Preparation of soybean seed samples for FT-IR microspectroscopy

Typical preparation of seed samples for infrared (IR) microspectroscopy involves imbibition of the seed for varying time periods followed by cryosectioning. Imbibition, however, may initiate germination even at 4° C with associated changes in the chemistry of the sample. We have found that it is possible to section seeds that are sufficiently hard, such as soybeans, on a standard laboratory microtome without imbibition. The use of dry sectioning of unimbibed seeds is reported here, as well as a comparison of different mounting media and modes of analysis. Glycerol, Tissue-Tek, and ethanol were used as mounting media, and the quality of the resulting spectra was assessed. Ethanol was the preferred mountant, because it dried quickly with no residue and thus did not interfere with the spectrum of interest. Analysis in transmission mode using barium fluoride windows to hold the samples was compared with transmission-reflection analysis with sections mounted on special infrared-reflecting slides. The two modes of analysis performed well in different regions of the spectrum. The mode of analysis (transmission vs. transmission-reflection) should be based on the components of greatest interest in the sample.     

Anti-tumor therapy with macroencapsulated endostatin producer cells

Background  

Theracyte is a polytetrafluoroethylene membrane macroencapsulation system designed to induce neovascularization at the tissue interface, protecting the cells from host's immune rejection, thereby circumventing the problem of limited half-life and variation in circulating levels. Endostatin is a potent inhibitor of angiogenesis and tumor growth. Continuous delivery of endostatin improves the efficacy and potency of the antitumoral therapy. The purpose of this study was to determine whether recombinant fibroblasts expressing endostatin encapsulated in Theracyte immunoisolation devices can be used for delivery of this therapeutic protein for treatment of mice bearing B16F10 melanoma and Ehrlich tumors.     

Differential Susceptibility of RAE-1 Isoforms to Mouse Cytomegalovirus

The NKG2D receptor is one of the most potent activating natural killer cell receptors involved in antiviral responses. The mouse NKG2D ligands MULT-1, RAE-1, and H60 are regulated by murine cytomegalovirus (MCMV) proteins m145, m152, and m155, respectively. In addition, the m138 protein interferes with the expression of both MULT-1 and H60. We show here that one of five RAE-1 isoforms, RAE-1δ, is resistant to downregulation by MCMV and that this escape has functional importance in vivo. Although m152 retained newly synthesized RAE-1δ and RAE-1γ in the endoplasmic reticulum, no viral regulator was able to affect the mature RAE-1δ form which remains expressed on the surfaces of infected cells. This differential susceptibility to downregulation by MCMV is not a consequence of faster maturation of RAE-1δ compared to RAE-1γ but rather an intrinsic property of the mature surface-resident protein. This difference can be attributed to the absence of a PLWY motif from RAE-1δ. Altogether, these findings provide evidence for a novel mechanism of host escape from viral immunoevasion of NKG2D-dependent control.Cytomegaloviruses (CMVs) are ubiquitous pathogens causing morbidity in immune suppressed and immunodeficient hosts (34). Since CMVs are strictly species-specific viruses, the infection of mice with murine CMV (MCMV) represents a widely used model for studying CMV infection and disease (22, 40).Natural killer (NK) cells play a crucial role in the control of many viruses and are among the first cells to sense proinflammatory cytokines, as well as the perturbations in the expression of major histocompatibility complex (MHC) class I molecules and other surface molecules induced by viral infection (13). Both human CMV (HCMV) and MCMV have evolved strategies to compromise innate immunity-mediated by NK cells (20, 49).Although proinflammatory cytokines released during the early stage of MCMV infection induce NK cell activation, this is usually not sufficient for virus control (11). Namely, most mouse strains fail to mount an effector phase of NK cell response against infected cells (42), in spite of the fact that MCMV infection causes the downmodulation of MHC I molecules (17), which should activate NK cells via a “missing-self” mechanism (28). The lack of NK cell activation by MCMV is even more puzzling considering that NK cells possess activating receptors that recognize cellular ligands induced by infection. Among these is the activating receptor NKG2D, a C-type lectinlike receptor encoded by a single gene in humans and rodents (39). Engagement of NKG2D transduces a strong activating signal to promote NK cell stimulation. NKG2D also serves as a costimulatory receptor on CD8⁺ T cells (2). Several NKG2D ligands have been described in mice: MULT-1, H60a, H60b, H60c, and RAE-1α, -1β, -1γ, -1δ, and -1ɛ isoforms (4-6, 10, 14, 32, 35, 44). What prevents the activation of NK cells via the NKG2D receptor during MCMV infection? We and others have characterized four MCMV proteins involved in the downmodulation of NKG2D ligands (15, 23, 24, 26, 29, 30). Furthermore, the deletion of any of the four MCMV inhibitors of NKG2D ligands rendered virus mutants susceptible to NK cell control in vivo. The MCMV immunoevasin of NKG2D described first was the glycoprotein gp40, encoded by the gene m152 (23). Note that m152 also compromises the CD8⁺ T-cell response by downregulation of MHC class I molecules (25, 54). Later, it was noticed that m152 also affects the expression of RAE-1 proteins (29). It is important to point out that mouse strains express different RAE-1 isoforms. Some strains, such as BALB/c, express RAE-1α, -1β, and -1γ, while others, such as C57BL/6, express RAE-1δ and -1ɛ (29). All five RAE-1 isoforms are glycosylphosphatidylinositol (GPI)-linked proteins and contain MHC class I-like α1 and α2 domains (6, 10, 14, 35).Based on our initial observation that there is NKG2D-dependent control of wild type (WT) MCMV in certain mouse strains, we postulated NKG2D ligands that resist virus mediated downmodulation. We show here that the RAE-1 proteins differ in their susceptibility to downregulation by MCMV. In contrast to RAE-1γ, representing the sensitive isoform, surface-resident RAE-1δ remains present on MCMV-infected cells. The differential downmodulation of RAE-1 isoforms during MCMV infection is caused by differences in the stability of the mature RAE-1 molecules associated with a sequence motif absent in RAE-1δ.