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Biomedical subjects

Kazunari Taira

Publications and source records attributed to Kazunari Taira.

At least 19 recordsLinked to original sources
PubMed2007

Genome-wide sreening by using small-interfering RNA expression libraries.

RNA interference (RNAi) is an evolutionarily conserved phenomenon in which gene expression is silenced by double-stranded RNA (dsRNA) in a sequence-specific manner. This technology has the potential to affect all aspects of target discovery and validation. With the completion of the human genome, it is now possible to design small-interfering RNA (siRNA) libraries targeting every human gene. Specific siRNAs, libraries containing a pathway, gene family, or gene set of interest, are expected to unsecure new targets in pathways of therapeutic interest. Here, we highlight the potential of siRNA screens for target identification by using cell-based assays.

Apoptosis↗
PubMed2007

Hammerhead ribozyme-based target discovery.

With the accumulation of vast amounts of data as a result of the sequencing of the human genome, it is necessary to identify human genes that are involved in various cellular, developmental, and disease-related processes and to clarify their functions and potential utility as targets in the treatment of disease. Identification methods based on the use of hammerhead and hairpin ribozymes have received increasing attention as possible tools for the rapid identification of key genes involved in biological processes. This chapter describes the method known as gene-discovery by a hammerhead ribozyme library for elucidation of the gene function. Use of this technology has already revealed new insights into several important biological phenomena.

Amino Acid Sequence↗
PubMed2007

Ribosome-inactivation display system.

We present a novel strategy for the connection of phenotype and genotype in vitro that can be used for the selection of functional proteins. The strategy involves the generation of a stable complex among a ribosome, an messenger RNA and its translated protein, without removal of the termination codon, as a result of the action of the ricin A chain during translation. The technique requires no transfection, no chemical synthesis, no ligation, and no removal of the termination codon. Thus, our novel ribosome-inactivation display system should provide, without loss of the pool population, a reliable, simple, and robust selection system for the in vitro evolution of the properties of proteins in a predictable direction by a combination of randomization and appropriate selection strategies.

Amino Acid Sequence↗
PubMed2006

Myosin motor Myo1c and its receptor NEMO/IKK-gamma promote TNF-alpha-induced serine307 phosphorylation of IRS-1.

Tumor necrosis factor-alpha (TNF-alpha) signaling through the IkappaB kinase (IKK) complex attenuates insulin action via the phosphorylation of insulin receptor substrate 1 (IRS-1) at Ser307. However, the precise molecular mechanism by which the IKK complex phosphorylates IRS-1 is unknown. In this study, we report nuclear factor kappaB essential modulator (NEMO)/IKK-gamma subunit accumulation in membrane ruffles followed by an interaction with IRS-1. This intracellular trafficking of NEMO requires insulin, an intact actin cytoskeletal network, and the motor protein Myo1c. Increased Myo1c expression enhanced the NEMO-IRS-1 interaction, which is essential for TNF-alpha- induced phosphorylation of Ser307-IRS-1. In contrast, dominant inhibitory Myo1c cargo domain expression diminished this interaction and inhibited IRS-1 phosphorylation. NEMO expression also enhanced TNF-alpha-induced Ser307-IRS-1 phosphorylation and inhibited glucose uptake. In contrast, a deletion mutant of NEMO lacking the IKK-beta-binding domain or silencing NEMO blocked the TNF-alpha signal. Thus, motor protein Myo1c and its receptor protein NEMO act cooperatively to form the IKK-IRS-1 complex and function in TNF-alpha-induced insulin resistance.

3T3-L1 Cells↗
PubMed2006

TRAIL-induced cell death cooperates with IFN-gamma activation in the graft-versus-tumor effect against colon tumors.

The graft-versus-tumor (GVT) effect that occurs following allogeneic bone marrow transplantation (BMT) and donor lymphocyte infusion (DLI) is currently being subjected to intensive investigation because of clinical evidence for GVT efficacy against leukemia. In this report, we investigate the efficacy and molecular mechanisms of GVT against solid tumors, using a modification of the mouse parent-to-F1 BMT model. Mouse Colon26 cells in which tumor necrosis factor related apoptosis-inducing ligand (TRAIL) receptor expression was stably knocked down were transplanted to investigate the role of the TRAIL-TRAIL receptor system in the GVT effect. In addition, Fas ligand-(FasL) deficient mice on a C57BL6 (B6) background were used as donors, to determine the significance of the Fas-FasL system for the antitumor effect. The group that received B6 DLI followed by preconditioning with 950 rad irradiation underwent tumor reduction associated with the induction of IFN-gamma, TRAIL and tumor-cell apoptosis. In vitro cultured Colon26 cells were resistant to TRAIL but susceptible to the combination of IFN-gamma and TRAIL in a TRAIL-dose-dependent manner. The infusion of lymphocytes from FasL-defective donors reduced the tumor progression, although efficacy was decreased in the TRAIL receptor knockdown tumors but not in wild-type ones, compared with infusion of B6-derived lymphocytes. The findings indicate that GVT activity against subcutaneous colon tumors is efficiently induced by preconditioning with irradiation and allogeneic DLI, and that TRAIL and IFN-gamma act cooperatively in the antitumor effect.

