Global analysis of gel mobility of proteins and its use in target identification

SDS-PAGE is a basic method that has long been used for separation of proteins according to their molecular sizes. Despite its simplicity, it provides information on characteristics of proteins beyond their molecular masses because gel mobility of proteins often reflects their physicochemical properties and post-translational modifications. Here we report on a global analysis of gel mobility of the proteome, which we term the "mobilitome," covering 93.4% of the fission yeast proteome. To our surprise, more than 40% of proteins did not migrate to their calculated positions. Statistical analyses revealed that the discrepancy was largely dependent on the hydrophobicity of proteins. This experimental data set, with a high coverage rate of real mobility, made it feasible to identify proteins detected on the gel without using any specialized techniques. This approach enabled us to detect previously unknown post-translational modifications of a protein; for example, we revealed that eIF5A is novel substrate of a Sir2-related deacetylase Hst2. Furthermore, we concomitantly identified twelve acetylated and eight methylated proteins using specific anti-acetylated and anti-methylated lysine antibodies, most of which had not been known to be subject to the modifications. Thus, we propose the general usefulness of the mobilitome and electrophoresis-based methodology for the identification and characterization of proteins detected on the gel.     

Pivotal role of Bcl-2 family proteins in the regulation of chondrocyte apoptosis

During endochondral ossification, chondrocytes undergo hypertrophic differentiation and die by apoptosis. The level of inorganic phosphate (P(i)) elevates at the site of cartilage mineralization, and when chondrocytes were treated with P(i), they underwent rapid apoptosis. Gene silencing of the proapoptotic Bcl-2 homology 3-only molecule bnip3 significantly suppressed P(i)-induced apoptosis. Conversely, overexpression of Bcl-xL suppressed, and its knockdown promoted, the apoptosis of chondrocytes. Bnip3 was associated with Bcl-xL in chondrocytes stimulated with P(i). Bcl-xL was expressed uniformly in the growth plate chondrocytes, whereas Bnip3 expression was exclusively localized in the hypertrophic chondrocytes. Finally, we generated chondrocyte-specific bcl-x knock-out mice using the Cre-loxP recombination system, and we provided evidence that the hypertrophic chondrocyte layer was shortened in those mice because of an increased apoptosis of prehypertrophic and hypertrophic chondrocytes, with the mice afflicted with dwarfism as a result. These results suggest the pivotal role of Bcl-2 family members in the regulation of chondrocyte apoptosis.     

Two patched protein subtypes and a conserved domain of group I proteins that regulates turnover

Patched (Ptc) is a 12-cross membrane protein that binds the secreted Hedgehog protein. Its regulation of the Hedgehog signaling pathway is critical to normal development and to a number of human diseases. This report analyzes features of sequence similarity and divergence in the Ptc protein family and identifies two subtypes distinguished by novel conserved domains. We used these results to propose a rational basis for classification. We show that one of the conserved sequence regions in the C-terminal domain of Ptch1 is responsible, at least in part, for rapid turnover. This sequence is absent in the stable Ptch2 protein.     

Brilliant Blue as an alternative dye to Fast Green for in ovo electroporation

Chick embryo electroporation is a powerful tool for the introduction of transgenes into tissues of interest for the study of developmental biology. This method often uses Fast Green to visualize the injected area by staining the solution containing DNA green. Here, we show that Fast Green fluoresces in a red color after electroporation, suggesting that researchers need to be cautious when detecting red fluorescence. Fast Green solution did not show any fluorescence before injection into chick embryos, but fluoresced red within 3 min post-injection into chick embryos. We identified Brilliant Blue as suitable alternative dye for use as an indicator of injection sites in ovo electroporation. We found that 0.2% of Brilliant Blue was sufficient to track the area of DNA injection. In addition, this chemical did not show red fluorescence after electroporation. Our findings demonstrate that Brilliant Blue can be used for detecting red fluorescent proteins introduced into chick embryos by electroporation. Our study also shows useful examples for the application of Brilliant Blue for the precise quantification of two fluorescence intensities after EGFP and mCherry co-electroporation.     

Concerted evolution of the primate immunoglobulin alpha-gene through gene conversion.

We determined four nucleotide sequences of the hominoid immunoglobulin alpha (C alpha) genes (chimpanzee C alpha 2, gorilla C alpha 2, and gibbon C alpha 1 and C alpha 2 genes), which made possible the examination of gene conversions in all hominoid C alpha genes. The following three methods were used to detect gene conversions: 1) phenetic tree construction; 2) detection of a DNA segment with extremely low variability between duplicated C alpha genes; and 3) a site by site search of shared nucleotide changes between duplicated C alpha genes. Results obtained from method 1 indicated a concerted evolution of the duplicated C alpha genes in the human, chimpanzee, gorilla, and gibbon lineages, while results obtained from method 2 suggested gene conversions in the human, gorilla, and gibbon C alpha genes. With method 3 we identified clusters of shared nucleotide changes between duplicated C alpha genes in human, chimpanzee, gorilla, and gibbon lineages, and in their hypothetical ancestors. In the present study converted regions were identified over the entire C alpha gene region excluding a few sites in the coding region which have escaped from gene conversion. This indicates that gene conversion is a general phenomenon in evolution, that can be clearly observed in non-functional regions.     

