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Biomedical subjects

K Sakaguchi

Publications and source records attributed to K Sakaguchi.

At least 73 records · Page 4Linked to original sources
PubMed2000

Studies on a novel DNA polymerase inhibitor group, synthetic sulfoquinovosylacylglycerols: inhibitory action on cell proliferation.

Some chemically synthesized sulfoquinovosylmonoacylglycerols (SQMG)-sulfoquinovosyldiacylglycerols (SQDG) have been reported to selectively and strongly inhibit the activities of mammalian DNA polymerases alpha and beta in vitro. In this study, using human cancer cell lines, we investigated the effects of SQMG-SQDG on the DNA polymerase in the cells. In the presence of n-decane, the IC(50) values on cell growth were approximately 1-5 microM for SQMG and about 0.3-1 microM for SQDG. The values were almost the same as the in vitro enzyme inhibitory levels. The cell lines were arrested in early S-phase by SQMG-SQDG at the concentrations of 0.1-4.7 microM in a manner dependent on incubation time, suggesting that SQMG-SQDG blocked the primary step of DNA replication by inhibiting DNA polymerase, possibly alpha-type. We also demonstrated the localization of SQMG in the cell using the fluorescent SQMG analog, SQMGalpha-NBDD, which was synthesized in our laboratory. SQMGalpha-NBDD was localized in the nucleus and on the nuclear surface, but the binding site seemed not to be the DNA/chromatin, suggesting that the SQMG-SQDG might interact with molecules located close to the DNA/chromatin and on the nuclear surface. These results suggested a correlation between the in vitro biochemical action of the SQMG-SQDGs and their intracellular mode of action.

Animals↗
PubMed2000

Damage-mediated phosphorylation of human p53 threonine 18 through a cascade mediated by a casein 1-like kinase. Effect on Mdm2 binding.

The p53 tumor suppressor protein is stabilized in response to ionizing radiation and accumulates in the nucleus. Stabilization is thought to involve disruption of the interaction between the p53 protein and Mdm2, which targets p53 for degradation. Here we show that the direct association between a p53 N-terminal peptide and Mdm2 is disrupted by phosphorylation of the peptide on Thr(18) but not by phosphorylation at other N-terminal sites, including Ser(15) and Ser(37). Thr(18) was phosphorylated in vitro by casein kinase (CK1); this process required the prior phosphorylation of Ser(15). Thr(18) was phosphorylated in vivo in response to DNA damage, and such phosphorylation required Ser(15). Our results suggest that stabilization of p53 after ionizing radiation may result, in part, from an inhibition of Mdm2 binding through a phosphorylation-phosphorylation cascade involving DNA damage-activated phosphorylation of p53 Ser(15) followed by phosphorylation of Thr(18).

Amino Acid Sequence↗
PubMed2000

Identification of gel-separated tumor marker proteins by mass spectrometry.

Two-dimensional gel electrophoresis with subsequent analysis by mass spectrometry was applied to study differences in protein expression between benign and malignant solid tumors from human beast, lung and ovary cells. Cells from freshly resected clinical material were lysed and the extracts were subjected to isoelectric focusing with immobilized pH gradients followed by second-dimensional separation on 10-13% sodium dodecyl sulfate (SDS)/polyacrylamide gels. Polypeptides were identified using matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry after in-gel protein digestion. Some of the upregulated polypeptides in malignant cells are of potential importance as markers of tumor proliferation. Twenty such proteins were identified, ten constituting novel identifications and ten sequence verifications of previously gel-matched proteins. The proteins identified span a wide range of functions, but several cases of protein truncation were found. Truncated forms of cytokeratins 6D and 8, and of cathepsin D were identified. Truncated froms of these over-expressed proteins support the presence of proteolytic processing steps in tumor material. The protein processing and the difference between protein and mRNA abundancies in tumors of different malignancy and origin suggest that studies at the protein level are important for an understanding of tumor phenotypes.

Biomarkers, Tumor↗
PubMed2000

Proteome mapping by two-dimensional polyacrylamide gel electrophoresis in combination with mass spectrometric protein sequence analysis.

