Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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We have previously shown that the polyethylene glycol conjugated superoxide dismutase (SOD), which has a plasma half-life of more than 24 h, protects the blood perfused rabbit heart against injury during ischaemia and reperfusion. However, the profile for the dose-dependency of protection was bell-shaped with loss of efficacy below 6000 and above 30,000 U/kg. In the present study, isolated rabbit hearts, perfused with blood from support rabbits, were subjected to a 2 min infusion with St Thomas' Hospital cardioplegic solution followed by 60 min of global ischaemia (37 degrees C) and 60 min of reperfusion. PEG-SOD was administered 1 h or 12-24 h before ischaemia. We assessed the effect of PEG-SOD on ischaemia- and reperfusion-induced changes in: (i) the tissue content of reduced glutathione (GSH), oxidized glutathione (GSSG) and malondialdehyde (MDA) and (ii) the activity of CuZn-SOD, Mn-SOD and glutathione peroxidase and reductase (GPD and GRD). Ischaemia and reperfusion reduced tissue GSH content by 70% and increased GSSG content by 400% (from their fresh aerobic values of 13.1.9 and 0.09 +/- 0.01 nmol/mg protein, respectively). PEG-SOD, given intravenously at various doses to donor and support rabbits 1 h or 12-24 h before ischaemia, protected against these changes with a bell-shaped dose-response relationship. Thus, with 0, 3000, 6000, 12,000, 30,000 and 60,000 U/kg, GSH content was 4.1 +/- 0.4, 4.8 +/- 0.4, 8.5 +/- 0.5, 12.3 +/- 1.6, 12.3 +/- 1.6 and 5.0 +/- 0.5 nmol/mg protein in the 1 h pretreatment group and 4.1 +/- 0.4, 4.2 +/- 0.5, 10.4 +/- 1.5, 11.2 +/- 1.1, 11.4 +/- 0.7 and 4.7 +/- 0.6 nmol/mg protein in the 12-24 h pretreatment group (means +/- S.E.M.). For GSSG the corresponding values were 0.36 +/- 0.04, 0.34 +/- 0.03, 0.12 +/- 0.01, 0.12 +/- 0.01, 0.11 +/- 0.01 and 0.41 +/- 0.03 nmol/mg protein for the 1 h group and 0.36 +/- 0.04, 0.35 +/- 0.02, 0.15 +/- 0.01, 0.12 +/- 0.01, 0.11 +/- 0.01 and 0.34 +/- 0.02 nmol/mg protein for the 12-24 h group. Ischaemia and reperfusion had no effect on tissue MDA content or CuZn-SOD, GDP and GRD activity, and in general, PEG-SOD also lacked significant effect on any of these variables at any dose studied. However, Mn-SOD activity was severely reduced by ischaemia and reperfusion (from 42 +/- 7 U/mg protein in fresh aerobic controls to 6 +/- 1 U/mg protein at the end of reperfusion).(ABSTRACT TRUNCATED AT 400 WORDS)
J Mol Cell Cardiol 1992 Sep
PMID:PEG-SOD and myocardial antioxidant status during ischaemia and reperfusion: dose-response studies in the isolated blood perfused rabbit heart. 143 18

A hammerhead ribozyme designed against the mRNA coding for the Escherichia coli beta-glucuronidase (GUS) reporter enzyme was constructed. The synthetic ribozyme appeared able to correctly cleave in vitro the target RNA. This catalytic molecule was then assayed for in vivo activity in plant protoplasts. Plasmids coding either for the ribozyme or for the GUS target gene were cotransfected into the cells by the PEG-calcium procedure and GUS gene expression monitored following transient expression by measuring the intracellular GUS enzymatic activity. Expression of the ribozyme to high molar excess over the GUS transcript did not lead to any significant decrease of GUS activity in the transfected protoplasts. Insertion of the ribozyme sequence in the 3'-untranslated region of the GUS mRNA also had no detectable effect on GUS reporter gene expression whereas the corresponding RNA appeared able to self-cleave in vitro. These results indicate that the ability of ribozymes to perform catalytic cleavage of their substrate mRNA in vitro is essential but clearly not sufficient to ensure that efficient inhibition of the corresponding target gene will occur upon endogenous expression of this catalytic RNA in the plant cell.
Plant Mol Biol 1992 Nov
PMID:Assaying synthetic ribozymes in plants: high-level expression of a functional hammerhead structure fails to inhibit target gene activity in transiently transformed protoplasts. 145 Mar 86

