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E Leung

Publications and source records attributed to E Leung.

At least 37 records · Page 2Linked to original sources
PubMed2000

A pathology-MRI study of the short-T2 component in formalin-fixed multiple sclerosis brain.

OBJECTIVE: To determine the pathologic basis of areas not exhibiting signal of the short-T2 component of the T2 relaxation distribution in MS, as studied in formalin-fixed brain. BACKGROUND: A myelin-specific MRI signal would be of great importance in assessing demyelination in patients with MS. Evidence indicates that the short-T2 (10 to 50 millisecond) component of the T2 relaxation distribution originates from water in myelin sheaths. The authors present two cases of MS in which the anatomic distribution of the short-T2 component was correlated with the pathologic findings in postmortem formalin-fixed brain. METHOD: One half of the formalin-fixed brain was suspended in a gelatin-albumin mixture cross-linked with glutaraldehyde, and scanned with a 32-echo MRI sequence. The brain was then cut along the center of the 5-mm slices scanned, photographed, dehydrated, and embedded in paraffin. Paraffin sections, stained with Luxol fast blue and immunocytochemically for 2',3'-cyclic nucleotide 3'-phosphohydrolase for myelin and by the Bielschowsky technique for axons, were compared with the distribution of the amplitude of the short-T2 component of the comparable image slices. RESULTS: The anatomic distribution of the short-T2 component signal corresponded to the myelin distribution. Chronic, silent MS plaques with myelin loss correlated with areas of absence of short-T2 signal. The numbers of axons within lesions were reduced, but many surviving axons were also seen in these areas of complete loss of myelin. CONCLUSION: In formalin-fixed MS brains the short-T2 component of the T2 relaxation distribution corresponds to the anatomic distribution of myelin. Chronic, silent demyelinated MS plaques show absence of the short-T2 component signal. These results support the hypothesis that the short-T2 component originates from water related to myelin.-1510

Aged↗
PubMed2000

Synthesis and biological effects of a new series of 2-amino-3-benzoylthiophenes as allosteric enhancers of A1-adenosine receptor.

New derivatives of PD 81,723, an allosteric enhancer of agonist binding to the A1-adenosine receptor, have been synthesized and evaluated in an intact cell assay. Compounds 3a, 3o and 3p appeared to be more potent than PD 81,723 and at a concentration of 0.1 microM caused significant reductions of cAMP content of CHO cells expressing the human A1-adenosine receptor. Compounds 4e and 4o appeared to be allosteric enhancers at a low concentration and antagonists at a higher concentration, whereas compounds 3c, 3g, 3s and 4l appeared to be weak antagonists that are also allosteric enhancers at the higher concentration of 10 microM.

Allosteric Regulation↗
PubMed2000

Beta7 integrins contribute to demyelinating disease of the central nervous system.

A role for alpha4 integrins in different forms of the multiple sclerosis-like disease experimental autoimmune encephalomyelitis (EAE) has been demonstrated, but the individual contributions of alpha4beta1, alpha4beta7, and the related alphaEbeta7 integrin have not been determined. The P7 integrins alpha4beta7 and alphaEbeta7 play a central role in chronic inflammation, mediating the trafficking, entry, and/or adhesion of lymphocytes in the inflamed pancreas and gut, and their ligands MAdCAM-1, VCAM-1 and E-cadherin are expressed on brain endothelial cells and/or on microvessels in the inflamed central nervous system. Here, we show that an antibody directed against the beta7 subunit greatly attenuates a non-remitting form of EAE, induced by adoptive transfer of myelin oligodendrocyte peptide (MOG35-55)-stimulated T cells. Combinational treatment with both anti-beta7 and alpha4 integrin subunit antibodies led to more rapid and complete remission than that obtained with anti-alpha4 antibody alone, potentially implicating a role for alphaEbeta7 in disease progression. Remission correlated with the down-regulation of the vascular addressins VCAM-1. MAdCAM-1, and ICAM-1 on cerebral blood vessels. Attenuated forms of disease were induced by adoptive transfer of either wild-type encephalitogenic T cells to beta7-deficient gene knockout mice, or of beta7-/-encephalitogenic T cells to wild-type recipients. The former finding indicates that beta7 + ve recruited cells contribute to disease progression. Thus alpha4beta1, alpha4beta7, and alphaEbeta7 integrins may all play a contributory role in the progression of chronic forms of demyelinating disease, and together with their ligands could represent potential targets for improved treatment of some forms of multiple sclerosis.

