Pokeweed antiviral protein cleaves double-stranded supercoiled DNA using the same active site required to depurinate rRNA.
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Ribosome-inactivating proteins (RIPs) are N-glycosylases that remove a specific adenine from the sarcin/ricin loop of the large rRNA in a manner analogous to N-glycosylases that are involved in DNA repair. Some RIPs have been reported to remove adenines from single-stranded DNA and cleave double-stranded supercoiled DNA. The molecular basis for the activity of RIPs on double-stranded DNA is not known. Pokeweed antiviral protein (PAP), a single-chain RIP from Phytolacca americana, cleaves supercoiled DNA into relaxed and linear forms. Double-stranded DNA treated with PAP contains apurinic/apyrimidinic (AP) sites due to the removal of adenine. Using an active-site mutant of PAP (PAPx) which does not depurinate rRNA, we present evidence that double-stranded DNA treated with PAPx does not contain AP sites and is not cleaved. These results demonstrate for the first time that PAP cleaves supercoiled double-stranded DNA using the same active site that is required for depurination of rRNA. (+info)
Changes in electrocortical power and coherence in response to the selective cholinergic immunotoxin 192 IgG-saporin.
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Changes in brain electrical activity in response to cholinergic agonists, antagonists, or excitotoxic lesions of the basal forebrain may not be reflective entirely of changes in cholinergic tone, in so far as these interventions also involve noncholinergic neurons. We examined electrocortical activity in rats following bilateral intracerebroventricular administration of 192 IgG-saporin (1.8 microg/ventricle), a selective cholinergic immunotoxin directed to the low-affinity nerve growth factor receptor p75. The immunotoxin resulted in extensive loss of choline acetyl transferase (ChAT) activity in neocortex (80%-84%) and hippocampus (93%), with relative sparing of entorhinal-piriform cortex (42%) and amygdala (28%). Electrocortical activity demonstrated modest increases in 1- to 4-Hz power, decreases in 20- to 44-Hz power, and decreases in 4- to 8-Hz intra- and interhemispheric coherence. Rhythmic slow activity (RSA) occurred robustly in toxin-treated animals during voluntary movement and in response to physostigmine, with no significant differences seen in power and peak frequency in comparison with controls. Physostigmine significantly increased intrahemispheric coherence in lesioned and intact animals, with minor increases seen in interhemispheric coherence. Our study suggests that: (1) electrocortical changes in response to selective cholinergic deafferentation are more modest than those previously reported following excitotoxic lesions; (2) changes in cholinergic tone affect primarily brain electrical transmission within, in contrast to between hemispheres; and (3) a substantial cholinergic reserve remains following administration of 192 IgG-saporin, despite dramatic losses of ChAT in cortex and hippocampus. Persistence of a cholinergically modulated RSA suggests that such activity may be mediated through cholinergic neurons which, because they lack the p75 receptor, remain unaffected by the immunotoxin. (+info)
Redundant basal forebrain modulation in taste aversion memory formation.
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Mnemonic deficits resulting from excitotoxic lesion of the basal forebrain have been classically attributed to the resulting depletion of cortical acetylcholine activity. It has been demonstrated that in spite of the strong cholinergic depletion after injections into the basal forebrain of the immunotoxin 192IgG-saporin, no detectable deficit can be found in the acquisition of several learning tasks, including conditioned taste aversion. Conversely, NMDA-induced lesions of the basal forebrain strongly impair taste aversion learning. In this study we show that 192IgG-saporin produces an efficient and selective cholinergic deafferentation of the rat neocortex but not the amygdala. Furthermore, a stronger relationship between severity of memory impairment after NMDA lesions and basoamygdaloid cholinergic deafferentation was found. Therefore, in a second experiment, we show that combining NMDA-induced lesions into the basolateral amygdala with 192IgG-saporin injections into the basal forebrain results in a strong disruption of taste aversion learning, whereas none of these treatments were by themselves capable of producing any detectable impairment in this learning task. The double lesion effect was only paralleled by simple NMDA lesions into the basal forebrain, suggesting that the learning deficits associated to excitotoxic lesions of the basal forebrain are the result of the simultaneous destruction of the corticopetal and basoamygdaloid interaction. A model is proposed, according to which the modulation of learning processes exerted by the basal forebrain can be redundantly performed by both the basocortical and basoamygdaloid pathway. (+info)
Expression of mature pokeweed antiviral protein with or without C-terminal extrapeptide in Escherichia coli as a fusion with maltose-binding protein.
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Genomic clones encoding the mature pokeweed antiviral protein with or without C-terminal extrapeptide (PAPMC and PAPM), which have been reported to be highly toxic to E. coli cells, were inserted into the expression vector pMAL-p2. The recombinant PAPs (rPAPMC and rPAPM) were successfully expressed in E. coli at 25 degrees C, being exported to the periplasm as soluble fusions with maltose-binding protein (MBP). The rPAPs were cleaved from MBP by treatment with factor Xa and subsequently purified with final yields of 4.0 mg/liter (rPAPMC) and 5.5 mg/liter (rPAPM). rPAPM was resistant to protease digestion, but the C-terminal extrapeptide appeared to be susceptible and was partially digested by some protease in E. coli. Both rPAPMC and rPAPM were as active as the native PAPM from pokeweed leaves in depurinating rat liver and E. coli ribosomes, while the activities of rPAPMC on both ribosomes were decreased at least 60-fold by fusion with MBP. (+info)
X-ray crystallographic analysis of the structural basis for the interactions of pokeweed antiviral protein with its active site inhibitor and ribosomal RNA substrate analogs.
