Hepatocellular carcinoma associated with adult-type citrullinemia.
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Adult↗
Biomedical subjects
K Sugimoto
Publications and source records attributed to K Sugimoto.
High-performance affinity beads for identifying drug receptors.
We have developed a method using novel latex beads for rapid identification of drug receptors using affinity purification. Composed of a glycidylmethacrylate (GMA) and styrene copolymer core with a GMA polymer surface, the beads minimize nonspecific protein binding and maximize purification efficiency. We demonstrated their performance by efficiently purifying FK506-binding protein using FK506-conjugated beads, and found that the amount of material needed was significantly reduced compared with previous methods. Using the latex beads, we identified a redox-related factor, Ref-1, as a target protein of an anti-NF-kappaB drug, E3330, demonstrating the existence of a new class of receptors of anti-NF-kappaB drugs. Our results suggest that the latex beads could provide a tool for the identification and analysis of drug receptors and should therefore be useful in drug development.
Amino Acid Sequence↗
Activation of an ataxia telangiectasia mutation-dependent intra-S-phase checkpoint by anti-tumour drugs in HL-60 and human lymphoblastoid cells.
In yeast cells, the intra-S-phase checkpoint slows down the rate of DNA replication in response to DNA damage. Here we showed that a similar checkpoint mechanism is present and activated by anti-tumour drugs in HL-60 and Epstein-Barr virus (EBV)-transformed human lymphoblastoid cells. Using bromodeoxyuridine (BrdU) pulse labelling combined with two-dimensional flow cytometric analysis, we clearly visualized the cell-cycle progression of the BrdU-positive population (cells originally belonging to the S phase) and detected even subtle changes in S-phase progression induced by mild drug treatment conditions free of apoptosis. The DNA topoisomerase II inhibitors, doxorubicin and etoposide (250 nmol/l and 400 nmol/l, respectively, for 8 h), retained the BrdU-positive HL-60 cells in the latter half of S and G2/M positions, and the pyrimidine analogue anti-metabolite, cytosine beta-D-arabinofuranose (Ara-C; 50 nmol/l), kept them in early-to-late S phase after 8 h of incubation. Because 10 micromol/l of caffeine added 2 h later attenuated the S-phase retardation by these drugs in HL-60 cells, slowing of the S-phase progression should be actively regulated. Furthermore, two ataxia telangiectasia (AT)-derived lymphoblastoid cell lines were impaired in the doxorubicin-induced S-phase retardation, which indicated that the process is at least partially dependent on ataxia telangiectasia mutated (ATM) gene product. The inhibitory mechanism on S-phase progression elicited by anti-tumour drugs in HL-60 and lymphoblastoid cells may therefore correspond to the intra-S-phase checkpoint of the yeast cells.
Antimetabolites↗
Autoepitopes on autoantigen centromere protein-A (CENP-A) are restricted to the N-terminal region, which has no homology with histone H3.
Anti-centromere autoantibodies (ACA) are commonly found in the serum of patients with a limited type of scleroderma and other systemic autoimmune diseases. CENP-A is one of the major antigens against ACA and a histone H3-like protein. To analyse the autoantigenic epitopes of CENP-A, a series of truncated peptides of human CENP-A were expressed in Escherichia coli and immunoblotting analysis was performed with 91 ACA+ sera. Eighty sera (88%) with the ACA reacted to the 52-amino acids N-terminal region which is not homologous to H3, while no sera reacted to the C-terminus which has a sequence similarity with H3. Moreover, ELISA was also employed in this study using two synthetic peptides corresponding to the amino acid sequences 3-17 (peptide A) and 25-38 (peptide B). Peptides A and B were reactive to 78 (86%) and 79 (87%) of ACA, respectively. Core antigens of hepatitis B virus (HBV) and hepatitis C virus (HCV) have similar sequences to peptide A and/or peptide B, but three sera containing HBV without ACA and five sera containing HCV without ACA were found to be reactive to neither peptide. Centromere localization of CENP-A is dependent on the H3-like C-terminal domain which is not autoantigenic, while the antigenic N-terminal domain, which might play unidentified functional roles, should be an important region for the induction of ACA.