Adoptive Transfer↗
PubMed2000

Synthesis and preliminary biological evaluation of [3H]-MRE 3008-F20: the first high affinity radioligand antagonist for the human A3 adenosine receptors.

The synthesis and the preliminary biological evaluation of the first high affinity radioligand antagonist for the human A3 adenosine receptor, named [3H]-MRE 3008-F20 are reported. [3H]-MRE 3008-20 bound human A3 receptors expressed in CHO cells with K(D) and Bmax value of 0.82 +/- 0.08 nM and 297 +/- 28 fmol/mg of protein, respectively. [3H]-MRE 3008-F20 represents a useful tool for a further characterization of A3 adenosine receptor subtype.

Animals↗
PubMed2000

[(3)H]MRE 3008F20: a novel antagonist radioligand for the pharmacological and biochemical characterization of human A(3) adenosine receptors.

The lack of a radiolabeled selective A(3) adenosine receptor antagonist is a major drawback for an adequate characterization of this receptor subtype. This paper describes the pharmacological and biochemical characterization of the tritiated form of a new potent A(3) adenosine receptor antagonist, the pyrazolo triazolo pyrimidine derivative [(3)H]5N-(4-methoxyphenylcarbamoyl)amino-8-propyl-2-(2-furyl )pyrazolo [4,3-e] -1,2,4- triazolo[1,5-c]pyrimidine ([(3)H]MRE 3008F20). [(3)H]MRE 3008F20 bound specifically to the human adenosine A(3) receptor expressed in CHO cells (hA(3)CHO), and saturation analysis revealed a single high affinity binding site, K(D) = 0.80 +/- 0.06 nM, with a B(max) = 300 +/- 33 fmol/mg protein. This new ligand displayed high selectivity (1294-, 165-, and 2471-fold) in binding assay to human A(3) versus A(1), A(2A), and A(2B) receptors, respectively, and binds to the rat A(3) receptors with a K(i) > 10 microM. The pharmacological profile of [(3)H]MRE 3008F20 binding to hA(3)CHO cells was evaluated using known adenosine receptor agonists and antagonists with a rank order of potency consistent with that typically found for interactions with the A(3) adenosine receptors. In the adenylyl cyclase assay the same compounds exhibited a rank order of potency identical with that observed in binding experiments. Thermodynamic data indicated that [(3)H]MRE 3008F20 binding to hA(3)CHO is entropy- and enthalpy-driven in agreement with the typical behavior of other adenosine antagonists to A(1) and A(2A) receptors. These results show that [(3)H]MRE 3008F20 is the first antagonist radioligand with high affinity and selectivity for the human A(3) adenosine receptor and may be used to investigate the physiopathological role of A(3) adenosine receptors.

Adenosine↗
PubMed1999

Cloning, sequence analysis, and chromosomal localization of the novel human integrin alpha11 subunit (ITGA11).

The integrins are a large family of cell adhesion molecules consisting of noncovalently associated alphabeta heterodimers. We have cloned and sequenced the cDNA of a novel human integrin alpha-subunit, designated alpha11. The alpha11 cDNA encodes a mature protein with a large 1120-residue extracellular domain that contains an I-domain of 207 residues and is linked by a transmembrane domain to a short cytoplasmic domain of 24 amino acids. The deduced alpha11 protein shows the typical structural features of integrin alpha-subunits and is similar to a distinct group of alpha-subunits from collagen-binding integrins. However, it differs from most integrin alpha-chains by an incompletely preserved cytoplasmic GFFKR motif. The human ITGA11 gene was localized to bands q22.3-q23 on chromosome 15, and its transcripts were found in a variety of tissues, but predominantly in bone, cartilage, cardiac muscle, and skeletal muscle. Expression of a 5.5-kb alpha11 mRNA was detectable in small intestine.

Amino Acid Sequence↗
PubMed1999

Cloning and characterization of a novel beta integrin-related cDNA coding for the protein TIED ("ten beta integrin EGF-like repeat domains") that maps to chromosome band 13q33: A divergent stand-alone integrin stalk structure.

