured tissue. Decreased ECM degradation results, in part, from increased expression of tissue inhibitor of metalloproteinase-1 (TIMP-1), which blocks matrix metalloproteinase (MMP) activity. TIMP-1 is also involved in promoting survival of activated hepatic stellate cells (HSCs), a major source of ECM. This study examined the effects of blocking TIMP-1 activity in a clinically relevant model of established liver fibrosis. Rats were treated with carbon tetrachloride (CCl(4)), or olive oil control, for 6 weeks; 24 days into the treatment, the rats were administered a neutralizing anti-TIMP-1 antibody derived from a fully human combinatorial antibody library (HuCAL), PBS, or an isotype control antibody. Livers from CCl(4)-treated rats exhibited substantial damage, including bridging fibrosis, inflammation, and extensive expression of smooth muscle alpha-actin (alpha-SMA). Compared to controls, rats administered anti-TIMP-1 showed a reduction in collagen accumulation by histological examination and hydroxyproline content. Administration of anti-TIMP-1 resulted in a marked decrease in alpha-SMA staining. Zymography analysis showed antibody treatment decreased the activity of MMP-2. In conclusion, administration of a TIMP-1 antibody attenuated CCl(4)-induced liver fibrosis and decreased HSC activation and MMP-2 activity.

Actins↗
PubMed2005

Molecular mechanisms of membrane perturbation by antimicrobial peptides and the use of biophysical studies in the design of novel peptide antibiotics.

Antibiotic resistant bacterial strains represent a global health problem with a strong social and economic impact. Thus, there is an urgent need for the development of antibiotics with novel mechanisms of action. There is currently an extensive effort to understand the mode of action of antimicrobial peptides which are considered as one alternative to classical antibiotics. The main advantage of this class of substances, when considering bacterial resistance, is that they rapidly, within minutes, kill bacteria. Antimicrobial peptides can be found in every organism and display a wide spectrum of activity. Hence, the goal is to engineer peptides with an improved therapeutic index, i.e. high efficacy and target specificity. For the rational design of such novel antibiotics it is essential to elucidate the molecular mechanism of action. Biophysical studies have been performed using to a large extent membrane model systems demonstrating that there are distinctive different mechanisms of bacterial killing by antimicrobial peptides. One can distinguish between peptides that permeabilize and/or disrupt the bacterial cell membrane and peptides that translocate through the cell membrane and interact with a cytosolic target. Lantibiotics exhibit specific mechanisms, e.g. binding to lipid II, a precursor of the peptidoglycan layer, either resulting in membrane rupture by pore formation or preventing cell wall biosynthesis. The classical models of membrane perturbation, pore formation and carpet mechanism, are discussed and related to other mechanisms that may lead to membrane dysfunction such as formation of lipid-peptide domains or membrane disruption by formation of non-lamellar phases. Emphasis is on the role of membrane lipid composition in these processes and in the translocation of antimicrobial peptides.

Anti-Bacterial Agents↗
PubMed2002

A comparison of combinatorial partitioning and linear regression for the detection of epistatic effects of the ACE I/D and PAI-1 4G/5G polymorphisms on plasma PAI-1 levels.

The detection and characterization of epistasis or non-additive gene-gene interactions remains a statistical challenge in genetic epidemiology. The recently developed combinatorial partitioning method (CPM) may overcome some of the limitations of linear regression for the exploratory analysis of non-additive epistatic effects. The goal of this study was to compare CPM with linear regression analysis for the exploratory analysis of non-additive interactive effects of the angiotensin converting enzyme (ACE) insertion/deletion (I/D) and plasminogen activator inhibitor 1 (PAI-1) 4G/5G polymorphisms on plasma PAI-1 levels in a sample of 50 unrelated African Americans and 117 unrelated Caucasians. Using linear regression, we documented the additive effects of the ACE and PAI-1 genes on plasma PAI-1 levels in African American females (R(2) = 0.10), African American males (R(2) = 0.16), Caucasian females (R(2) = 0.11), and Caucasian males (R2 = 0.09). Using CPM, we found evidence for non-additive effects of the ACE and PAI-1 genes in both African American females (R(2) = 0.22) and African American males (R(2) = 0.24) but not in Caucasian females (R(2) = 0.10) or Caucasian males (R(2) = 0.11). The results of this exploratory data analysis support previous experimental, clinical, and epidemiological studies that have proposed as a working hypothesis that the ACE gene mediates interaction effects of the fibrinolytic and renin-angiotensin systems on plasma levels of PAI-1.

Black People↗
PubMed2002

High-throughput mass-directed parallel purification incorporating a multiplexed single quadrupole mass spectrometer.

We report on the development of a parallel HPLC/MS purification system incorporating an indexed (i.e., multiplexed) ion source. In the method described, each of the flow streams from a parallel array of HPLC columns is directed toward the multiplexed (MUX) ion source and sampled in a time-dependent, parallel manner. A visual basic application has been developed and monitors in real-time the extracted ion current from each sprayer channel. Mass-directed fraction collection is initiated into a parallel array of fraction collectors specific for each of the spray channels. In the first embodiment of this technique, we report on a four-column semipreparative parallel LC/MS system incorporating MUX detection. In this parallel LC/MS application (in which sample loads between 1 and 10 mg on-column are typically made), no cross talk was observed. Ion signals from each of the channels were found reproducible over 192 injections, with interchannel signal variations between 11 and 17%. The visual basic fraction collection application permits preset individual start collection and end collection thresholds for each channel, thereby compensating for the slight variation in signal between sprayers. By incorporating postfraction collector UV detection, we have been able to optimize the valve-triggering delay time with precut transfer tubing between the mass spectrometer and fraction collectors and achieve recoveries greater than 80%. Examples of the MUX-guided, mass-directed fraction purification of both standards and real library reaction mixtures are presented within.

