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

Several studies have suggested that pulmonary toxicity to asbestos and silica may be mediated through oxidant-induced cell injury. We have reported recently that surface radicals associated with freshly fractured silica may be an important factor in cell injury and induction of pulmonary disease. Although the generation of oxygenated radicals in dust-cell interactions has been demonstrated, there are no data correlating the toxicity of a dust with the level of oxygen radical generation by the dust during its interaction with phagocytic cells. In the present study, we have investigated the in vitro generation of oxygen free radicals from human neutrophils and rat alveolar macrophages stimulated with freshly fractured silica, aged silica, amosite, crocidolite, chrysotile, and nontoxic dust, barite. Electron spin resonance (ESR) with the aid of a spin trap phenyl-N-tert-butyl nitrone (PBN) was used to measure the oxygen radicals generated during phagocytosis of the dusts. The relative toxicity index and ESR peak heights, on an equal surface area basis and normalized to barite as one, showed a direct relationship. The normalized toxicity indices and peak heights were: silica, 3.5 versus 2; chrysotile, 4 versus 2; crocidolite, 11 versus 8; and amosite, 26 versus 13. Addition of hydroxyl radical scavengers such as catalase, dimethyl sulfoxide, 1,3 dimethyl-2-thiourea (DMTU), sodium benzoate, and mannitol prevented the radical generation. Carmustine, a glutathione reductase-glutathione peroxidase inhibitor, caused a 5-fold increase in the radical generation. These results indicate that a nontoxic dust such as barite generates toxic oxygen radicals at a minimal level that can be quenched by the normal cellular defense system. For toxic dusts such as silica, amosite, chrysotile, and crocidolite, the potential for oxygen radical generation is enhanced by their surface properties, physical dimensions, and the surface-based radical-generating redox sites. The enhanced radical generation may impair the cellular defense system, resulting in cell injury. Use of scavengers, chelators, and potentiating agents suggests the membrane-based oxidase system as the probable primary source of the radical-generating system. The data presented herein suggest the generation of oxygen free radicals as an important primary event in silica- as well as asbestos-induced cell injury.
Am J Respir Cell Mol Biol 1992 Apr
PMID:Enhanced generation of free radicals from phagocytes induced by mineral dusts. 131 51

We have cloned and examined the 5' flanking regions of the heavy (NF-H), light (NF-L) and mid-sized (NF-M) mouse neurofilament (NF) genes in order to begin to characterize the regions of each gene that regulate NF transcription. Chimeric plasmids bearing the CAT reporter gene and deletion mutants of the upstream NF genes were transiently transfected into neuronal (PC12 and Neuro 2A) and non-neuronal (HeLa) cell lines. Constructs bearing upstream regions to -4000 in NF-H, to -5600 in NF-L and to -4500 in NF-M were expressed at low levels in neuronal and in non-neuronal cells. Progressive deletion of 5' flanking sequence to -385 in NF-H, to -325 in NF-L and to -505 in NF-M caused a several-fold increase of transcription from the transfected plasmids. Increases of transcription by deletion mutants followed a similar pattern in neuronal and in non-neuronal cell lines. Negative upstream regions are located between -1314 and -385 in NF-H, between -936 and -325 in NF-L and between -874 and -505 in NF-M. Additional negative regions are present further upstream in NF-L and in NF-H. The negative regions of NF-H and of NF-L suppress transcription when placed in either orientation in front of the SV40 or a heterologous NF promoter. These studies demonstrate that the three mouse NF genes possess similar functional features, namely, that of a relatively strong and promiscuous promoter with negative upstream elements. The role of the negative elements in regulating NF expression remains unclear.
Brain Res Mol Brain Res 1992 Mar
PMID:Negative regulatory regions are present upstream in the three mouse neurofilament genes. 131 9

