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Biomedical subjects

A Shaw

Publications and source records attributed to A Shaw.

At least 127 records · Page 7Linked to original sources
PubMed1993

Stereoselective blockade of amphibian epithelial sodium channels by amiloride analogs.

The blockade of amphibian epithelial sodium channels by amiloride has been shown to not be a simple diffusion-limited encounter but rather a selective binding process with the rate of approach of the molecule for the channel site determined by the side chain at position 2 of the pyrazine ring and the stability of the blocking complex provided largely by the bond of the ligand at position 6. The presence of such a putative channel-associated receptor suggests that the sodium channel might have chiral recognition for its blockers. Using the short-circuit technique to measure the amiloride-sensitive sodium current through the apical membrane of the toad urinary bladder and frog skins, we evaluated the sodium channel-blocking potency of three enantiomeric pairs of amiloride analogs with chiral centers on the side chain. We demonstrated the stereoselectivity in sodium channel blockade of those analogs and confirmed the existence of a second attachment site on the channel for interacting with the lipophilic ligand on the side chain of the analog molecule to enhance its blocking activity. Depending on the separation between the chiral carbon and the pyrazine ring and the chemical groups on the chiral carbon, the eudismic ratio for the 50% inhibition constant of the enantiomers, i.e., the ratio of the sodium channel-blocking potency of the more active enantiomer to that of the less active enantiomer, was found to range from slightly more than 1 to more than 100.

Amiloride↗
PubMed1992

High levels of IL-2 alter signal transduction in cloned IL-4-producing CD4 T cells.

Cloned CD4 T cells of the Th2 type make IL-4 and related cytokines upon receptor cross-linking, whereas cloned CD4 T cells of the Th1 type make IL-2, IFN-gamma, and TNF-beta. These two types of CD4 T cell are also reported to use distinct mechanisms of signal transduction. It has been reported that Th1 cells flux Ca2+ upon receptor cross-linking, whereas Th2 cells do not. We have noted that when cloned Th2 cells are exposed to high levels (20 U/ml) of IL-2, they show an altered phenotype. Such cells are much more sensitive to activation by certain antireceptor antibodies, they flux calcium upon receptor ligation without additional cross-linking with anti-Ig antibodies, and they make much larger amounts of IL-4. In addition, the organization of their TCR is altered, with increased levels of the TCR-eta chain and an increase in the extent of association of CD4 with CD3 and CD45, changes similar to those found in Th1 cells. These results suggest that there is no fundamental difference in the signal transduction apparatus of Th1 and Th2 cells; rather, the IL-2 made by Th1 cells may create similar phenotypic changes in these cells and thus create the impression of altered signal transduction mechanisms. These results do show that exposure to high levels of IL-2 can profoundly affect signal transduction in T cells. Furthermore, we found that the Ca2+ signal caused by CD3 antibodies seemed to differ in character from that caused by TCR antibodies suggesting that the use of CD3 antibodies is not always a good model for activation through the TCR.

Biological Assay↗
PubMed1992

Cloning and expression of a cell surface receptor for advanced glycosylation end products of proteins.

