Specifically, F344 rats vaccinated i.t. with 105 CFU U112 in this study were visibly stressed and ill for 7�C10 days following immunization, with symptoms including,10% weight loss, ruffled fur, hunched posture, and periorbital porphyrin production. Such severe morbidity in immunocompetent hosts would likely prevent administration of U112 to immunocompromised individuals. In contrast, vaccination with a hundred-fold higher dose of U112DiglB caused no adverse effects or visible morbidity to rats, and yet this mutant was still able to induce antigen-specific cellular and humoral responses which generated protection against subsequent SCHU S4 challenge. It is likely that booster doses of this mutant strain would increase the degree of protective efficacy. These results collectively suggest the feasibility of developing targeted oral-based attenuated mutant vaccine strains for immunization against F. tularensis and provide impetus for further refinement of novicida-based vaccines, given the ease of its genetic manipulation. Furthermore, M-cells have distinctive morphological features such as a poorly organized brush border, irregular microvilli, and a thin glycocalyx suggesting that they do not play a role in intestinal digestion or absorption. Importantly, M-cells can serve as antigen sampling sites and contain a distinct basal invagination in which live and non-replicating pathogens are presented to lymphocytes, dendritic cells, and macrophages. Synthesis of recombinant extracellular proteins in the human 293 embryonic kidney 
cell expression system enables appropriate Mechlorethamine hydrochloride folding and post-translational modifications, thus generating secreted proteins that are functional and structurally similar to the native molecules. To purify the recombinant protein, a common technique includes the addition of a poly-histidine tag at either terminus of the protein; this small tag does typically not alter protein conformation and the imidazole functional group on histidine residues allows for coordination with divalent metal ions and thus purification by immobilized metal affinity chromatography. The histidine-tagged protein binds to nickel and other transition metals immobilized on either an imminodiacetic acid or nitrilo-tri-acetic acid modified chromatography column with high affinity, whereas protein contaminants without the histidine-tag bind weakly or not at all. Histidine-tagged proteins are eluted with imidazole in the range of 20�C200 mM, which competitively displaces proteins bound to the immobilized metal ions. This scheme is widely used to generate numerous secreted proteins including stroma proteins, basement membrane proteins, matricellular proteins, blood proteins and signaling molecules. The Catharanthine sulfate purity of the recombinant protein is typically assessed by SDS-PAGE, Western blotting and mass spectrometry. However, this quality control may be insufficient when the objective of the study is to investigate cell signaling pathways regulated by the protein of interest. In this study, we tested the hypothesis that potent signaling molecules of the TGF-b superfamily are co-purified in this general purification scheme using extracellular fibrillin-1 as an example. If this is the case, it would have important consequences for the quality control of purification schemes and for the design of experiments using recombinant secreted proteins produced in this fashion. Fibrillin-1 is a 350 kDa glycoprotein that multimerizes to form microfibril suprastructures in elastic and non-elastic tissues. Due to its modular domain structure, fragments of fibrillins can be produced and purified conveniently as correctly folded proteins using the HEK293 expression system. Direct interactions between TGF-b and fibrillin-1 have not been documented. However, other members of the TGF-b superfamily including GDF-5 can interact directly.