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E Leung

Publications and source records attributed to E Leung.

At least 73 records · Page 4Linked to original sources
PubMed1994

The delta-subunit of murine guanine nucleotide exchange factor eIF-2B. Characterization of cDNAs predicts isoforms differing at the amino-terminal end.

Protein synthesis in mammalian cells is regulated at the level of the guanine nucleotide exchange factor, eIF-2B, which catalyzes the exchange of eukaryotic initiation factor 2-bound GDP for GTP. We have isolated and sequenced cDNA clones encoding the delta-subunit of murine eIF-2B. The cDNA sequence encodes a polypeptide of 544 amino acids with molecular mass of 60 kDa. Antibodies against a synthetic polypeptide of 30 amino acids deduced from the cDNA sequence specifically react with the delta-subunit of mammalian eIF-2B. The cDNA-derived amino acid sequence shows significant homology with the yeast translational regulator Gcd2, supporting the hypothesis that Gcd2 may be the yeast homolog of the delta-subunit of mammalian eIF-2B. Primer extension studies and anchor polymerase chain reaction analysis were performed to determine the 5'-end of the transcript for the delta-subunit of eIF-2B. Results of these experiments demonstrate two different mRNAs for the delta-subunit of eIF-2B in murine cells. The isolation and characterization of two different full-length cDNAs also predicts the presence of two alternate forms of the delta-subunit of eIF-2B in murine cells. These differ at their amino-terminal end but have identical nucleotide sequences coding for amino acids 31-544.

Alternative Splicing↗
PubMed1994

Cloning of a gene encoding a human leukocyte protein characterised by extensive heptad repeats.

Complementary DNA (cDNA) clones, encoding a fusion protein that was recognised by an antiserum raised against a purified polypeptide fragment of a 180-kDa human leukocyte protein, were isolated from a lambda gt11 expressed library. The clones encoded a unique amino acid (aa) sequence interspersed with heptad repeats that typify coiled-coil proteins, and hybridised to a 5-kb transcript universally expressed in a panel of eight human tissues. Comparatively high levels of RNA expression were seen in testis, ovary and mitogen-activated peripheral blood leukocytes (PBLs). The deduced 1300-aa sequence reveals a protein with a typical signal peptide, a hydrophilic domain containing an N-terminal globular head with a nuclear localization signal sequence, a C-terminal region of coiled-coil structure, a candidate transmembrane domain, and a short 10-aa C-terminal domain. Rabbit polyclonal antisera raised against a truncated lambda gt11 fusion protein recognized a 150-170-kDa protein (non-reduced) in mitogen-activated PBLs. The protein designated here as CG-1 may exist as a homodimer destined for translocation to the nucleus, with a role in leukocyte differentiation and/or effector function.

Amino Acid Sequence↗
PubMed1994

Drug interactions with radiopharmaceuticals.

Considerable information on documented drug and radiopharmaceutical interactions has been assembled in a tabular form, classified by the type of nuclear medicine study. The aim is to provide a rapid reference for nuclear medicine staff to look for such interactions. The initiation of drug chart monitoring or drug history taking of nuclear medicine patients and the reporting of such events are encouraged.

Drug Interactions↗
PubMed1994

Nitric oxide-dependent efflux of cGMP in rat cerebellar cortex: an in vivo microdialysis study.

The stimulation of excitatory amino acid receptors in the cerebellar cortex results in the Ca2+/calmodulin-dependent activation of nitric oxide synthase. This leads to an increase in tissue levels of cGMP following the interaction of nitric oxide with soluble guanylyl cyclase. The cerebellar cortex has the highest levels of nitric oxide synthase and cGMP in the brain; however, the levels of guanylyl cyclase and cGMP-phosphodiesterase are remarkably low. Thus, the mechanisms regulating cGMP levels in cerebellar cells are unclear. One report has noted that cGMP can be released from cerebellar slices. We have therefore used intracerebellar microdialysis in awake, freely moving rats to test the hypothesis that activation of nitric oxide synthase in the cerebellar cortex results in the release of cGMP. Climbing fibers, which release excitatory amino acids in the cerebellum, were activated with systemic harmaline. This resulted in an immediate increase in extracellular cGMP, which was blocked by TTX or the removal of extracellular Ca2+, and attenuated by prior lesion of the climbing fibers. Blockade of N-type calcium channels with omega-conotoxin also antagonized the harmaline-induced increase. In contrast, blockade of L-type calcium channels, or inhibition of anion transport with probenecid or bromosulfophthalein, potentiated the increase in cGMP seen in response to harmaline. Inhibitors of nitric oxide synthase or guanylyl cyclase prevented the harmaline-induced increase in extracellular cGMP, while phosphodiesterase inhibitors potentiated the increase. Local application of the NMDA antagonist 2-amino-5-phosphonopentanoic acid or the AMPA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione attenuated the effect of harmaline.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Oxidoreductases↗
PubMed1994