Macrophage cytocidal activation requires the sequential impingement on the macrophage of a priming stimulus (interferon [IFN] alpha, beta, or gamma) and a triggering stimulus (such as polyinosinic acid:polycytidylic acid [poly [I:C]] or bacterial lipopolysaccharide). The mechanism of progression from the IFN-primed state to the cytocidal state is poorly understood. By quantifying the level of expression of a gene product (complement component factor B [Bf]) associated with cytocidal activation and through the use of phenotypically distinct populations of macrophages (unprimed and IFN-primed), we have investigated the functional necessity of changes in intracellular concentration of free calcium ions ([Ca2+]i) in signaling the transition from the primed to the cytocidal state. Elevating the [Ca2+]i by incubation of unprimed macrophages with the calcium ionophore, ionomycin, failed to induce the expression of Bf. By contrast, Bf was expressed at high levels when IFN-primed macrophages were exposed to ionomycin, suggesting that priming induced within the macrophages the capacity to respond to a nonspecific change in [Ca2+]i. Quantification of the [Ca2+]i in response to exposure to ionomycin revealed an initial transient elevation, followed by a secondary sustained component. No differences in these changes were observed between unprimed and IFN-primed macrophages. We therefore questioned if changes in [Ca2+]i were also implicated in the transition between the primed and the cytocidal state using the ligand, poly [I:C]. In contrast to ionomycin, incubation of IFN-primed macrophages with poly [I:C] did not sustain measurable increases in [Ca2+]i, yet fully stimulated the transition from the IFN primed to the cytocidal state. However, incubation of IFN-primed macrophages with poly [I:C] in the presence of 1) a Ca2+/ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid buffer calculated to clamp the extracellular concentration of free calcium ions to a value approximately equal to the resting [Ca2+]i; 2) the calcium channel blocker verapamil; or 3) the intracellular Ca2+ antagonists (W-7, W-13, and TMB-8) substantially inhibited the induction of Bf. Collectively, these data support the following conclusions. First, that changes in [Ca2+]i comprise an important element in the induction of progression from the IFN-primed to the cytocidal state. Second, the failure to detect global changes in [Ca2+]i in response to the ligand, poly [I:C], suggests that changes in [Ca2+]i or Ca2+ movement may occur in either a spatially restricted or in an asynchronous cyclical fashion and are not detected by population fluorescence measurements. Third, the source of the relevant Ca2+ is extracellular. Fourth, our findings suggest that priming influences macrophage functional responses at a locus that is distal to the changes in [Ca2+]i, thereby potentially allowing signaling processes to be utilized to initiate different cellular responses.
Mol Biol Cell 1992 Mar
PMID:Transmembrane-mediated changes in [Ca2+] are involved in the signaling pathway leading to macrophage cytocidal differentiation: implications of localized changes in intracellular [Ca2+] and of interferon priming on Ca2+ utilization. 162 33

The alcohol dehydrogenase (Adh) gene of Arabidopsis is expressed constitutively in immature seedlings and cells in suspension, and may be induced by hypoxic stress only in roots of mature plants. Deletions and G-box mutations of the Adh promoter were assayed in Arabidopsis protoplasts by PEG-mediated transient expression. Sequence domains necessary for full gene activity are confined to the 384 bp immediately 5' to the transcription start site, and deletion to -177 results in greater than 90% reduction in promoter activity. Site-specific mutations of G-box bases result in greater than 60% reduction in activity and disrupt G-box factor binding in vitro.
Plant Mol Biol 1992 Aug
PMID:Functional elements of the Arabidopsis Adh promoter include the G-box. 164 86

