Identification of peripherin as a Akt substrate in neurons

Activation of Akt-mediated signaling pathways is crucial for survival and regeneration of injured neurons. In this study, we attempted to identify novel Akt substrates by using an antibody that recognized a consensus motif phosphorylated by Akt. PC12 cells that overexpressed constitutively active Akt were used. Using two-dimensional PAGE, we identified protein spots that exhibited increased immunostaining of the antibody. Mass spectrometry revealed several major spots as the neuronal intermediate filament protein, peripherin. Using several peripherin fragments, the phosphorylation site was determined as Ser(66) in its head domain in vitro. Furthermore, a co-immunoprecipitation experiment revealed that Akt interacted with the head domain of peripherin in HEK 293T cells. An antibody against phosphorylated peripherin was raised, and induction of phosphorylated peripherin was observed not only in Akt-activated cultured cells but also in nerve-injured hypoglossal motor neurons. These results suggest that peripherin is a novel substrate for Akt in vivo and that its phosphorylation may play a role in motor nerve regeneration.     

Effect of Nicking the C-terminal Region of the <Emphasis Type="Italic">Clostridium botulinum</Emphasis> Serotype D Neurotoxin Heavy Chain on its Toxicity and Molecular Properties

A unique strain of Clostridium botulinum serotype D 4947 produces toxin complexes that are composed of un-nicked components, including a neurotoxin (BoNT) and auxiliary proteins. This BoNT showed aberrant elution upon Superdex gel filtration, indicating a much lower molecular weight, due to hydrophobic interaction with the column. Limited trypsin proteolysis of BoNT produces two nicks; first nick yielded a BoNT 50 kDa light chain disulfide linked to a 100 kDa heavy chain (Hc), and a second nick arose in Hc C-terminal 10 kDa. The second nick occurred in the putative binding domain of the BoNT molecule and induced alterations in its secondary structure, leading to a significant reduction of mouse toxicity in comparison with that of the fully-activated singly nicked BoNT. These results help to clarify the role of the C-terminal half of the Hc in the oral toxicity of single-chain and more complex forms of BoNT.     

Embryonic and fetal beta-globin gene repression by the orphan nuclear receptors, TR2 and TR4

The TR2 and TR4 orphan nuclear receptors comprise the DNA-binding core of direct repeat erythroid definitive, a protein complex that binds to direct repeat elements in the embryonic and fetal beta-type globin gene promoters. Silencing of both the embryonic and fetal beta-type globin genes is delayed in definitive erythroid cells of Tr2 and Tr4 null mutant mice, whereas in transgenic mice that express dominant-negative TR4 (dnTR4), human embryonic epsilon-globin is activated in primitive and definitive erythroid cells. In contrast, human fetal gamma-globin is activated by dnTR4 only in definitive, but not in primitive, erythroid cells, implicating TR2/TR4 as a stage-selective repressor. Forced expression of wild-type TR2 and TR4 leads to precocious repression of epsilon-globin, but in contrast to induction of gamma-globin in definitive erythroid cells. These temporally specific, gene-selective alterations in epsilon- and gamma-globin gene expression by gain and loss of TR2/TR4 function provide the first genetic evidence for a role for these nuclear receptors in sequential, gene-autonomous silencing of the epsilon- and gamma-globin genes during development, and suggest that their differential utilization controls stage-specific repression of the human epsilon- and gamma-globin genes.     

The functions of UCH-L1 and its relation to neurodegenerative diseases

Parkinson's disease (PD) and Alzheimer's disease (AD), the most common neurodegenerative diseases, are caused by both genetic and environmental factors. Ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) is a deubiquitinating enzyme that is involved in the pathogenesis of both of these neurodegenerative diseases. Several functions of UCH-L1, other than as an ubiquitin hydrolase, have been proposed; these include acting as an ubiquitin ligase and stabilizing mono-ubiquitin. This review focuses on recent findings on the functions and the regulation of UCH-L1, in particular those that relate to PD and AD.     