[3H]RS-23597-190, a potent 5-hydroxytryptamine4 antagonist labels sigma-1 but not sigma-2 binding sites in guinea pig brain.

Recent findings have suggested a relationship between 5-hydroxytryptamine (5-HT)4 receptors and sigma binding sites. To test this idea, the affinity of 5-HT4 receptor ligands for sigma binding sites was examined. In contrast to the 5-HT4 receptor ligands BIMU-1 [endo-N-(8-methyl-8-azabicyclo[3.2.1]oct-3-yl)-2,3- dihydro-3-ethyl-2-oxo-1H-benzimidazole-1-carboxamide hydrochloride] and BIMU-8 [endo-N-(8-methyl-8-azabicyclo[3.2.1]oct-3- yl)-2,3-dihydro-(1-methyl)ethyl-2-oxo-1H-benzamidazole-1-carbox ami de hydrochloride], DAU 6215 ]N-(endo-8-methyl-8-azabicyclo[3.2.1.]oct-3-yl)-2,3-dihydro-2-oxo-1H- benzimidazole-1-carboxamide hydrochloride], 5-HT and 5-methoxytryptamine had low affinity for sigma binding sites (pKi < 6). Conversely, the sigma ligands haloperidol and pentazocine had low affinity for 5-HT4 receptors. Thus, no relationship was found between the affinity of ligands at 5-HT4 receptors and sigma binding sites. However, one potent 5-HT4 receptor antagonist, RS-23597-190 [3-(piperidine-1-yl)propyl-4-amino-5-chloro-2-methoxybenzoate hydrochloride], had high affinity for sigma-1 (pKi = 8.4) but not sigma-2 (pKi = 6.2) binding sites. [3H]RS-23597-190 bound to a saturable site with the pharmacology of a sigma-1 binding site: (pIC50) haloperidol (9.0) > (+)-pentazocine (8.8) > (+)-3-(hydroxyphenyl)-N-(1-propyl)piperidine (8.2) > 1,3-di-o-tolyl-guanidine (8.0) > (-)-pentazocine (7.8) = (+)-SKF 10,047 [N-allylnormetazocine] > (-)-SKF 10,047 (6.2) > BIMU-1 (5.3) > 5-HT and 5-methoxytryptamine. The distribution of [3H]RS-23597-190 binding sites was similar to that described for other sigma radioligands, with the greatest binding densities in cranial nerve nuclei, the tegmental nucleus and in the mamillary nucleus. In contrast to (+)-3-(hydroxyphenyl)-N-(1-propyl)piperidine, [3H]RS-23597-190 binding was not allosterically modulated by phenytoin. These studies do not support the notion of an obvious relationship between sigma and 5-HT4 receptors, but they provide additional insight into the structure/affinity relationship of ligands at specific sigma binding sites, and they uncover a novel sigma-1 receptor ligand whose binding is insensitive to the action of phenytoin.

Aminobenzoates↗
PubMed1993

The mouse beta 7 integrin gene promoter: transcriptional regulation of the leukocyte integrins LPAM-1 and M290.

