Development of defective and persistent Sendai virus vector: a unique gene delivery/expression system ideal for cell reprogramming.

The ectopic expression of transcription factors can reprogram differentiated tissue cells into induced pluripotent stem cells. However, this is a slow and inefficient process, depending on the simultaneous delivery of multiple genes encoding essential reprogramming factors and on their sustained expression in target cells. Moreover, once cell reprogramming is accomplished, these exogenous reprogramming factors should be replaced with their endogenous counterparts for establishing autoregulated pluripotency. Complete and designed removal of the exogenous genes from the reprogrammed cells would be an ideal option for satisfying this latter requisite as well as for minimizing the risk of malignant cell transformation. However, no single gene delivery/expression system has ever been equipped with these contradictory characteristics. Here we report the development of a novel replication-defective and persistent Sendai virus (SeVdp) vector based on a noncytopathic variant virus, which fulfills all of these requirements for cell reprogramming. The SeVdp vector could accommodate up to four exogenous genes, deliver them efficiently into various mammalian cells (including primary tissue cells and human hematopoietic stem cells) and express them stably in the cytoplasm at a prefixed balance. Furthermore, interfering with viral transcription/replication using siRNA could erase the genomic RNA of SeVdp vector from the target cells quickly and thoroughly. A SeVdp vector installed with Oct4/Sox2/Klf4/c-Myc could reprogram mouse primary fibroblasts quite efficiently; ∼1% of the cells were reprogrammed to Nanog-positive induced pluripotent stem cells without chromosomal gene integration. Thus, this SeVdp vector has potential as a tool for advanced cell reprogramming and for stem cell research.

Optimization of the virus concentration method using polyethyleneimine-conjugated magnetic beads and its application to the detection of human hepatitis A, B and C viruses.

To enhance the sensitivity of virus detection by polymerase chain reaction (PCR) and reverse transcription PCR (RT-PCR), a novel virus concentration method using polyethyleneimine (PEI)-conjugated magnetic beads was developed in our previous study. However, several viruses could not be concentrated by this method. In this paper, the conditions of virus concentration were optimized to concentrate a wide range of viruses more efficiently. The PEI beads adsorbed viruses more efficiently than other cationic polymers, and the optimum virus concentration was obtained under weak acidic conditions. Mass spectrometric analysis revealed that several serum proteins, such as complement type 3, complement type 4 and immunoglobulin M (IgM), were co-adsorbed by the PEI beads, suggesting that the beads may adsorb viruses not only by direct adsorption, but also via immune complex formation. This hypothesis was confirmed by the result that poliovirus, which PEI beads could not adsorb directly, could be concentrated by the beads via immune complex formation. On the other hand, hepatitis A (HAV) and hepatitis C (HCV) viruses were adsorbed directly by PEI beads almost completely. Like poliovirus, hepatitis B virus (HBV) was concentrated efficiently by the addition of anti-HBV IgM. In conclusion, virus concentration using PEI beads is a useful method to concentrate a wide range of viruses and can be used to enhance the sensitivity of detection of HAV, HBV and HCV.

The role of NADRIN, a Rho GTPase-activating protein, in the morphological differentiation of astrocytes.

Reorganization of the actin cytoskeleton caused by inactivation of the Rho GTPase RhoA is critical for the morphological differentiation of astrocytes into process-bearing stellate cells. The molecular mechanisms underlying the RhoA inactivation and, in particular, the factors that inactivate RhoA, remain to be elucidated. We show here that the expression of a GTPase-activating protein (GAP) for Rho GTPases, neuron-associated developmentally regulated protein (NADRIN) also known as RICH and ARHGAP17, was significantly increased in stellate astrocytes and induced expression of NADRIN accelerated the morphological differentiation of cultured astrocytes into stellate cells. A GAP activity-negative mutant or truncated forms of NADRIN failed to induce the stellation. Immunoprecipitation analyses revealed that, in response to inductive signals such as dibutyryl cyclic AMP and epidermal growth factor, NADRIN formed a complex with ezrin-radixin-moesin (ERM) protein by interacting with ERM-binding phosphoprotein 50 via its carboxy-terminal PSD95/DlgA/ZO-1-binding motif. We also showed that NADRIN formed a dimer via the interaction between the amino- and carboxy-terminal domains, which was disrupted in response to the inductive signals. These results suggest that the inductive signals cause the structural change of NADRIN, which allows NADRIN to associate with the ERM protein complex, where it inactivates RhoA and leads to the morphological differentiation of astrocytes.

Identification and functional characterization of nadrin variants, a novel family of GTPase activating protein for rho GTPases.

Nadrin is a GTPase-activating protein (GAP) for the rho family of GTPases that controls Ca2+-dependent exocytosis in nerve endings. In this study, three novel splice variants of nadrin were identified and the variants were designated as nadrin-102, -104, -116 and -126 according to their relative molecular masses. All nadrin variants share the GAP domain, coiled-coil domain, serine/threonine/proline-rich domain, SH3-binding motif, and a successive repeat of 29 glutamines. Tissue distribution analyses using polyclonal antibodies that can discriminate each variant showed that the expression of nadrin-102, -104 and -116 was dominant in neuronal tissues and correlates well with the differentiation of neurons while nadrin-126 was strongly expressed in embryonic brain. Expression of nadrin-116 in PC12 cells strongly inhibited NGF-dependent neurite outgrowth and this effect was dependent on its GAP activity. In contrast, no significant effect on either cell morphology or neurite outgrowth was observed with other variants. All variants showed punctate appearance throughout the cytoplasm, while the 66-kDa carboxyl-terminal fragment of nadrin-102 and/or nadrin-116 was localized to the nucleus and its nuclear translocation was accelerated by NGF-induced differentiation of the cells. These results suggested that nadrin variants are different in their ability to regulate rho-mediated signaling and that, in addition to being a GTPase-activating protein, nadrin-102 and -116 have other distinct functions in the nucleus of the cell, implying a possible role in the cross-talk between the cytoskeleton and the nucleus.