Advanced glycosylation end products of proteins (AGEs) are nonenzymatically glycosylated proteins which accumulate in vascular tissue in aging and at an accelerated rate in diabetes. A approximately 35-kDa polypeptide with a unique NH2-terminal sequence has been isolated from bovine lung and found to be present on the surface of endothelial cells where it mediates the binding of AGEs (receptor for advanced glycosylation end product or RAGE). Using an oligonucleotide probe based on the amino-terminal sequence of RAGE, an apparently full-length cDNA of 1.5 kilobases was isolated from a bovine lung cDNA library. This cDNA encoded a 394 amino acid mature protein comprised of the following putative domains: an extracellular domain of 332 amino acids, a single hydrophobic membrane spanning domain of 19 amino acids, and a carboxyl-terminal domain of 43 amino acids. A partial clone encoding the human counterpart of RAGE, isolated from a human lung library, was found to be approximately 90% homologous to the bovine molecule. Based on computer analysis of the amino acid sequence of RAGE and comparison with databases, RAGE is a new member of the immunoglobulin superfamily of cell surface molecules and shares significant homology with MUC 18, NCAM, and the cytoplasmic domain of CD20. Expression of the RAGE cDNA in 293 cells allowed them to bind 125I-AGE-albumin in a saturable and dose-dependent manner (Kd approximately 100 nM), blocked by antibody to RAGE. Western blots of 293 cells transfected with RAGE cDNA probed with anti-RAGE IgG demonstrated expression of immunoreactive protein compared to its absence in mock-transfected cells. These results suggest that RAGE functions as a cell surface receptor for AGEs, which could potentially mediate cellular effects of this class of glycosylated proteins.

Albumins↗
PubMed1992

Physical association of CD4 with the T cell receptor.

The coreceptor hypothesis postulates that physical association of CD4 with the TCR is required for effective signaling for T cell activation. A variety of studies has suggested that the coreceptor function of CD4 allows responses to 10- to 100-fold lower levels of peptide:self MHC class II ligand. We test the hypothesis of CD4 physical association with the TCR in two different ways. First, we use a panel of soluble antibodies directed at different TCR epitopes to activate a cloned T cell line, and show that activation by antibodies directed at a particular TCR epitope can be inhibited by anti-CD4 antibodies binding to a certain CD4 epitope. These effects establish that the interaction of CD4 and the TCR occurs in a specific orientation. Second, we use the same system to provide evidence that the physical association of CD4 with the TCR is required for effective tyrosine phosphorylation of the TCR zeta-chain subunit, presumably reflecting delivery of p56lck (lck) to the TCR. Only anti-TCR antibodies that induce physical association of CD4 with the TCR as monitored by cocapping can induce efficient tyrosine-phosphorylation of the TCR zeta-chain, unless second antibodies are used to force CD4 and the TCR to associate. Furthermore, the phosphorylation of the TCR zeta-chain exactly parallesl physical association in time and drug sensitivity. We conclude from these studies that stimuli that drive physical association of CD4 and the TCR strongly favor T cell activation, supporting the coreceptor hypothesis of CD4 function.

Animals↗
PubMed1992

Extensive CD4 cross-linking inhibits T cell activation by anti-receptor antibody but not by antigen.

Anti-CD4 mAbs have been shown to inhibit T cell activation in a variety of ways. We have tested a panel of IgG and IgM anti-CD4 mAbs for their effects on the activation of a cloned T cell line by antigen presented by syngeneic accessory cells, by soluble anti-T cell receptor antibodies, and by mitogenic lectins. Both IgM and IgG mAbs to CD4 inhibit responses to mitogenic lectins. However, IgM, but not IgG, anti-CD4 antibodies inhibit T cell activation by mAbs specific for the TCR. This inhibitory activity appears to be due to the signaling effects of IgM mAbs, as cross-linked IgG antibodies mimic the behavior of the IgM anti-CD4 antibodies. Inhibition of T cell activation correlates with the ability of IgM and of cross-linked IgG anti-CD4 antibodies to induce tyrosine phosphorylation of the CD4-associated tyrosine kinase p56lck and an unknown substrate, pp32. Surprisingly, we find that the IgM anti-CD4 mAbs tested had no effect on the specific antigen recognition, despite their potent inhibitory effects on the other responses of the same cloned T cell line. These results suggest that multivalent CD4 interactions with ligands such as MHC class II molecules are inhibitory of T cell activation, but that this inhibition can be reversed when CD4 and the TCR bind the same ligand. We discuss the possible implications for positive intrathymic selection of these findings on signaling through CD4.

Animals↗
PubMed1991

Analysis of mutations in the putative nuclear localization sequence of interleukin-1 beta.

