<<Disclaimer: Verify this information before applying it to your situation.>> Finding Gluten Peptides Inside Bacteria - Part 2 ---------- Examples of Immunogold Electron Microscopy The following articles contain micrograpic images of immunogold-labelled ultrathin sections of bacteria as well as discussions of the methods used to prepare the specimens. The older articles are stored as PDF files of scanned pages which take a long time to download. (Paste web addresses together on one line): Kruger E, Witt E, Ohlmeier S, Hanschke R, Hecker M. The clp proteases of Bacillus subtilis are directly involved in degradation of misfolded proteins. J Bacteriol. 2000 Jun;182(11):3259-65. http://jb.asm.org/cgi/content/full/182/11/3259?view=full&pmid=10809708 Newman G, Crooke E. DnaA, the initiator of Escherichia coli chromosomal replication, is located at the cell membrane. J Bacteriol. 2000 May;182 (9):2604-10. http://jb.asm.org/cgi/content/full/182/9/2604?view=full&pmid=10762265 Walderich B, Holtje JV. Subcellular distribution of the soluble lytic transglycosylase in Escherichia coli. J Bacteriol. 1991 Sep;173(18):5668- 76. http://www.pubmedcentral.nih.gov/articlerender.fcgi? tool=pubmed&pubmedid=1885544 Kroncke KD, Orskov I, Orskov F, Jann B, Jann K. Electron microscopic study of coexpression of adhesive protein capsules and polysaccharide capsules in Escherichia coli. Infect Immun. 1990 Aug;58(8):2710-4. http://www.pubmedcentral.nih.gov/articlerender.fcgi? tool=pubmed&pubmedid=1973415 Bayer MH, Keck W, Bayer ME. Localization of penicillin-binding protein 1b in Escherichia coli: immunoelectron microscopy and immunotransfer studies. J Bacteriol. 1990 Jan;172(1):125-35. http://www.pubmedcentral.nih.gov/articlerender.fcgi? tool=pubmed&pubmedid=2403537 Paques M, Teppema JS, Beuvery EC, Abdillahi H, Poolman JT, Verkleij AJ. Accessibility of gonococcal and meningococcal surface antigens: immunogold labeling for quantitative electron microscopy. Infect Immun. 1989 Feb;57 (2):582-9. http://www.pubmedcentral.nih.gov/articlerender.fcgi? tool=pubmed&pubmedid=2492264 Diaz E, Garcia E, Ascaso C, Mendez E, Lopez R, Garcia JL. Subcellular localization of the major pneumococcal autolysin: a peculiar mechanism of secretion in Escherichia coli. J Biol Chem. 1989 Jan 15;264(2):1238-44. http://www.jbc.org/cgi/reprint/264/2/1238 Acker G, Bitter-Suermann D, Meier-Dieter U, Peters H, Mayer H. Immunocytochemical localization of enterobacterial common antigen in Escherichia coli and Yersinia enterocolitica cells. J Bacteriol. 1986 Oct;168(1):348-56. http://www.pubmedcentral.nih.gov/articlerender.fcgi? tool=pubmed&pubmedid=3531175 ---------- Microscopy Links: Advanced Electron Microscopy & Imaging http://www.hei.org/research/depts/aemi/tec.htm Optical Microscopy Primer http://microscopy.fsu.edu/primer/index.html SPI-Mark Colloidal Gold Conjugates for Immunogold Labelling - SPI Supplies Introductory articles on gold labelling http://www.2spi.com/catalog/chem/gold.html SPI Microscopy Library - Books http://www.2spi.com/catalog/books/bookmain.html Tokuyasu KT. A technique for ultracryotomy of cell suspensions and tissues. J Cell Biol. 1973 May;57(2):551-65. http://www.jcb.org/cgi/reprint/57/2/551 Keller GA, Tokuyasu KT, Dutton AH, Singer SJ. An improved procedure for immunoelectron microscopy: ultrathin plastic embedding of immunolabeled ultrathin frozen sections. Proc Natl Acad Sci U S A. 1984 Sep;81(18):5744- 7. http://www.pubmedcentral.nih.gov/articlerender.fcgi? tool=pubmed&pubmedid=6435119 ---------- Dealing with Fecal Samples: Here are some examples where bacteria from fecal samples are studied, illustrating techiniques used to separate bacteria from feces: Wang RF, Cao WW, Cerniglia CE. PCR detection and quantitation of predominant anaerobic bacteria in human and animal fecal samples. Appl Environ Microbiol. 1996 Apr;62(4):1242-7. http://aem.asm.org/cgi/reprint/62/4/1242?view=reprint&pmid=8919784 MacKay WG, Williams CL, McMillan M, Ndip RN, Shepherd AJ, Weaver LT. Evaluation of protocol using gene capture and PCR for detection of Helicobacter pylori DNA in feces. J Clin Microbiol. 