Characterization of the light-induced increase in the Michaelis constant of the cGMP phosphodiesterase in frog rod outer segments

Activation of cGMP phosphodiesterase in rod disk membrane in the light is thought to be an intermediary process of phototransduction. In various preparations of frog rod outer segments, the Michaelis constant (Km) of the phosphodiesterase was measured with pH assay method. On illumination, the Km increased from the value of the dark (130 microM) by about 8-fold (1 mM) in crude preparations, but did not change appreciably in purified disk membranes, confirming the previous finding by Robinson et al. (Robinson, P.R., Kawamura, S., Abramson, B. and Bownds, M.D. (1980) J. Gen. Physiol. 76, 631-645). The present work further showed that the light-induced Km increase is labile to various experimental manipulations such as sonication, freeze-thawing, etc. However, the Km in the light was relatively high in a crude disk membrane preparation and in a lyzed preparation. In addition, reconstitution experiments revealed that the Km increase does not require soluble components. Both proteolytic digestion and phospholipase treatment reduced the light Km of the phosphodiesterase in crude disk membranes. The above results suggest that there is a labile factor(s) responsible for the light-induced Km increase and that the factor is a membrane-bound protein and requires structural integrity of the disk membrane to exert its function. The latency of the Km increase after light stimulation was less than 2 s.     

Enhancement of antigenic potency in vitro and immunogenicity in vivo by coupling the antigen to anti-immunoglobulin

We have examined the effect of targeting an antigen to the immune system, by covalently coupling it to anti-immunoglobulin (Ig), on its efficacy for T cell stimulation in vitro and its immunogenicity for antibody production in vivo. In vitro, we compared the potency (for stimulation of a ferritin-specific T cell line) of free ferritin, ferritin coupled to goat antimouse IgM (heavy (H) chain specific), ferritin coupled to anti-IgG (H and light (L) chain specific), or ferritin coupled to anti-IgA (H chain specific), as well as a mixture of free ferritin plus goat anti-IgG. The ferritin coupled to anti-IgM or to anti-IgG (H + L), which could bind to surface Ig of B cells, stimulated T cell proliferation at concentrations of ferritin at least 10-fold lower than those required for the other forms of the antigen over the entire time course of the response, with 1000 rad-irradiated spleen cells as presenting cells. Because the goat antibodies were all of the same IgG isotype and coupling ratio, the failure of goat anti-IgA to enhance potency served as a control to exclude Fc receptor binding as the mechanism. The effect was not due to the nonspecific activation of B cells to become more efficient antigen-presenting cells, because mixtures of ferritin plus anti-IgG (H + L) had no effect, and the anti-IgG coupled to ferritin did not enhance presentation of myoglobin to a myoglobin-specific T cell line. The enhanced presentation of ferritin conjugated to goat anti-IgG (H + L) or to anti-IgM was sensitive to radiation doses greater than 2000 R, and was effective at less than one-tenth the number of spleen cells, consistent with the predominance of B cells as antigen-presenting cells for this form of the antigen rather than macrophages and dendritic cells only. When B cells and accessory cells were purified from T-depleted spleen cells, only the B cell preparation but not the accessory cell population manifested enhanced presentation of ferritin coupled to anti-IgG compared with free ferritin, and it was radiosensitive. Finally, allogeneic B cells could not mediate the enhancement in the presence of syngeneic splenic accessory cells (SAC); therefore, the enhancement was not due to shedding of immune complexes from B cells and subsequent presentation by SAC. We conclude that targeting the antigen to B cells as presenting cells greatly enhances its efficacy in vitro.(ABSTRACT TRUNCATED AT 400 WORDS)     

In vivo influence of the anti-B220 monoclonal antibody administration of the T cell differentiation in mice

B220, a pan-B marker, is known to be also expressed on immature T cells of MRL/1pr or other congenic 1pr mice and minor population of immature thymocytes but not on peripheral T cells. In this study, we investigated in vivo the possibility whether B220 is one kind of premature T or prethymic T precursor cell marker as well as a pan-B cell marker. A monoclonal antibody against B220 glycoprotein was intravenously injected every 2 days into various strains of mice. After the third administration of this antibody, thymocytes decreased remarkably compared with those from the rat IgG-treated group, and spleen cells were also reduced significantly. Further, the number of cells expressing Thy-1, Ly-1, Lyt-2, and asialo GM1 (asGM1) in the spleen were significantly reduced. On the contrary, the number of cells expressing surface IgM (sIgM) or B220 were increased by this treatment, especially after the 8th treatment. Some T-dependent immunological functions including mitogenic responses to lectins and cytotoxic T cell activity were markedly suppressed but mixed lymphocyte reaction (MLR) and natural killer (NK) activity were rather augmented. Thus, B220 may be expressed on some kinds of T cell progenitor. Taken together, in vivo treatment with anti-B220 antibody may influence differentiating stages of some T cells from bone marrow progenitors before or just after their homing into the thymus.