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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To study the control of acetylcholine release from airway parasympathetic neurons, primary cultures of these cells were established. Guinea pig tracheas were disaggregated with collagenase and plated onto matrigel-coated plates in medium that contained cytosine arabinoside to inhibit growth of dividing cells. Over 7 to 10 days neurites grow from the cell bodies, reaching a length of 2 mm. The vast majority of the cells in these cultures were neurons, as identified by morphology and staining with Neurotag and with antibody to neuron-specific antigen protein gene product 9.5. Cultured neurons contained acetylcholine, which was released by electrical field stimulation. Thus these were parasympathetic neurons. Staining with antibodies to M1, M2, and M4 muscarinic receptors revealed the presence of only M2 receptors. Likewise, reverse transcription-polymerase chain reaction using primers for M1, M2, and M4 muscarinic receptors revealed mRNA only for M2 receptors. Blocking these M2 receptors using atropine potentiated the stimulated release of acetylcholine, demonstrating that the M2 receptors inhibit acetylcholine release, as they have been shown to do in vivo. Thus airway parasympathetic neurons can be grown in culture, they retain the ability to synthesize and release acetylcholine, and they express functional inhibitory M2 muscarinic receptors.
Am J Respir Cell Mol Biol 1996 Dec
PMID:Cultures of airway parasympathetic nerves express functional M2 muscarinic receptors. 896 65

Mycobacterium tuberculosis survives and replicates within human macrophages, but the mechanisms whereby tubercle bacilli resist killing are incompletely understood. We tested the general model in which M. tuberculosis evades killing by entering naive macrophages through receptors that are unable to activate cellular microbicidal activities. Complement receptor types 1 (CR1), 3 (CR3), and 4 (CR4) were blocked with monoclonal antibodies, and mannose receptors were blocked with a competitive ligand, mannosylated bovine serum albumin (MBSA). Survival and replication of M. tuberculosis (Erdman) were evaluated after the bacteria were phagocytosed in the presence of blocking agents (directing binding to the unblocked receptors). Although there was significant variation in the growth rate of virulent M. tuberculosis in monocyte-derived macrophages from different donors, the intracellular survival and replication of mycobacteria were equivalent regardless of the receptor(s) used for binding and phagocytosis. We conclude that the mechanisms whereby M. tuberculosis evades killing by human macrophages are independent of the receptor-mediated route of entry, and operate at one or more steps common to all entry pathways. Blocking complement and mannose receptors in combination did not completely abrogate binding of M. tuberculosis to macrophages. However, we found that two polyanionic scavenger-receptor ligands exhibited a concentration-dependent ability to block binding of M. tuberculosis to macrophages. Moreover, blocking class A scavenger receptors abrogated nearly all binding that persisted after blocking complement and mannose receptors. This indicates that class A scavenger receptors are quantitatively important mediators of M. tuberculosis-macrophage interactions. M. tuberculosis has evolved multiple mechanisms to promote its efficient entry into macrophages. This suggests that passage of the organism through macrophages may be an essential early step in the pathogenesis of tuberculosis.
Am J Respir Cell Mol Biol 1996 Dec
PMID:Selective receptor blockade during phagocytosis does not alter the survival and growth of Mycobacterium tuberculosis in human macrophages. 896 71

