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Biomedical subjects

I Collins

Publications and source records attributed to I Collins.

At least 19 recordsLinked to original sources
PubMed2005

Pharmacologic profiling of transcriptional targets deciphers promoter logic.

The blueprint for cellular diversity and response to environmental change is encoded in the cis-acting regulatory sequences of most genes. Deciphering this 'cis-regulatory code' requires multivariate data sets that examine how these regions coordinate transcription in response to diverse environmental stimuli and therapeutic treatments. We describe a transcriptional approach that profiles the activation of multiple transcriptional targets against combinatorial arrays of therapeutic and signal transducing agents. Application of this approach demonstrates how cis-element composition and promoter context combine to influence transcription downstream of mitogen-induced signaling networks. Computational dissection of these transcriptional profiles in activated T cells uncovers a novel regulatory synergy between IGF-1 and CD28 costimulation that modulates NF-kappaB and AP1 pathways through signaling cascades sensitive to cyclosporin A and wortmannin. This approach provides a broader view of the hierarchical signal integration governing gene expression and will facilitate a practical design of combinatorial therapeutic strategies for exploiting critical control points in transcriptional regulation.

Algorithms↗
PubMed2004

Salicylidene salicylhydrazide, a selective inhibitor of beta 1-containing GABAA receptors.

1. A high-throughput assay utilizing the voltage/ion probe reader (VIPR) technology identified salicylidene salicylhydrazide (SCS) as being a potent selective inhibitor of alpha2beta1gamma1 GABA(A) receptors with a maximum inhibition of 56+/-5% and an IC(50) of 32 (23, 45) nm. 2. Evaluation of this compound using patch-clamp electrophysiological techniques demonstrated that the compound behaved in a manner selective for receptors containing the beta1 subunit (e.g. maximum inhibition of 68.1+/-2.7% and IC(50) value of 5.3 (4.4, 6.5) nm on alpha2beta1gamma1 receptors). The presence of a beta1 subunit was paramount for the inhibition with changes between alpha1 and alpha2, gamma1 and gamma2, and the presence of a subunit having little effect. 3. On all subtypes, SCS produced incomplete inhibition with the greatest level of inhibition at alpha1beta1gamma1 receptors (74.3+/-1.4%). SCS displayed no use or voltage dependence, suggesting that it does not bind within the channel region. Concentration - response curves to GABA in the presence of SCS revealed a reduction in the maximum response with no change in the EC(50) or Hill coefficient. In addition, SCS inhibited pentobarbitone-induced currents. 4. Threonine 255, located within transmembrane domain (TM) 1, and isoleucine 308, located extracellularly just prior to TM3, were required for inhibition by SCS. 5. SCS did not compete with the known allosteric modulators, picrotoxin, pregnenolone sulphate, dehydroepiandrosterone 3-sulphate, bicuculline, loreclezole or mefenamic acid. Neither was the inhibition by SCS influenced by the benzodiazepine site antagonist flumazenil. 6. In conclusion, SCS is unique in selectively inhibiting GABA(A) receptors containing the beta1 subunit via an allosteric mechanism. The importance of threonine 255 and isoleucine 308 within the beta1 subunit and the lack of interaction with a range of GABA(A) receptor modulators suggests that SCS is interacting at a previously unidentified site.

Amino Acid Sequence↗
PubMed2001

Postoperative changes in visual evoked potentials and cognitive function tests following sevoflurane anaesthesia.

We tested the hypothesis that minor disturbance of the visual pathway persists following general anaesthesia even when clinical discharge criteria are met. To test this, we measured visual evoked potentials (VEPs) in 13 ASA I or II patients who did not receive any pre-anaesthetic medication and underwent sevoflurane anaesthesia. VEPs were recorded on four occasions, before anaesthesia and at 30, 60, and 90 min after emergence from anaesthesia. Patients completed visual analogue scales (VAS) for sedation and anxiety, a Trieger Dot Test (TDT) and a Digit Symbol Substitution Test (DSST) immediately before each VEP recording. These results were compared using Student's t-test. P<0.05 was considered significant. VEP latency was prolonged (P<0.001) and amplitude diminished (P<0.05) at 30, 60, and 90 min after emergence from anaesthesia, when VAS scores for sedation and anxiety, TDT, and DSST had returned to pre-anaesthetic levels.

Adolescent↗
PubMed2001

Transcriptional consequences of topoisomerase inhibition.

