Biomedical subjects
Publications and source records attributed to K Taira.
We present here a novel approach to connect phenotype and genotype in vitro by exploiting a strong interaction between RNA and protein. This strong interaction was used for the selection of functional proteins, such as dihydrofolate reductase (DHFR) and streptavidin, which were chosen as examples. The noncovalent but strong interaction should be useful as a novel tool for the future selection of functional proteins.
The substitution effect on hydrogen bond energy of the Watson-Crick type base pair between 9-methylguanine (G) and chemically modified 1-methylcytosine (CX) derivatives was evaluated by ab initio molecular orbital theory. A remarkable trend was observed in the substitution effect of the hydrogen bond stability: Cytosine derivatives possessing an electron-donating group form stable base pairs with guanine. However, both the hydrogen bond distance and the charge distribution were not good indexes for the hydrogen bond status in CX-G base pairing.
Multiple cell and stimulus specific apoptosis pathways have been identified (1) revealing a complexity of molecular events involved. Tumor necrosis factor (TNF alpha) is known to activate mitochondria dependent or independent pathways for cell death. These pathways converge to activate effector caspases, caspases 3, 6 and 7 (2). The intermediate events including the substrates and effectors however are not clearly understood. In this report, we have employed ribozyme technology to elucidate some of these intermediate events. We show here that Bid, a pro-apoptotic facilitator that plays an important role in the mitochondrial pathway, can be targeted by Bid-specific ribozymes. MCF7 breast carcinoma cells stably transfected with expression plasmids encoding active (but not inactive or mock) Bid ribozymes showed delayed response to TNF alpha. This was accompanied by decreased activation of caspases 7 and 9, but not of caspase 8. The data assign caspase 9 as an upstream activator of caspase 7 in TNF alpha-induced Bid-mediated apoptosis.
Hammerhead ribozymes have a metal-ion-binding motif in the conserved core region, and this motif can capture several kinds of metal ions. In order to determine what kind of metal ions can be recognized by the motif, we measured 2D 1H-1H NOESY spectra in the presence of various metal ions, such as Cd2+, Na+ and Co(NH3)6(3+) ions, as well as in the absence of these ions. We have completed the assignment of the resonances of the base protons and anomeric protons in all cases, and determined their chemical shift perturbations.
Interferon gamma (IFN gamma) is an important immunoreguratory protein and is secreted from CD4+, CD8+ T cells and NK cells. Expression of IFN gamma gene is regulated by either DNA-methylation or cis-elements of transcriptional factors such as AP-1, NFAT, AP-4, YY-1, ATF and GATA. At a putative ATF binding site in a promoter region of IFN gamma gene, the position from -59 to -43, is important for the IFN gamma promoter activity. Our gel-shift competition studies demonstrated that addition of antibodies against ATF-2 caused super-shifts of DNA-protein complexes. In addition, it was clarified that p300 was involved in DNA-protein complexes by in vitro "pull-down" experiments. Moreover, a co-expression of p300 and ATF-2 enhanced the promoter activity of IFN gamma gene. Thus, these results indicate that p300 and ATF-2 play critical rules in a basal transcription of IFN gamma gene in Jurkat T cells.
The ribosome display system is a very powerful too for in vitro screening of mRNAs that encode proteins (or peptides) with specific (known and unknown) functions. The ribosome display system depends on the stability of ribosome-mRNA complexes that have been achieved by the removal of a stop codon. An additional interaction we employed was between tandem fused MS2 coat protein (MSp) dimer and the RNA of its specific motif called a "C-variant" (or "Cv"). The MSp dimer and the Cv were placed at the N-terminal end of a nascent protein translated in vitro and the 5'-end of its mRNA, that might circularize the polysome complex and, as a result, further stabilize the ribosome-mRNA complex. The selectivity of mRNAs after selection was increased 2-fold by the introduction of the interaction between the MSp dimer and Cv.
The efficacy of two different cationic liposomes, Lipofectin and hemagglutinating virus of Japan (HVJ)-cationic liposomes, on nuclear uptake of fluorescence-labeled phosphorothioate oligodeoxyribonucleotide (S-ODN) by ligament scar fibroblasts and suppression of decorin mRNA expression when antisense decorin S-ODN was transferred was investigated. There was no significant difference in nuclear uptake of fluorescent ODN between the two methods. However, only HVJ-cationic liposomes had a significant effect on suppression of decorin mRNA expression levels. To address the discrepancy, the molecular integrity of the transferred ODN in the cells was assessed by analysis of fluorescence resonance energy transfer (FRET) within double-fluorescence-labeled S-ODN. More than 70% of the ODN transfected by HVJ-cationic liposomes remained intact within the nucleus at 20 h after transfection, while the majority of the ODN transferred by Lipofectin was degraded at this point. These results suggest a strong relationship between the nuclear integrity of transfected antisense ODN and its suppression of target mRNA expression.
Okinawans, who have a different ethnicity and food cultural history to other Japanese nationals, and an exceptional longevity have been studied at home and as migrant groups in Hawaii and Brazil. Biomarkers for fish and soy intake and intervention studies indicate that these foods, along with seaweed and green vegetables are candidates for chronic non-communicable disease prevention.
