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Query: UNIPROT:P06889 (Mol)
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CAR and C1, two carrot (Daucus carota L.) suspension cultures of different genotypes, were subjected to stepwise selection for tolerance to the herbicide glyphosate [(N-phosphonomethyl)glycine]. The specific activity of the target enzyme, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), as well as the mRNA level and copy number of the structural gene increased with each glyphosate selection step. Therefore, the tolerance to glyphosate is due to stepwise amplification of the EPSPS genes. During the amplification process, DNA rearrangement did not occur within the EPSPS gene of the CAR cell line but did occur during the selection step from 28 to 35 mM glyphosate for the C1 cell line, as determined by Southern hybridization of selected cell DNA following EcoRI restriction endonuclease digestion. Two cell lines derived from a previously selected glyphosate-tolerant cell line (PR), which also had undergone EPSPS gene amplification but have been maintained in glyphosate-free medium for 2 and 5 years, have lost 36 and 100% of the increased EPSPS activity, respectively. Southern blot analysis of these lines confirms that the amplified DNA is relatively stable in the absence of selection. These studies demonstrate that stepwise selection for glyphosate resistance reproducibly produces stepwise amplification of the EPSPS genes. The relative stability of this amplification indicates that the amplified genes are not extrachromosomal.
Mol Gen Genet 1992 Apr
PMID:Glyphosate selected amplification of the 5-enolpyruvylshikimate-3-phosphate synthase gene in cultured carrot cells. 137 13

Expression of the RNA replicase domain of tobacco mosaic virus (TMV) and certain protein-coding regions in other plant viruses, is mediated by translational readthrough of a leaky UAG stop codon. It has been proposed that normal tobacco tyrosine tRNAs are able to read the UAG codon of TMV by non-conventional base-pairing but recent findings that stop codons can also be bypassed as a result of extended translocational shifts (tRNA hopping) have encouraged a re-examination. In light of the alternatives, we investigated the sequences flanking the leaky UAG codon using an in vivo assay in which bypass of the stop codon is coupled to the transient expression of beta-glucuronidase (GUS) reporter genes in tobacco protoplasts. Analysis of GUS constructions in which codons flanking the stop were altered allowed definition of the minimal sequence required for read through as UAG-CAA-UUA. The effects of all possible single-base mutations in the codons flanking the stop indicated that 3' contexts of the form CAR-YYA confer leakiness and that the 3' context permits read through of UAA and UGA stop codons as well as UAG. Our studies demonstrate a major role for the 3' context in the read through process and do not support a model in which teh UAG is bypassed exclusively as a result of anticodon-codon interactions. No evidence for tRNA hopping was obtained. The 3' context apparently represents a unique sequence element that affects translation termination.
J Mol Biol 1991 Mar 20
PMID:The signal for a leaky UAG stop codon in several plant viruses includes the two downstream codons. 201 Sep 14

Human and animal cell cultures were evaluated for their susceptibility to two environmental toxins found as contaminants in human food supplies: aflatoxin B1, a hepatotoxin produced by the mold Aspergillus flavus, and saxitoxin, a paralytic neurotoxin produced by the marine dinoflagellate Gonyaulax catenella. Both toxins cause food poisoning in humans and other animals. The acute cytotoxicity of both toxins was measured and compared by inhibition of cell growth and by progressive cytopathogenicity resulting in cell destruction. Aflatoxin B1 was cytotoxic to all of the 11 primary kidney cultures derived from susceptible animals. The cell growth inhibition 10% values (TD10) ranged from 0.02 to 6.0 micrograms/ml: mouse (TD10 = 0.02 micrograms), guinea pig (0.03 micrograms), rat (0.07 micrograms), hamster (0.16 micrograms), monkey (0.1 microgram), human (0.7-1.5 micrograms), chick (0.05 micrograms), and duck (6.0 micrograms). The corresponding TD50 levels were about 10 times higher concentrations and caused cell destruction within 2 d. Saxitoxin did not induce cytotoxicity manifestations in cultures derived from susceptible species--mouse kidney, human carcinoma HeLa line, chick embryo, and goldfish fin (CAR) cell line--at high concentration levels up to 5 micrograms/ml. When the same toxin preparation at only 1 microgram was injected into mice, the animals died immediately. The results indicate that animal cell cultures are useful for studies of general cytotoxins that affect common essential metabolism but cannot be used to detect environmental toxins that cause toxic manifestations by an interference with specific physiological functions of organ systems.
Mol Toxicol
PMID:Comparative cytotoxicity of aflatoxin B1 and saxitoxin in cell cultures. 313 May 68