To study the mechanisms involved in the movement of neutrophils from the blood stream into the lung airways, we investigated human neutrophil transmigration across a monolayer of human airway epithelial cells, both in the apical-to-basolateral direction and in the more physiologic basolateral-to-apical direction. Migration of human neutrophils across monolayers of human airway epithelial H292 cell-line cells and primary bronchial epithelial cells occured most efficiently in the basolateral-to-apical direction, both after the addition of chemoattractants to resting epithelial cells and across interleukin-1beta (IL-1beta)-stimulated epithelial cells. Blocking studies with monoclonal antibodies revealed that the migration of neutrophils was mediated by the CR3 adhesion molecule (CD11b/CD18) on the neutrophils. IL-1beta-treated epithelial cells caused neutrophil movement via the secretion of chemoattractants. The most potent chemoattractant released by the epithelial cells was found to be IL-8, because the IL-1beta-induced migration was inhibited for 75 +/- 10% by the addition of an antibody against IL-8. After apical stimulation of the epithelial cells with an optimal concentration of IL-1beta, 27 +/- 4 ng/ml IL-8 was found in the supernatant at the apical side of epithelial cells. Platelet-activating factor (PAF) synthesis by the epithelial cells did not play a role in neutrophil transmigration, as was demonstrated by the lack of inhibition of this process after addition of the PAF-receptor antagonist WEB 2086. We conclude that the movement of neutrophils across airway epithelial cell monolayers occurs preferentially in the physiologic basolateral-to-apical direction, indicating that the polarity of epithelial cells is important for neutrophil transmigration.
Am J Respir Cell Mol Biol 1996 Dec
PMID:Transmigration of human neutrophils across airway epithelial cell monolayers is preferentially in the physiologic basolateral-to-apical direction. 896 72

Tissue plasminogen activator (tPA), the serine protease that converts inactive plasminogen to the protease plasmin, was recently shown to mediate neurodegeneration in the mouse hippocampus. Mice deficient in tissue plasminogen activator (tPA) display a dramatic resistance to a paradigm of excitotoxic neuronal death that involves intrahippocampal injection of the excitotoxin. This model is thought to reproduce the mechanism of neuronal death observed during acute (such as ischemic stroke) and degenerative (such as amyotrophic lateral sclerosis) diseases of the nervous system. The requirement for the proteolytic activity of tPA to mediate neuronal death is acute in the adult mouse. Serine protease inhibitors, specific for tPA or the tPA/plasmin proteolytic cascade, are effective in conferring extensive neuroprotection following the excitotoxic injection. These findings suggest possible new ways for interfering with the neuronal death observed in the hippocampus as a result of excitotoxicity. In addition, tPA is produced in the hippocampus primarily by microglial cells, which become activated in response to the neuronal injury. Blocking microglial activation has been shown in other injury paradigms to protect against neuronal death, therefore suggesting another way to retard neurodegeneration in the CNS. Furthermore, after the insult has been inflicted and in the presence of a compromised blood-brain barrier macrophages (cells deriving from the same lineage as microglia) migrate into the brain, where they are thought to contribute to the neuronal cell loss by secreting neurotoxic molecules. If these macrophages/microglia expressed, however, a tPA inhibitor, rather than the possibly neurotoxic tPA, they might be able to protect the neurons from dying.
J Mol Med (Berl) 1997 May
PMID:Clinical implications of the involvement of tPA in neuronal cell death. 918 75

Cultured human and rat endothelial cells were used to study cellular toxicity and Ca2+ signalling upon exposure to reactive oxygen species. Superoxide and hydrogen peroxide (O2.-/H2O2) were produced by the hypoxanthine/xanthine oxidase system (HX/XO) and caused intracellular Ca2+ concentration ([Ca2+]i) to rise steadily when activities above 2 mU/ml were used. These Ca2+ increases were also measured when the glucose/glucose oxidase (G/GO) system above 5 mU/ml was used to produce hydrogen peroxide (H2O2). Gross morphological changes appeared to parallel elevated [Ca2+]i levels preceding cell death. However, when HX/XO or G/GO were used at non toxic doses rapid and transient changes in [Ca2+]i were measured. These treatments did not alter subsequent receptor mediated Ca2+ signalling induced by ATP (10 microM) or histamine (100 microM). Superoxide dismutase (50 U/ml), which dismutates O2.- into H2O2 also had no influence, whereas catalase (50 U/ml), which removes H2O2, completely diminished transient [Ca2+]i responses. H2O2 added directly was able to induce similar Ca2+ transients when concentrations of at least 500 microM were used. Buffering trace amounts of iron (o-phenanthroline; 200 microM) in order to inhibit .OH radical formation was not effective to alter Ca2+ changes. Experiments performed in Ca(2+)-free buffer showed a similar rise in [Ca2+]i and readdition of Ca2+ to the extracellular medium indicated the activation of store operated Ca2+ entry. Blocking Ca(2+)-ATPases of the endoplasmatic reticulum with thapsigargin (1 microM) inhibited ROS induced transient increases and cells preincubated with pertussis toxin (200 nM) showed unchanged Ca2+ transients after exposure to both enzyme systems. Phospholipase C inhibitor U73122 (2 microM) effectively reduced hydrogen peroxide induced emptying of intracellular stores. Taken together, we demonstrate that enzymatically produced non-toxic H2O2 rather than O2.- or .OH causes calcium signalling from thapsigargin sensitive stores, and activates store operated Ca2+ entry at least partially by activating phospholipase C. These changes clearly differ from pathological 'oxidative stress' associated with a progressive increase in [Ca2+]i.
Mol Cell Biochem 1997 Jun
PMID:Transient Ca2+ changes in endothelial cells induced by low doses of reactive oxygen species: role of hydrogen peroxide. 920 90