Animals↗
PubMed2006

Cutting Edge: Lentiviral short hairpin RNA silencing of PTEN in human mast cells reveals constitutive signals that promote cytokine secretion and cell survival.

Engagement of the FcepsilonRI expressed on mast cells induces the production of phosphatidylinositol 3, 4, 5-trisphosphate by PI3K, which is essential for the functions of the cells. PTEN (phosphatase and tensin homologue deleted on chromosome ten) directly opposes PI3K by dephosphorylating phosphatidylinositol 3, 4, 5-trisphosphate at the 3' position. In this work we used a lentivirus-mediated short hairpin RNA gene knockdown method to study the role of PTEN in CD34(+) peripheral blood-derived human mast cells. Loss of PTEN caused constitutive phosphorylation of Akt, p38 MAPK, and JNK, as well as cytokine production and enhancement in cell survival, but not degranulation. FcepsilonRI engagement of PTEN-deficient cells augmented signaling downstream of Src kinases and increased calcium flux, degranulation, and further enhanced cytokine production. PTEN-deficient cells, but not control cells, were resistant to inhibition of cytokine production by wortmannin, a PI3K inhibitor. The findings demonstrate that PTEN functions as a key regulator of mast cell homeostasis and FcepsilonRI-responsiveness.

Calcium↗
PubMed2006

Down-regulation of endogenous Wt1 expression by Sry transgene in the murine embryonic mesonephros-derived M15 cell line.

Wt1 is one of numerous candidate genes comprising the hypothetical chain of gene expression essential for male sex differentiation of the bipotential indifferent gonads during embryogenesis. However, the evidence in the literature is ambivalent regarding the position of Wt1 relative to Sry in this scheme; Wt1 might act either upstream or downstream of Sry. In the present study, the effects of Sry expression upon Wt1 were investigated using M15 cells (XX karyotype), which are derived from murine embryonic mesonephros and express endogenous Wt1. In 3 stably-transformed Sry-expressing M15 cell lines, we showed that the expression levels of the mRNAs coding for all 4 isoforms of the WT1 proteins were down-regulated. Similarly, Wnt 4 expression was down-regulated in these cell lines. Silencing of Sry in the transformed cell lines using ribozymes or short hairpin RNAs (shRNAs) resulted in elevated levels of Wt1 and Wnt4 expression. These results strongly indicate that Wt1 might be under the control of Sry during gonadal differentiation in the mouse. In electrophoretic mobility shift assays (EMSA), we demonstrated that the 3.7 kb 5'-upstream DNA stretch of Wt1 containing potential Sry binding sites was capable of forming molecular complexes with nuclear protein(s) from Sry expressing cells but not with those from control non-Sry expressing cells. In summary, our present results support the notion that Wt1 is located downstream of Sry and down-regulated by the sex determining gene. Although the precise biological meaning of the present findings have yet to be clarified, it is possible that Wt1 plays a dual role during gonadal differentiation, i. e., turning on Sry expression on one hand, and being down-regulated by its product, Sry, on the other, possibly forming a type of negative feed-back mechanism. Further work is needed to substantiate this view.

Animals↗
PubMed2006

Sequence-specific interference by small RNAs derived from adenovirus VAI RNA.

A virus-associated RNA (VAI) of adenoviruses is a cytoplasmic non-coding RNA and it plays an important role for viral replication in infected cells. VAI RNA transcripts, produced by RNA polymerase III (pol III), form tightly structured stems, which confer resistance to cellular defense systems. We demonstrate here that small RNAs of approximately 22 nucleotides are produced from a terminal stem region but not from an apical stem of VAI RNA. We determined the processing sites of VAI RNA by S1 nuclease mapping and further confirmed that the processed small RNA can act as small interfering RNAs (siRNAs) or as microRNAs (miRNAs) in transient transfection assays and during viral infection. Our data demonstrate that non-coding RNAs synthesized by pol III can be substrates for Dicer, and diced small RNAs might regulate cellular phenomena as siRNAs and miRNAs.

Adenoviridae↗
PubMed2006

Effective inhibition of cell growth and invasion of melanoma by combined suppression of BRAF (V599E) and Skp2 with lentiviral RNAi.

p27Kip1 that regulates the G1/S transition of cell cycle and inhibits Rho A signaling is frequently lost in several cancers leading to the deregulation of cell growth and cell motility. Mitogen-activated protein kinases (MAPK) regulate the export of p27Kip1 from nucleus to cytoplasm, followed by the degradation with proteases. Skp-2, a subunit of an SCF ubiquitin-protein ligase complex responsible for the ubiquitination of p27Kip1, is upregulated frequently in several cancers, leading to the decrease of p27Kip1. We applied human immunodeficiency virus (HIV) lentivirus-mediated RNA interference (RNAi) to melanoma cells with the BRAF mutation (V599E) and overexpressed Skp-2 and found that the simultaneous suppression of these activated oncogenes resulted in the effective inhibition of in vitro cell growth and invasive ability of melanoma cells accompanied by the additional increase of p27Kip1. Our results suggest that gene therapy against melanoma with the enhanced MAPK and ubiquitin-proteasomal pathways could be a specific and effective therapeutic strategy for cancers.