The high resolving power of two-dimensional polyacrylamide gel electrophoresis 2D-PAGE and its full analytical and preparative potential have been described with special emphasis on reproducibility and standardization of protein spot patterns, enhanced protein detection sensitivity, and computer analysis database development. New methodologies for peptide mass fingerprinting, peptide, sequence, and fragmentation tagging have been highlighted. Major challenges associated with 2D-PAGE/mass spectrometric protein sequencing were outlined which need to be addressed in the future, including sample enrichment, use of alternative gel matrices, improvements in separation systems interfaced directly to the mass spectrometer, and design of high-sensitivity instruments with very high mass ranges. It is hoped that comparative studies to identify, quantitate, and characterize proteins differentially expressed in normal versus diseased cells would give insight into mechanisms of pathogenesis and allow the development of a way to control both the etiology and the course of diseases.

Electrophoresis, Polyacrylamide Gel↗
PubMed2000

DNA polymerases and Ki-67 nuclear antigen are induced in correlation with the resected mass of rat liver up to 90%.

BACKGROUND AND AIMS: We studied the regeneration potential by measuring induction of DNA polymerases in the remnant rat liver after a partial hepatectomy (PHx) that is maximal but compatible with survival. METHODS: The regenerating rat liver was obtained after the 90% PHx. The induction of activities of DNA polymerase alpha, delta, and epsilon were measured after partial purification. The Ki-67 nuclear antigen was also detected histochemically. These parameters were compared with those after both 30% and 70% PHx. RESULTS: The 90% hepatectomy resulted in the strong inductions of DNA polymerase alpha, delta, and epsilon, at 48 h after operation, in association with increases in wet weight and total DNA in the remnant liver. The enzyme induction was much higher after 90% PHx than after 30% and 70% hepatectomy, in correlation with the resection volume. At 48 h after 90% hepatectomy, the Ki-67 positive cells increased up to 47.2% of hepatocytes in the remnant liver. CONCLUSION: The higher induction of replication enzymes by 90% hepatectomy reflects more cells entering mitogenic cell cycle, which supports the fast regeneration of the remnant liver. The number of proliferating hepatocytes is stringently controlled by an unknown mechanism sensing the mass of resected liver parenchyma.

Animals↗
PubMed2000

Differential suppression of liver-specific genes in regenerating rat liver induced by extended hepatectomy.

BACKGROUND/AIMS: The function of the remnant liver is critical to survival of patients following an extended hepatectomy. The aim of this study was to determine whether proliferating hepatocytes in the remnant liver preserve the expression of liver-specific genes. METHODS: Using regenerating rat livers after 30, 70, and 90% partial hepatectomy (PHx), Northern blot analyses were performed with probes for seven liver-specific genes, six growth-related genes, two housekeeping genes and two acute phase reactant protein genes. RESULTS: During the regeneration after 90% PHx, the transcription of liver-specific genes showed three chronological patterns: transcription of serum albumin and cytochrome P450 2B decreased rapidly and reached a nadir at 6 to 24 h after PHx; those of apolipoprotein A-1, phosphoenolpyruvate carboxykinase and ornithine transcarbamylase decreased gradually until 24 to 48 h; those of UDP-glucuronosyltransferase and hepatocyte nuclear factor 4 did not show any changes until 48 h after PHx. In contrast, expression levels of all the growth-related genes and of housekeeping genes increased rapidly after PHx. After 30 and 70% PHx, expression of these genes changed in a similar manner to the 90% PHx case but to a lower extent. CONCLUSIONS: Based upon the fractions of Ki-67 positive hepatocytes in remnant livers, we could estimate the degree of expression of each liver-specific gene in the proliferating hepatocytes. The serum albumin gene was completely suppressed, while that encoding UDP-glucuronosyltransferase was not affected. These results correlated well with the patterns of albumin and bilirubin in rat serum after PHx. Other liver-specific genes were moderately suppressed in proliferating hepatocytes. Thus, expression of liver-specific gene is differentially suppressed when hepatocytes enter a proliferation cycle. Those that are unaffected may be indispensable for maintaining the homeostasis of the living organism.