Amino Acid Sequence↗
Intrathoracic kidney combined with pneumomediastinum in an asphyxiated neonate.
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Asphyxia Neonatorum↗
Survivin promotes cell proliferation in human hepatocellular carcinoma.
Survivin is a recently described inhibitor of apoptosis. Because suppression of apoptosis is important for carcinogenesis and tumor growth, we investigated the expression and function of survivin in human hepatocellular carcinomas (HCCs). We have shown that 4 HCC cell lines and 7 out of 8 human HCC tissues expressed survivin messenger RNA (mRNA), whereas expression of survivin mRNA was not detected in normal liver and nontumor areas of these tissues using the reverse transcription polymerase chain reaction. Survivin was detected primarily in the nucleus by immunofluorescence staining of HCC cells. In addition, 14 of 20 (70%) HCC tissues showed positive nuclear staining for survivin, whereas nontumor tissues showed little detectable staining by immunohistochemistry. Survivin expression strongly correlated with the proliferation index but not significantly with the apoptosis index in HCC tissues. Therefore, we performed cell cycle analysis after survivin transfection and showed that overexpression of survivin resulted in a decrease in the G(0)/G(1) phase and an increase in the S phase in all 4 HCC cell lines. Furthermore, we have found that survivin interacted with cyclin-dependent kinase 4 (Cdk4) and overexpression of survivin released p21(WAF1/Cip1) (p21) from Cdk4. From these results, we conclude that survivin promotes cell proliferation by interacting with Cdk4 and releasing p21 from Cdk4. This may play an important role in carcinogenesis and progression of human HCCs.
Apoptosis↗
Chemotherapeutic agents augment TRAIL-induced apoptosis in human hepatocellular carcinoma cell lines.
TNF-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis in various transformed cell lines but not in almost-normal tissues. It is regulated by 2 death receptors, TRAIL receptor 1 (TRAIL-R1) and TRAIL-R2, and 2 decoy receptors, TRAIL-R3 and TRAIL-R4. We investigated the expression of TRAIL-R- and TRAIL-induced apoptosis in human hepatocellular carcinomas (HCCs). TRAIL-R1, -R2, and -R4 were expressed in 6 HCC cell lines examined, but TRAIL-R3 was expressed in only 2 of the 6 cell lines. In addition, immunohistochemical results revealed a high and prevalent expression of TRAIL-R1 and -R2 in human HCC tissues. Despite the expression of TRAIL-R1 and -R2, all 6 HCC cell lines showed resistance to TRAIL-induced apoptosis with no relation to nuclear factor kappa B (NF-kappaB) levels induced by TRAIL. TRAIL-induced death signal was inhibited with both decreased caspase-8 and caspase-3 activity. However, TRAIL induced significant apoptosis in the presence of a subtoxic level of actinomycin D, indicating that the TRAIL-induced apoptotic pathway is in place in these cell lines. In addition, we found that treatment with conventional chemotherapeutic agents, doxorubicin and camptothecin, dramatically augmented TRAIL-induced cytotoxicity in most of the HCC cell lines. Actinomycin D and camptothecin almost completely suppressed NF-kappaB induction by TRAIL, whereas doxorubicin had little effect. These results indicate that TRAIL, in combination with chemotherapeutic agents, may have therapeutic potential in the treatment of human HCC.
Antibiotics, Antineoplastic↗
Hepatocyte growth factor promotes cell survival from fas-mediated cell death in hepatocellular carcinoma cells via Akt activation and Fas-death-inducing signaling complex suppression.