Previous studies have shown that, after receptor-mediated endocytosis, interleukin-1 alpha (IL1 alpha) and interleukin-1 beta (IL1 beta) are translocated to the nucleus, where they appear to accumulate. It has been suggested that nuclear translocation may be involved in the biological responsiveness of target cells to IL1 stimulation. The human IL1 beta molecule contains a seven-amino-acid sequence (-Pro208-Lys-Lys-Lys-Met-Glu-Lys-) that shows some sequence identity with the nuclear localization sequence of the simian-virus-40 large T-antigen. The effects of point mutations within this putative nuclear localization sequence on IL1 beta binding, receptor-mediated endocytosis and biological activity have been characterized. Mutants M49 (Lys210----Ala), M50 (Lys211----Ala) and M51 (Pro208----Ala) all retained the ability to bind to the IL1 receptor, albeit with lower affinity than the wild-type molecules. However, mutants M49, M50 and M51 showed greater biological potency than wild-type IL1 alpha or IL1 beta, as measured by the induction of IL2 secretion. However, receptor-mediated endocytosis and nuclear accumulation of M50 were comparable with those in the wild-type. These observations suggest that the putative nuclear localization sequence may play an important role in the generation of biological responses to IL1 stimulation, even though it may not influence internalization of the ligand.

Amino Acid Sequence↗
PubMed1991

Involvement of the liver, but not of IL-6, in IL-1-induced desensitization to the lethal effects of tumor necrosis factor.

C57BL/cnb mice were found to be protected against a lethal combination of recombinant murine (m) TNF and GalN by pretreatment with several cytokines. At certain doses, rmTNF and human (h) TNF protected completely. The clearest protection was induced by rIL-1: all four rIL-1 species (both m and h, as well as alpha and beta) protected when given 12 h before the challenge. LPS and rmIFN-gamma protected weakly, whereas rmIL-6 and rhIL-6 did not protect at all. Also adrenocorticotropic hormone, dexamethasone, or dexamethasone in combination with rhIL-6 could not protect. A single IL-1 injection also completely protected mice against a lethal dose of mTNF in the absence of GalN sensitization. The desensitization by IL-1 cannot be explained by a faster clearance of the challenge TNF. In addition, we demonstrate that the IL-1-induced desensitization was only observed when a functioning liver was present, that IL-1-pretreated animals did not show decreased numbers of hepatocyte TNF receptors, and that the amount of TNF-induced IL-6 was not reduced.

Adrenocorticotropic Hormone↗
PubMed1991

24-hour survival of autotransfused red cells in elective aortic surgery: a comparison of two intraoperative autotransfusion systems.

Twelve patients undergoing elective aortic surgery had intraoperative autotransfusion using either the Haemonetics Cell Saver or the Solcotrans device. The 24-h post-transfusion survival of the patients' preoperative red cells, labelled with indium-111, was compared with the survival of salvaged red cells labelled with chromium-51. A correction coefficient for the instability of the indium-111 label was calculated from the study of red cell survival in six healthy volunteers. There was no significant difference in the volume of circulating red cells measured using each isotope, indicating that extensive early destruction of the autotransfused red cells does not occur. The percentage survival of autotransfused red cells at 24 h was similar to that of the red cells that had not undergone salvage. Both devices were comparable with respect to the effects of cell salvage on the 24-h survival rate of autotransfused red cells.

Aortic Diseases↗
PubMed1991

Prediction of in situ exposure to ultrasound: an acoustical attenuation method.