An 11-year-old girl was admitted to our hospital with complaint of disturbance of consciousness and muscle weakness. We diagnosed her as having meningoencephalitis because of the pleocytosis in the cerebrospinal fluid (CSF) and diffuse slow EEG waves. Laboratory tests in admission showed that serum passive hemagglutinin titer to Mycoplasma pneumoniae (M. pneumoniae) was 1:5,120, serum antibody titer to galactocerebroside (Gc) was 1:160, and CSF interleukin-6 (IL-6) level was 20,500 pg/ml, but a specific DNA to M. pneumoniae was not detected in CSF using the polymerase chain reaction. Cranial and whole spine MRI were unremarkable. These results suggest that anti-Gc antibody and IL-6 play some roles in the development of mycoplasmal central nervous system involvement.
Since the molecular identification of the low density lipoprotein receptor (LDLR), an ever increasing number of related proteins have been discovered. These receptors belonging to the LDLR family are thought to play key roles in lipoprotein metabolism in a variety of tissues, including the arterial wall. We have discovered that the expression of a 250-kDa mosaic LDLR-related protein, which we termed LR11 for the presence of 11 LDLR ligand-binding repeats, is markedly induced in smooth muscle cells in the hyperplastic intima of animal models used for the study of atherosclerosis. Here, we demonstrate that the human LR11, when overexpressed in hamster cells, binds and internalizes 39-kDa receptor-associated protein (RAP), an in vitro ligand for all receptors belonging to the LDLR family. Furthermore, LR11 binds the apolipoprotein E (apoE)-rich lipoproteins, beta-very low density lipoproteins (VLDLs), with a high affinity similar to that of other members, such as the LDLR and VLDL receptor. RAP and beta-VLDL compete with each other; however, other serum lipoproteins are not able to inhibit their binding. LR11 shows specific binding of apoE-enriched HDL prepared from human cerebrospinal fluid as well as of beta-VLDL, suggesting that the apoE content of lipoproteins is most likely important for mediating the high-affinity binding to the receptor. LR11-overexpressing cells are able to internalize and degrade the bound beta-VLDL; these cells also show increased accumulation of cholesteryl esters when incubated with beta-VLDL. Incubation for 48 hours with beta-VLDL of LR11-overexpressing cells, but not of control cells, promotes the appearance of numerous intracellular lipid droplets. Taken together, LR11, a mosaic LDLR family member whose expression in smooth muscle cells is markedly induced in atheroma, has all the properties of a receptor for the endocytosis of lipoproteins, particularly for the incorporation of apoE-rich lipoproteins.
Biotinylation of a protein generally involves chemical modification of a translated protein. Using this methodology, however, biotinylation at a specific position remains difficult. We investigated whether it would be possible to use an Escherichia coli initiator tRNA(fmet) aminoacylated with methionine biotinylated at the alpha-amino group to introduce a biotin tag specifically at the N terminus. We report here that a biotin tag could be incorporated into the green fluorescent protein (GFP) at the N-terminal site, in the presence of an E. coli initiator tRNA(fmet) aminoacylated with methionine biotinylated at the alpha-amino group. The biotinylated GFP was purified by simple monomeric streptavidin-agarose affinity column chromatography. Based on the total amount of GFP molecules, the purification yield and the biotin labelling efficiency of this system were approximately 7% and 10-20%, respectively, according to the densitometric analysis of Western blots. Judging from the results of a fluorescence imaging experiment, almost all the purified GFP molecules retained the native fluorescence activity. Importantly, the present results support the hypothesis that the E. coli initiator tRNA(fmet) aminoacylated with a relatively large substituent can be recognized by an E. coli ribosome and adequately placed at the P site to initiate translation.
We report a 43-year-old Japanese man with hypobetalipoproteinemia likely due to apolipoprotein (apo) B-76, a new truncation of apo B, and with homozygosity for the apo E2 isoform. He had no history suggestive of fat malabsorption and no sign of neurological disorder. His fasting baseline serum low-density lipoprotein (LDL) cholesterol and apo B levels were approximately half of normal. His plasma apo E level was elevated and its phenotype showed the E2/E2 homozygote. SDS-polyacrylamide gel electrophoresis of delipidated LDL fraction revealed a new truncated apo B, designated as apo B-76 according to the centile system of nomenclature. The postprandial lipid metabolism of the patient showed an almost normal response after fat loading.
We performed concomitant graft replacement for descending thoracic aortic aneurysm and pulmonary resection for squamous cell carcinoma of the left upper lobe in a 79-year-old man. The tumor reached the parietal pleura. No distance metastasis was found, and the tumor was diagnosed preoperatively as a stage IIB (N0, M0, T3) tumor. The descending thoracic aortic aneurysm was saccular, with greatest diameter being 55 mm, and extending from TH5 to TH8. A left upper lobectomy was performed, and after irrigation with a large volume of saline diluted with povidone iodine, graft replacement for the aortic aneurysm was performed under femoro-femoral partial bypass. To prevent postoperative graft infection, the greater omentum was dissected and placed over the resected pulmonary hilum and the graft. The patient's postoperative course was uneventful. There was no sign of infection, and the patient was discharged 1 month after surgery. Artificial graft wrapping with the greater omentum was useful for the prevention of the postoperative graft infection in this case of surgical treatment of lung cancer and descending thoracic aortic aneurysm.