The Fab fragment of an antibody, made against an E2-specific feline infectious peritonitis virus neutralizing antibody, has been crystallized in a form suitable for X-ray diffraction analysis from PEG 4000 using vapor diffusion methods. The Fab fragment crystals diffract to about 2.9 A resolution and are of triclinic space group P1. Unit cell dimensions, by which the reciprocal lattice can be indexed, are a = 57.16 A, b = 70.85 A, c = 75.81 A, alpha = 85.11 degrees, beta = 121.28 degrees and gamma = 116.33 degrees. There are two Fab fragments comprising the asymmetric unit of the crystals. The presence of a pseudo-mirror plane in the diffraction pattern suggests the presence of at least an approximate dyad axis relating the two Fab fragments within the asymmetric unit.
J Mol Biol 1991 Dec 05
PMID:Characterization of crystals of an Fab fragment of a murine monoclonal antibody. 166 Sep 22

Two monoclonal IgG3 syngeneic anti-idiotypes are described which form soluble and insoluble complexes with anti-alpha(1----6)dextran hybridoma and myeloma proteins. Specific precipitation was seen when purified anti-alpha(1----6)dextrans were added to ascitic fluid containing IgG3 kappa anti-idiotype. Analysis of the supernatants of the idiotype-anti-idiotype precipitates demonstrated the presence of soluble complexes whose mobilities in polyacrylamide gels could, in some cases, be distinguished from that of free anti-idiotype. An IgG1 kappa anti-idiotype is described which did not form precipitates with anti-alpha(1----6)dextrans unless 3% PEG 6000 was added.
Mol Immunol 1990 Jun
PMID:An immunochemical analysis of precipitating and non-precipitating idiotype-anti-idiotype reactions. 169 51

Cell suspension-derived rice (Oryza sativa L.) protoplasts were transformed by direct gene uptake. PEG-mediated transformation was more efficient than electroporation. Plasmid DNA containing a hygromycin phosphotransferase (HPT) gene (which confers hygromycin resistance) driven by the CaMV 35S promoter and a beta-D-glucuronidase (GUS) gene under control of the 1', 2' double promoter of the mannopine synthase (mas) locus of Agrobacterium tumefaciens was introduced into rice protoplasts. Southern analysis of DNA from transformed cell lines showed that the HPT and GUS genes were present intact. Both genes were expressed in transgenic cell suspensions. GUS activity was detected by histochemical staining of the cells and by enzyme assays. During a 12-day culture period the proportion of stained cells rose to a maximum and then decreased again. Considerably higher numbers of blue-stained cells were obtained when the transgenic cell lines were grown in the presence of 5-azacytidine. Transcripts of the GUS gene could not be detected, in contrast with the HPT gene. Plantlets were regenerated from one transgenic cell line. GUS activity was found in both leaf and root tissues of these plants, particularly, but not exclusively, in vascular bundles. A mouse dihydrofolate reductase coding sequence (DHFR), conferring methotrexate resistance, fused to the CaMV 35S promotor and the wild-type nopaline synthase (NOS) gene of A. tumefaciens were also introduced into rice protoplasts. Stable integration of both genes was confirmed by Southern analysis. Expression of the DHFR gene was demonstrated by high levels of resistance to methotrexate of the transgenic cell suspensions and by the presence of DHFR transcripts. Expression of the NOS gene at enzyme or RNA level was not detected. Southern analysis suggests that this gene was probably either methylated or scrambled in these lines.
Plant Mol Biol 1991 May
PMID:Transgenic rice cell lines and plants: expression of transferred chimeric genes. 171 79

