RNAi and epigenetics: Pol IV is a matchmaker of small RNAs meeting with chromatin.
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Animals↗
Biomedical subjects
K Taira
Publications and source records attributed to K Taira.
siRNAs generated by recombinant human Dicer induce specific and significant but target site-independent gene silencing in human cells.
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Retraction Notice↗
Screening and determination of gene function using randomized ribozyme and siRNA libraries.
Rapid progress in the sequencing of the genomes of model organisms, such as the mouse, rat, nematode, fly, and Arabidopsis, as well as the human genome, has provided abundant sequence information, but functions of long stretches of these genomes remain to be determined. RNA-based technologies hold promise as tools that allow us to identify the specific functions of portions of these genomes. In particular, catalytic RNAs, known also as ribozymes, can be engineered for optimization of their activities in the intracellular environment. The introduction of a library of active ribozymes into cells, with subsequent screening for phenotypic changes, can be used for the rapid identification ofa gene function. Ribozyme technology complements another RNA-based tool for the determination of gene function, which is based on libraries of small interfering RNAs (siRNAs).
Animals↗
Small interfering RNA expression vector targeting hypoxia-inducible factor 1 alpha inhibits tumor growth in hepatobiliary and pancreatic cancers.
Hepatobiliary and pancreatic carcinomas are hypovascular tumors that can proliferate under hypoxic conditions. Recent reports have demonstrated that hypoxia-inducible factor 1 alpha (HIF1alpha) plays an important role in the survival of these cancers. Given these findings, the inhibition of the HIF1alpha pathway might prove to be a powerful tool in the treatment of these cancers. To inhibit HIF1alpha expression, we used small interference RNA (siRNA) expression vectors in this study. The transient transfection of siRNA expression vectors significantly reduced both HIF1alpha mRNA levels (13% of control) and protein levels (41% of control) and significantly inhibited the growth of cancer cell lines (P<0.05). VEGF, Glut1, and aldorase A expressions were also significantly reduced by transfection with these vectors (P<0.05), and we found that these vectors induced apoptosis but not cell cycle arrest. In a subcutaneous tumor model using nude mice, transfected MIA PaCa-2 cells, stably expressing siRNAs, barely formed tumors compared to control (P<0.05). This study thus demonstrates the usefulness of siRNA expression vector in targeting HIF1alpha and points to a potential clinical role in the treatment of pancreatic and hepatobiliary carcinomas.
Animals↗
Intracellular-diced dsRNA has enhanced efficacy for silencing HCV RNA and overcomes variation in the viral genotype.
RNA interference (RNAi) can be used to inhibit viral replication in mammalian cells and therefore could be a powerful new antiviral therapy. Small interfering RNA (siRNA) may be effective for RNAi, but there are some technical problems that must be solved in each case, for example, predicting the effective siRNA target site and targeting heterogeneous sequences in a virus population. We show here that diced siRNA generated from long double-stranded RNA (dsRNA) is highly effective for inducing RNAi in HuH-7 cells harboring hepatitis C virus (HCV) replicons and can overcome variations in the HCV genotype. However, in mammalian cells, long dsRNA induced an interferon response and caused cell death. Here we describe an improvement of this method, U6 promoter-driven expression of long hairpin-RNA with multiple point mutations in the sense strand. This can efficiently silence HCV RNA replication and HCV protein expression without triggering the interferon response or cell death normally caused by dsRNA. In conclusion, intracellular-diced dsRNA efficiently induces RNAi, and, despite the high rate of mutation in HCV, it should be a feasible therapeutic strategy for silencing HCV RNA.
Base Sequence↗
Changes in the number of Merkel cells with the hair cycle in hair discs on rat back skin.
BACKGROUND: Hair discs are known to contain a large number of Merkel cells and are ideal for investigating Merkel cell biology. Hair follicles, which are important elements of hair discs, undergo unique cyclical morphological and biological changes. OBJECTIVES: To define the relationships between the number and the morphology of Merkel cells within the hair disc in association with the hair cycle on rat back skin. METHODS: Merkel cells in hair discs were observed three-dimensionally using immunohistochemistry. Epidermal sheets were incubated with monoclonal murine antibody to CK20. As a result, Merkel cells in hair discs were clearly demonstrated as whole shapes and were counted under a light microscope. RESULTS: Merkel cells in hair discs increased during the early to middle phase of anagen and decreased during the middle phase of anagen to catagen and telogen in perinatal and postnatal rat back skin. We observed the morphological variation of Merkel cells in hair discs of rat back skin, and consequently divided them into two subtypes at the light microscopic level: the oval type and the dendritic type. The number of oval-type Merkel cells was not markedly affected by the hair cycle. In contrast, the number of dendritic-type Merkel cells markedly changed with the hair cycle. CONCLUSIONS: This difference of the hair cycle dependency between oval and dendritic-type Merkel cells suggests some functional differences, such as a secretory function, related to the hair cycle.