In principle, the generation, transmission, and dissipation of supercoiling forces are determined by the arrangement of the physical barriers defining topological boundaries and the disposition of enzymes creating (polymerases and helicases, etc.) or releasing (topoisomerases) torsional strain in DNA. These features are likely to be characteristic for individual genes. By using topoisomerase inhibitors to alter the balance between supercoiling forces in vivo, we monitored changes in the basal transcriptional activity and DNA conformation for several genes. Every gene examined displayed an individualized profile in response to inhibition of topoisomerase I or II. The expression changes elicited by camptothecin (topoisomerase I inhibitor) or adriamycin (topoisomerase II inhibitor) were not equivalent. Camptothecin generally caused transcription complexes to stall in the midst of transcription units, while provoking little response at promoters. Adriamycin, in contrast, caused dramatic changes at or near promoters and prevented transcription. The response to topoisomerase inhibition was also context dependent, differing between chromosomal or episomal c-myc promoters. In addition to being well-characterized DNA-damaging agents, topoisomerase inhibitors may evoke a biological response determined in part from transcriptional effects. The results have ramifications for the use of these drugs as antineoplastic agents.

Antineoplastic Agents↗
PubMed2000

N-(indol-3-ylglyoxylyl)piperidines: high affinity agonists of human GABA-A receptors containing the alpha1 subunit.

A new class of N-(indol-3-ylglyoxylyl)piperidines are high affinity agonists at the benzodiazepine binding site of human GABA-A receptor ion-channels, with modest selectivity for receptors containing the alpha1 subunit over alpha2 and alpha3. All three receptor subtypes discriminate substantially between the two enantiomers of the chiral ligand 10.

GABA-A Receptor Agonists↗
PubMed2000

Loss of FBP function arrests cellular proliferation and extinguishes c-myc expression.

The c-myc regulatory region includes binding sites for a large set of transcription factors. The present studies demonstrate that in the absence of FBP [far upstream element (FUSE)-binding protein], which binds to the single-stranded FUSE, the remainder of the set fails to sustain endogenous c-myc expression. A dominant-negative FBP DNA-binding domain lacking effector activity or an antisense FBP RNA, expressed via replication-defective adenovirus vectors, arrested cellular proliferation and extinguished native c-myc transcription from the P1 and P2 promoters. The dominant-negative FBP initially augmented the single-stranded character of FUSE; however, once c-myc expression was abolished, melting at FUSE could no longer be supported. In contrast, with antisense FBP RNA, the single-stranded character of FUSE decreased monotonically as the transcription of endogenous c-myc declined. Because transcription is the major source of super-coiling in vivo, we propose that by binding torsionally strained DNA, FBP measures promoter activity directly. We also show that FUSE is predicted to behave as a torsion-regulated switch poised to regulate c-myc and to confer a higher order regulation on a large repertoire of factors.

Animals↗
PubMed2000

The FBP interacting repressor targets TFIIH to inhibit activated transcription.

FUSE-binding protein (FBP) binds the single-stranded far upstream element of active c-myc genes, possesses potent transcription activation and repression domains, and is necessary for c-myc expression. A novel 60 kDa protein, the FBP interacting repressor (FIR), blocked activator-dependent, but not basal, transcription through TFIIH. Recruited through FBP's nucleic acid-binding domain, FIR formed a ternary complex with FBP and FUSE. FIR repressed a c-myc reporter via the FUSE. The amino terminus of FIR contained an activator-selective repression domain capable of acting in cis or even in trans in vivo and in vitro. The repression domain of FIR targeted only TFIIH's p89/XPB helicase, required at several stages in transcription, but not factors required for promoter selection. Thus, FIR locks TFIIH in an activation-resistant configuration that still supports basal transcription.

Amino Acid Sequence↗
PubMed1999

Fluorination of 3-(3-(piperidin-1-yl)propyl)indoles and 3-(3-(piperazin-1-yl)propyl)indoles gives selective human 5-HT1D receptor ligands with improved pharmacokinetic profiles.

It has previously been reported that a 3-(3-(piperazin-1-yl)propyl)indole series of 5-HT1D receptor ligands have pharmacokinetic advantages over the corresponding 3-(3-(piperidin-1-yl)propyl)indole series and that the reduced pKa of the piperazines compared to the piperidines may be one possible explanation for these differences. To investigate this proposal we have developed versatile synthetic strategies for the incorporation of fluorine into these ligands, producing novel series of 4-fluoropiperidines, 3-fluoro-4-aminopiperidines, and both piperazine and piperidine derivatives with one or two fluorines in the propyl linker. Ligands were identified which maintained high affinity and selectivity for the 5-HT1D receptor and showed agonist efficacy in vitro. The incorporation of fluorine was found to significantly reduce the pKa of the compounds, and this reduction of basicity was shown to have a dramatic, beneficial influence on oral absorption, although the effect on oral bioavailability could not always be accurately predicted.