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The pokeweed antiviral protein (PAP) belongs to a family of ribosome-inactivating proteins (RIP), which depurinate ribosomal RNA through their site-specific N-glycosidase activity. We report low temperature, three-dimensional structures of PAP co-crystallized with adenyl-guanosine (ApG) and adenyl-cytosine-cytosine (ApCpC). Crystal structures of 2.0-2.1 A resolution revealed that both ApG or ApCpC nucleotides are cleaved by PAP, leaving only the adenine base clearly visible in the active site pocket of PAP. ApCpC does not resemble any known natural substrate for any ribosome-inactivating proteins and its cleavage by PAP provides unprecedented evidence for a broad spectrum N-glycosidase activity of PAP toward adenine-containing single stranded RNA. We also report the analysis of a 2.1 A crystal structure of PAP complexed with the RIP inhibitor pteoric acid. The pterin ring is strongly bound in the active site, forming four hydrogen bonds with active site residues and one hydrogen bond with the coordinated water molecule. The second 180 degrees rotation conformation of pterin ring can form only three hydrogen bonds in the active site and is less energetically favorable. The benzoate moiety is parallel to the protein surface of PAP and forms only one hydrogen bond with the guanido group of Arg135. (+info)
Lesions of the C1 catecholaminergic neurons of the ventrolateral medulla in rats using anti-DbetaH-saporin.
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Phenylethanolamine-N-methyltransferase (PNMT)-containing neurons in the rostral ventrolateral medulla (RVLM) are believed to play a role in cardiovascular regulation. To determine whether injection of anti-dopamine beta-hydroxylase (DbetaH)-saporin directly into the RVLM in rats could selectively destroy these cells and thereby provide an approach for evaluating their role in cardiovascular regulation, we studied rats 2 wk after unilateral injection of 21 ng anti-DbetaH-saporin into the RVLM. There was an approximately 90% reduction in the number of PNMT-positive neurons in the RVLM, although the number of non-C1, spinally projecting barosensitive neurons of this area was not altered. The A5 cell group was the only other population of DbetaH-containing cells that was significantly depleted. The depressor response evoked by injection of tyramine into the RVLM was abolished by prior injection of toxin. The pressor response evoked by injection of glutamate into the RVLM was attenuated ipsilateral to the toxin injection but was potentiated contralateral to the toxin injection. Thus anti-DbetaH-saporin can be used to make selective lesions of PNMT-containing cells, allowing for the evaluation of their role in cardiovascular regulation. (+info)
Cognitive changes and modified processing of amyloid precursor protein in the cortical and hippocampal system after cholinergic synapse loss and muscarinic receptor activation.
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A number of in vitro studies have shown that activation of muscarinic receptors by cholinergic agonists stimulates the nonamyloidogenic, alpha-secretase-processing pathway of amyloid precursor protein (APP). To determine whether increased cholinergic neurotransmission can modify the APP processing in vivo, we administered a muscarinic receptor agonist (RS86) to normal or aged rats and rats with severe basal forebrain cholinergic deficits (induced by 192 IgG-saporin). The levels of the cell-associated APP in neocortex, hippocampus, and striatum, as well as the secreted form of APP (APPs) in cerebrospinal fluid, were examined by Western blots. Additionally, we investigated the association between the altered APP levels and behavioral deficits caused by cholinergic lesions. We found that treatment with muscarinic receptor agonist resulted in decreased APP levels in neocortex and hippocampus and increased levels of APPs in cerebrospinal fluid. Regulation of APP processing by the muscarinic agonist treatment occurred not only in normal rats, but also in aged and cholinergic denervated rats that model this aspect of Alzheimer's disease. Interestingly, we found that elevation of APP in neocortex correlated with the cognitive deficits in water-maze testing of rats with cholinergic dysfunction. These data indicate that increased cholinergic neurotransmission can enhance nonamyloidogenic APP processing in intact and lesioned rats and that APP may be involved in cognitive performance. (+info)
Shock wave permeabilization with ribosome inactivating proteins: a new approach to tumor therapy.
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Extracorporeal shock waves are high-pressure pulses of microsecond duration clinically used for lithotripsy. Recently, shock waves been shown to cause a transient increase of the permeability of the cell membrane. We therefore hypothesized that shock waves might be able to transfer tumoricidal agents into tumor cells and examined this in vitro and in vivo. In vitro, the ribosome inactivating proteins gelonin and saporin were transferred into L1210, SSK2, and HeLa cells, and dose-response curves were established. The drug concentration that reduced the cell proliferation by 50% (IC50) was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and the enhancement factors from shock wave application were calculated. It was found that shock waves enhanced the action of gelonin from 900-fold in L1210 cells to 40,000-fold in HeLa cells and the action of saporin from 300-fold in L1210 cells to 15,000-fold in HeLa cells. In vivo, the effect of gelonin and saporin was assessed in a murine tumor model. SSK2 fibrosarcoma tumors locally grown in C3H mice were treated with shock waves after i.p. administration of gelonin or saporin. Shock wave application delayed the tumor growth, and long-term remissions lasting >180 days were induced in 40% of the animals. In conclusion, shock waves enhanced the action of ribosome inactivating proteins and led to complete tumor remissions. The local transfer of toxic substances by shock waves into tumors constitutes a new approach to a local tumor therapy. (+info)