Alveolar macrophages (AM) exposed to cytokines or bacterial lipopolysaccharide (LPS) produce the free radical nitric oxide (NO.) by an inducible nitric oxide synthase (iNOS). They also release reactive oxygen free radicals following exposure to silica dust. The purpose of the present study was to determine whether NO. is produced by rat AM and/or recruited leukocytes following the intratracheal (IT) instillation of silica. Male Sprague-Dawley rats (175 to 225 g) were IT instilled with either silica dust (10 mg/100 g body wt) or LPS (0.25 mg/100 g body wt). After 24 h, bronchoalveolar lavage cells (BALC) and lavaged lung tissue were assayed for iNOS mRNA. Cell counts of BALC and iNOS-dependent (N omega-nitro-L-arginine methyl ester [L-NAME]-inhibitable) chemiluminescence generated by AM were also determined. Northern blot analysis demonstrated that the steady-state levels of BALC iNOS mRNA were significantly increased by 3-fold following IT silica and by 7-fold following IT LPS. Partially enriched fractions of either AM or leukocytes from silica-treated rats both exhibited significantly elevated iNOS mRNA in Northern analysis. iNOS-dependent chemiluminescence was significantly increased in AM by 36-fold following IT silica and by 89-fold following IT LPS. Differential counts of BALC showed that AM numbers did not change in any of the treatments; however, red blood cells increased by 30-fold following IT silica and by 23-fold following IT LPS. Total leukocytes (polymorphonuclear leukocytes plus lymphocytes) increased by 58-fold following IT silica and by 274-fold following IT LPS.(ABSTRACT TRUNCATED AT 250 WORDS)
Am J Respir Cell Mol Biol 1994 Oct
PMID:Intratracheal instillation of silica up-regulates inducible nitric oxide synthase gene expression and increases nitric oxide production in alveolar macrophages and neutrophils. 752 85

We compared inhibitory nonadrenergic noncholinergic (i-NANC) neural relaxations, evoked by electrical field stimulation (EFS), at three levels (main [MA], proximal [PA], and distal [DA] airways) of isolated human airways and correlated these with nitric oxide synthase-immunoreactive (NOS-IR) nerves, using antiserum raised to rat cerebellar NOS. Maximal relaxations to papaverine (100 microM) were reduced in PA and DA (MA: 1,712 +/- 219 mg, n = 12; DA: 862 +/- 69 mg, n = 5, P < 0.05 versus MA); hence, subsequent relaxations were expressed as a percentage of the papaverine maximum. EFS elicited frequency-dependent relaxations that were largest in MA and reduced in PA and DA, especially at high stimulation frequencies (10 Hz EFS: MA: 51.6 +/- 3.7%, n = 12; PA: 30.5 +/- 6.0%, n = 6, P < 0.01 versus MA; DA: 17.8 +/- 3.6%, n = 5, P < 0.001 versus MA). The NOS inhibitor L-NG-nitroarginine methyl ester (L-NAME) (100 microM) and tetrodotoxin (3 microM) significantly inhibited i-NANC responses at all frequencies, leaving an L-NAME-resistant non-neural relaxation at frequencies > 5 Hz which was reduced in PA and DA. Cumulative concentration-response studies to sodium nitroprusside (1 nM to 0.1 mM) and the NO donor 3-morpholinosydnonimine (1 nM to 1 mM) were not significantly different in PA and DA, suggesting impaired relaxation is not caused by impaired guanylyl cyclase activity. Total nerve density, shown by protein gene product 9.5 staining, was not significantly different in PA and DA; however, NOS-IR nerve density was reduced in PA and DA (NOS-IR [intercepts/mm2]: MA: 705 +/- 98, n = 6; DA: 284 +/- 32, n = 6, P < 0.01 versus MA). These studies demonstrate that i NANC neural relaxations are reduced in DA, apparently due to a decrease in the density of nitrergic innervation.
Am J Respir Cell Mol Biol 1995 Aug
PMID:Distribution of human i-NANC bronchodilator and nitric oxide-immunoreactive nerves. 754 97