Administration, Oral↗
PubMed1998

3-(1-piperazinyl)-4,5-dihydro-1H-benzo[g]indazoles: high affinity ligands for the human dopamine D4 receptor with improved selectivity over ion channels.

3-(4-Piperidinyl)-5-arylpyrazoles, such as 1, were selective for the cloned human dopamine D4 receptor (hD4), but also showed affinity at voltage sensitive calcium, sodium and potassium ion channels. A combination of substituent changes to reduce the basicity of the piperidine nitrogen and conformational restriction to give 4,5-dihydro-1H-benzo[g]indazoles reduced this ion channel affinity at the expense of selectivity for hD4 over other dopamine receptors. Incorporation of piperazine into the 4,5-dihydro-1H-benzo[g]indazoles in place of piperidine gave a novel series of high affinity, selective, orally bioavailable hD4 ligands, such as 16, with improved selectivity over ion channels.

Administration, Oral↗
PubMed1998

Substituted pyrazoles as novel selective ligands for the human dopamine D4 receptor.

Two novel series of 3-(heterocyclylmethyl)pyrazoles have been synthesised and evaluated as ligands for the human dopamine D4 receptor. Compounds in series I (exemplified by 8k) have a phenyl ring joined to the 4-position of the pyrazole while those in series II (exemplified by 15j) have a 5-phenyl ring linked by a saturated chain to the 4-position of the pyrazole. Both series supplied compounds with excellent affinity for the human D4 and good selectivity over other dopamine receptors. Excellent selectivity over calcium, sodium, and potassium ion channels was also achieved.

Antipsychotic Agents↗
PubMed1998

Photoaffinity labeling of the benzodiazepine binding site of alpha1beta3gamma2 gamma-aminobutyric acidA receptors with flunitrazepam identifies a subset of ligands that interact directly with His102 of the alpha subunit and predicts orientation of these within the benzodiazepine pharmacophore.

Photoincorporation of ligands into the benzodiazepine site of native gamma-aminobutyric acidA (GABAA) receptors provides useful information about the nature of the benzodiazepine (BZ) binding site. Photoincorporation of flunitrazepam into a single population of GABAA receptors, recombinant human alpha1beta3gamma2, was investigated to probe further the mechanism and orientation of flunitrazepam and other ligands in the BZ binding site. It was concluded that the receptor is primarily derivatized with the entire, unfragmented, flunitrazepam molecule, which undergoes a conformational change during photolysis and largely vacates the benzodiazepine binding site. Investigation of the BZ site after photoincorporation of [3H]flunitrazepam confirmed that binding of other radioligands was unaffected by incorporation of flunitrazepam. This did not correlate with their efficacy but depended on the presence of particular structural features in the molecule. It was observed that affected compounds have a pendant phenyl moiety, analogous to the 5-phenyl group of flunitrazepam, which are proposed to overlap and interact with the same residue or residues in the BZ binding site. Because the major site of flunitrazepam photoincorporation has been shown to be His102, we propose that this group of compounds interacts directly with His 102, whereas compounds of other structural types have no direct interaction with this amino acid. The orientation of ligands within the BZ binding site and their specific interaction with identified amino acids are not well understood. The data in the current study indicate that His102 interacts directly with the pendant phenyl group of diazepam, and further implications for the pharmacophore of the BZ binding site are discussed.

Azides↗
PubMed1997

4-Heterocyclylpiperidines as selective high-affinity ligands at the human dopamine D4 receptor.