The leukocyte adhesion receptors M290 (alpha M290/beta 7) and LPAM-1 (alpha 4 beta 7) comprise the beta 7-subfamily of integrins, which are constitutively expressed on subsets of lymphocytes populating the mouse small intestine. They are induced de novo after in vitro activation of lymphocytes and hence may serve a more general role in inflammation. In order to understand how beta 7 integrins are regulated during an immune response, we isolated and characterized the promoter region of the beta 7 gene. Primer extension and rapid amplification of cDNA ends identified one major transcriptional start site in a favourable context, which resembles the initiator of terminal deoxynucleotidyl transferase. Transfection assays with a luciferase reporter gene revealed that cell-specific expression in vitro was retained in a 292 bp sequence, which contained several consensus binding motifs for transcriptional factors preferentially expressed in cells of the lymphoid lineages. Multiple retinoic acid receptor sites for steroid/thyroid hormone receptors which typify the leukocyte cell adhesion molecule subset of integrins are present. The beta 7 promoter, like its alpha 4 chain partner, contains the E box core sequence CACCTG found within the muscle creatine kinase enhancer which binds MyoD in vitro. The number of potential DNA binding sites for transcriptional factors in the beta 7 promoter parallels the complex regulation of expression of M290 and LPAM-1 in inflammation and gut mucosal immunity.

Amino Acid Sequence↗
PubMed1993

The action of SDZ 205,557 at 5-hydroxytryptamine (5-HT3 and 5-HT4) receptors.

1. The interaction of the novel antagonist, SDZ 205,557 (2-methoxy-4-amino-5-chloro benzoic acid 2-(diethylamino) ethyl ester), at 5-HT3 and 5-HT4 receptors has been assessed in vitro and in vivo. 2. In guinea-pig hippocampus and in the presence of 0.4 microM 5-carboxamidotryptamine, 5-HT4-mediated stimulation of adenylyl cyclase was competitively antagonized by SDZ 205,557, with a pA2 value of 7.5, and a Schild slope of 0.81. In rat carbachol-contracted oesophagus, 5-HT4-receptor mediated relaxations were surmountably antagonized by SDZ 205,557 with a similar pA2 value (7.3). This value was agonist-independent with the exception of (R)-zacopride, against which a significantly lower value (6.4) was observed. 3. In functional studies of 5-HT3 receptors, SDZ 205,557 exhibited an affinity of 6.2 in guinea-pig ileum compared with 6.9 at binding sites labelled by [3H]-quipazine in NG108-15 cells. In the anaesthetized, vagotomized micropig, SDZ 205,557 produced only a transient blockade of 5-HT4-mediated tachycardia. This contrasted with tropisetron, which was active for over 60 min after administration. The half-lives for the inhibitory responses of SDZ 205,557 and tropisetron were 23 and 116 min, respectively. 4. In conclusion, SDZ 205,557 has similar affinity for 5-HT3 and 5-HT4 receptors. The apparent selectivity observed in guinea-pig is due to the atypical nature of the 5-HT3 receptor in this species. The short duration of action of this novel antagonist may complicate its use in vivo. SDZ 205,557 should, therefore, be used with appropriate caution in studies defining the 5-HT4 receptor.

4-Aminobenzoic Acid↗
PubMed1993

RS 23597-190: a potent and selective 5-HT4 receptor antagonist.