We studied the effect of Saiboku-to (TJ-96), an anti-allergic herbal medicine, on transepithelial potential difference of rabbit trachea and possible involvement of nitric oxide (NO) generation in vivo. Perfusion of TJ-96 on the tracheal mucosal surface increased PD in a concentration-dependent manner, the maximal increase from the baseline level and the concentration of TJ-96 required to produce a half-maximal effect (EC50) being 8.1 +/- 1.4 mV (mean +/- SE, P < 0.001) and 47 micrograms/ml. This effect was abolished by pretreatment with the Na channel blocker amiloride. NG-nitro-L-arginine methylester (L-NAME) but not NG-nitro-D-arginine methylester (D-NAME) inhibited TJ-96-induced increase in PD, and this inhibition was selectively reversed by L-arginine. These results suggest that TJ-96 stimulates Na absorption by airway epithelial cells probably through NO generation.
Res Commun Mol Pathol Pharmacol 1995 Apr
PMID:Effect of TJ-96, an anti-allergic herbal medicine, on tracheal transepithelial potential difference in vivo. 762 Aug 37

Respiratory syncytial virus (RSV) is an important respiratory pathogen that preferentially infects epithelial cells in the airway, and causes a local inflammatory response. Although it has been previously demonstrated that RSV-infected airway epithelial produce cytokines, including interleukin-8 (IL-8), which contributes to the inflammatory response, the regulation of this effect of RSV is unknown. To further characterize the mechanisms by which RSV infection triggers release of IL-8, we first exposed cultured A549 cells to RSV, and measured IL-8 release via enzyme-linked immunosorbent assays (ELISA), and IL-8 messenger RNA (mRNA) induction via Northern blot analysis. We observed a dose- and time-dependent release of IL-8 in response to RSV. The optimal dose of RSV was 10(4) TCID50/ml, and maximal release of IL-8 was measured at 72 to 96 h after infection. RSV induced a biphasic (early and late) increase in IL-8 mRNA. The early phase was independent of viral infection, whereas the more pronounced late phase required the presence of live virus and infection of the epithelium. Partial (< 50%) cytopathic effects were noted at 48 h and progressed to 75% at 96 h. The monolayer was still intact at 96 h. Inhibitors of nitric oxide, including NG-monomethyl-L-arginine (L-NMMA), NG-nitro-L-arginine methyl ester (L-NAME), and aminoguanidine had no effect on IL-8 release or IL-8 mRNA induction. We did, however, demonstrate a dose-dependent decrease in IL-8 release and IL-8 mRNA induction in RSV-infected epithelial treated with the antioxidants dimethyl sulfoxide (DMSO) or 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). Peak effects were noted at a concentration of 2% DMSO and 50 microM DMPO. The antioxidants did not inhibit viral replication or infection. This data suggest that RSV-induced IL-8 production in airway epithelium is mediated via changes in oxidant tone. The data also suggest a potential therapeutic role for antioxidants in RSV infections.
Am J Respir Cell Mol Biol 1995 Aug
PMID:Oxidant tone regulates IL-8 production in epithelium infected with respiratory syncytial virus. 762 91

