scholarly journals CRISPR/Cas9-Mediated Genome Editing in Comfrey (Symphytum officinale) Hairy Roots Results in the Complete Eradication of Pyrrolizidine Alkaloids

Molecules ◽  
2021 ◽  
Vol 26 (6) ◽  
pp. 1498
Author(s):  
Mahmoud M. Zakaria ◽  
Brigitte Schemmerling ◽  
Dietrich Ober
Keyword(s):  
Medicinal Plant ◽  
Pyrrolizidine Alkaloids ◽  
Gene Encoding ◽  
Pharmaceutical Application ◽  
Symphytum Officinale ◽  
In Vitro Systems ◽  
Target Effect ◽  
Homospermidine Synthase ◽  
Potential Use

Comfrey (Symphytum officinale) is a medicinal plant with anti-inflammatory, analgesic, and proliferative properties. However, its pharmaceutical application is hampered by the co-occurrence of toxic pyrrolizidine alkaloids (PAs) in its tissues. Using a CRISPR/Cas9-based approach, we introduced detrimental mutations into the hss gene encoding homospermidine synthase (HSS), the first pathway-specific enzyme of PA biosynthesis. The resulting hairy root (HR) lines were analyzed for the type of gene-editing effect that they exhibited and for their homospermidine and PA content. Inactivation of only one of the two hss alleles resulted in HRs with significantly reduced levels of homospermidine and PAs, whereas no alkaloids were detectable in HRs with two inactivated hss alleles. PAs were detectable once again after the HSS-deficient HRs were fed homospermidine confirming that the inability of these roots to produce PAs was only attributable to the inactivated HSS and not to any unidentified off-target effect of the CRISPR/Cas9 approach. Further analyses showed that PA-free HRs possessed, at least in traces, detectable amounts of homospermidine, and that the PA patterns of manipulated HRs were different from those of control lines. These observations are discussed with regard to the potential use of such a CRISPR/Cas9-mediated approach for the economical exploitation of in vitro systems in a medicinal plant and for further studies of PA biosynthesis in non-model plants.

The leucyl aminopeptidase from Helicobacter pylori is an allosteric enzyme

Microbiology ◽  
2005 ◽  
Vol 151 (6) ◽  
pp. 2017-2023 ◽  
Author(s):  
Lei Dong ◽  
Ni Cheng ◽  
Ming-Wei Wang ◽  
Junfeng Zhang ◽  
Chang Shu ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Biochemical Characterization ◽  
Gene Encoding ◽  
Allosteric Enzyme ◽  
H Pylori ◽  
Potential Use ◽  
Leucyl Aminopeptidase ◽  
Cobalt And Nickel

This study describes the cloning, genetic analysis and biochemical characterization of a leucyl aminopeptidase (LAP) from Helicobacter pylori. A gene encoding LAP was cloned from H. pylori and the expressed 55 kDa protein displayed homology to aminopeptidases from Gram-negative bacteria, plants and mammals. This LAP demonstrated amidolytic activity against l-leucine-p-nitroanilide. Optimal activity was observed at pH 8·0 and 45 °C, with V max of 232 μmol min−1 (mg protein)−1 and S 0·5 of 0·65 mM. The data suggest that LAP could be allosteric (n H=2·27), with regulatory homohexamers, and its activity was inhibited by ion chelators and enhanced by divalent manganese, cobalt and nickel cations. Bestatin inhibited both LAP activity (IC50=49·9 nM) and H. pylori growth in vitro. The results point to the potential use of LAP as a drug target to develop novel anti-H. pylori agents.

scholarly journals Reduction of Pyrrolizidine Alkaloid Levels in Comfrey (Symphytum officinale) Hairy Roots by RNAi Silencing of Homospermidine Synthase

Planta Medica ◽  
2019 ◽  
Vol 85 (14/15) ◽  
pp. 1177-1186 ◽  
Author(s):  
Lars H. Kruse ◽  
Thomas Stegemann ◽  
Julia Jensen-Kroll ◽  
Annika Engelhardt ◽  
Anne-Maria Wesseling ◽  
...  
Keyword(s):  
Hairy Roots ◽  
Pyrrolizidine Alkaloid ◽  
Primary Metabolism ◽  
In Planta ◽  
Deoxyhypusine Synthase ◽  
Gene Encoding ◽  
Symphytum Officinale ◽  
Medicinal Plant Extracts ◽  
Cost Efficient ◽  
Homospermidine Synthase