Nonstructural protein 3 (NS3) of hepatitis C virus (HCV) is a trypsin-like protease and is essential for processing of viral polyprotein. Accordingly, it is a potential target for anti-HCV drugs. Recently we could isolate RNA aptamers (G9-I, II, and III) which bind and inhibit NS3 protease using in vitro selection strategy. In addition, G9-I aptamer showed noncompetitive inhibition. In order to elucidate the binding site of G9-I aptamer in NS3 protease domain (deltaNS3), we carried out alanine scanning mutagenesis at positive charged residues on the surface of deltaNS3. The result of binding analysis by surface plasmon resonance measurements and protease inhibition assay clarified that Arg161 as well as Arg130 of deltaNS3 are essential for interaction with G9-I aptamer. This region appears to be a potential targeting site for anti-HCV drugs.
A glucocorticoid-induced target gene expression system was used to control the expression of the uidA gene, whose product was beta-glucuronidase (GUS), in tobacco BY-2 cell suspension culture. This targeting system showed quick, sensitive, and reversible response to dexamethazone (DEX), an artificial glucocorticoid hormone. Addition of DEX greatly and quickly enhanced uidA gene expression, whose level was as high as that under the control of the CaMV 35S promoter whereas in the absence of DEX, the GUS specific activity was suppressed to be as low as that of nontransformed BY-2 cells. The dilution of DEX decreased GUS specific activity showing that the concentration of DEX plays a major role in controlling the expression level of the target. The use of the glucocorticoid-induced system in plant cell suspension culture was demonstrated to precisely control target gene expression.
RNA helicase A (RHA) has two double-stranded (ds) RNA-binding domains (dsRBD1 and dsRBD2). These domains are conserved with the cis-acting transactivation response element (TAR)-binding protein (TRBP) and dsRNA-activated protein kinase (PKR). TRBP and PKR are involved in the regulation of HIV-1 gene expression through their binding to TAR RNA. This study shows that RHA also plays an important role in TAR-mediated HIV-1 gene expression. Wild-type RHA preferably bound to TAR RNA in vitro and in vivo. Overexpression of wild type RHA strongly enhanced viral mRNA synthesis and virion production as well as HIV-1 long terminal repeat-directed reporter (luciferase) gene expression. Substitution of lysine for glutamate at residue 236 in dsRBD2 (RHA(K236E)) reduced its affinity for TAR RNA and impaired HIV-1 transcriptional activity. These results indicate that TAR RNA is a preferred target of RHA dsRBDs and that RHA enhances HIV-1 transcription in vivo in part through the TAR-binding of RHA.
The appropriate folding of catalytic RNA is a prerequisite for effective catalysis. A novel ribozyme, the maxizyme, has been generated and its activity can be controlled allosterically. The maxizymes work both in vitro and in vivo indicating the potential utility of this novel class of ribozyme as a gene-inactivating agent with a biosensor function.
Postprandial hyperlipidemia is frequently accompanied with intra-abdominal visceral accumulation in human subjects. We have found that the decreased lipoprotein lipase (LPL) mass and activity is negatively associated with the amount of visceral fat accumulation. Here, we studied the postprandial hyperlipidemia using the OLETF rat, a model with visceral obesity, in order to clarify the molecular mechanism causing postprandial hyperlipidemia accompanied with visceral obesity. At the same age of 32 weeks, the OLETF rats showed obviously higher plasma leptin, total cholesterol, triglyceride, and HDL-cholesterol levels than the control LETO rats, although the plasma glucose level was not significantly different. Fat-loading test revealed the delayed metabolism of exogenous fat in the OLETF rats compared to the LETO rats, similar to human subjects with visceral obesity. In the obese rats, plasma levels of LPL mass and activities were 60 and 49% of control rats. The expression of LPL gene was decreased in subcutaneous adipose tissues and skeletal muscle of OLETF rats to 40 and 52% compared to those of LETO rats. In OLETF rats, plasma tumor necrosis factor-alpha (TNF-alpha) and insulin levels were increased to 2.0- and 2.3-folds compared to those in control rats. Furthermore, plasma insulin and TNF-alpha levels in OLETF rats were negatively correlated with the expression levels of LPL gene in subcutaneous fat and muscle. These results indicate that decreased LPL mass and activity in the animal model with visceral obesity is possibly caused by decreased expression of LPL gene in tissues mediated by the increased levels of insulin and TNF-alpha. The different expression of LPL gene in tissues associated with the increased levels of insulin and TNF-alpha possibly elucidate the underlying mechanisms involving the postprandial hyperlipidemia observed in visceral obesity.