दीमक प्रोटोजोअन आंत के खिलाफ ligand-lytic पेप्टाइड के Protozoacidal गतिविधि परीक्षण इन विट्रो में</em(प्रोटोजोअन संस्कृति) और Vivo में (दीमक Hindgut में Microinjection)
Summary
हम प्रदर्शन के लिए प्रक्रियाओं है कि ligands Formosan भूमिगत फ्लोरोसेंट माइक्रोस्कोपी और कि ligands lytic पेप्टाइड के साथ युग्मित का उपयोग दीमक के पेट में प्रोटोजोआ सेलूलोज़ पचाने को मारने प्रोटोजोआ इन की सतह झिल्ली करने के लिए बाध्य वर्तमान<em> इन विट्रो में</em(Anaerobic प्रोटोजोआ संस्कृति) और<em> Vivo में</em> (दीमक hindgut में इंजेक्शन).
Abstract
We are developing a novel approach to subterranean termite control that would lead to reduced reliance on the use of chemical pesticides. Subterranean termites are dependent on protozoa in the hindguts of workers to efficiently digest wood. Lytic peptides have been shown to kill a variety of protozoan parasites (Mutwiri et al. 2000) and also protozoa in the gut of the Formosan subterranean termite, Coptotermes formosanus (Husseneder and Collier 2009). Lytic peptides are part of the nonspecific immune system of eukaryotes, and destroy the membranes of microorganisms (Leuschner and Hansel 2004). Most lytic peptides are not likely to harm higher eukaryotes, because they do not affect the electrically neutral cholesterol-containing cell membranes of higher eukaryotes (Javadpour et al. 1996). Lytic peptide action can be targeted to specific cell types by the addition of a ligand. For example, Hansel et al. (2007) reported that lytic peptides conjugated with cancer cell membrane receptor ligands could be used to destroy breast cancer cells, while lytic peptides alone or conjugated with non-specific peptides were not effective. Lytic peptides also have been conjugated to human hormones that bind to receptors on tumor cells for targeted destruction of prostate and testicular cancer cells (Leuschner and Hansel 2004).
In this article we present techniques used to demonstrate the protozoacidal activity of a lytic peptide (Hecate) coupled to a heptapeptide ligand that binds to the surface membrane of protozoa from the gut of the Formosan subterranean termite. These techniques include extirpation of the gut from termite workers, anaerobic culture of gut protozoa (Pseudotrichonympha grassii, Holomastigotoides hartmanni,
Spirotrichonympha leidyi), microscopic confirmation that the ligand marked with a fluorescent dye binds to the termite gut protozoa and other free-living protozoa but not to bacteria or gut tissue. We also demonstrate that the same ligand coupled to a lytic peptide efficiently kills termite gut protozoa in vitro (protozoa culture) and in vivo (microinjection into hindgut of workers), but is less bacteriacidal than the lytic peptide alone. The loss of protozoa leads to the death of the termites in less than two weeks.
In the future, we will genetically engineer microorganisms that can survive in the termite hindgut and spread through a termite colony as “Trojan Horses” to express ligand-lytic peptides that would kill the protozoa in the termite gut and subsequently kill the termites in the colony. Ligand-lytic peptides also could be useful for drug development against protozoan parasites.
Protocol
Discussion
Ligand-lytic पेप्टाइड सफलतापूर्वक किया गया है को प्रभावी ढंग से लक्ष्य और कैंसर की कोशिकाओं (बयाना और Leuschner 2004, बयाना एट अल 2007.) को नष्ट करने के लिए इस्तेमाल किया. इस अवधारणा के आधार पर, हम एक heptapeptide ligand है कि की सतह के…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
हम फ्लोरोसेंट ligand संश्लेषण, Interdisciplinaray जैव प्रौद्योगिकी अनुसंधान के लिए केंद्र, ligand-lytic पेप्टाइड संश्लेषण के लिए UF, और प्रतिदीप्ति सूक्ष्मदर्शी का उपयोग प्रदान करने के लिए Socolovsky माइक्रोस्कोप सुविधा के लिए LSU पेप्टाइड सुविधा के पूर्व निदेशक डा. एलीसन रिचर्ड, धन्यवाद. अनुदान SERDP खोजपूर्ण रक्षा विभाग के विकास कार्यक्रम (बीज), ऊर्जा और पर्यावरण संरक्षण एजेंसी विभाग, जैव प्रौद्योगिकी AgCenter अंतःविषय टीम कार्यक्रम और लुइसियाना के राज्य द्वारा प्रदान किया गया.
Materials
| Material Name | Tipo | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| Sigmacote | Sigma Aldrich | SL-2 | ||
| EDANS | Novabiochem | |||
| Anaerobic glove box | Coy Laboratories, Inc. | Custom made | ||
| Intellus environmental controller | Percival | I36NL | ||
| PC-10 Glass micropipette puller | Narishige Scientific Instrument Lab | PC-10 | ||
| Glass needles (Model GD-1, 1 X 900 mm) | Narishige Scientific Instrument Lab | GD-1 | ||
| Leitz micromanipulators | Vermont Optechs, Inc. | ACS01 | ||
| Microinjector | Tritech Research, Inc. | MINJ-1 | ||
| Microcaps | Drummond Scientific Company | 1-000-0005 | ||
| LEICA fluorescence imaging system | Leica | DMRxA2 | ||
| LEICA dissecting scope | Leica | MZ16 | ||
| LEICA microscope | Leica | DMLB | ||
| Olympus dissecting scope | Olympus | SZ61 |
Riferimenti
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- Leuschner, C., Hansel, W. Membrane disrupting lytic peptides for cancer treatments. Curr. Pharm. Des. 10, 2299-2310 (2004).
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