The Akt/PI-3 kinase pathway is a system essential for cell survival. In the current study, we showed that hepatocyte growth factor (HGF) activates the Akt/PI-3 kinase pathway to suppress Fas-mediated cell death in human hepatocellular carcinoma (HCC; 3 lines; SK-Hep1, HLE, and Chang Liver cell lines), hepatoblastoma (1 line; HepG2), and embryonic hepatocyte (1 line; WRL). Five tested cell lines showed the resistance to Fas-mediated cell death by the pretreatment of HGF. This HGF-induced cell survival was suppressed by wortmannin (Akt/PI-3 kinase pathway inhibitor), suggesting an involvement of Akt. When cells were pretreated with HGF, Fas-mediated cell death was suppressed, followed by Akt phosphorylation at Ser473. Fas-death-inducing signaling complex (DISC) formation, especially FADD and caspase 8 interaction, was suppressed by HGF and the suppression of the Akt/PI-3 kinase pathway by transient expression of PTEN, resulting in acquisition of Fas-DISC formation and Fas-mediated cell death in HGF-treated cells. We suggest that HGF promotes cell survival in hepatocyte-derived cell lines (HCC, hepatoblastoma, and embryonic hepatocyte) from Fas-mediated cell death via Fas-DISC suppression as a result of Akt activation.
Carcinoma, Hepatocellular↗
Crystal structure of recombinant native SDF-1alpha with additional mutagenesis studies: an attempt at a more comprehensive interpretation of accumulated structure-activity relationship data.
Crystal structures, forms 1 and 2, of recombinant native stromal cell-derived factor-1alpha (SDF-1alpha), expressed using the Sendai virus expression vector system, have been determined by x-ray crystallography at 2.0 A resolution. The crystal of form 1 is almost isomorphous with that used in the previous crystal structure analysis of the synthetic [N33A] mutant of SDF-1alpha (Dealwis, C., et al. Proc. Natl. Acad. Sci. USA 1998;95, 6941-6946). However, the present structure analysis led to considerably better refinement statistics, revealing an error in the structural assignment of N-terminal residues in the previous report. Comparison of the solution structure, as previously determined by nuclear magnetic resonance (NMR) spectroscopy, and the present structure, with two monomers in the asymmetric unit, reveals several local conformational differences. Alanine scan mutagenesis studies for each residue in the so-called RFFESH motif revealed that only the first residue, Arg12, is effective in enhancing receptor binding (and successive activation). A new notion that steric restraint between Arg8 and Arg12 is favorable (if not vital) for retaining SDF activities appears to explain more consistently the structure-activity relationship data accumulated to date. Four guiding principles are presented that may be useful for designing potent therapeutic compounds interfering with HIV-1 infection through competition at the CXCR4 coreceptor.
Amino Acid Motifs↗
Differences in specificities of anti-centromere sera for the monomeric and dimeric C-terminal peptides of human centoromere protein C.
Centromere protein-C (CENP-C), one of the centromere autoantigens and components of the inner plate of the kinetochore, is suggested to make a dimer at the C-terminus. In order to investigate the presence of conformation-specific anti-centromere antibodies (ACA) to the dimer form, the C-terminal 124 amino acids (CF-124) were expressed in Escherichia coli, affinity purified and chemically cross-linked. Immunoblotting was utilized to compare the reactivities between the dimers and the monomers against 58 ACA(+) sera. The reactivities of the dimers were obviously higher in both IgG and IgM responses. The dimer was still more reactive than the glutathione S-transferase-fused monomer in some sera. Two kinds of CF-124 mutant (each contained one amino acid change at the N-terminal region of CF-124) and two cut segments of CF-124 (67 N-terminal amino acids and 58 C-terminal amino acids) were also examined. The former two mutants decreased the dimerization activity. The latter two mutants lost both activities except for the faint dimerization activity of the N-terminal half. Affinity-purified antibodies with CF-124 in a liquid phase containing the co-purified GroE protein of E. coli, GroEL, reacted to the centromere in culture cells. In conclusion, there are heterogeneous autoepitopes including some conformational epitopes at the C-terminal CENP-C.