Alveolar macrophages (AM) from sarcoid patients have been shown to be good antigen presenting cells (APC) unlike normal AM which are usually ineffective. We demonstrate in ten consecutive sarcoid patients that most of their AM, unlike normal AM, do coexpress high levels of CD86, CD40, and CD30L, all known to be important for T-cell activation. CD80 is also slightly more expressed on sarcoid AM than on normal AM, but is detected on only 26 +/- 6% (mean +/- SEM) of sarcoid AM. A good correlation is present between the percentage of sarcoid AM expressing CD86 and CD40 or CD86 and CD30L. However, no correlation is found between the percentage of CD80 and CD86 positive AM in these same patients. Blocking antibodies against CD86 were able to reduce by more than 80% allogeneic T-cell proliferation induced by the AM of sarcoid patients. This study provides evidence that AM can, in pathologic states such as sarcoidosis, express functional costimulatory molecules for T-cell activation such as CD86, thought to be rather specific for more professional APC such as dendritic cells.
Am J Respir Cell Mol Biol 1997 Jul
PMID:Alveolar macrophages in sarcoidosis coexpress high levels of CD86 (B7.2), CD40, and CD30L. 922 14

A competitive enzyme linked immunosorbent assay with antigen immobilized on the solid phase was developed to measure alpha 2-macroglobulin in rat serum. The cross reactivity with albumin and hemoglobin was eliminated by use of IgG fractions that were isolated after chromatography on Cibacron Blue F3-GA Sepharose immobilized rat whole serum and hemoglobin Sepharose. Blocking materials and pH of the coating buffer had no effect on the amount of alpha 2-macroglobulin that binds to the plate. When the coating amount of alpha 2-macroglobulin was 100 ng/well, at pH 7.4, 10 mM Tris-HCl containing 150 mM sodium chloride and the IgG amount added was 60 ng/well, then albumin and hemoglobin did not affect the assay at concentrations of 150 micrograms/ml or 200 micrograms/ml. This assay is useful for measuring the concentration of alpha 2-macroglobulin in normal and irradiated rat serum.
Biochem Mol Biol Int 1997 Nov
PMID:Development of a competitive enzyme linked immunosorbent assay to measure alpha 2-macroglobulin in irradiated rat serum. 938 29

Excitatory amino acid (EAA) receptors play an important role in neuronal cell death in acute cerebral ischemia. Blocking the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) subtype of EAA receptor has been shown to reduce cell death in global cerebral ischemia. However their role in focal stroke, although suggestive, has remained more contentious. To clarify this issue, we generated transgenic mice overexpressing the AMPA receptor (AMPAR) subunit GluR2-flip which would increase AMPAR-mediated currents. Excitatory neurons in these transgenic mice are thus predicted to be more susceptible than wild-type neurons to EAA (glutamate)-induced excitotoxic damage. Consistent with this prediction, cultured neurons from transgenic mice had a lower LD50 for exposure to glutamate (10(-3)-10(-5) M for 5 min) compared to wild-type neurons. Moreover, transgenic mice subjected to permanent focal ischemia of the middle cerebral artery (MCA) using the intralumenal filament model sustained larger infarctions compared to wild-type controls. Hence we have developed a genetic mouse model that demonstrates the crucial role of AMPAR containing GluR2-flip in the pathogenesis of focal hypoxic-ischemic neuronal cell death. This model will be a valuable tool in elucidating molecular mechanisms of glutamate excitotoxicity and evaluating the efficacy of glutamate receptor antagonists in attenuating post-ischemic neuronal cell death.
Brain Res Mol Brain Res 1997 Dec 15
PMID:Enhanced neuronal death from focal ischemia in AMPA-receptor transgenic mice. 949 44

