Neuroglycan C,A Brain-Specific Chondroitin Sulfate Proteoglycan,Interacts with Pleiotrophin,A Heparin-Binding Growth Factor

Neuroglycan C (NGC) is a transmembrane-type chondroitin sulfate proteoglycan that promotes neurite outgrowth. To identify the ligand of NGC, we applied a detergent-solubilized membrane fraction of fetal rat brains to an NGC-immobilized affinity column. Several proteins were eluted from the column including an 18 kDa-band protein recognized by an anti-pleiotrophin antibody. The binding of pleiotrophin (PTN) to NGC was confirmed by a quartz crystal microbalance method and had a Kd of 8.7 nM. PTN bound to the acidic amino acid cluster of the NGC extracellular domain. In addition, PTN bound to both chondroitin sulfate-bearing NGC and chondroitinase-treated NGC prepared from the neonatal rat brain. These results suggest that NGC interacts with PTN.     

In vivo molecular imaging analysis of a nasal vaccine that induces protective immunity against botulism in nonhuman primates

Nasal administration is an effective route for a needle-free vaccine. However, nasally administered Ags have the potential to reach the CNS directly from the nasal cavity, thus raising safety concerns. In this study, we performed real-time quantitative tracking of a nasal vaccine candidate for botulism, which is a nontoxic subunit fragment of Clostridium botulinum type A neurotoxin (BoHc/A) effective in the induction of the toxin-neutralizing immune response, by using (18)F-labeled BoHc/A-positron-emission tomography, an in vivo molecular imaging method. This method provides results that are consistent with direct counting of [(18)F] radioactivity or the traditional [(111)In]-radiolabel method in dissected tissues of mice and nonhuman primates. We found no deposition of BoHc/A in the cerebrum or olfactory bulb after nasal administration of (18)F-labeled BoHc/A in both animals. We also established a real-time quantitative profile of elimination of this nasal vaccine candidate and demonstrated that it induces highly protective immunity against botulism in nonhuman primates. Our findings demonstrate the efficiency and safety of a nasal vaccine candidate against botulism in mice and nonhuman primates using in vivo molecular imaging.     

Not lipoteichoic acid but lipoproteins appear to be the dominant immunobiologically active compounds in Staphylococcus aureus

Lipoteichoic acid (LTA) derived from Staphylococcus aureus is reported to be a ligand of TLR2. However, we previously demonstrated that LTA fraction prepared from bacterial cells contains lipoproteins, which activate cells via TLR2. In this study, we investigated the immunobiological activity of LTA fraction obtained from S. aureus wild-type strain, lipoprotein diacylglycerol transferase deletion (delta lgt) mutant, which lacks palmitate-labeled lipoproteins, and its complemented strain and evaluated the activity of LTA molecule. LTA fraction was prepared by butanol extraction of the bacteria followed by hydrophobic interaction chromatography. Although all LTA fractions activated cells through TLR2, the LTA from delta lgt mutant was 100-fold less potent than those of wild-type and complemented strains. However, no significant structural difference in LTA was observed in NMR spectra. Further, alanylation of LTA molecule showed no effect in immunobiological activity. These results showed that not LTA molecule but lipoproteins are dominant immunobiologically active TLR2 ligand in S. aureus.     

Novel role for RbAp48 in tissue-specific, estrogen deficiency-dependent apoptosis in the exocrine glands

Although tissue-specific apoptosis in the exocrine glands in estrogen-deficient mice may contribute to the development of autoimmune exocrinopathy, the molecular mechanism responsible for tissue-specific apoptosis remains obscure. Here we show that RbAp48 overexpression induces p53-mediated apoptosis in the exocrine glands caused by estrogen deficiency. RbAp48-inducible transfectant results in rapid apoptosis with p53 phosphorylation (Ser9) and alpha-fodrin cleavage. Reducing the expression of RbAp48 through small interfering RNA inhibits the apoptosis. Prominent RbAp48 expression with apoptosis was observed in the exocrine glands of C57BL/6 ovariectomized (OVX) mice but not in OVX estrogen receptor alpha(-/-), p53(-/-), and E2F-1(-/-) mice. Indeed, transgenic expression of the RbAp48 gene induced apoptosis in the exocrine glands but not in other organs. These findings indicate that estrogen deficiency initiates p53-mediated apoptosis in the exocrine gland cells through RbAp48 overexpression and exerts a possible gender-based risk of autoimmune exocrinopathy in postmenopausal women.     