1. The pharmacological properties of RS 23597-190 (3-(piperdine-1-yl)-propyl-4-amino-5-chloro-2-methoxy benzoate hydrochloride) have been studied in vitro and in vivo. 2. RS 23597-190 competitively antagonized 5-HT4 receptor-mediated relaxations of rat, carbachol precontracted oesophageal muscularis mucosae, (pA2 = 7.8 +/- 0.1; Schild slope = 1.2 +/- 0.2). Affinity estimates (-log KB) at 5-HT4 receptors using either renzapride or SC-53116 as agonists yielded a -log KB value of 8.0 +/- 0.01. In contrast, RS 23597-190 failed to antagonize contractile responses to 5-HT of guinea-pig ileal 5-HT3 receptors, even at concentrations up to 10 microM. 3. Increases in short-circuit current, induced by 5-HT, were studied in guinea-pig ileal mucosal sheets. Concentration-response curves to 5-HT were biphasic, with the high potency phase to 5-HT inhibited by RS 23597-190 and mimicked by 5-methoxytryptamine. The -log KB value for RS 23597-190 at the high potency phase was 7.3 confirming that 5-HT4 receptors mediated the high potency phase. 4. In rat isolated vagus nerve, 5-HT elicited a slow, maintained depolarization at low concentrations and a rapid, transient depolarization at higher concentrations. The high potency, slow depolarizing phase to 5-HT was abolished selectively in the presence of 1 microM RS 23597-190 and the low potency phase was abolished selectively in the presence of 1 microM ondansetron. These data confirm that 5-HT4 and 5-HT3 receptors mediated slow and fast depolarization responses, respectively. 5. At 5-HT3 binding sites in membranes from NG 108-15 cells, labelled by [3H]-quipazine, RS 23597-190 exhibited an apparent affinity (- log Ki) of 5.7 +/- 0.1. At 5-HT3 receptors in membranes from rat cerebral cortex, labelled by [3H]-RS 42358-197, the apparent affinity (- log Ki) of RS 23597-190 was also 5.7 +/- 0.1. In both studies, Hill coefficients were not significantly different from unity. At 5-HT1A, 5-HT2,muscarinic M1, M2, M3, M4 and dopamine D1 and D2 receptors, RS 23597-190 exhibited low apparent affinities, with all - log Ki values less than 5.5.6. Intravenous infusion of RS 23597-190 in the conscious, restrained rat antagonized the von Bezold Jarisch reflex induced by 2-methyl 5-HT, with an ID50 of 300 microg kg-1 min-1, i.v. In the anaesthetized,bilaterally vagotomized micropig, RS 23597-190 (6 mg kg-1, i.v.) antagonized 5-HT-induced tachycardia with a half-life of 77 (63-99) min. Transient arrhythmic effects were noted after administration of the compound.7. In conclusion, RS 23597-190 acts as a high affinity, selective competitive antagonist at 5-HT4 receptors. Thus, the compound appears to be a useful tool for 5-HT4 receptor identification in vitro. In vivo, the compound is rapidly metabolized in pigs such that 5-HT4 blockade is not maintained. However,in the rat, when given by infusion, RS 23597-190 antagonizes 5-HT3 mediated responses, at doses consistent with a low affinity 5-HT3 receptor. These data suggest that, under appropriate experimental conditions, RS 23597-190 may also be used in vivo to characterize further 5-HT4 receptor function.

4-Aminobenzoic Acid↗
PubMed1993

Characterization of angiotensin II receptors in smooth muscle preparations of the guinea pig in vitro.

Angiotensin II (AII) receptors in guinea pig isolated esophageal muscularis mucosae (EMM), stomach fundus, gall bladder, ileum, colon and thoracic aorta have been characterized by peptide agonists and nonpeptide antagonists in the presence of peptidase inhibitors. Angiotensin peptides contracted every preparation studied; the potency order typically was [Sar1]AII > or = AII > angiotensin III (AIII) > or = [Val4]AIII >> AI >>> [des Phe8]AII. AI was ineffective everywhere except the gall bladder, where it acted as a full agonist. Tetrodotoxin (1 microM) and atropine (1 microM) did not affect the AII response in EMM, fundus and gall bladder. In ileum, AII and AIII were equieffective, and both the maximal response and potency were decreased by tetrodotoxin and atropine. Indomethacin (3 microM) abolished response to AII in the fundus but had little effect on the gall bladder and the atropine-resistant component of the ileal response. The AT1-selective antagonist losartan (DuP 753) antagonized responses to AII in all tissues with similar affinities when there was no depression of maximal response (pKB = approximately 8-8.3). The AT2-selective antagonist PD123177 (10 microM) failed to antagonize responses to AII in any tissue. These data suggest the presence of AT1 receptors in intestinal and vascular smooth muscles of the guinea pig. It is unclear whether all AT1 receptors are similar because of the differential potency order observed in the presence of peptidase inhibitors. Of the isolated tissue investigated, responses to AII are robust and reproducible in the ileum, fundus and gall bladder.

Angiotensin Receptor Antagonists↗
PubMed1992

Molecular cloning of the mouse integrin beta 7 subunit.