Herein we describe the cDNA sequence of a novel human gene, ITGBL1, encoding a beta integrin-related protein termed TIED [for ten beta integrin epidermal growth factor (EGF)-like repeat domains]. Overlapping cDNA clones from fetal lung, HUVEC, and osteoblast cDNA libraries encode a sequence comprising a typical signal peptide, followed by a hydrophilic 471-amino-acid domain containing 10 tandem EGF-like repeats strikingly similar to those found in the cysteine-rich "stalk-like" structure of integrin beta subunits. The EGF-like repeats of TIED and beta integrins are unique in that they alternate in homology and possess two additional cysteines (eight in total) whose positions differ from those in the other eight-cysteine EGF-like domains of laminin, fibrillin, and the latent TGF-beta binding proteins. TIED mRNA transcripts of 2.8 kb were detected in aorta, thymus, and osteogenic sarcoma cells. The ITGBL1 gene was mapped to human chromosome 13, band 13q33. We suggest that ITGBL1 may be linked in some way with the evolution of the integrin beta subunits.

3' Untranslated Regions↗
PubMed1999

The small GTP-binding proteins Rho and Rac induce T cell adhesion to the mucosal addressin MAdCAM-1 in a hierarchical fashion.

Here we report that an activator (AIF4-) of heterotrimeric GTP-binding proteins (G-proteins) and inhibitors (lovastatin and C3 exoenzyme) of small GTP-binding proteins regulate the induction of alpha4beta7-mediated adhesion of TK-1 T lymphoma cells (alpha4+beta7+beta1-) to the mucosal addressin cell adhesion molecule MAdCAM-1. Activation of cell adhesion by AIF4- was abrogated by lovastatin, thereby establishing a link between heterotrimeric G-proteins and small GTP-binding proteins in the regulation of alpha4beta7-mediated cell adhesion. Increased numbers of cells bound MAdCAM-1-coated microspheres following activation with AIF4-, discounting an obligatory role for cell spreading in alpha4beta7-mediated cell adhesion. MAdCAM-1-Fc dimers triggered ligand-induced clustering of alpha4beta7 in response to AIF4- and Mn2+-induced activation of integrins. Hence alpha4beta7 cluster formation may be responsible, at least in part, for inducing cell adhesion in response to both extracellular and intracellular signals that impact on integrin function. Electroporation of constitutively active V14RhoA and V12Rac1 recombinant proteins into TK-1 cells revealed that both RhoA and Rac1 induce alpha4beta7 adhesion to MAdCAM-1. Activation is hierarchical since Rac1 is unable to directly activate alpha4beta7, but induces cell adhesion via RhoA, whereas the transient induction of cell adhesion mediated by RhoA is dependent on the activities of protein tyrosine kinases and protein kinase(s) C.

3T3 Cells↗
PubMed1999

RS-127445: a selective, high affinity, orally bioavailable 5-HT2B receptor antagonist.

Efforts to define precisely the role of 5-HT2B receptors in normal and disease processes have been hindered by the absence of selective antagonists. To address this deficiency, we developed a series of naphthylpyrimidines as potentially useful 5-HT2B receptor antagonists. RS-127445 (2-amino-4-(4-fluoronaphth-1-yl)-6-isopropylpyrimidine) was found to have nanomolar affinity for the 5-HT2B receptor (pKi = 9.5+/-0.1) and 1,000 fold selectivity for this receptor as compared to numerous other receptor and ion channel binding sites. In cells expressing human recombinant 5-HT2B receptors, RS-127445 potently antagonized 5-HT-evoked formation of inositol phosphates (pK(B) = 9.5+/-0.1) and 5-HT-evoked increases in intracellular calcium (pIC50 = 10.4+/-0.1). RS-127445 also blocked 5-HT-evoked contraction of rat isolated stomach fundus (pA2 = 9.5+/-1.1) and (+/-)alpha-methyl-5-HT-mediated relaxation of the rat jugular vein (pA2 = 9.9+/-0.3). RS-127445 had no detectable intrinsic activity in these assays. In rats, the fraction of RS-127445 that was bioavailable via the oral or intraperitoneal routes was 14 and 60% respectively. Intraperitoneal administration of RS-127445 (5 mg kg(-1)) produced plasma concentrations predicted to fully saturate accessible 5-HT2B receptors for at least 4 h. In conclusion, RS-127445 is a selective, high affinity 5-HT2B receptor antagonist suitable for use is vivo. The therapeutic potential of this molecule is being further evaluated.

Animals↗
PubMed1999

The effect of Glu75 of staphylococcal nuclease on enzyme activity, protein stability and protein unfolding.