Animals↗
PubMed2000

Plant homologue of flap endonuclease-1: molecular cloning, characterization, and evidence of expression in meristematic tissues.

Flap endonuclease-1 (FEN-1) is an important enzyme involved in DNA replication and repair. We isolated a 1.4 kb cDNA from rice (Oryza sativa), termed OsFEN-1, encoding a protein which shows homology with the eukaryotic FEN-1 proteins. OsFEN-1 protein was overexpressed in Escherichia coli and purified to near homogeneity. DNA cleavage analysis using different branched DNA structures indicated that OsFEN-1 protein possesses both 5'-flap endonuclease and 5' to 3' double-stranded DNA exonuclease activities. OsFEN-1 protein incises a 5'-flap and 5'-pseudo Y structure one base 3' of the branched point in the duplex region. The enzymatic properties indicated that we succeeded in obtaining the gene and the protein of a plant counterpart of FEN-1. OsFEN-1 transcripts were expressed strongly in proliferating tissues such as root tips and young leaves that contain root apical meristem and marginal meristem, respectively. No expression was detected in mature leaves although the leaves were exposed to UV. We analyzed the spatial distribution pattern of OsFEN-1 transcripts by in situ hybridization. In the shoot apex, OsFEN-1 mRNA was abundant in the shoot apical meristem, tiller bud, leaf primordia, ligule primordia and marginal meristem of young leaves. In the roots, the transcript accumulated to high levels in the root apical meristem. Our results indicate that OsFEN-1 is expressed in tissues rich in proliferating cells, and its expression may be required for cell growth and organ formation.

Amino Acid Sequence↗
PubMed2000

Pulsed electromagnetic fields promote bone formation around dental implants inserted into the femur of rabbits.

The present study examined the effect of applying a pulsed electromagnetic field (PEMF) on bone formation around a rough-surfaced dental implant. A dental implant was inserted into the femur of Japanese white rabbits bilaterally. A PEMF with a pulse width of 25 microseconds and a pulse frequency of 100 Hz was applied. PEMF stimulation was applied for 4 h or 8 h per day, at a magnetic intensity of 0.2 mT, 0.3 mT or 0.8 mT. The animals were sacrificed 1, 2 or 4 weeks after implantation. After staining the resin sections with 2% basic fuchsin and 0.1% methylene blue, newly formed bone around the implant on tissue sections was evaluated by computer image analysis. The bone contact ratios of the PEMF-treated femurs were significantly larger than those of the control groups. Both the bone contact ratio and bone area ratio of the 0.2 mT- and 0.3 mT-treated femurs were significantly larger than the respective value of the 0.8 mT-treated femurs (P < 0.001). No significant difference in bone contact ratio or bone area ratio was observed whether PEMF was applied for 4 h/day or 8 h/day. Although a significantly greater amount of bone had formed around the implant of the 2-week treated femurs than the 1-week treated femurs, no significant difference was observed between the 2-week and 4-week treated femurs. These results suggest that PEMF stimulation may be useful for promoting bone formation around rough-surfaced dental implants. It is important to select the proper magnetic intensity, duration per day, and length of treatment.

Alloys↗
PubMed2000

Increase in CD95 (Fas/APO-1)-positive CD4+ and CD8+ T cells in peripheral blood derived from patients with autoimmune hepatitis or chronic hepatitis C with autoimmune phenomena.