The complete analysis of a cDNA clone encoding the mouse integrin beta 7 subunit that was isolated from a lambda gt10 cDNA library prepared from interleukin-4-activated mouse spleen B cells is reported. The 805-amino acid sequence deduced from the cDNA revealed a signal peptide, a 704-amino acid extracellular domain with eight N-glycosylation sites, a transmembrane domain, and a 61-amino acid intracellular domain. Several structural features were conserved with other integrin beta chains including the four cysteine-rich epidermal growth factor-like repeat sequences in the extracellular domain. Comparative analysis of the mouse and human beta 7 subunits revealed 87% sequence identity with conservation of three potential intracellular tyrosine phosphorylation sites. The cDNA hybridized to two mRNA transcripts of 3 and 2 kilobases (kb) in size. The 3-kb transcript, which is considered the productive form, was found in T and B leukemic cell lines, a macrophage line, and a mastocytoma cell line. Suprisingly, the smaller 2-kb transcript, which seems unlikely to encode a complete beta 7 protein, was found expressed in epithelial cells cultured from mouse skin. The integrin beta 7 subunit displays sequence identity with the beta subunit of the M290 antigen found expressed almost exclusively on intraepithelial lymphocytes in the small intestine suggesting that beta 7 may play an adhesive role in intraepithelial lymphocytes immunosurveillance of the gut mucosa. The cellular distribution of beta 7 transcripts is more extensive than the T-lineage-restricted distribution of the M290 antigen, indicating that beta 7 may be the common beta subunit for more than one receptor or that translation of beta 7 transcripts varies in different cell types.

Amino Acid Sequence↗
PubMed1992

Immunologic and structural relatedness of the integrin beta 7 complex and the human intraepithelial lymphocyte antigen HML-1.

We recently cloned the newest human integrin beta subunit, termed beta 7, from a cDNA library constructed from SEA-activated T lymphocytes. In this communication, we report on the structure of the human integrin beta 7 protein complex determined using a rabbit anti-beta 7 peptide antibody raised to an N-terminal 22 amino acid residue sequence deduced from the human beta 7 subunit cDNA. The beta 7 subunit (Mr 116,000) expressed on PHA lymphoblasts associates with a single major alpha subunit (alpha H) that is distinct from the prominent T cell marker, integrin alpha 4. The alpha H subunit (Mr 180,000 nonreduced) displays a distinctive shift in size on reduction to an apparent Mr of 150,000. We show that these structural properties of the integrin beta 7 complex are shared with the cell surface antigen HML-1 found highly expressed on T cells which populate the intestinal epithelium and are proposed to be involved in mucosal immunity. Sequential immunoprecipitation and Western blotting demonstrate identity or close homology between the alpha H beta 7 and HML-1 proteins.

Amino Acid Sequence↗
PubMed1992

Analysis of concentration-response relationships by seemingly unrelated nonlinear regression (SUNR) technique.

In performing statistical evaluation of concentration-response relationship in pharmacological studies, all the commercially available statistical packages assume each data point is an independent measure of the drug response, and do not account for the dependence between the multiple measurements taken from the same subject (tissue, animal, or sample). Seemingly unrelated nonlinear regression (SUNR) is a statistical technique that takes into account both within-and between-subject variance. This technique has been implemented in an SAS-based interactive program called SUNR. The statistical analyses are based upon the original work by Gallant (Gallant, 1975, J Econometrics 3:35-50; Gallant and Goebel, 1976, JASA 71:961-967), which has been further developed by Muller and Helms (1984) (Presented at ASA meeting in Washington, D.C.) To test this program, we have analyzed both simulated and actual data with SUNR, comparing our results to those of several popular statistical programs. All the programs yielded essentially the same estimates for the EC50, minimum and maximum response in both the simulated and experimental data sets. However, our results differed markedly from the commercial packages in the estimates of standard errors associated with the estimated maxima. When analyzing simulated data, which were far less noisy than the experimental data, differences between the analyses were minimal. However, in the analyses of experimental data, the standard errors calculated by the commercial programs appear to significantly underestimate the standard error. Using SUNR, however, the 95% confidence limits on the maxima are markedly wider, and, importantly, always cover the observed actual data range.

Animals↗
PubMed1992

The gene organization of the human beta 7 subunit, the common beta subunit of the leukocyte integrins HML-1 and LPAM-1.

