Biomedical subjects

Tomoko Kubo

Publications and source records attributed to Tomoko Kubo.

6 recordsLinked to original sources

PubMed2005

Choice of the adequate detection time for the accurate evaluation of the efficiency of siRNA-induced gene silencing.

RNA interference (RNAi) mediated by small interfering RNA (siRNA) has become a popular tool of examining the function of various genes. However, many studies have failed to identify any inhibitory effect of the siRNAs on the expression of the target gene, even though the siRNA being tested had been designed sequence-specifically. In order to determine if this failure is due to the incorrect choice of observation time rather than that of the target site of the gene of interest, this study examined the RNAi efficiency of a vector-driven siRNA targeting two different reporter proteins, EGFP and d2EGFP, whose targeted sequences were identical but the half-lives within the cells differed remarkably from each other (>24h versus 2h), during the time course after transfection. The EGFP expression levels in both cells were reduced in time-dependent manner but the reduction patterns were quite different from each other. The RNAi efficiency varied among the different observation time points and the time required for the maximum RNAi efficiency was proportional to the half-life of the target protein. Stable knocked down cell lines for EGFP expression were then established and the reduced EGFP expression levels in these cell lines were retained for a long period. These results suggest that the choice of an adequate observation time or the establishment of stable knocked down cells by antibiotic selection might be required for making an accurate evaluation of the RNAi effect on the target protein possessing a long half-life.

Animals↗

PubMed2004

Delta-short consensus repeat 4-decay accelerating factor (DAF: CD55) inhibits complement-mediated cytolysis but not NK cell-mediated cytolysis.

NK cells play a critical role in the rejection of xenografts. In this study, we report on an investigation of the effect of complement regulatory protein, a decay accelerating factor (DAF: CD55), in particular, on NK cell-mediated cytolysis. Amelioration of human NK cell-mediated pig endothelial cell (PEC) and pig fibroblast cell lyses by various deletion mutants and point substitutions of DAF was tested, and compared with their complement regulatory function. Although wild-type DAF and the delta-short consensus repeat (SCR) 1-DAF showed clear inhibition of both complement-mediated and NK-mediated PEC lyses, delta-SCR2-DAF and delta-SCR3-DAF failed to suppress either process. However, delta-SCR4-DAF showed a clear complement regulatory effect, but had no effect on NK cells. Conversely, the point substitution of DAF (L147 x F148 to SS and KKK(125-127) to TTT) was half down-regulated in complement inhibitory function, but the inhibition of NK-mediated PEC lysis remained unchanged. Other complement regulatory proteins, such as the cell membrane-bound form factor H, fH-PI, and C1-inactivator, C1-INH-PI, and CD59 were also assessed, but no suppressive effect on NK cell-mediated PEC lysis was found. These data suggest, for DAF to function on NK cells, SCR2-4 is required but no relation to its complement regulatory function exists.

Amino Acid Substitution↗

PubMed2004

Bisphenol A, an environmental endocrine-disrupting chemical, inhibits hypoxic response via degradation of hypoxia-inducible factor 1alpha (HIF-1alpha): structural requirement of bisphenol A for degradation of HIF-1alpha.

Bisphenol A (BpA), an endocrine-disrupting chemical, is known to be a xenoestrogen and to affect the reproductive functions of animals. Recent reports have documented BpA-induced developmental abnormalities in the neuronal systems of humans and animals, and these effects appear to be non-estrogenic. In this study, we found that BpA inhibited the hypoxic response of human hepatoma cells. The expression of hypoxic response genes such as the erythropoietin (EPO) gene is done via a hypoxia inducible factor 1 (HIF-1)-dependent signaling pathway. To investigate possible structural requirements for this inhibitory effect, several BpA analogs were synthesized and added to this system. The blocking of two phenol groups in BpA did not change the effect, but the inhibition completely disappeared by the removal of two central methyl groups in BpA (the resulting compound is designated BpF). BpA, but not BpF, promoted degradation of the HIF-1alpha protein, which is a component of HIF-1, followed by inhibition of EPO induction. An immunoprecipitation assay indicated that BpA dissociated heat shock protein 90 (Hsp90) from HIF-1alpha and destabilized HIF-1alpha protein. HIF-1alpha is usually degraded first by ubiquitination and then by the proteasome pathway. Cobalt ion inhibits ubiquitination of HIF-1alpha and stabilizes it. In the present study, BpA promoted HIF-1alpha degradation in the presence of cobalt and in the presence of proteasome inhibitor. These results suggest that BpA degraded HIF-1alpha via a currently unknown pathway, and that this phenomenon required two methyl groups in BpA.