Staphylococcal nuclease mutants, E57G and E75G, were generated. A comparison of the kinetic parameters both for mutants and wild-type protein shows that the Michaelis constants (Km) were almost identical for the wild-type protein and E57G mutant. An approximately 30-fold decrease in Km compared with the wild-type protein was observed for the E75G mutant. The turnover numbers for the enzyme (kcat) were higher with both the wild-type protein and the E57G mutant (3.88 +/- 0.21 x 103 s-1 and 3.71 +/- 0.28 x 103 s-1) than with the E75G mutant (3.04 +/- 0.02 x 102 s-1). The results of thermal denaturation with differential scanning microcalorimetry indicate that the excess calorimetric enthalpy of denaturations, DeltaHcal, was almost identical for the wild-type protein and E57G mutant (84.1 +/- 6.2 kcal.mol-1 and 79.3 +/- 7.1 kcal.mol-1, respectively). An approximately twofold decrease in DeltaHcal compared with the wild-type protein was observed for the E75G mutant (42.7 +/- 5.5 kcal.mol-1). These outcomes imply that Glu at position 75 plays a significant role in maintaining enzyme activity and protein stability. Further study of the unfolding of the wild-type protein and E75G mutant was conducted by using time-resolved fluorescence with a picosecond laser pulse. Two fluorescent lifetimes were found in the subnanosecond time range. The faster lifetime (tau2) did not generally vary with either pH or the concentration of guanidinium hydrochloride (GdmHCl) in the wild-type protein and the E75G mutant. The slow lifetime (tau1), however, did vary with these parameters and was faster as the protein is unfolded by either pH or GdmHCl denaturation. The midpoints of the transition for tau1 are pH 3.5 and 5.8 for the wild-type protein and E75G mutant, respectively, and the GdmHCl concentrations are 1.1 m and 0.6 m for the wild-type protein and E75G mutant, respectively. Parallel steady-state fluorescence measurements have also been carried out and the results are in general agreement with the time-resolved fluorescence experiments, indicating that Glu at position 75 plays an important role in protein unfolding.

Calorimetry, Differential Scanning↗
PubMed1999

The integrin alpha10 subunit: expression pattern, partial gene structure, and chromosomal localization.

Herein we report the cloning of cDNAs and incompletely processed hnRNAs from endothelia and heart that encode the alpha10 subunit forming part of the novel collagen type II-binding integrin alpha10beta1 of chondrocytes. Analysis of hnRNA clones and reported expressed sequence tags revealed the positions of 17 putative intron-exon splice junctions shared with those of the p150,95 (ITGAX) gene. Human alpha10 transcripts of 5.4 and 1.8 kb were not restricted to chondrocytes but, instead, were widely expressed in a panel of 24 tissue types, where the highest expression was found in muscle and heart. The human alpha10 subunit gene (ITGA10) was localized to band q21 of chromosome 1.

Amino Acid Sequence↗
PubMed1998

Sleep architecture in a canine model of obstructive sleep apnea.

Obstructive sleep apnea (OSA) causes recurrent sleep disruption that is thought to contribute to excessive daytime sleepiness in patients with this disorder. The purpose of this study was to determine the specific effects of OSA on overall sleep architecture in a canine model of OSA. The advantage of this model is that sleep during long-term OSA can be compared to both normal sleep before OSA and recovery sleep after OSA. Studies were performed in four dogs in which sleep-wake state was monitored continuously by a computer that received telemetered EEG and EMG signals. Whenever sleep was detected, the computer sent a signal to close a valve through which the dog breathed; when the dog awoke the occlusion was released. In each dog, data were analyzed from 4 consecutive nights in three phases: a control phase before induction of OSA, a phase during long-term OSA (mean = 85 days, apnea index = 59/hour), and a recovery phase after cessation of OSA. During recovery there was a significant increase in the amount of rapid-eye-movement (REM) sleep compared to the OSA phase (p < 0.01), as well as significant increases in sleep efficiency and decreases in wakefulness (p < 0.01), similar to that reported in OSA patients. The REM rebound during recovery, however, could not be attributed to overall REM deprivation since the amount of REM sleep during the OSA phase was not different from the control phase (p = 0.708). This finding suggests that REM rebound during recovery from OSA is not the result of an overall REM sleep deficit per se. Rather, repeated sleep disruption due to the effects of repetitive apneas and hypoxia may lead to an increased REM sleep drive that manifests itself as a REM sleep rebound during recovery sleep after OSA.