The purpose of this study was to explore the role of singlet oxygen in cardiovascular injury. To accomplish this objective, we investigated the effect of singlet oxygen [generated from photoactivation of rose-bengal] on the calcium transport and Ca(2+)-ATPase activity of cardiac sarcoplasmic reticulum and compared these results with those obtained by superoxide radical, hydrogen peroxide and hydroxyl radical. Isolated cardiac SR exposed to rose bengal (10 nM) irradiated at (560 nm) produced a significant inhibition of Ca2+ uptake; from 2.27 +/- 0.05 to 0.62 +/- 0.05 mumol Ca2+/mg.min (mean +/- SE) (P less than 0.01) and Ca(2+)-ATPase activity from 2.08 +/- 0.05 mumol Pi/min.mg to 0.28 +/- 0.04 mumol Pi/min.mg (mean +/- SE) (P less than 0.01). The inhibition of calcium uptake and Ca(2+)-ATPase activity by rose bengal derived activated oxygen (singlet oxygen) was dependent on the duration of exposure and intensity of light. The singlet oxygen scavengers ascorbic acid and histidine significantly protected SR Ca(2+)-ATPase against rose bengal derived activated oxygen species but superoxide dismutase and catalase did not attenuate the inhibition. SDS-polyacrylamide gel electrophoresis of SR exposed to photoactivated rose bengal up to 14 min, demonstrated complete loss of Ca(2+)-ATPase monomer band which was significantly protected by histidine. Irradiation of rose bengal also caused an 18% loss of total sulfhydryl groups of SR. On the other hand, superoxide (generated from xanthine oxidase action on xanthine) and hydroxyl radical (0.5 mM H2O2 + Fe(2+)-EDTA) as well as H2O2 (12 mM) were without any effect on the 97,000 dalton Ca(2+)-ATPase band of sarcoplasmic reticulum.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Cell Biochem 1992 Apr
PMID:Singlet oxygen: a potential culprit in myocardial injury? 131 3

The oxidation of NADH and accompanying reduction of oxygen to H2O2 stimulated by polyvanadate was markedly inhibited by SOD and cytochrome c. The presence of decavanadate, the polymeric form, is necessary for obtaining the microsomal enzyme-catalyzed activity. The accompanying activity of reduction of cytochrome c was found to be SOD-insensitive and therefore does not represent superoxide formation. The reduction of cytochrome c by vanadyl sulfate was also SOD-insensitive. In the presence of H2O2, all the forms of vanadate were able to oxidize reduced cytochrome c, which was sensitive to mannitol, tris and also catalase, indicating H2O2-dependent generation of hydroxyl radicals. Using ESR and spin trapping technique only hydroxyl radicals, but not superoxide anion radicals, were detected during polyvanadate-dependent NADH oxidation.
Mol Cell Biochem 1992 Apr
PMID:Characterization of oxygen free radicals generated during vanadate-stimulated NADH oxidation. 131 4

We investigated the susceptibility of sarcolemmal Na+K(+)-ATPase to singlet oxygen. The role of this enzyme is regulation of Na+ concentration and thereby membrane potential. Inhibition of Na+ pump would lead to intracellular Ca2+ overload therefore further aggravating the injury caused by free radicals. Incubation of isolated sarcolemmal vesicles with irradiated rose bengal (150 nM) resulted in 86 +/- 1% inhibition of Na+K(+)-ATPase activity and histidine (25-100 mM) protected the enzyme in a dose-dependent fashion whereas SOD, catalase or mannitol (.OH radical scavenger) did not have any effect. Also, the inhibition of Na+K(+)-ATPase activity was dependent on rose bengal concentration, intensity of irradiation, duration of light exposure, showing that inhibition was directly related to amount of singlet oxygen generated. These results show that singlet oxygen may have significant disruptive effects on sarcolemmal function and may represent an important mechanism by which the oxidative injury to the myocardium induces arrhythmogenesis.
J Mol Cell Cardiol 1992 May
PMID:Singlet oxygen-induced inhibition of cardiac sarcolemmal Na+K(+)-ATPase. 132 12