Air Pollutants, Occupational↗

PubMed2004

Only the light chain is sufficient for the serine protease function of the membrane bound form of factor I on a xeno-surface.

The cell membrane-bound forms of whole factor I (fI-PI), the light chain of the serine protease (SP) domain (SP-PI), and the light chain plus the COOH-terminal 45 amino acid (AA) of the heavy chain (SP+45-PI) were constructed. Chinese hamster ovary (CHO) cells, expressing these molecules were established by transfection of cDNA and confirmed by flow cytometry. Amelioration of complement-mediated cell lysis and complement fragment deposition on the cell surface by the transfectant molecules was tested in each CHO cell by means of a lactate dehydrogenase (LDH) assay and flow cytometry, respectively. A highly expressed fI-PI blocked human complement-mediated cell lysis by approximately 84% of the cells. CHO cell transfectants with SP-PI also showed a clear inhibition in cell lysis by human serum, whereas CHO cell transfectants with SP+45-PI showed no inhibition. In addition, fI-PI and SP-PI, but not SP+45-PI, suppressed C5b-9 deposition on CHO cell surface. These data indicate that the last 45 amino acid of the heavy chain, including a disulfide bridge area, did not participate in the serin protease function of factor I. The results suggest that SP-PI has potential for use in clinical xenotransplantation.

Animals↗

PubMed2004

A study of the xenoantigenicity of adult pig islets cells.

BACKGROUND: The pig pancreas is considered to be the most suitable source of islets for xenotransplantation into patients with type I diabetes. The purpose of this study was to assess the antigenicity of pig islets, including the Galalpha1-3Galbeta1-4GlcNAc-R (the alpha-Gal) and Hanganutziu-Deicher (H-D) antigens, and the pathway involved in human complement activation. METHODS: The expression of alpha-Gal on islets from adult pigs was investigated by immunohistochemical staining and flowcytometric analysis. The alpha1,3 galactosyltransferase (alpha1,3GT) activity of islets was determined by high-performance liquid chromatography. Antigenicity to human natural antibodies, including the H-D antigen of pig islets was next examined by treatment of pig islets with tunicamycin, D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) and/or neuraminidase. In addition, complement-mediated islets lysis was examined using factor D-deficient and C1-deficient sera. RESULTS: Adult pig islets expressed negligible amounts of alpha-Gal epitope, and alpha1,3GT activity was also undetectable. However, human natural antibodies, immunoglobulin G and M, and the anti H-D antibody react to the adult islet. Treatment of pig islets with tunicamycin, but not PDMP, led to a drastic reduction in antigenicity to human serum, indicating the importance of N-linked sugars on the islets. Neuraminidase treatment indicated the presence of, not only the H-D antigen, but also other sialic acid antigens that reacted with the human natural antibody. The complement deposition of C4, C3 and factor B on islets was demonstrated. The alternative pathway-mediated pig islet killing accounted for approximately 30% of that by the total complement pathway. CONCLUSION: The origin of antigenicity of pig islets is mainly N-linked sugars including sialic acid antigens, but not the alpha-Gal, and pig islets can be injured by both the classical and the alternative complement pathway in human serum.

ABO Blood-Group System↗

PubMed2003

Effect of hybrid complement regulatory proteins on xenogeneic cells.

To suppress C3 fragment deposition in the classical pathway complement activation on xenogeneic membranes, decay accelerating factor (DAF) was the most effective molecule among the complement regulatory proteins (CRPs) used in the present study. C3 fragment deposition was closely related to subsequent xenogeneic cell lysis. However, other molecules were also very effective in different ways and include phosphatidylinositol (PI)-anchored short consensus repeat (SCR) 2-4 of membrane cofactor protein (MCP-PI), PI-anchored C1 esterase inhibitor (C1-INH-PI), and PI-anchored SCR8-11 of complement receptor type 1 (CR1-PI). On the other hand, regarding a strategy for downregulating C4 fragment deposition, the use of only C1-INH-PI and PI-anchored SCR1-3 of the C4b-binding protein (C4bp-PI) was found to be effective.

Animals↗