Generation of hybrid hepatocytes by cell fusion from monkey embryoid body cells in the injured mouse liver

Monkey embryonic stem (ES) cells have characteristics that are similar to human ES cells, and might be useful as a substitute model for preclinical research. When embryoid bodies (EBs) formed from monkey ES cells were cultured, expression of many hepatocyte-related genes including cytochrome P450 (Cyp) 3a and Cyp7a1 was observed. Hepatocytes were immunocytochemically observed using antibodies against albumin (ALB), cytokeratin-8/18, and α1-antitrypsin in the developing EBs. The in vitro differentiation potential of monkey ES cells into the hepatic lineage prompted us to examine the transplantability of monkey EB cells. As an initial approach to assess the repopulation potential, we transplanted EB cells into immunodeficient urokinase-type plasminogen activator transgenic mice that undergo liver failure. After transplantation, the hepatocyte colonies expressing monkey ALB were observed in the mouse liver. Fluorescence in-situ hybridization revealed that the repopulating hepatocytes arise from cell fusion between transplanted monkey EB cells and recipient mouse hepatocytes. In contrast, neither cell fusion nor repopulation of hepatocytes was observed in the recipient liver after undifferentiated ES cell transplantation. These results indicate that the differentiated cells in developing monkey EBs, but not contaminating ES cells, generate functional hepatocytes by cell fusion with recipient mouse hepatocytes, and repopulate injured mouse liver.     

Reduction of nonspecific binding proteins to self-assembled monolayer on gold surface

We developed a gold coated glass chip bearing a poly(ethyleneglycol) (PEG) type compound as hydrophilic spacer for surface plasmon resonance studies, which enabled adequate estimation of K(d) value between FK506 and FKBP12 not only using purified FKBP12 (K(d)=22 nM) but also using Escherichia coli lysate expressing FKBP12 (K(d)=15 nM). These results indicated effectiveness of the PEG spacer for reduction of nonspecific interactions. Chemical stability and simple surface-structure of the novel chip are also attractive.     

AIB1 promotes DNA replication by JNK repression and AKT activation during cellular stress

Amplified in breast cancer 1 (AIB1) is a member of the p160 family of nuclear receptor coactivator protein. Recent studies have reported that high-level AIB1 production is involved in the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway for progression to malignant carcinoma in a steroid-independent manner. Here we demonstrate that, in AIB1-knockout DT40 chicken B-lymphocytes, loss of AIB1 results in induction of phosphorylation of c-Jun N-terminal kinase (JNK) and c-Jun, in addition to the inhibition of DNA replication. In contrast, high-level AIB1 production prevents proapoptotic activation of the JNK/c-Jun signal transduction pathway and induces DNA replication through phosphorylation of the Akt/p65 NF-kappaB subunit RelA under cellular stresses such as UV irradiation or serum deprivation. Moreover, we have found that AIB1 is essential for the phosphorylation of histone H3 at serine 10, which is associated with the signal transduction to chromatin, leading to the transient expression of immediate-early genes in response to UV stimulation. Our results therefore suggest that AIB1 directly links to cell cycle control mechanisms in concern with the balance between apoptosis and proliferation.     

Nefiracetam activation of CaM kinase II and protein kinase C mediated by NMDA and metabotropic glutamate receptors in olfactory bulbectomized mice

Aberrant behaviors related to learning and memory in olfactory bulbectomized (OBX) mice have been documented in the previous studies. We reported that the impairment of long-term potentiation (LTP) of hippocampal CA1 regions from OBX mice was associated with down-regulation of CaM kinase II (CaMKII) and protein kinase C (PKC) activities. We now demonstrated that the nootropic drug, nefiracetam, significantly improved spatial reference memory-related behaviors as assessed by Y-maze and novel object recognition task in OBX mice. Nefiracetam also restored hippocampal LTP injured in OBX mice. Nefiracetam treatment restored LTP-induced PKCα (Ser657) and NR1 (Ser896) phosphorylation as well as increase in their basal phosphorylation in the hippocampal CA1 region of OBX mice. Likewise, nefiracetam improved LTP-induced CaMKIIα (Thr286) autophosphorylation and GluR1 (Ser831) phosphorylation and increased their basal phosphorylation. The enhancement of PKCα (Ser657) and CaMKIIα (Thr286) autophosphorylation by nefiracetam was inhibited by treatment with (±)-α-Methyl-(4-carboxyphenyl)glycine and DL-2-Amino-5-phosphonovaleric acid, respectively. The enhancement of LTP induced by nefiracetam is inhibited by treatment with 2-methyl-6-(phenylethynyl)-pyridine, but not by treatment with LY367385, suggesting that metabotropic glutamate receptor 5 (mGluR5) but not mGluR1 is involved in the nefiracetam-induced LTP enhancement. Taken together, nefiracetam ameliorates OBX-induced deficits in memory-related behaviors and impairment of LTP in the hippocampal CA1 region through activation of NMDAR and mGluR5, thereby leading to an increase in activities of CaMKIIα (Thr286) and PKCα (Ser657), respectively.     

A Lectin from the Mussel Mytilus galloprovincialis Has a Highly Novel Primary Structure and Induces Glycan-mediated Cytotoxicity of Globotriaosylceramide-expressing Lymphoma Cells

A novel lectin structure was found for a 17-kDa α-d-galactose-binding lectin (termed “MytiLec”) isolated from the Mediterranean mussel, Mytilus galloprovincialis. The complete primary structure of the lectin was determined by Edman degradation and mass spectrometric analysis. MytiLec was found to consist of 149 amino acids with a total molecular mass of 16,812.59 Da by Fourier transform-ion cyclotron resonance mass spectrometry, in good agreement with the calculated value of 16,823.22 Da. MytiLec had an N terminus of acetylthreonine and a primary structure that was highly novel in comparison with those of all known lectins in the structure database. The polypeptide structure consisted of three tandem-repeat domains of ∼50 amino acids each having 45–52% homology with each other. Frontal affinity chromatography technology indicated that MytiLec bound specifically to globotriose (Gb3; Galα1–4Galβ1–4Glc), the epitope of globotriaosylceramide. MytiLec showed a dose-dependent cytotoxic effect on human Burkitt lymphoma Raji cells (which have high surface expression of Gb3) but had no such effect on erythroleukemia K562 cells (which do not express Gb3). The cytotoxic effect of MytiLec was specifically blocked by the co-presence of an α-galactoside. MytiLec treatment of Raji cells caused increased binding of anti-annexin V antibody and incorporation of propidium iodide, which are indicators of cell membrane inversion and perforation. MytiLec is the first reported lectin having a primary structure with the highly novel triple tandem-repeat domain and showing transduction of apoptotic signaling against Burkitt lymphoma cells by interaction with a glycosphingolipid-enriched microdomain containing Gb3.