Neuropeptide Y (NPY) is the most abundant neuropeptide detected in the mammalian brain, and is found throughout the central and peripheral nervous system. This peptide is a proposed regulator of appetite, blood pressure, and pituitary hormone release. Previous experiments have demonstrated the ability of 5' sequences within the human NPY gene to promote transcription in cultured neuronal cells. To identify sequences in this gene that regulate tissue-specific expression, a NPY/CAT fusion gene, containing approximately 850 bp of NPY sequences, was microinjected into fertilized mouse ova. Five lines of transgenic mice were derived from these ova and several tissues from mice of each line were tested for transgene expression using the CAT assay. One line demonstrated X-chromosome-linked transmission of the transgene while the other lines demonstrated autosomally-linked transmission. Three lines demonstrated transgene expression with significant levels of CAT activity detectable only in tissues which have been shown to express endogenous NPY. One autosomally-linked line did not demonstrate significant levels of transgene activity because the transgene appeared to have undergone structural alteration during genomic integration. No transgene activity was detected in either male of female mice from the X-linked line, suggesting a positional regulation of the transgene locus other than X-inactivation in this line. The present research demonstrated the NPY regulatory sequences included in pCATNPY delta 796 sufficiently directed tissue-appropriate gene expression in transgenic mice.
Brain Res Mol Brain Res 1992 Jun
PMID:Tissue-specific expression of the human neuropeptide Y gene in transgenic mice. 132 21

A hormone-inducible transcriptional system has been established, based on the stable transfection of the rat androgen receptor (rAR) and a reporter plasmid containing the mouse mammary tumour virus promoter linked to the chloramphenicol acetyltransferase gene (pMMTV-CAT) into steroid receptor-negative CV-1 cells. First, the rAR was stably introduced into CV-1 cells. Single clones were tested for stable expression of functionally active AR by analysing the effect of dihydrotestosterone on induction of transiently transfected pMMTV-CAT. Stable transfection and the expression of AR was confirmed by steroid-binding assays. In a second step, a clone expressing physiological amounts of AR protein (30 fmol/mg protein) was stably transfected with pMMTV-CAT to yield a permanent cell line that stably expresses functional AR and MMTV-CAT sequences. This cell line provides a powerful tool for the efficient and accurate determination and quantification of the effects of androgens and anti-androgens on reporter gene transcription. This was demonstrated by investigating the action of the three anti-androgens hydroxyflutamide, casodex and cyproterone acetate. The three compounds were shown to reverse the effects of the androgen R1881 on gene expression but were themselves devoid of agonistic activity.
J Steroid Biochem Mol Biol 1992 Sep
PMID:Stable transfection of androgen receptor and MMTV-CAT into mammalian cells: inhibition of cat expression by anti-androgens. 132 16

1. The skeletal muscle acetylcholine receptor comprises several subunits whose coordinated expression during myogenesis is probably controlled by cis elements in the individual subunit genes. We have previously analyzed promoter regions of the alpha and delta genes (Wang et al., 1988, 1990); to gain further insight into receptor regulation, we have now studied the promoter of the chick muscle gamma-subunit gene. 2. This analysis was faciliated by the close upstream proximity of the coding region of the delta-subunit gene and the consequent brevity (740 bp) of the untranslated linker connecting the two genes (Nef et al., 1984). Nuclease protection and primer extension analysis revealed that transcription of the gamma-subunit gene starts at position 56 upstream of the translational initiation site. 3. Nested deletions of the promoter region were employed to identify functionally important elements. A 360-bp sequence (-324 to +36) was found to activate transcription, in a position- and orientation-independent manner, during myotube formation. This sequence comprises 5 M-CAT (Nikovits et al., 1986) similarities and contains, at positions -52/-47 and -33/-28, two CANNTG (Lassar et al., 1989) motifs. 4. Binding experiments were performed by means of gel retardation, gel shift competition, and footprint analysis. The CANNTG motifs were found to bind MyoD and myogenin fusion proteins and to interact with proteins in nuclear extracts from cultured myotubes. 5. Point mutations in the CANNTG motifs revealed that these elements are crucial for full promoter activity in myotubes and essential in fibroblasts cotransfected with a myogenin expression vector. 6. We conclude that the activity of the gamma-subunit gene is determined largely by E boxes, which in vivo are likely to be activated by MyoD family proteins; in addition, other transactivators such as the M-CAT binding protein presumably play a role. Both CANNTG elements and M-CAT motifs are also present in the alpha- and delta-subunit enhancer and may therefore account for the coordinate expression of the three subunits during muscle differentiation.
Cell Mol Neurobiol 1992 Jun
PMID:Analysis of binding and activating functions of the chick muscle acetylcholine receptor gamma-subunit upstream sequence. 133 Mar 9