The Xdsg protein in presumptive primordial germ cells (pPGCs) is essential to their differentiation into PGCs in Xenopus

In order to know the role of the Xdsg gene in presumptive PGCs (pPGCs) of Xenopus, we attempted to inhibit the translation of Xdsg mRNA in pPGCs by injecting antisense morpholino oligo (asMO), together with Fluorescein Dextran-Lysine (FDL), into single germ plasm-bearing cells of 32-cell embryos. Among three types of asMOs complementary to different parts of the 5'-untranslated region of Xdsg mRNA tested, only one asMO, designated as Xdsg-3, inhibited the translation of the mRNA in FDL-labeled pPGCs, resulting in the absence of labeled PGCs in experimental tadpoles. On the other hand, two other asMOs, Xdsg-1 and -2, did not inhibit the translation, so that a similar number of labeled PGCs found in FDL-injected but asMO-uninjected control tadpoles were observed in experimental tadpoles derived from asMO-injected embryos. Surprisingly, use of Xdsg-3 asMO resulted in the disappearance of the protein of Xenopus vasa homolog (Xenopus vasa-like gene 1, XVLG1) from FDL-labeled pPGCs by inhibiting the translation of XVLG1 mRNA. However, the effect of Xdsg-3 asMO on the translation of Xdsg and XVLG1 mRNAs and PGC formation could be canceled by the coinjection with Xdsg mRNA. Consequently, the Xdsg protein in pPGCs may play an important role in the formation of PGCs by regulating the production of XVLG1 protein.     

Smad7 and protein phosphatase 1α are critical determinants in the duration of TGF-β/ALK1 signaling in endothelial cells

Background  

In endothelial cells (EC), transforming growth factor-β (TGF-β) can bind to and transduce signals through ALK1 and ALK5. The TGF-β/ALK5 and TGF-β/ALK1 pathways have opposite effects on EC behaviour. Besides differential receptor binding, the duration of TGF-β signaling is an important specificity determinant for signaling responses. TGF-β/ALK1-induced Smad1/5 phosphorylation in ECs occurs transiently.     

Application of microchip electrophoresis in the analysis of RNA aptamer-protein interactions

DNA and RNA can be separated by microchip electrophoresis (ME) and detected using an intercalating fluorescent dye. The advantages of this method are short sensing times (<3 min), avoidance of a radioisotope labeling detection system, relatively low costs, and reduced labor intensity. In the present study, RNA aptamer-protein or -peptide interactions were analyzed using ME and the regression of free aptamers corresponding to unbound RNA was detected as the target protein or peptide increased in a dose-dependent manner. Our results demonstrate the applicability of this method to simple, rapid ligand screening in the interactions between oligonucleotides and their targets.     

Asymmetric expression of antivin/lefty1 in the early chick embryo

Mammalian lefty and zebrafish antivin, highly related to lefty, are shown to be expressed asymmetrically and involved in the specification of the left body side of early embryos. We isolated a chick homologue of the antivin/lefty1 cDNA and studied its expression pattern during early chick development. We found that antivin/lefty1 is expressed asymmetrically on the left side of the prospective floorplate, notochord and lateral plate mesoderm of the chick embryo.     

Transducible form of p47phox and p67phox compensate for defective NADPH oxidase activity in neutrophils of patients with chronic granulomatous disease

Protein delivery to primary cells by protein transduction domain (PTD) serves as a novel measure for manipulation of the cells for biological study and for the treatment of various human conditions. Although the method has been employed to modulate cellular function in vitro, only limited reports are available on its application in the replacement of deficient signaling molecules into primary cells. We examined the potential of recombinant proteins to compensate for defective cytosolic components of the NADPH oxidase complex in chronic granulomatous disease (CGD) neutrophils in both p47(phox) and p67(phox) deficiency. The p47(phox) or p67(phox) protein linked to Hph-1 PTD was effectively expressed in soluble form and transduced into human neutrophils efficiently without eliciting unwanted signal transduction or apoptosis. The delivered protein was stable for more than 24h, expressed in the cytoplasm, translocated to the membrane fraction upon activation, and, most importantly able to restored reactive oxygen species (ROS) production. Although research on human primary neutrophils using the protein delivery system is still limited, our data show that the protein transduction approach for neutrophils may be applicable to the control of local infections in CGD patients by direct delivery of the protein product.