Avermectins are a family of potent broad-spectrum anthelmintic compounds, which bind with high affinity to membranes isolated from the free-living nematode Caenorhabditis elegans. Binding of avermectins is thought to modulate chloride channel activity, but the exact mechanism for anthelmintic activity remains to be determined. In this report, the properties of an avermectin-sensitive membrane current were evaluated in Xenopus laevis oocytes that were injected with poly(A)+ RNA from C. elegans. In such oocytes, avermectins increased inward membrane current at a holding potential of -80 mV. An avermectin analog without anthelmintic activity had no effect. Half-maximal activation of current was observed with 90 nM avermectin. The reversal potential for avermectin-sensitive current was -19.3 +/- 1.9 mV, and it shifted with external chloride, as expected for a chloride current. Avermectin increased membrane current in C. elegans-injected oocytes that were also injected with the Ca2+ chelator ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. The response to avermectin was greatest in the 1.0-2.5-kilobase class of size-fractionated C. elegans poly(A)+ RNA. Oocytes that responded to avermectin were insensitive to gamma-aminobutyric acid and the avermectin-induced current was blocked by picrotoxin.
Mol Pharmacol 1991 Sep
PMID:Avermectin-sensitive chloride currents induced by Caenorhabditis elegans RNA in Xenopus oocytes. 171 30

Polychlorinated hydrocarbons known to be nongenotoxic carcinogens were screened as activators of protein kinase C (PKC)-beta 1 either at high concentrations of Ca2+ or in the absence of Ca2+ (i.e., with 1 mM ethylene glycol-bis(beta-aminoethyl ether) N,N,N',N',-tetraacetic acid). Of those compounds tested, kepone and dicofol significantly stimulated PKC activity in the absence, but not the presence, of Ca2+. PKC activation was most pronounced in the presence of phosphatidylserine. Kepone and dicofol stimulated PKC activity 26% and 13%, respectively, as compared with the PKC activity (100%) stimulated by the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Northern blot analysis of expression of TPA-inducible genes by kepone showed slight expression of phorbin and ornithine decarboxylase in murine embryo fibroblasts. Future studies are required to determine the relevance of PKC activation by kepone and dicofol to the known carcinogenicity of these compounds.
Mol Carcinog 1991
PMID:Two polychlorinated hydrocarbons cause phospholipid-dependent protein kinase C activation in vitro in the absence of calcium. 172 72

The requirements for homologous recombination between plasmid DNA molecules have been studied using the PEG (polyethylene glycol)-mediated transformation system of maize (Zea mays L.) protoplasts coupled with the transient expression assay for beta-glucuronidase (GUS). Two plasmids were introduced into maize protoplasts; one plasmid (pB x 26) contained a genomic clone of the Adh1 maize gene; the other plasmid (piGUS) was a promoterless construction containing part of intron A of the Adh1 gene fused to the gusA coding sequence. Thus, the two vectors shared an effective homologous region consisting of a 459 bp (HindIII-PvuII) fragment of the Adh1 intron A sequence. An active gusA fusion gene would result upon homologous recombination between the plasmids within the intron A sequence, and indeed GUS activity was observed in extracts following co-transformation of maize protoplasts with the two plasmids. The presence of recombinant DNA molecules in protoplast DNA isolated 1 day after co-transformation was verified using polymerase chain reactions (PCR) and Southern blots. For efficient homologous recombination, both plasmids had to be linearized. The recombination reaction was induced by restriction of the plasmid molecules either inside the effective homologous region or at the borders of the intron sequence. However, the presence of even small, terminal, nonhomologous sequences at the 3' end of the pB x 26 fragment inhibited the recombination reaction. Also, both ends of the linearized piGUS DNA molecules were involved in the recombination reaction. The results revealed some features of homologous recombination reactions occurring in plant cells which cannot be accommodated by mechanisms postulated for similar reactions in animal system and in lower eukaryotes.
Mol Gen Genet 1991 Nov
PMID:Homologous recombination between plasmid DNA molecules in maize protoplasts. 174 30


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