Animals↗
PubMed1998

A novel extracellular domain variant of the human integrin alpha 7 subunit generated by alternative intron splicing.

The integrin alpha 7 beta 1 laminin receptor, which is expressed on replicating myoblasts, and upregulated during myogenic differentiation, is involved in cell adhesion and communication between muscle cells and the extracellular matrix. It is a major cell-surface substrate in skeletal muscle cells for the cell-surface, argininespecific, ADP-ribosyltransferase. Both the extracellular and cytoplasmic domains of the mouse alpha 7 subunit undergo alternative splicing during development, generating differentially expressed variants with presumably unique ligand-binding and signalling properties. Here human cDNA clones isolated from a fetal heart lambda gt10 cDNA library encoded the complete sequence of the alpha 7 subunit and hybridised to a single major 4.4 kb alpha 7 subunit transcript abundantly expressed in human skeletal muscle, moderately expressed in heart, and weakly expressed in most other tissues. One clone out of four contained a novel 225-nucleotide in-frame deletion corresponding to 75 amino acids in the C-terminal region of the extracellular domain. The variant, whose expression appears to be tissue-specific, is created by alternative splicing at sites flanking an intron in the alpha 7 gene. A related mouse form was identified in P19 embryonal carcinoma cells. Deletion of the spliced region, which either contains or is in very close proximity to the major ADP-ribosylation site of the alpha 7 subunit, may serve to modulate the effects of ADP-ribosylation, or alternatively molecular associations, and receptor-ligand affinity.

Alternative Splicing↗
PubMed1998

The beta7 integrin gene (Itgb-7) promoter is responsive to TGF-beta1: defining control regions.

The beta7 integrins LPAM-1 (alpha4beta7) and M290 (alphaEbeta7) mediate the homing of lymphocytes to gut-associated lymphoid tissue, and the proposed retention of intraepithelial lymphocytes (IEL), respectively. Here we show that the gut mucosal cytokine TGF- beta1 increases the expression of beta7 and alphaE subunit mRNA transcripts and the cell-surface expression of M290 on T cells, and that it decreases the level of alpha4 integrin transcripts. Induced beta7 integrin gene expression was inhibited by the protein tyrosine kinase inhibitor genistein, implicating a role for tyrosine phosphorylation. An analysis of the beta7 integrin gene promoter revealed three DNAse I hypersensitivity sites, two of which mapped to the 5' and 3' ends of a promoter fragment (nucleotides +690 to +63) that directed both the basal and the TGF-beta1-induced expression of a heterologous reporter gene. Deletion analysis identified two TGF-beta1 response regions encompassing nucleotides -509 to -398 (TGFBRR1), and -122 to +32 (TGFBRR2). TGFBRR1 interacted with at least five protein complexes, whose binding could be induced with TGF-beta1 stimulation and could be antagonized by TGFBRR2 which harbored both similar and distinctive cis-elements. TGFBRR2 interacted specifically with at least two major nuclear protein complexes, whose binding was phosphorylation dependent. These data provide new insights into the mechanism by which TGF-beta may switch LPAM-1(+ve) migrating T cells to express M290, facilitating their retention in the gut.

Animals↗
PubMed1998

Identification of multiple forms of 180-kDa ribosome receptor in human cells.

Herein, we describe the analysis and mapping of cDNA clones encoding variant forms of the human homolog of the canine 180-kDa ribosome receptor (p180). One form, similar to the chicken ES/130 homolog, possesses a large uninterrupted C-terminal region composed predominantly of heptad repeats predicted to form an alpha-helical double-stranded coiled-coil rod. Other forms contain in addition a 10-amino acid consensus motif, NQGKKAEGAQ, repeated up to 54 times in tandem close to the N-terminus. Such repeats in canine p180 represent a ribosome-binding domain. The cDNA hybridized to a major 6-kb transcript in all tissues examined, where very high expression was observed in tissues that carry out a high level of secretion such as pancreas, liver, and placenta. The ES130/p180 gene was mapped to chromosome 20p12, and a potential pseudogene appears to reside on chromosome 7. In summary, the data suggest that p180 exists in humans in different forms because of complete removal of tandem repeats, or partial intraexonic splicing, creating different repeat lengths with potentially novel ribosome-binding characteristics.

Alternative Splicing↗