The aim of these studies was to investigate the mechanism underlying the haemodynamic changes associated with brain death. The initial series of studies were to assess whether these changes involved some blood-borne factor. When control rats (n = 6) were exsanguinated whilst being simultaneously transfused with blood from rats that had been brain-dead for 60 min, their haemodynamic function did not deteriorate. Likewise, when brain-dead rats (n = 6) were exsanguinated and transfused with blood from control rats there was no improvement of haemodynamic function. The absence of any blood-borne factor was further confirmed in studies in which isolated hearts from control rats were perfused with blood from a support rat which had been brain-dead for 15 min (n = 6/group). The brain-death-induced haemodynamic changes in the support rat (mean arterial pressure increased from 98 +/- 6 to 176 +/- 9 mm Hg after 30 s and then fell to 44 +/- 5 mm Hg after 5 min) were not associated with changes in cardiac function of the perfused heart (left ventricular developed pressure was 146 +/- 4 mmHg before the induction of brain death and 147 +/- 4 and 151 +/- 7 mm Hg at 30 s and 5 min, respectively, after the induction). In further in vivo studies, we assessed the involvement of the autonomic nervous system in brain-death-induced haemodynamic instability. We achieved this by employing beta-adrenoreceptor blockade or bilateral vagotomy (n = 6/group); propranolol (1 mg/kg given as a bolus 6 min before brain death followed by 0.5 mg/kg/h continuous infusion) abolished the early transient tachycardia and positive inotropic response to brain death but did not alter the subsequent deterioration of function (mean arterial pressure fell from 75 +/- 7 mmHg before brain death to 49 +/- 5 mmHg after 30 min). Bilateral vagotomy had no effect on the functional changes induced by brain death. The effect of catecholamine depletion was then investigated; 6-hydroxydopamine (given over 15 days) depleted myocardial norepinephrine content by approximately 90% (from 2.3 +/- 0.1 to 0.3 +/- 0.1 nmol/g wet wt; P < 0.05). Depletion of cardiac catecholamines reduced brain-death-induced mortality to zero but did not affect cardiac dysfunction. Finally, we used L-NAME and naloxone in an attempt to identify roles for nitric oxide and endogenous opioid peptides but were again unable to influence the cardiac events. In conclusion, the initial transient hyperdynamic response induced by brain death appears to be mediated through cardiac innervation and can be inhibited by beta-adrenoreceptor blockade. However, the autonomic nervous system, nitric oxide, endogenous opioid peptides and blood-borne factors do not appear to be involved in the subsequent deterioration of cardiac function.
J Mol Cell Cardiol 1994 Apr
PMID:Brain-death-induced cardiac contractile dysfunction: studies of possible neurohormonal and blood-borne mediators. 791 33

Previous studies proposed the involvement of the N-methyl-D-aspartate (NMDA) type of glutamate receptors in the development of sensitization to the convulsive effect of cocaine (cocaine kindling). The present study was undertaken to determine, first, if cocaine kindling is associated with enhanced sensitivity of the NMDA receptor to the convulsive response of N-methyl-D,L-aspartate (NMDLA), and second, whether in vivo modulation of nitric oxide synthase (NOS) function regulates the development of cocaine kindling. The following results were observed: 1. Cocaine-kindled animals were significantly more susceptible to the convulsive effect of the NMDA receptor agonist NMDLA than saline controls; 2. Pretreatment with the NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME; 100 mg/kg; ip) blocked the development of cocaine kindling; 3. The protective effect of L-NAME was partially reversed with the coadministration of the NOS substrate, L-arginine (300 mg/kg; ip), but not D-arginine; and 4. L-Arginine (300 mg/kg; ip), but not D-arginine, amplified the development of cocaine kindling. Taken together, these findings suggest that supersensitivity of the NMDA receptor and activation of NOS may underlie the development of cocaine kindling.
Mol Neurobiol
PMID:Cocaine kindling in mice. Responses to N-methyl-D,L-aspartate (NMDLA) and L-arginine. 856 64

Recent reports suggest that endothelial-dependent relaxant factor, recognized as nitric oxide (NO), reduces myocardial contractility. Here, we showed that both exposures to acetylcholine and bradykinin for 30 min increased cyclic guanylate monophosphate (cyclic GMP) in isolated rat cardiomyocytes. These increases in cyclic GMP were blunted by NW-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NO synthase. Hypoxia augmented the cyclic GMP accumulation due to exposures to acetylcholine and bradykinin, which were blunted by L-NAME. The increases in cyclic GMP due to acetylcholine and bradykinin during normoxic and hypoxic conditions were not blunted by aminoguanidine, an inhibitor of inducible NO synthase. These findings revealed that NO is produced in cardiomyocytes due to stimulation of NO synthase and modulates their own guanylate cyclase, which was augmented by hypoxia. NO production, through NO synthase in cardiomyocytes, may constitute autocrine regulations of myocardial contractility and paracrine regulations of coronary vasodilation and platelet aggregation.
J Mol Cell Cardiol 1995 Oct
PMID:Evidence for nitric oxide generation in the cardiomyocytes: its augmentation by hypoxia. 857 31


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