Aging↗
Gene therapy for human small-cell lung carcinoma by inactivation of Skp-2 with virally mediated RNA interference.
Increase of Skp-2, which is involved in the degradation of cell cycle regulators including p27Kip1, p21 and c-myc, is one of the important mechanisms for dysregulation of cell cycles in various cancers. We applied RNA interference (RNAi) for Skp-2 by using HIV-lentiviral or adenoviral vectors for a human small-cell lung carcinoma cell line with increased Skp-2 to evaluate RNAi strategy for cancer gene therapy. HIV-lentivirus-mediated RNAi for Skp-2 resulted in efficient inhibition of the in vitro cell growth of cancer cells with increased Skp-2 through the increase of p27Kip1 and p21, but no significant effect on the growth of cells without high Skp-2 expression. Furthermore, intratumoral administration of adenovirus siRNA vector for Skp-2 efficiently inhibited growth of established subcutaneous tumor on NOD/SCID mice. These results indicate that the Skp-2 RNAi may be a useful strategy for gene therapy of cancers with high Skp-2 expression.
Adenoviridae↗
[Nutritional status survey in 200 of the Korean and Han nationality elderly in Yanji].
OBJECTIVE: To assess the nutritional status and dietary intake of the Korean and Han nationality elderly in Yanji. METHODS: We selected 200 of the Korean and Han nationality adults aged 60 and older and measured their blood pressure. Dietary survey was performed with 24-hour dietary recall method. RESULTS: (1) The daily average intake of energy surpassed recommended nutrient intake (RNI) value in male and amounted RNI in women. In the male, Breakfast energy intake ratio was significantly lower and supper energy intake ratio was significantly higher than female. Supper energy intake in male with hypertension was significantly higher than normal blood pressure. (2) The daily average intake of fat in the Korean was significantly lower than in Han nationality (P < 0.01), and also was lower than RNI value. The daily average intake of carbohydrate in the Korean was significantly higher than in Han nationality (P < 0.01). (3) The daily average intake of protein exceed RNI value in the Korean male and was slightly lower than RNI value in Han male and both nationality women. The daily average intake of protein in the Korean males was significantly higher than in Han males (P < 0.01). The ratio of good protein was 35%-45% and bean protein exceed 15%. (4) The daily average intakes of calcium and vitamin A were only half RNI value and vitamin B2 lower than RNI values. CONCLUSION: The consumption of some nutrients among the Korean and Han nationality is inequality. The daily intake of calcium, vitamin A and vitamin B2 in elderly is seriously inadequate. Distribution of three meal energy is irrational and the high ratio of supper energy in male relates to hypertension.
Aged↗
Zoonotic risk of Toxocara canis infection through consumption of pig or poultry viscera.
The potential zoonotic risk of Toxocara canis infections from consumption of swine or poultry viscera containing larvae was assessed using a pig model. Two groups of six pigs were fed either fresh swine viscera (group FS) or poultry viscera (FP) containing around 3500 Toxocara larvae. Another two groups of six pigs were fed swine viscera (PS) or poultry viscera (PP) preserved at 4 degrees C for 1 week. All pigs were necropsied 14 days after the exposure. Liver white spots were counted and T. canis specific IgG antibodies were measured by ELISA. Larval burdens were assessed in the mesenteric lymph nodes, liver, lungs, brain, tongue, and eyes. All recipient pigs exhibited several white spots on the liver surface and detectable antibody levels. Larvae were recovered predominantly from the lungs, but also from the mesenteric lymph nodes and the liver, a few larvae were found in the brain and tongue of the pigs. Two larvae were found in the eyes of two pigs in group FS. Mean percentages of total larval recoveries in groups FS, FP, PS, and PP were 75.3, 63.6, 42.6, and 18.8%, respectively. Significantly higher numbers of larvae were recovered from pigs given swine viscera than pigs given poultry viscera. The preservation at 4 degrees C for 1 week caused a significant reduction in the larval infectivity overall, nevertheless, the recoveries remained substantial. The fact that larvae migrating in swine or poultry organs and tissues have high infectivity in pigs even after preservation at 4 degrees C for 1 week, suggests that human infection with T. canis might easily occur following consumption of raw or undercooked dishes, either fresh or refrigerated, prepared from swine or poultry organs and tissues harbouring T. canis larvae.