2003 Oct;41(10):4589- 93. http://aem.asm.org/cgi/reprint/62/4/1242?view=reprint&pmid=8919784 ---------- An example of incubating feces with an added substance to study bacterial takeup: J Agric Food Chem. 2004 May 5;52(9):2689-96. Human fecal metabolism of soyasaponin I. Hu J, Zheng YL, Hyde W, Hendrich S, Murphy PA. Department of Food Science and Human Nutrition, and College of Veterinary Medicine, Iowa State University, 2312 Food Science Building, Ames, Iowa 50011, USA. The metabolism of soyasaponin I (3-O-[alpha-L-rhamnopyranosyl-beta-D- galactopyranosyl-beta-D-glucuronopyranosyl]olean-12-ene-3beta,22beta,24- triol) by human fecal microorganisms was investigated. Fresh feces were collected from 15 healthy women and incubated anaerobically with 10 mmol soyasaponin I/g feces at 37 degrees C for 48 h. The disappearance of soyasaponin I in this in vitro fermentation system displayed apparent first- order rate loss kinetics. Two distinct soyasaponin I degradation phenotypes were observed among the subjects: rapid soyasaponin degraders with a rate constant k = 0.24 +/- 0.04 h(-)(1) and slow degraders with a k = 0.07 +/- 0.02 h(-)(1). There were no significant differences in the body mass index, fecal moisture, gut transit time, and soy consumption frequency between the two soyasaponin degradation phenotypes. Two primary gut microbial metabolites of soyasaponin I were identified as soyasaponin III (3-O-[beta- D-galactopyranosyl-beta-D-glucuronopyranosyl]olean-12-ene-3beta,22beta,24- triol) and soyasapogenol B (olean-12-ene-3beta,22beta,24-triol) by NMR and electrospray ionized mass spectroscopy. Soyasaponin III appeared within the first 24 h and disappeared by 48 h. Soyasapogenol B seemed to be the final metabolic product during the 48 h anaerobic incubation. These results indicate that dietary soyasaponins can be metabolized by human gut microorganisms. The sugar moieties of soyasaponins seem to be hydrolyzed sequentially to yield smaller and more hydrophobic metabolites. PMID: 15113177 [PubMed - in process] ---------- Centrifugation Techniques: Centrifugation http://ntri.tamuk.edu/centrifuge/centrifugation.html Percoll http://www.cellseparation.nu/media/18-1115-69AC.pdf __________ Book Review: I have been considering the possibilities of carrying out an immunogold electron microscope investigation to find gluten peptides within gut bacteria myself, but I lack hands-on experience and access to laboratory facilities. I do note that my former university, about 1.5 hours away, has lab facilities available for use by NIH funded projects and does have a transmission electron microscope available with quite a bit of time open on its scheduling calender. Quite tempting... I have gone so far as to purchase a couple of books on electron microscopy which I can recommend to any one interested. These books are: Electron Microscopy: Principles and Techniques for Biologists by John J. Bozzola, Lonnie D. Russell Hardcover: 670 pages ; Dimensions (in inches): 1.50 x 11.25 x 9.00 Publisher: Jones & Bartlett Pub; 2nd edition (January 1999) List Price: $91.95 (WalMart Price: $83.38) Principles and Techniques of Electron Microscopy: Biological Applications by M. A. Hayat Hardcover: 543 pages ; Dimensions (in inches): 1.08 x 10.32 x 8.33 Publisher: Cambridge University Press; 4th edition (April 15, 2000) List Price: $110.00 (Amazon Price: $80.30) There a number of good books on electron microscopy... all expensive. These 2 stood out. These books complement each other very well, covering material that neither one alone covers. Bozzola is an excellent introductory book covering all the basics of both transmission and scanning electron microscopes. Hayat goes into depth on the chemistry of specimen preparation, staining, and labelling covering advanced techniques not found in Bozzola including a full chapter on immunogold labelling. Hayat is also the editor of the authorative (and expensive) 3 volume set, Colloidal Gold: Principles, Methods and Applications. * * * *Support summarization of posts, reply to the SENDER not the CELIAC List*