SUMMARY The grey mould Botrytis cinerea is an economically important plant pathogen. Previously we found that null mutants of bcg1 encoding one of the two Galpha subunits of heterotrimeric GTP-binding proteins differed in colony morphology and showed reduced pathogenicity. To further understand the mechanisms involved in infection, we cloned the bac gene encoding adenylate cyclase, the enzyme that catalyses production of cAMP from ATP. The deduced protein sequence consists of 2300 amino acids, the ORF is interrupted by three conserved introns, and there is a high degree of similarity with the catalytic domains of other fungal adenylate cyclases. Gene replacement resulted in reduced vegetative growth and a morphology similar to that of bcg1 mutants. The wild-type (WT) colony morphology was partially restored by feeding exogenous cAMP. These bac mutants still had a low but constant level of cAMP, despite deletion of the complete catalytic domain of the enzyme. Conidia from bac mutants germinated, penetrated the leaves of Phaseolus vulgaris and caused spreading soft rot lesions (in contrast to bcg1 mutants), although these were slower to develop than in WT controls. Compared to the latter, the most striking difference was that no sporulation occurred on leaves inoculated with bac mutant conidia. These results confirm that the cAMP signalling pathway plays an important role in vegetative growth and pathogenicity in B. cinerea. On the other hand, a much stronger effect of bcg1 mutation on pathogenicity in comparison to the effects of bac mutations suggests that BCG1 controls at least one more signalling component other than adenylate cyclase, and that the cAMP signalling pathway is not the only one responsible for pathogenicity.
Peptide nucleic acids (PNAs) are DNA analogs that hybridize to complementary nucleic sequences with high affinity and stability. In our previous work, we showed that a PNA complementary to a 12-base pair (bp) sequence of the coding region of the rat neurotensin receptor (rNTR1) mRNA is effective in significantly blocking a rat's central responses to neurotensin (NT), even when the PNA is injected intraperitoneally (i.p.). Using a novel gel shift detection assay to detect PNA, we have now used this same PNA sequence to derive its pharmacokinetic variables and its tissue distribution in the rat. The PNA has a distribution half-life of 3 +/- 3 minutes and an elimination half-life of 17 +/- 3 minutes. The total plasma clearance and volume of distribution of this PNA were 3.4 +/- 0.9 ml/min x kg and 60 +/- 30 ml/kg. Two hours after dosing, the PNA was found at detectable but low levels in all organs examined-in order of decreasing concentration: kidney, liver, heart, brain, and spleen. Approximately 90% of the PNA dose was recovered as unchanged parent compound in the urine 24 hours after administration.
Intraperitoneal injection of an unmodified antisense peptide nucleic acid (PNA) complementary to mRNA of the rat neurotensin (NT) receptor (NTR1) was demonstrated by a gel shift assay to be present in brain, thus indicating that the PNA had in fact crossed the blood-brain barrier. An i.p. injection of this antisense PNA specifically inhibited the hypothermic and antinociceptive activities of NT microinjected into brain. These results were associated with a reduction in binding sites for NT both in brain and the small intestine. Additionally, the sense-NTR1 PNA, targeted to DNA, microinjected directly into the brain specifically reduced mRNA levels by 50% and caused a loss of response to NT. To demonstrate the specificity of changes in behavioral, binding, and mRNA studies, animals treated with NTR1 PNA were tested for behavioral responses to morphine and their mu receptor levels were determined. Both were found to be unaffected in these NTR1 PNA-treated animals. The effects of both the antisense and sense PNAs were completely reversible. This work provides evidence that any antisense strategy targeted to brain proteins can work through i.p. delivery by crossing the normal blood-brain barrier. Equally important was that an antigene strategy, the sense PNA, was shown in vivo to be a potentially effective therapeutic treatment.Peptide nucleic acids (PNAs), a new type of DNA analog ( Fig. 1), hold great promise as antisense or antigene drugs, because they are electrically neutral oligomers that are stable against nucleases and proteases, bind independently of salt concentration to their complementary nucleic acids, and have higher affinity for nucleic acids than do DNA͞DNA duplexes (1, 2). Additionally, PNA͞DNA duplexes are much more gene specific, because they are less tolerant of mismatches than are DNA͞DNA duplexes (3). Initial enthusiasm for their use as antisense or antigene drugs was dampened by the fact that these molecules pass poorly into cells (4, 5). Our laboratory reported that unmodified (carrier-free) PNAs, on their direct injection into rat brain, enter neuronal cells and inhibit protein synthesis in a gene-specific manner (6).To determine both the mechanism of action of PNAs and whether PNAs could pass the blood-brain barrier (BBB), brain neurotensin (NT) receptors (NTR1) again were targeted. After a single i.p. injection of antisense PNA to NTR1 (targeted to mRNA) behavioral and physiological responses to NT (antinociception and hypothermia) were specifically and almost completely lost. These results were accompanied by specific reductions in receptor sites as determined by radioligand binding assays. However, there were no changes in mRNA levels. A sensitive assay developed to detect the amount of PNAs in tissue (gel shift assay) confirmed the presence of PNA in brain after i.p. injection. Therefore, these results provided evidence that any antisense strategy targeted to brain proteins can work by i.p. delivery and by crossing the normal (i.e., not compromised by malignancy) BBB. Also, of ...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.