Animal Feed↗
Evaluation of the anti-proliferative and anti-oxidative activities of leaf extract from in vivo and in vitro raised Ashwagandha.
Withania somnifera (Ashwagandha) is used in Indian traditional medicine, Ayurveda and is believed to have a variety of health promoting effects. Molecular mechanisms and pathways underlying these effects have not been studied. We tried to characterize various activities of leaf extract of Ashwagandha (Lash) raised in the field and in the laboratory. We found that the Lash from field-raised plants has a significant anti-proliferative activity in human tumorigenic cells. However, it did not impart any protection against the oxidative damage caused by high glucose and hydrogen peroxide to human tumor cells suggesting that it can be used as an anti-tumor, but not as an anti-oxidant, substance.
Antineoplastic Agents, Phytogenic↗
Autoimmune thyroid disease in anti-Ro/SS-A-positive children with annular erythema: report of two cases.
Anti-Ro/SS-A-associated recurrent annular erythema is a rare disorder, and represents a cutaneous manifestation of primary Sjögren's syndrome (SS). We report two childhood cases complicated with autoimmune thyroid disease, one with Graves' disease and the other with autoimmune thyroiditis. Both children were positive for anti-Ro/SS-A and anti-La/SS-B antibodies. One patient was lacking clinical SS with objective evidence of salivary gland involvement, while the other was diagnosed with primary SS. Our observation suggests that autoimmune thyroid disease in the subset of anti-Ro/SS-A-positive children with annular erythema might occur with similar frequency to that in adult primary SS.
Adolescent↗
The ARF-p53 senescence pathway in mouse and human cells.
Mouse and human cells have most frequently been used for studies that have led to the elucidation of various molecular pathways involved in senescence. The ARF-p53 pathway has been assigned as one of the major protagonists in these phenomena. ARF is an alternative reading frame protein encoded along with p16INK4A by the INK4a locus on human chromosome 9p21 and the corresponding locus on mouse chromosome 4. Whereas the mouse ARF (p19ARF) consists of 169 amino acids, the human ARF (p14ARF) consists of 132 amino acids, truncated at the C-terminus. Molecular studies on the regulation of ARF activity by its binding partners have revealed that mouse ARF protein, but not human ARF protein, interacts with a cytoplasmic protein, Pex19p. This interaction of mouse ARF with Pex19p results in its milder p53 activation function in mouse cells as compared to human cells and thus accounts, at least in part, for the weaker tumor surveillance and frequent immortalization of mouse cells.
Animals↗
Population dynamics of Toxocara canis in pigs receiving a single or multiple infection.
The population dynamics of Toxocara canis in pigs, and their immune response to a primary and a challenge infection, were studied by parasitological, haematological and serological parameters. Seventy pigs were divided into 4 groups; 35 pigs received a primary infection (group A), 15 pigs received both a primary and a challenge infection (group B), 15 pigs received the challenge infection only (group C), and 5 pigs served as helminth-free controls (group NC). A dose of 50,000 eggs was administered for the primary infection (day 0) and a dose of 10,000 eggs was given for the challenge infection (day 28). On days 7, 14, 21 and 28 p.i., 5 pigs of group A, and on days 35, 42 and 49 p.i., 5 pigs from each of groups A, B and C were necropsied. Numbers of recovered larvae varied widely among the 5 pigs of each group on all days of necropsy. Toxocara canis larvae were recovered predominantly from the lungs; migration of larvae to other organs or tissues from the lungs was restricted. In group A, the larval burden in the lungs peaked on day 14 p.i., and the larval densities decreased significantly over time. Thereafter, the majority of larvae were recovered from the lungs until the end of the experiment (day 49 p.i.). A few larvae were found in the muscles and brain until day 42 p.i., and 2 larvae were found in the eyes of 2 pigs on day 35 p.i. There was little evidence of protective immunity to a challenge infection in this experiment. The eosinophil levels tended not to increase in pigs receiving a challenge infection, in contrast to the challenge control pigs. The fact that T. canis larvae migrate and persist in the tissues of pigs for more than 1 month suggests a zoonotic risk in infected pigs. The relevance of these data to the population biology and immunology of porcine and human toxocarosis is discussed.
Animals↗
Mechanism of action of hammerhead ribozymes and their applications in vivo: rapid identification of functional genes in the post-genome era by novel hybrid ribozyme libraries.