Myocardial infarctions and stroke arise primarily as a result of hypoxia/ischemia-induced cell injury. However, the molecular mechanism of cardiac cell death due to hypoxia has not been elucidated. We showed here that chemical hypoxia induced by 1 mM azide triggered apoptosis of isolated neonatal rat ventricular cardiac myocytes but had no effect on cardiac fibroblasts. The azide-induced cardiomyocyte apoptosis could be characterized by a reversible initiation phase (0-46 h after azide exposure) during which cytosolic ATP levels remained little affected. This was followed by an irreversible execution phase (12-18 h) exhibiting prominent internucleosomal DNA fragmentation, cell membrane leakage, mitochondrial dysfunction, and increased calpain messenger RNA. Blocking extracellular calcium influx or intracellular calcium release was each effective in suppressing myocyte apoptosis. Cell death was also found to be mediated by calcium sensitive signal transduction events based on the use of specific antagonists. Consistent with the induction of calpain expression during apoptosis, blocking de novo protein synthesis and calpain activity inhibited cell death. These regulatory features coupled with the ease of the cell system suggest that the myocyte apoptosis model described here should be useful in the study of events leading to the demise of the myocardium.
Mol Cell Biochem 1998 Jan
PMID:Chemical hypoxia triggers apoptosis of cultured neonatal rat cardiac myocytes: modulation by calcium-regulated proteases and protein kinases. 954 93

The intratesticular concentration of progesterone (P) rises up to the micromolar range during high-dose luteinizing hormone (LH)/hCG stimulation. The aim of this study was to examine whether P is involved in the concomitant down-regulation of the LH receptor (R) function. The effects were tested in a mouse Leydig tumor cell line (mLTC-1) and in Percoll-purified adult mouse Leydig cells. Pre-incubation of the mLTC-1 cells for 48 h with P (1-10 micromol/l) decreased in dose-dependent fashion their specific binding of [125I]iodo-hCG as well as the hCG-induced cAMP production (down to 65 and 40% respectively, of controls, P < 0.01). Similar effect of P on hCG-induced cAMP production was observed in adult mouse Leydig cells following a 24 h incubation in the presence of P (0.3-10 micromol/l). In addition, P treatment significantly inhibited the expression of a transiently transfected murine LHR promoter (715 or 950 bp of the 5' untranslated region)-luciferase fusion constructs in mLTC-1 cells (down to 50% of control, P < 0.01). In accordance, a 6-12 h culture in the presence of 5-10 micromol/l of P showed significant down-regulatory effects on the steady state levels of LHR-mRNA in mLTC-1 cells. These inhibitory effects of P on the LHR expression and function were mimicked by similar concentrations of cortisol, but not by testosterone or estradiol. Blocking the steroid synthesis of mLTC-1 cells with 86 micromol/l of aminoglutethimide (AMG) partially reversed the down-regulating effect of hCG on the LHR-mRNA. Moreover, a 24 h culture in the presence of AMG showed an up-regulating effect on expression of the LHR promoter-luciferase constructs, and including hCG (50 microg/l) in the culture medium enhanced this effect. Hence, in the absence of steroidogenesis, hCG up-regulates the LHR promoter expression. In conclusion, we present here a novel short-loop regulatory mechanism in murine Leydig cells where P exerts a negative effect on LHR expression and function. Since Leydig cell P production is dramatically increased during high-dose stimulation with LH/hCG, due to blockade of C21 steroid side chain cleavage, the present findings offer a function for this steroid in the LHR down-regulation.
Mol Cell Endocrinol 1998 Feb
PMID:Progesterone can participate in down-regulation of the luteinizing hormone receptor gene expression and function in cultured murine Leydig cells. 960 14


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