Liposomes with differential lipid components exert differential adjuvanticity in antigen-liposome conjugates via differential recognition by macrophages

We previously reported that liposomes having differential lipid components displayed differential adjuvant effects when antigen was coupled with liposomes via glutaraldehyde. In the present study, antigen-liposome conjugates prepared using liposomes having differential lipid components were added to the macrophage culture, and phagocytosis and the antigen digest of liposome-coupled antigen by macrophages were then investigated. Antigen presentation by macrophages to an antigen-specific T-cell clone was further investigated using the same conjugates. Antigen-liposome conjugates which induced higher levels of antibody production in vivo were recognized more often, and the liposome-coupled antigen was digested to a greater degree by macrophages than antigen-liposome conjugates which induced lower levels of antibody production. These results correlated closely with those regarding antigen presentation by macrophages; when antigen was coupled to liposomes showing higher adjuvant effect, macrophages cocultured with antigen-liposome conjugates activated antigen-specific T-cells at a higher degree. The concentration of OVA in the macrophage culture added as antigen-liposome conjugates was approximately 32 microg/mL. However, the extent of T-cell activation was almost equal to that when 800 microg/mL of soluble OVA was added to the culture. The results of the present study demonstrated that the adjuvant activity of liposomes observed primary in vivo correlated closely with the recognition of antigen-liposome conjugates and antigen presentation of liposome-coupled antigen by macrophages, suggesting that the adjuvant effects of liposomes are exerted at the beginning of the immune response, i.e., recognition of antigen by antigen-presenting cells.     

Clearance rates of biotinylated ferritins in canine: A possible physiological role of canine serum ferritin as an iron transporter

To elucidate the physiological role of canine serum ferritin, we measured clearance rates of biotinylated ferritins in beagle. Biotinylated canine tissue ferritins were cleared rapidly from circulation. The clearance time (T_1/2) of liver ferritin (H/L subunit ratio=0.43) was 6.8 to 11.8 min, and that of heart ferritin (H/L=3.69) was 9.3 to 25.0 min. T_1/2 of biotinylated canine liver ferritin was independent of iron content, whereas canine heart apoferritin (T_1/2=31.2 and 32.7 min) was more slowly removed from circulation than the holoferritin. On the other hand, biotinylated recombinant bovine H-chain ferritin homopolymer show a much slower rate of removal (T_1/2=153.8 and 155.0 min) compared with the L-chain ferritin homopolymer (T_1/2=26.4 and 31.3 min). The rapid clearance of canine tissue ferritin suggests that serum ferritin is an iron transporter in canines.     

Origin of higher affinity to RNA of the N-terminal RNA-binding domain than that of the C-terminal one of a mouse neural protein,musashi1, as revealed by comparison of their structures,modes of interaction,surface electrostatic potentials,and backbone dynamics

Musashi1 is an RNA-binding protein abundantly expressed in the developing mouse central nervous system. Its restricted expression in neural precursor cells suggests that it is involved in maintenance of the character of progenitor cells. Musashi1 contains two ribonucleoprotein-type RNA-binding domains (RBDs), RBD1 and RBD2, the affinity to RNA of RBD1 being much higher than that of RBD2. We previously reported the structure and mode of interaction with RNA of RBD2. Here, we have determined the structure and mode of interaction with RNA of RBD1. We have also analyzed the surface electrostatic potential and backbone dynamics of both RBDs. The two RBDs exhibit the same ribo-nucleoprotein-type fold and commonly make contact with RNA on the beta-sheet side. On the other hand, there is a remarkable difference in surface electrostatic potential, the beta-sheet of RBD1 being positively charged, which is favorable for binding negatively charged RNA, but that of RBD2 being almost neutral. There is also a difference in backbone dynamics, the central portion of the beta-sheet of RBD1 being flexible, but that of RBD2 not being flexible. The flexibility of RBD1 may be utilized in the recognition process to facilitate an induced fit. Thus, comparative studies have revealed the origin of the higher affinity of RBD1 than that of RBD2 and indicated that the affinity of an RBD to RNA is not governed by its fold alone but is also determined by its surface electrostatic potential and/or backbone dynamics. The biological role of RBD2 with lower affinity is also discussed.     

Pyruvate:NADP+ oxidoreductase is stabilized by its cofactor,thiamin pyrophosphate,in mitochondria of Euglena gracilis

Pyruvate:NADP(+) oxidoreductase (PNO) is a thiamin pyrophosphate (TPP)-dependent enzyme that plays a central role in the respiratory metabolism of Euglena gracilis, which requires thiamin for growth. When thiamin was depleted in Euglena cells, PNO protein level was greatly reduced, but its mRNA level was barely changed. In addition, a large part of PNO occurred as an apoenzyme lacking TPP in the deficient cells. The PNO protein level increased rapidly, without changes in the mRNA level, after supplementation of thiamin into its deficient cells. In the deficient cells, in contrast to the sufficient ones, a steep decrease in the PNO protein level was induced when the cells were incubated with cycloheximide. Immunofluorescence microscopy indicated that most of the PNO localized in the mitochondria in either the sufficient or the deficient cells. These findings suggest that PNO is readily degraded when TPP is not provided in mitochondria, and consequently the PNO protein level is greatly reduced by thiamin deficiency in E. gracilis.     