A hammerhead ribozyme was demonstrated to be a metalloenzyme. By controlling the metal-binding ability of the hammerhead ribozyme in the presence or absence of a specific sequence of interest, we engineered an allosterically controllable ribozyme, designated the maxizyme. Hybrid ribozymes were then constructed by coupling the site-specific cleavage activity of a hammerhead ribozyme with the unwinding activity of an endogenous RNA helicase. This leads to extremely efficient cleavage of target mRNA, not only in vitro, but also in vivo, and eliminates one of the major problems arising in the application of ribozymes for cleavage of mRNA in vivo : that many target sites on the RNA were previously inaccessible to cleavage owing to secondary and/or tertiary structure formation. Since hybrid ribozymes can efficiently attack target sites within mRNA, libraries were made of hybrid ribozymes with randomized binding arms, which were then introduced into cells. This procedure made it possible to readily identify the relevant genes associated with a specific phenotype, such as in apoptosis and cancer metastasis pathways. This application of a randomized library of hybrid ribozymes represents a simple, yet powerful, method for the identification of genes associated with specific phenotypes in the post-genome era. Moreover, vector-based siRNA (short-interfering RNA for RNA interference, RNAi) can also be used for the creation of the libraries and for the subsequent confirmation of the identified genes, relevant in the examined phenotype.
Gene Expression Profiling↗
Mortalin: a potential candidate for biotechnology and biomedicine.
Mortalin is a novel member of the hsp70 family of proteins that exhibits a different staining pattern in normal and immortal cells. It was also cloned as glucose regulated protein, GRP75 and peptidebinding protein, PBP74. It has been assigned multiple functions ranging from stress response, intracellular trafficking, antigen processing, control of cell proliferation, differentiation and tumorigenesis. The present article compiles and reviews information on multiple sites and functions of mortalin. In view of its upregulation in many tumors and transcriptional inactivation function of p53, its potential use in biotechnology and biomedicine is discussed.
Animals↗
Molecular tryst peeping: detection of interactions between nonlabeled nucleic acids by fluorescence resonance energy transfer.
We have developed a new method for monitoring the interactions between nonlabeled RNAs that involves detection of fluorescence resonance energy transfer (FRET) between two DNA probes with different fluorescent label. The sequences of the probes are complementary to those of the RNAs. In this study, we examined the interaction between a portion of the LTR RNA of HIV-1 and the corresponding antisense RNA. The antisense RNA was designed not to bind to the fluorescent DNA without prior hybridization to the target RNA. A mixture of RNAs and DNA probes with fluorescent labels was fractionated by electrophoresis on a nondenaturing polyacrylamide gel and then the gel was analyzed with a fluorescence imaging analyzer. FRET was observed only in the presence of target RNA, antisense RNA, and both of the fluorescent DNA probes. This strategy should be useful for the detection of interactions between nucleic acids that cannot be subjected to chemical modification, such as RNA transcripts inside cells.
Base Sequence↗
A non-natural amino acid for efficient incorporation into proteins as a sensitive fluorescent probe.
A small and highly fluorescent non-natural amino acid that contains an anthraniloyl group (atnDap) was incorporated into various positions of streptavidin. The positions were directed by a CGGG/CCCG four-base codon/anticodon pair. The non-natural mutants were obtained in excellent yields and some of them retained strong biotin-binding activity. The fluorescence wavelength as well as the intensity of the anthraniloyl group at position 120 were sensitive to biotin binding. These unique properties indicate that the atnDap is the most suitable non-natural amino acid for a position-specific fluorescent labeling of proteins that is highly sensitive to microenvironmental changes.
Amino Acids↗
Significantly higher activity of a cytoplasmic hammerhead ribozyme than a corresponding nuclear counterpart: engineered tRNAs with an extended 3' end can be exported efficiently and specifically to the cytoplasm in mammalian cells.
Hammerhead ribozymes were expressed under the control of similar tRNA promoters, localizing transcripts either in the cytoplasm or the nucleus. The tRNA(Val)-driven ribozyme (tRNA-Rz; tRNA with extra sequences at the 3' end) that has been used in our ribozyme studies was exported efficiently into the cytoplasm and ribozyme activity was detected only in the cytoplasmic fraction. Both ends of the transported tRNA-Rz were characterized comprehensively and the results confirmed that tRNA-Rz had unprocessed 5' and 3' ends. Furthermore, it was also demonstrated that the activity of the exported ribozyme was significantly higher than that of the ribozyme which remained in the nucleus. We suggest that it is possible to engineer tRNA-Rz, which can be exported to the cytoplasm based on an understanding of secondary structures, and then tRNA-driven ribozymes may be co-localized with their target mRNAs in the cytoplasm of mammalian cells.
Base Sequence↗