Chromatography, High Pressure Liquid↗
PubMed2006

The role of the striatum in processing language rules: evidence from word perception in Huntington's disease.

On the assumption that linguistic faculties reflect both lexical storage in the temporal cortex and combinatorial rules in the striatal circuits, several authors have shown that striatal-damaged patients are impaired with conjugation rules while retaining lexical knowledge of irregular verbs [Teichmann, M., Dupoux, E., Kouider, S., Brugières, P., Boissé, M. F., Baudic, S., Cesaro, P., Peschanski, M., & Bachoud-Lévi, A. C. (2005). The role of the striatum in rule application. The model of Huntington's disease at early stage. Brain, 128, 1155-1167; Ullman, M. T., Corkin, S., Coppola, M., Hickok, G., Growdon, J. H., Koroshetz, W. J., & Pinker, S. (1997). A neural dissociation within language: Evidence that the mental dictionary is part of declarative memory, and that grammatical rules are processed by the procedural system. Journal of Cognitive Neuroscience, 9, 266-276]. Yet, such impairment was documented only with explicit conjugation tasks in the production domain. Little is known about whether it generalizes to other language modalities such as perception and whether it refers to implicit language processing or rather to intentional rule operations through executive functions. We investigated these issues by assessing perceptive processing of conjugated verb forms in a model of striatal dysfunction, namely, in Huntington's Disease (HD) at early stages. Rule application and lexical processes were evaluated in an explicit task (acceptability judgments on verb and nonword forms) and in an implicit task (lexical decision on frequency-manipulated verb forms). HD patients were also assessed in executive functions, and striatal atrophy was evaluated with magnetic resonance imaging (bicaudate ratio). Results from both tasks showed that HD patients were selectively impaired for rule application but lexical abilities were spared. Bicaudate ratios correlated with rule scores on both tasks, whereas executive parameters only correlated with scores on the explicit task. We argue that the striatum has a core function in linguistic rule application generalizing to perceptive aspects of morphological operations and pertaining to implicit language processes. In addition, we suggest that the striatum may enclose computational circuits that underpin explicit manipulation of regularities.

Adult↗
PubMed2005

The role of the striatum in rule application: the model of Huntington's disease at early stage.

The role of the basal ganglia, and more specifically of the striatum, in language is still debated. Recent studies have proposed that linguistic abilities involve two distinct types of processes: the retrieving of stored information, implicating temporal lobe areas, and the application of combinatorial rules, implicating fronto-striatal circuits. Studies of patients with focal lesions and neurodegenerative diseases have suggested a role for the striatum in morphological rule application, but functional imaging studies found that the left caudate was involved in syntactic processing and not morphological processing. In the present study, we tested the view that the basal ganglia are involved in rule application and not in lexical retrieving in a model of striatal dysfunction, namely Huntington's disease at early stages. We assessed the rule-lexicon dichotomy in the linguistic domain with morphology (conjugation of non-verbs and verbs) and syntax (sentence comprehension) and in a non-linguistic domain with arithmetic operations (subtraction and multiplication). Thirty Huntington's disease patients (15 at stage I and 15 at stage II) and 20 controls matched for their age and cultural level were included in this study. Huntington's disease patients were also assessed using the Unified Huntington's Disease Rating Scale (UHDRS) and MRI. We found that early Huntington's disease patients were impaired in rule application in the linguistic and non-linguistic domains (morphology, syntax and subtraction), whereas they were broadly spared with lexical processing. The pattern of performance was similar in patients at stage I and stage II, except that stage II patients were more impaired in all tasks assessing rules and had in addition a very slight impairment in the lexical condition of conjugation. Finally, syntactic rule abilities correlated with all markers of the disease evolution including bicaudate ratio and performance in executive function, whereas there was no correlation with arithmetic and morphological abilities. Together, this suggests that the striatum is involved in rule processing more than in lexical processing and that it extends to linguistic and non-linguistic domains. These results are discussed in terms of domain-specific versus domain-general processes of rule application.

Adult↗
PubMed2002

Complexities in ETS-domain transcription factor function and regulation: lessons from the TCF (ternary complex factor) subfamily. The Colworth Medal Lecture.

The ETS-domain transcription factor family can be divided into a series of subfamilies. Elk-1 represents the founding member of the ternary complex factor (TCF) subfamily. By focusing on the TCF subfamily, we can demonstrate the complexities that exist in the function and regulation of ETS-domain transcription factors. This article focuses on Elk-1 in detail and summarizes the functions of other TCFs. The key themes covered include the domain structure of the TCFs, the mechanisms of complex formation with serum response factor, regulation of TCFs by mitogen-activated protein kinase cascades, and transcriptional regulatory properties of the TCFs. Finally, the emerging role of the TCFs in vivo is discussed. A picture is developing indicating that, while these proteins exhibit significant sequence and functional conservation, key differences in their structure and regulation are being identified which may relate to unique functions of these proteins in vivo.

Amino Acid Sequence↗