An endogenous electrophile that modulates the regulatory mechanism of protein turnover: inhibitory effects of 15-deoxy-Delta 12,14-prostaglandin J2 on proteasome

Prostaglandin D(2) (PGD(2)), a major cyclooxygenase product in a variety of tissues and cells, readily undergoes dehydration to yield electrophilic PGs, such as 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)). We have previously shown that 15d-PGJ(2) potently induces apoptosis of SH-SY5Y human neuroblastoma cells via accumulation of the tumor suppressor gene product p53. In the study presented here, we investigated the molecular mechanisms involved in the 15d-PGJ(2)-induced accumulation of p53. It was observed that 15d-PGJ(2) potently induced p53 protein expression but scarcely induced p53 gene expression. In addition, exposure of the cells to 15d-PGJ(2) resulted in an accumulation of ubiquitinated proteins and in a significant inhibition of proteasome activities, suggesting that 15d-PGJ(2) acted on the ubiquitin-proteasome pathway, a regulatory mechanism of p53 turnover. The effects of 15d-PGJ(2) on the protein turnover were attributed to its electrophilic feature, based on the observations that (i) the reduction of the double bond in the cyclopentenone ring of 15d-PGJ(2) virtually abolished the effects on protein turnover, (ii) overexpression of an endogenous redox regulator, thioredoxin 1, significantly retarded the inhibition of proteasome activities and accumulations of p53 and ubiquitinated proteins induced by 15d-PGJ(2), and (iii) treatment of SH-SY5Y cells with biotinylated 15d-PGJ(2) indeed resulted in the formation of a 15d-PGJ(2)-proteasome conjugate. These data suggest that the modulation of proteasome activity may be involved in the mechanism responsible for the accumulation of p53 and subsequent induction of apoptotic cell death induced by 15d-PGJ(2).     

Fabrication of nickel and chromium nanoparticles using the protein cage of apoferritin

The iron storage protein, apoferritin, has a cavity in which iron is oxidized and stored as a hydrated oxide core. The size of the core is about 7 nm in diameter and is regulated by the cavity size. The cavity can be utilized as a nanoreactor to grow inorganic crystals. We incubated apoferritin in nickel or chromium salt solutions to fabricate hydroxide nanoparticles in the cavity. By using a solution containing dissolved carbon dioxide and by precisely controlling the pH, we succeeded in fabricating nickel and chromium cores. During the hydroxylation process of nickel ions a large portion of the apoferritin precipitated through bulk precipitation of nickel hydroxide. Bulk precipitation was suppressed by adding ammonium ions. However, even in the presence of ammonium ions the core did not form using a degassed solution. We concluded that carbonate ions were indispensable for core formation and that the ammonium ions prevented precipitation in the bulk solution. The optimized condition for nickel core formation was 0.3 mg/mL horse spleen apoferritin and 5 mM ammonium nickel sulfate in water containing dissolved carbon dioxide. The pH was maintained at 8.65 using two buffer solutions: 150 mM HEPES (pH 7.5) and 195 mM CAPSO (pH 9.5) with 20 mM ammonium at 23 degrees C. The pH had not changed after 48 h. After 24 h of incubation, all apoferritins remained in the supernatant and all of them had cores. Recombinant L-ferritin showed less precipitation even above a pH of 8.65. A chromium core was formed under the following conditions: 0.1 mg/mL apoferritin, 1 mM ammonium chromium sulfate, 100 mM HEPES (pH 7.5) with a solution containing dissolved carbon dioxide. About 80% of the supernatant apoferritin (0.07 mg/mL) formed a core. In nickel and chromium core formation, carbonate ions would play an important role in accelerating the hydroxylation in the apoferritin cavity compared to the bulk solution outside.     

The kic1 kinase of schizosaccharomyces pombe is a CLK/STY orthologue that regulates cell-cell separation

The CLK/STY kinases are a family of dual-specificity protein kinases implicated in the regulation of cellular growth and differentiation. Some of the kinases in the family are shown to phosphorylate serine-arginine-rich splicing factors and to regulate pre-mRNA splicing. However, the actual cellular mechanism that regulates cell growth, differentiation, and development by CLK/STY remains unclear. Here we show that a functionally conserved CLK/STY kinase exists in Schizosaccharomyces pombe, and this orthologue, called Kic1, regulates the cell surface and septum formation as well as a late step in cytokinesis. The Kic1 protein is modified in vivo, likely by phosphorylation, suggesting that it can be involved in a control cascade. In addition, kic1(+) together with dsk1(+), which encodes a related SR-specific protein kinase, constitutes a critical in vivo function for cell growth. The results provide the first in vivo evidence for the functional conservation of the CLK/STY family through evolution from fission yeast to mammals. Furthermore, since cell division and cell-cell interaction are fundamental for the differentiation and development of an organism, the novel cellular role of kic1(+) revealed from this study offers a clue to the understanding of its counterparts in higher eukaryotes.     

Growth and differentiation potential of main- and side-population cells derived from murine skeletal muscle

Skeletal muscle-derived CD34+/45- (Sk-34) cells were identified as a new candidate for stem cells. However, the relationship between Sk-34 cells and side-population (SP) cells is unknown. Here, we demonstrate that Sk-34 cells prepared from murine skeletal muscles consist wholly of main-population (MP) cells. The Sk-34 cells included only a few SP cells (1:1000, SP:MP). Colony-forming units of Sk-34 cells of both SP and MP possessed the same potential to differentiate into adipocytes, endothelial, and myogenic cells and showed the same colony-forming activity (1.6%). In addition, the colony-forming units of the CD34-/45- (double negative: DN) population were found to begin CD34 expression and to possess the potential to differentiate into myogenic and endothelial cells. We also found that expression of CD34 antigen precedes MyoD expression during the myogenic process of DN cells. Furthermore, both Sk-34 and DN cell populations were mostly negative for CD73 (93-95%), whereas the CD45+ cell population was >25% positive for CD73, and this trend was also seen in bone marrow-derived CD45+ cells. These results indicate that the MP cell population is about 99.9% responsible for the reported in vitro myogenic-endothelial responses of skeletal muscle-derived cells.