BACKGROUND: The expressions of CD95 (Fas/APO-1) and Bcl-2 are determinants of apoptosis in normal lymphocytes, and abnormalities in their expressions might contribute to the induction of autoimmunity. In this study, we examined the expressions of CD95 and Bcl-2 on freshly isolated T and B cells from patients with autoimmune hepatitis (AIH) or chronic hepatitis C associated with autoimmune phenomena (CH-C(AI)). METHODS: The CD95 and Bcl-2 expressions within CD4+ T, CD8+ T, and CD19+ B cell subsets were analysed by two-colour flow cytometry. RESULTS: The surface expression of CD95 was significantly high in both the CD4+ T and CD8+ T cell subsets derived from the patients with AIH and those with CH-C(AI), compared with expression in patients with CH-C and normal subjects. The increase in CD95 expression was associated with the phenotypic conversion of naive CD45RO- to primed CD45RO+ CD4+ T cells. Bcl-2 was detected in the vast majority of peripheral T and B cells. There was no significant difference in the percentage of Bcl-2-positive cells in the CD4+ T cell, CD8+ T cell and CD19+ B cell subsets among the patient groups and normal subjects. CONCLUSIONS: These results indicate that an increase in CD4+ T cells expressing CD45RO and CD95 marks an important subset of AIH and CH-C(AI) patients. These expanded CD95+ CD45RO+ primed T cells most likely reflect a continuous antigen-specific or non-specific activation of T lymphocytes, and/or the persistent presence of activated lymphocytes as a consequence of abnormalities in the peripheral deletion of activated lymphocytes. These persistently activated lymphocytes might play a role in the induction of autoimmunity in AIH and CH-C(AI).

Adult↗
PubMed2000

Relationship between the ability to support differentiation of osteoclast-like cells and adipogenesis in murine stromal cells derived from bone marrow.

In vitro osteoclast differentiation is supported by stromal cells. In order to isolate a stromal cell line that can support osteoclast differentiation, 22 cell lines were cloned from mouse bone marrow. One of these clones, TMS-14, is a line of preadipocytes that supports osteoclast-like cell formation without any bone resorbing factors; and another, TMS-12, is a line of preosteoblasts that supports osteoclast-like cell formation with bone resorbing factors such as prostaglandin E(2)(PGE(2)). The difference of these two lines for osteoclast formation was not related with their abilities of PGE(2)production, but with the expression of osteoclast differentiation factor (ODF, also called OPGL, RANKL, and TRANCE), which detected with RT-PCR, in both cell lines. In TMS-14 cells, ODF mRNA was detected with or without PGE(2). In TMS-12 cells, ODF expression was detected in the PGE(2)-treated cells alone. When TMS-14 cells were induced to undergo adipogenic differentiation in response to treatment with thiazolidinedione, a ligand and activator of peroxisome proliferator-activated receptor gamma (PPARgamma), the ability of TMS-14 cells to support osteoclast-like cell formation was prevented in the presence or absence of 1,25(OH)(2)D(3). The gene expression of ODF in TMS-14 cells was also inhibited by treatment with thiazolidinedione. These results suggest that adipogenesis in bone marrow cells is related to the ability to support osteoclast differentiation. This is the first report of a cloned stromal cell line that can support osteoclastogenesis without the treatment with any osteotropic factors. Furthermore, this murine clonal preadipose cell line may be useful for studying senescence-dependent osteoporosis.

Adipocytes↗
PubMed2000

Early clearance of circulating hepatitis C virus enhanced by induction therapy with twice-a-day intravenous injection of IFN-beta.

To improve the long-term efficacy of interferon (IFN) for treatment of chronic hepatitis C virus (HCV) infection, we proposed induction therapy with twice-a-day IFN-beta injection. This study was intended to clarify the antiviral mechanism. Thirty patients were randomly assigned to two groups: group A (twice-a-day therapy) received 3 MU IFN-beta intravenously (i.v.) twice a day for 2 weeks; group B (once-a-day therapy) received 6 MU of IFN-beta daily. HCV RNA, IFN-beta, alanine aminotransferase (ALT), 2'5'-oligoadenylate synthetase (2'5'-AS) activity, and beta2-microglobulin in serum were compared between the two groups during the first 2 weeks of IFN therapy. The clearance rate of serum HCV RNA in group A (86.7%) was significantly higher than that in group B (13.3%) at day 3 (p = 0.0006). No accumulation of IFN-beta was shown in serum throughout the therapy. The ratio (day 3/day 1) of 2'5'-AS activity was significantly higher in group A. Multivariate analysis indicated twice-a-day IFN-beta injection therapy led to significantly early clearance of circulating HCV. Twice-a-day IFN-beta injection therapy could induce biologically enhanced antiviral activities and be an efficient induction therapy for eradication of HCV.