Activin Receptors, Type I↗
PubMed2006

Screening of siRNA target sequences by using fragmentized DNA.

BACKGROUND: RNA interference (RNAi) has become a powerful tool in silencing target genes in various organisms. In mammals, RNAi can be induced by using short interfering RNA (siRNA). The efficacy of inducing RNAi in mammalian cells by using siRNA depends very much on the selection of the target sequences. METHODS: We developed an siRNA target sequence selection system by first constructing parallel-type siRNA expression vector libraries carrying siRNA expression fragments originating from fragmentized target genes, and then using a group selection system. For a model system, we constructed parallel-type siRNA expression vector libraries against DsRed and GFP reporter genes. RESULTS: We carried out the first screening of groups containing more than 100 random siRNA expression plasmids in total for each target gene, and successfully obtained target sequences with very strong efficacy. Furthermore, we also obtained some clones that express dsRNAs of various lengths that might induce cytotoxicity. CONCLUSIONS: This system should allow us to perform screening for powerful target sequences, by including all possible target sequences for any gene, even without knowing the whole sequence of the target gene in advance. At the same time, target sequences that should be avoided due to cytotoxicity can be identified.

Base Sequence↗
PubMed2006

Ligand-targeted delivery of therapeutic siRNA.

RNA interference (RNAi) is a post-transcriptional gene-silencing phenomenon that is triggered by double-stranded RNA (dsRNA). Since many diseases are associated with the inappropriate production of specific proteins, attempts are being made to exploit RNAi in a clinical settings. However, before RNAi can be exploited as therapeutically, several obstacles must be overcome. For example, small interfering RNA (siRNA) is unstable in the blood stream so any effects of injected siRNA are only transient. Accordingly, methods must be developed to prolong its activity. Furthermore, the efficient and safe delivery of siRNA into target tissues and cells is critical for successful therapy. Any useful delivery method should be designed to target siRNA to specific cells and to promote gene-silencing activity once the siRNA is inside the cells. Recent chemical modifications of siRNA have overcome problems associated with the instability of siRNA, and various ligands, including glycosylated molecules, peptides, proteins, antibodies and engineered antibody fragments, appear to be very useful or have considerable potential for the targeted delivery of siRNA. The use of such ligands improves the efficiency, specificity and, as a consequence, the safety of the corresponding delivery systems.

Animals↗
PubMed2006

Molecular design and delivery of siRNA.

Double stranded short interfering RNAs (siRNAs) mediate gene silencing in a sequence specific manner. By virtue of their specific gene silencing activity and owing to the recent discoveries on their plasmid and virus driven expression, siRNAs are being widely adopted in research and therapeutics. Efforts were made to optimize the siRNA expression system for the application in therapy. One major obstacle in developing RNA interference (RNAi) therapy is the delivery of siRNAs to the target cells. Combination of novel molecular targeting technologies, such as recombinant protein technology and ribosome display technology, will enable to deliver gene silencing agents to target cells specifically and efficiently.

Animals↗
PubMed2006

Correlation of structure and activity of short hairpin RNA.

To explore the relationship between the structure of the precursor of short hairpin RNA (shRNA) and the activity of the shRNA-expression vector, 32 kinds of vectors, which expressed shRNAs with different flanking sequences, were tested. Addition of the poly(A) enhanced the activity of shRNA driven by U6 promoter. The activity of poly (A) tagged U6 promoter driven shRNA was lower than that of the CMV promoter driven constructs, in spite of the identical sequence of both transcripts. The result indicated that it is not only the structure but also the cascade of the transcripts that affects the RNAi activity.

Base Sequence↗
PubMed2006

Involvement of template-activating factor I/SET in transcription of adenovirus early genes as a positive-acting factor.

The adenovirus genome complexed with viral core protein VII (adenovirus DNA-protein VII complex) at least is the bona fide template for transcription of adenovirus early genes. It is believed that the highly basic protein VII, like cellular histones, is a negative regulator for genome functions. Analyses with in vitro replication and transcription systems using the adenovirus DNA-protein VII complex have revealed that remodeling of the complex is crucial for efficient DNA replication and transcription. We identified host acidic proteins, template-activating factor I (TAF-I), TAF-II, and TAF-III as stimulatory factors for replication from the adenovirus DNA-protein VII complex. Recently, it was reported that the adenovirus DNA interacts with TAF-I and pp32, another host acidic protein (Y. Xue, J. S. Johnson, D. A. Ornelles, J. Lieberman, and D. A. Engel, J. Virol. 79:2474-2483, 2005). We found that TAF-I interacts and colocalizes with protein VII in adenovirus-infected cells during the early phases of infection, but pp32 does not. Although pp32 had the potential ability to interact with protein VII, pp32 did not remodel the adenovirus DNA-protein VII complex in vitro. Small interfering RNA-mediated knockdown of TAF-I expression leads to the delay of the transcription timing of early genes. These results provide evidence that TAF-I plays an important role in the early stages of the adenovirus infection cycle.

Adenoviridae↗