Successful induction of clinically competent dendritic cells from granulocyte colony-stimulating factor-mobilized monocytes for cancer vaccine therapy

Recent studies have suggested that dendritic cell (DC)-based immunotherapy is one promising approach for the treatment of cancer. We previously studied the clinical toxicity, feasibility, and efficacy of cancer vaccine therapy with peptide-pulsed DCs. In that study, we used granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood monocytes as a cell source of DCs. However, previous investigations have suggested that G-CSF-mobilized peripheral blood monocytes produce reduced levels of proinflammatory cytokines such as interleukin (IL)-12 and tumor necrosis factor (TNF)-α. These T helper (Th)-1-type cytokines are thought to promote antitumor immune response. In this study, we assessed the functional abilities of DCs generated from G-CSF-mobilized monocytes obtained from 13 patients with CEA-positive advanced solid cancers. Peripheral blood mononuclear cells were obtained from leukapheresis products collected before and after systemic administration of G-CSF (subcutaneous administration of high-dose [5–10 μg/kg] human recombinant G-CSF for five consecutive days). In vitro cytokine production profiles after stimulation with lipopolysaccharide (LPS) were compared between monocytes with and without G-CSF mobilization. DCs generated from monocytes were also examined with respect to cytokine production and the capacity to induce peptide-specific T cell responses. Administration of G-CSF was found to efficiently mobilize peripheral blood monocytes. Although G-CSF-mobilized monocytes (G/Mo) less effectively produced Th-1-type cytokines than control monocytes (C/Mo), DCs generated from G/Mo restored the same level of IL-12 production as that seen in DCs generated from C/Mo. T cell induction assay using recall antigen peptide and phenotypic analyses also demonstrated that DCs generated from G/Mo retained characteristics identical to those generated from C/Mo. Our results suggest that G-CSF mobilization can be used to collect monocytes as a cell source for the generation of DCs for cancer immunotherapy. DCs generated in this fashion were pulsed with HLA-A24-restricted CEA epitope peptide and administered to patients safely; immunological responses were induced in some patients.     

In vitro Th1 cytokine-independent Th2 suppressive effects of bifidobacteria

A comparison between 17 strains of lactic acid bacteria and 15 strains of bifidobacteria indicated that bifidobacteria induced significantly lower levels of interleukin-12 (IL-12) in murine splenic cells. The present study aims to evaluate the effect and mechanism of Bifidobacterium longum BB536, a probiotic strain, in suppressing antigen-induced Th2 immune response in vitro. BB536 suppressed immunoglobulin (Ig) E and IL-4 production by ovalbumin-sensitized splenic cells, but induction of Th1-inducing cytokine production, such as IL-12 and gamma interferon (IFN-gamma) tended to be lower compared with lactic acid bacteria. Neutralization with antibodies to IL-12, IFN-gamma, IL-10 and transforming growth factor beta indicated negative involvement of Th1-inducing cytokines and regulatory cytokines in the suppression of Th2 immune response by BB536, especially when treated at higher doses of BB536 (>10 microg cells/ml). Furthermore, BB536 induced the maturation of immature bone marrow-derived dendritic cells (BM-DCs), and suppressed antigen-induced IL-4 production mediated by BM-DCs. These results suggested that BB536 suppressed Th2 immune responses, partially independent of Th1-inducing cytokines and independent of regulatory cytokines, mediated by antigen-presenting cells such as dendritic cells.     

A multi-body model for comparative study of cervical traction simulation – development,improvement and validation

A computer simulation model was developed to study the dynamic behavior of the cervical spine during cervical traction therapy in inclined and sitting traction positions. The model improved upon an old model with additional components to represent the behavior of the intervertebral discs and the posterior ligaments. The simulation result of the new model was compared against the cervical traction data from a radiographic experiment in both positions. The simulation results of the old model and new model were compared to illustrate the improvement. Using the new model, we compared the timing response of cervical traction in the inclined and sitting positions.     