The integrin beta 7 subunit associates with two alternative alpha subunits termed alpha HML-1 and alpha 4 to give the lymphocyte activation and homing receptors HML-1 and LPAM-1. Overlapping genomic clones that encoded the human beta 7 subunit gene were isolated from cosmid and phage lambda libraries. The coding portion of the gene spanned approximately 10 kb and was composed of 14 exons. Exon 1 (123 bp) encoded 5' untranslated sequences; exon 2 (204 bp) encoded the initiation codon, signal peptide, and 50 amino acid residues of the N-terminus of the mature protein; 7 exons (exons 3-9), ranging in size from 90 bp to 242 bp, encoded most of the extracellular domain proximal to the four cysteine-rich repeats; the region corresponding to the beta 3 arginine-glycine-aspartic acid (RGD)-binding domain was divided amongst exons 4 and 5; the four cysteine-rich repeats were encoded by 3 exons (exons 10-12) with intron insertion into the first and third repeats; exon 13 (209 bp) provided a spacer between the cysteine-rich domains and the transmembrane domain; exon 14 (161 bp) encoded the transmembrane domain and exactly half of the cytoplasmic domain; the remainder of the cytoplasmic domain and most of the 3' untranslated region was contained in the largest 313 bp exon 15. Comparison of integrin beta subunit genes revealed that the gene organization of beta 7 was almost identical to that of beta 2, but had diverged from that of beta 3. Amplification of integrin DNAs directly from genomic DNA, using PCR primers based on beta subunit consensus sequences corresponding to the beta 3 RGD-binding domain, yielded partial gene sequences for the beta 3, beta 5, and beta 6 subunits only. Inspection of the amplified sequences revealed that, as for beta 3, the regions in beta 5 and beta 6 corresponding to the beta 3 RGD-binding domain lacked the intron present in beta 7, beta 1, and beta 2, which divides this region in beta 2 into two subdomains that contribute to subunit assembly. This study provides genetic evidence for at least two major branches to the integrin beta subunit evolutionary tree, with beta 7, beta 2, and probably beta 1 in one branch, and the cytoadhesin beta 3 and probably also beta 5 and beta 6 in the other.

Amino Acid Sequence↗
PubMed1991

Identity between the novel integrin beta 7 subunit and an antigen found highly expressed on intraepithelial lymphocytes in the small intestine.

A cDNA clone encoding the N-terminal sequence of the murine integrin beta 7 subunit, a novel member of the leukocyte cell adhesion molecule subset (Leu-CAM), has been isolated. An N-terminal region of 13 contiguous amino acids deduced from the cDNA shows complete identity with the N-terminus of the 120 kDa subunit of the M290 antigen, a surface molecule found highly expressed on mouse intestinal intraepithelial lymphocytes (IEL). This unexpected result focuses two previously unconnected areas of research and suggests that integrins may have a special role to play in the defence of the gut mucosa.

Amino Acid Sequence↗
PubMed1991

Apparent heterogeneity of cardiac A1 adenosine receptors as revealed by radioligand binding experiments on N-ethylmaleimide-treated membranes.

While G protein-coupled receptors are often studied by analyzing antagonist radioligand: "cold" agonist inhibition curves using an independent site model, it is now clear that KL and KH values determined in these analyses are not reliable estimates of the affinities of the agonists for "free" and G protein-coupled forms of the receptor. Thus, such experiments cannot be used to contrast the characteristics of a given type of receptor in different tissues, i.e., to probe for the existence of receptor subtypes. Since treatment with N-ethylmaleimide treatment blocks receptor: Gi/Go protein interactions, such analyses on N-ethylmaleimide-pretreated membranes should allow direct assessment of the affinities of competing ligands for the free receptor or for multiple receptor subtypes. As A1 adenosine receptors couple to Gi, and perhaps to Go, we have performed A1 adenosine receptor radioligand "competition" studies first on control, then on N-ethylmaleimide-pretreated bovine cardiac and cerebral cortical membranes. Results of experiments with the antagonist radioligand [3H]xanthine amine congener appeared to be confounded by ligand binding to A2 adenosine receptors present in the cardiac membrane preparations. Further experiments utilized the A1-specific radioligand [3H]1,3-dipropyl-8-cyclopentylxanthine. These experiments confirmed once more that the KL values determined by computer analysis of "competition" curves performed on control membranes are not reliable estimates of the affinities of the competing ligand for free receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine↗