AbstractComfrey is a medicinal plant, extracts of which are traditionally used for the treatment of painful inflammatory muscle and joint problems, because the plant contains allantoin and rosmarinic acid. However, its medicinal use is limited because of its toxic pyrrolizidine alkaloid (PA) content. PAs encompass more than 400 different compounds that have been identified from various plant lineages. To date, only the first pathway-specific enzyme, homospermidine synthase (HSS), has been characterized. HSS catalyzes the formation of homospermidine, which is exclusively incorporated into PAs. HSS has been recruited several times independently in various plant lineages during evolution by duplication of the gene encoding deoxyhypusine synthase (DHS), an enzyme of primary metabolism. Here, we describe the establishment of RNAi knockdown hairy root mutants of HSS in Symphytum officinale. A knockdown of HSS by 60 – 80% resulted in a significant reduction of homospermidine by ~ 86% and of the major PA components 7-acetylintermedine N-oxide and 3-acetylmyoscorpine N-oxide by approximately 60%. The correlation of reduced transcript levels of HSS with reduced levels of homospermidine and PAs provides in planta support for HSS being the central enzyme in PA biosynthesis. Furthermore, the generation of PA-depleted hairy roots might be a cost-efficient way for reducing toxic by-products that limit the medicinal applicability of S. officinale extracts.

Dental optical coherence tomography: a comparison of two in vitro systems

1999 ◽  
Vol 29 (2) ◽  
pp. 85-89 ◽  
Author(s):  
L L Otis ◽  
B W Colston ◽  
M J Everett ◽  
H Nathel
Keyword(s):  
Optical Coherence Tomography ◽  
Optical Coherence ◽  
In Vitro Systems

Rhenium-188 Labeled Hydroxyapatite and Rhenium-188 Sulfur Colloid

1997 ◽  
Vol 36 (02) ◽  
pp. 71-75 ◽  
Author(s):  
S. Glatz ◽  
S. N. Reske ◽  
K. G. Grillenberger
Keyword(s):  
Synovial Fluid ◽  
Ph Value ◽  
Surgical Removal ◽  
Radiation Synovectomy ◽  
Sulfur Colloid ◽  
Target Organs ◽  
Aseptic Conditions ◽  
In Vitro Stability ◽  
Potential Use

Summary Aim: One therapeutic approach to rheumatoid arthritis and other inflammatory arthropathies besides surgical removal of inflamed synovium is radiation synovectomy using beta-emitting radionuclides to destroy the affected synovial tissue. Up to now the major problem associated with the use of labeled particles or colloids has been considerable leakage of radionuclides from the injected joint coupled with high radiation doses to liver and other non target organs. In this study we compared 188Re labeled hydroxyapatite particles and 188Re rhenium sulfur colloid for their potential use in radiation synovectomy. Methods: To this end we varied the labeling conditions (concentrations, pH-value, heating procedure) and analyzed the labeling yield, radiochemical purity, and in vitro stability of the resulting radiopharmaceutical. Results: After optimizing labeling conditions we achieved a labeling yield of more than 80% for 188Re hydroxyapatite and more than 90% for the rhenium sulfur colloid. Both of the radiopharmaceuticals can be prepared under aseptic conditions using an autoclav for heating without loss of activity. In vitro stability studies using various challenge solutions (water, normal saline, diluted synovial fluid) showed that 188Re labeled hydroxyapatite particles lost about 80% of their activity within 5 d in synovial fluid. Rhenium sulfur colloid on the other hand proved to be very stable with a remaining activity of more than 93% after 5 d in diluted synovial fluid. Conclusion: These in vitro results suggest that 188Re labeled rhenium sulfur colloid expects to be more suitable for therapeutic use in radiation synovectomy than the labeled hydroxyapatite particles.

Sign in / Sign up

Export Citation Format

Share Document