Is the Setal Patch on the Chelae of Hemigrapsus takanoi and Hemigrapsus sinensis (Crustacea,Brachyura, Varunidae) Advantageous in Fighting and Mating?

Many animals have unique morphological characters that function in social behavior. Sexual selection can affect the expression of such traits in males and females, leading to sexual dimorphism. We investigated the social function of setal patches on the chelae of two species of varunid crabs, one in which males, but not females, have setal patches (Hemigrapsus takanoi), and one in which both sexes have setal patches (Hemigrapsus sinensis). We experimentally removed setal patches and compared fighting and mating behavior of individuals with and without setal patches. In H. sinensis, males with setal patches removed were inferior fighters compared to intact males. In male H. takanoi and female H. sinensis, setal removal did not influence the outcome of fights. In mating, males lacking setal patches had a similar ability to copulate with females as intact males in both species. However, male H. takanoi with their setae removed tended to take more time to initiate copulation than did intact males. When females were given the opportunity to choose intact males or males without setal patches, females of H. takanoi did not discriminate between the two. Female H. sinensis, however, copulated with intact males more frequently compared to males lacking setal patches. Male H. sinensis showed no preferences for the presence of setal patches or the body size of females. Thus, our results indicate that setal patches have a social function in male H. takanoi and male H. sinensis, but not in female H. sinensis, suggesting that the setal patches of male crabs are a sexually selected trait in both species. However, the social function of male setal patches was more prominent in the species in which both sexes possess setal patches than in the species in which only males bear setal patches.     

The Structural Differences between a Glycoprotein Specific F-Box Protein Fbs1 and Its Homologous Protein FBG3

The Skp1-Cul1-F-box protein (SCF) complex catalyzes protein ubiquitination in diverse cellular processes and is one of the best-characterized ubiquitin ligases. F-box proteins determine the substrate specificities of SCF ubiquitin ligases. Among these, Fbs1/FBG1/FBXO2, Fbs2/FBG2/FBXO6, and Fbs3/FBG5/FBXO27 recognize the N-glycans of glycoproteins, whereas FBG3/FBXO44 has no sugar-binding activity, despite the high sequence homology and conservation of the residues necessary for oligosaccharide binding between Fbs1–3 and FBG3. Here we determined the crystal structure of the Skp1–FBG3 complex at a resolution of 2.6 Å. The substrate-binding domain of FBG3 is composed of a 10-stranded antiparallel β-sandwich with three helices. Although the overall structure of FBG3 is similar to that of Fbs1, the residues that form the Fbs1 carbohydrate-binding pocket failed to be superposed with the corresponding residues of FBG3. Structure-based mutational analysis shows that distinct hydrogen bond networks of four FBG3 loops, i.e., β2-β3, β5-β6, β7-β8, and β9-β10, prevent the formation of the carbohydrate-binding pocket shown in Fbs1.     

Scaffold-Free Tubular Tissues Created by a Bio-3D Printer Undergo Remodeling and Endothelialization when Implanted in Rat Aortae

BackgroundSmall caliber vascular prostheses are not clinically available because synthetic vascular prostheses lack endothelial cells which modulate platelet activation, leukocyte adhesion, thrombosis, and the regulation of vasomotor tone by the production of vasoactive substances. We developed a novel method to create scaffold-free tubular tissue from multicellular spheroids (MCS) using a “Bio-3D printer”-based system. This system enables the creation of pre-designed three-dimensional structures using a computer controlled robotics system. With this system, we created a tubular structure and studied its biological features.ConclusionsThe scaffold-free tubular tissues made of MCS using a Bio-3D printer underwent remodeling and endothelialization. Further studies are warranted to elucidate the underlying mechanism of endothelialization and its function, as well as the long-term results.