Adult↗
PubMed2000

Cloning and expression of gene encoding a novel endoglycoceramidase of Rhodococcus sp. strain C9.

Endoglycoceramidase (EGCase) is an enzyme capable of cleaving the glycosidic linkage between oligosaccharides and ceramides of various glycosphingolipids. We previously reported that the Asn-Glu-Pro (NEP) sequence is part of the active site of EGCase of Rhodococcus sp. strain M-777. This paper describes the molecular cloning of a new EGCase gene utilizing the NEP sequence from the genomic library of Rhodococcus sp. strain C9, which was clearly distinguishable from M-777 by 16S rDNA analysis. C9 EGCase possessed an open reading frame of 1,446 bp encoding 482 amino acids, and showed 78% and 76% identity to M-777 EGCase II at the nucleotide and amino acid levels, respectively. Interestingly, C9 EGCase showed the different specificity to the M-777 enzyme: it hydrolyzed b-series gangliotetraosylceramides more slowly than the M-777 enzyme, whereas both enzymes hydrolyzed a-series gangliosides and neutral glycosphingolipids to the same extent.

Amino Acid Sequence↗
PubMed2000

Structure-activity relationship of a novel group of mammalian DNA polymerase inhibitors, synthetic sulfoquinovosylacylglycerols.

We reported previously that sulfolipids in the sulfoquinovosylacylglycerol class from a fern and an alga are potent inhibitors of DNA polymerase alpha and beta and potent anti-neoplastic agents. In developing a procedure for chemical synthesis of sulfolipids, we synthesized many derivatives and stereoisomers of sulfoquinovosylmonoacylglycerol (SQMG) / sulfoquinovosyldiacylglycerol (SQDG). Some of these molecules were stronger inhibitors than the SQMG / SQDG originally reported as natural compounds. In this study, we examined the structure-inhibitory function relationship of synthetic SQMG / SQDG and its relationship to cytotoxic activity. The inhibitory effect is probably mainly dependent on the fatty acid effect, which we reported previously, although each of the SQMG / SQDG was a much stronger inhibitor than the fatty acid alone that was present in the SQMG / SQDG. The inhibitory effect could be influenced by the chain size of fatty acids in the SQMG / SQDG. The sulfate moiety in the quinovose was also important for the inhibition. Lineweaver-Burk plots of SQMG / SQDG indicated that DNA polymerase alpha was non-competitively inhibited, but the SQMG / SQDG were effective as antagonists of both template-primer DNA-binding and nucleotide substrate-binding of DNA polymerase beta. The SQMG had an cytotoxic effect, but the SQDG tested did not. The SQDG might not be able to penetrate into cells. Based on these results, we discuss the molecular action of SQMG / SQDG and propose drug design strategies for developing new anti-neoplastic agents.

Antineoplastic Agents↗
PubMed1999

Phosphorylation of human p53 by p38 kinase coordinates N-terminal phosphorylation and apoptosis in response to UV radiation.

Components of the ras signaling pathway contribute to activation of cellular p53. In MCF-7 cells, p38 kinase activated p53 more effectively than other members of the ras pathway. p53 and p38 kinase exist in the same physical complex, and co-expression of p38 stabilized p53 protein. In vitro, p38 kinase phosphorylated p53 at Ser33 and Ser46, a newly identified site. Mutation of these sites decreased p53-mediated and UV-induced apoptosis, and the reduction correlated with total abrogation of UV-induced phosphorylation on Ser37 and a significant decrease in Ser15 phosphorylation in mutant p53 containing alanine at Ser33 and Ser46. Inhibition of p38 activation after UV irradiation decreased phosphorylation of Ser33, Ser37 and Ser15, and also markedly reduced UV-induced apoptosis in a p53-dependent manner. These results suggest that p38 kinase plays a prominent role in an integrated regulation of N-terminal phosphorylation that regulates p53-mediated apoptosis after UV radiation.

Alanine↗