Testosterone and its 5 alpha-reduced derivative 5 alpha-dihydrotestosterone exert different actions in the male during embryogenesis and in postnatal life. Nevertheless the two hormones bind to the same intracellular androgen receptor, and genetic and endocrinological studies in the Tfm mouse suggest that the actions of both hormones are mediated by this single receptor. Previous studies indicate that dihydrotestosterone binds more tightly to the androgen receptor but that the Bmax of binding of the two hormones is the same. To determine whether these differences in binding parameters could explain the mechanism by which the two hormones exert different physiological actions via the same receptor, we introduced a plasmid encoding the androgen receptor cDNA and a reporter plasmid encoding MMTV-CAT into Chinese hamster ovary cells. These cells do not express endogenous androgen receptor and do not convert testosterone to dihydrotestosterone. Therefore, it was possible to examine the relation between the concentration of each of the steroids and reporter gene expression. Both hormones enhanced CAT activity, but dihydrotestosterone was approximately 10 times as potent (half maximal of 0.018 nM) as testosterone (half maximal of 0.2 nM); the maximal activity achieved was the same for the two androgens. These findings are nearly identical to the apparent Kd values for the interaction of the two hormones with the androgen receptor. Although testosterone and dihydrotestosterone may influence the expression of other genes differently, these findings are compatible with a model system in which the differential effects can be explained as a consequence of different binding affinities to the receptor.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Cell Endocrinol 1992 Oct
PMID:Testosterone and 5 alpha-dihydrotestosterone interact differently with the androgen receptor to enhance transcription of the MMTV-CAT reporter gene. 133 7

The protective action of deferoxamine, an iron chelator, against functional and metabolic deteriorations of ventricular muscle, induced by ischaemia-reperfusion, was investigated in Langendorff-perfused hearts of neonatal rabbits in comparison with superoxide dismutase (SOD) plus catalase. The perfused hearts were subjected to normothermic (37 degrees C) global ischaemia for 45 min following cardiac arrest with St Thomas cardioplegic solution and then reperfused with oxygenated Krebs-Henseleit solution. In control hearts, the recovery of the left ventricular developed pressure (LVDP) after 30 min reperfusion was 50.7 +/- 3.1% (mean +/- SE, n = 5) of the pre-ischaemic value. The LVDP recovery was significantly improved in the hearts treated with deferoxamine at 10-100 microM (89.4 +/- 1.4% at 30 microM, P < 0.01 vs. control). The improvement in LVDP was less prominent when treated with 30 x 10(4) U/l SOD plus 30 x 10(4) U/l catalase (67.9 +/- 2.0%, P < 0.01 vs. deferoxamine at 30 microM). CPK leakage into the coronary effluent during the initial 5 min of reperfusion was reduced to around half of the control value with 30 microM deferoxamine (P < 0.05 vs. control), while unaffected by the addition of SOD plus catalase. Free radicals in the coronary effluent were measured with electron spin resonance spectroscopy in separate experiments by using a spin-trapping agent, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). A burst of DMPO-OH signal was detected during the initial minutes of reperfusion. The intensity of DMPO-OH signal was significantly reduced by 30 microM deferoxamine to about one-third of control.(ABSTRACT TRUNCATED AT 250 WORDS)
J Mol Cell Cardiol 1992 Nov
PMID:Deferoxamine, an iron chelator, reduces myocardial injury and free radical generation in isolated neonatal rabbit hearts subjected to global ischaemia-reperfusion. 133 63


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