The prediction of the acoustic levels occurring in a patient during ultrasound examination is important for the assessment of equipment safety. While considerable effort has been devoted to theoretical methods of predicting exposure levels, there is a need to develop simple experimental methods which are universally applicable to the wide range of ultrasonic fields generated by medical ultrasonic equipment. This article outlines a number of methods that have been proposed and explores in detail a new experimental method based on the use of acoustical attenuators, made of low-density polyethylene, which are placed in the ultrasound beam between the transducer and the measuring hydrophone. Measurements of important acoustical quantities have been made using a measurement system based on a multielement hydrophone and comparisons made between this new method and other methods such as electrical attenuation and linear derating theory. The acoustical attenuation technique has been systematically studied, and results are compared with reference levels obtained using tissue-mimicking material. A procedure involving making measurements with the attenuator placed first at the face of the transducer and then at the hydrophone has been developed. By taking the mean value of these two measurements, it has been shown that this new method can simulate within +/- 10% the acoustic levels obtained using the reference. It is concluded that it could be the basis of a universally applicable method.

Absorption↗
PubMed1991

Prediction of in situ exposure to ultrasound: a proposed standard experimental method.

This article presents results for the prediction of in situ exposure levels using a new experimental technique based on the use of acoustical attenuators. These are made of low-density polyethylene and are placed in the ultrasound beam between the transducer face and the measuring hydrophone. The method has been tested for a number of different ultrasonic fields including those generated by diagnostic medical ultrasonic equipment. Using a tissue-mimicking material as a reference, it is shown that it is possible to use this new method to simulate acoustic pressure levels to within +/- 10% and acoustic intensity and power levels within +/- 20%. The method is proposed as the basis for a standard test method with wide applicability.

Acoustics↗
PubMed1991

Changes in quantitative SPECT thallium-201 results associated with the use of energy-weighted acquisition.

The effect of utilizing energy-weighted acquisition on quantitative analysis of SPECT thallium-201 images was evaluated by simultaneously acquiring energy-weighted and windowed projection images in ten patients. The paired image sets were processed identically and evaluated by probability analysis of defect magnitude as indicated by a commercially available software analysis package. It was predicted that defect magnitude would increase as a result of improved image contrast. This was confirmed experimentally. One should be cautious in relying on strict quantitative criteria in cardiac studies with thallium-201, especially when major changes in the imaging system or technique are introduced.

Coronary Disease↗
PubMed1990

Coordinate secretion of interleukin-1 beta and granulocyte-macrophage colony-stimulating factor by the blast cells of acute myeloblastic leukemia: role of interleukin-1 as an endogenous inducer.

Acute myeloblastic leukemia (AML) blasts have been shown to produce a variety of cytokines in culture such as interleukin-1 (IL-1), IL-6, granulocyte-, macrophage-, and granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor-alpha (TNF alpha). Using two sensitive and specific enzyme-linked immunosorbent assays for IL-1 beta and GM-CSF, we document in the present study that the production of the two cytokines by AML blasts in culture is coordinated. First, we observe a striking correlation between the levels of GM-CSF and IL-1 beta released by the cells. Thus, a high production of IL-1 beta is always concordant with a high production of GM-CSF and, conversely, low production of IL-1 beta is concordant with low levels of GM-CSF. Second, neutralization of intrinsic IL-1 using antibodies that are specific for IL-1 alpha and -1 beta suppresses the release of GM-CSF by the cells. Third, neutralization of the endogenous source of IL-1 also results in an abrogation of GM-CSF mRNA. Fourth, the production of both IL-1 beta and GM-CSF is up-regulated by exposing AML blasts to an exogenous source of IL-1, suggesting a positive regulation of autocrine growth factor production. Taken together, our results indicate that GM-CSF production by AML blasts is mediated by endogenously produced IL-1. Both IL-1 beta and -1 alpha are produced by AML blasts, although IL-1 beta appears to be more abundant. Spontaneous colony formation by AML blasts is abrogated by the addition of neutralizing antibodies against IL-1 beta and GM-CSF, whereas each antibody alone has little effect on blast proliferation. Taken together, our results are consistent with the view that the production of IL-1 beta by AML blasts supports autocrine growth in culture, through induction of CSFs or other cytokines that stimulate blast proliferation.

Antibodies↗