5-(4-Chlorophenyl)-3-(1-(4-chlorobenzyl)piperidin-4-yl)pyrazole (3) was identified from screening of the Merck sample collection as a human dopamine D4 (hD4) receptor ligand with moderate affinity (61 nM) and 4-fold selectivity over human D2 (hD2) receptors. Four separate parts of the molecule have been examined systematically to explore structure-activity relationships with respect to hD4 affinity and selectivity over other dopamine receptors. It was found that the 4-chlorophenyl group attached to the pyrazole is optimal, as is the 4-substituted piperidine. The lipophilic group on the basic nitrogen is more amenable to change, with the optimal group found to be a phenethyl. The aromatic heterocyle can be altered to a number of different groups, with isoxazoles and pyrimidines showing improved affinities. This heterocycle can also be advantageously alkylated, improving the selectivity of the compounds over D2 receptors. It is hypothesized that the conformation around the bond joining the aromatic heterocycle to the piperidine is important for D4 affinity, based on crystal structures of isoxazoles (29 and 30) and on a conformationally constrained compound (28). Putting all the favorable changes together led to the discovery that 5-(4-chlorophenyl)-4-methyl-3-(1-(2-phenylethyl)piperidin-4-yl)iso xazole (36) is a nanomolar antagonist at human dopamine D4 receptors with > 500-fold selectivity over hD2 and > 200-fold selectivity over hD3. Compound 36 is an antagonist of hD4 receptors with good oral bioavailability of 38%, a half life of 2 h, and brain levels 10-fold higher than plasma levels.

Cell Line↗
PubMed1997

A distinct profile of six soluble adhesion molecules (ICAM-1, ICAM-3, VCAM-1, E-selectin, L-selectin and P-selectin) in rheumatoid arthritis.

Soluble forms of ICAM-1, VCAM-1, E-selectin, L-selectin, P-selectin and, more recently, ICAM-3 are known to exist in human serum and have elevated levels in numerous diseases. Previous studies have demonstrated that in rheumatoid arthritis (RA) the levels of circulating sICAM-1 and sE-selectin are elevated relative to healthy controls. We have compared the serum profiles of these six soluble adhesion molecules in patients with RA (n = 22) to those seen in healthy controls (n = 10) using sandwich ELISA. In the patients, there were significant elevations of serum sICAM-1 (P < 0.0001), sICAM-3 (P = 0.0327), sVCAM-1 (P = 0.0025), sL-selectin (P = 0.0194) and sP-selectin (P = 0.0025), but not E-selectin (P = 0.0672). However, only sP-selectin was found to correlate with disease activity in the patients (r = 0.461, P < 0.05). Thus, there is a distinct profile of soluble adhesion molecules in RA of which only sP-selectin correlates with disease activity.

Adult↗
PubMed1996

A unique transactivation sequence motif is found in the carboxyl-terminal domain of the single-strand-binding protein FBP.

The far-upstream element-binding protein (FBP) is one of several recently described factors which bind to a single strand of DNA in the 5' region of the c-myc gene. Although cotransfection of FBP increases expression from a far-upstream element-bearing c-myc promoter reporter, the mechanism of this stimulation is heretofore unknown. Can a single-strand-binding protein function as a classical transactivator, or are these proteins restricted to stabilizing or altering the conformation of DNA in an architectural role? Using chimeric GAL4-FBP fusion proteins we have shown that the carboxyl-terminal region (residues 448 to 644) is a potent transcriptional activation domain. This region contains three copies of a unique amino acid sequence motif containing tyrosine diads. Analysis of deletion mutants demonstrated that a single tyrosine motif alone (residues 609 to 644) was capable of activating transcription. The activation property of the C-terminal domain is repressed by the N-terminal 107 amino acids of FBP. These results show that FBP contains a transactivation domain which can function alone, suggesting that FBP contributes directly to c-myc transcription while bound to a single-strand site. Furthermore, activation is mediated by a new motif which can be negatively regulated by a repression domain of FBP.

Amino Acid Sequence↗
PubMed1995

Epitope mapping and functional properties of anti-intercellular adhesion molecule-3 (CD50) monoclonal antibodies.

Intercellular adhesion molecule-3 (ICAM-3, CD50), a member of the immunoglobulin gene superfamily, is a major ligand for the lymphocyte function-associated antigen 1 (LFA-1, CD18/CD11a) in the resting immune system and plays a role as a signaling and costimulatory molecule on T lymphocytes. In this study we have generated a large panel of anti-ICAM-3 monoclonal antibodies (mAb) and show that the biological effects of these antibodies are critically dependent on the epitope recognized. By using an adhesion assay employing COS cells expressing LFA-1 binding to recombinant chimeric ICAM-3-Fc proteins (which overcomes the confounding effects of interleukocyte LFA-1/ICAM binding events), we have been able to examine the effects of these antibodies in blocking LFA-1/ICAM-3 adhesion. Our data suggests that only a small minority of ICAM-3 mAb, recognizing a distinct epitope, are able to mimic the effects of LFA-1 binding to ICAM-3. Moreover these antibodies are functionally distinct as defined by their costimulatory activity and ability to elicit interleukin-2 production and cell proliferation in T lymphocytes.

Animals↗