Amino Acid Sequence↗
Preoperative diagnosis of ileal lipoma by endoscopic ultrasonography probe.
The occurrence of tumor in the small intestine is relatively rare. It has been demonstrated that lipoma of the ileum is a cause of intussusception. We report a 59-year-old man admitted to our hospital for lower abdominal pain. Diagnosis of intussusception was made by abdominal x-ray and ultrasonography. Enema contrast studies revealed ileocolic intussusception. Colonoscopy revealed a tumor with an submucosal tumor (SMT)-like head and coil-spring appearance in the ascending colon. Endoscopic ultrasonography (EUS) revealed a hyperechoic submucosal lesion with features compatible with lipoma. Subsequently, this was confirmed histopathologically after resection. To our knowledge, this is the first report of preoperative diagnosis of ileal lipoma by EUS.
Endosonography↗
Polymorphonuclear leukocytes (PMNs) functions in SHR, L-NAME- and DOCA/salt-induced hypertensive rats.
OBJECTIVES: To clarify ex-vivo polymorphonuclear leukocytes (PMNs) functions, we examined superoxide anion (O2-) production and adhesion to a plastic plate of isolated PMNs obtained from spontaneously hypertensive rats (SHR/lzm), NG-nitro-L-arginine methyl ester (L-NAME)- and deoxycorticosterone acetate (DOCA)/salt-induced hypertensive rats. DESIGN: Sixteen week-old male SHR/Izm and Wistar-Kyoto rats (WKY/Izm) were used as a model of hypertension and its control, respectively. L-NAME-hypertension was induced by oral administration of 100 mg/kg per day of L-NAME twice daily for 4 weeks using 4-week-old male Wistar rats. DOCA/salt-hypertension was induced by once daily subcutaneous injection of 1 mg DOCA with 1% NaCl drinking water for 2 weeks using 8-week-old male Wistar rats with heminephrectomy. METHODS: Heparinized whole blood was obtained from abdominal aorta. PMNs were isolated by density gradient following dextran sedimentation. A production of superoxide anion (O2-) by PMNs stimulated with phorbol ester myristate acetate (PMA, 100 ng/ml) was determined by a superoxide dismutase (SOD)-inhibitable cytochrome-C reduction method. Adhesion of PMNs was evaluated by their protein content on a plastic plate measured by Lowry method. RESULTS: SHR/Izm showed a significant enhancement of O2- production by isolated PMNs compared with WKY/Izm. Rats treated with L-NAME showed a lower O2- production by PMNs compared to control animals. In DOCA/salt hypertensive rats, O2- production was not different from that in the control rats. Adherent function of isolated PMNs did not differ significantly among these hypertensive animal models. CONCLUSIONS: These results suggest that O2- production by circulatory PMNs is augmented in SHR, but not in L-NAME and DOCA/salt hypertensive rats. This enhanced function, which is also observed in human essential hypertension, might contribute to the development of cardiovascular damage in genetically determined hypertension.
Animals↗
Preferential consumption of coagulation factors I, V, and VIII in rat endotoxemia.
To ascertain the time course of prolonged coagulation time and the coagulation factors that were consumed preferentially after injection of Escherichia coli endotoxin (ETX, 3 mg/kg, intravenously) in rats, the activated partial thromboplastin time (aPTT) and prothrombin time (PT) were measured. Using aPTT and PT, the residual levels of the major coagulation factors were quantified by partial replacement of ETX-injected rat plasma with individual factor-deficient human plasma. The residual levels of prekallikrein and high molecular weight (HMW) kininogen were also measured. After ETX injection, aPTT and PT showed gradual increasing prolongation, which was marked at 3-5 h after the injection. The residual level of fibrinogen was markedly reduced between 1 and 3 h after ETX injection and dropped to the determination limit 7 h after the injection. Ratios of the consumed coagulation factors, prekallikrein, and HMW kininogen in rat plasma collected 7 h after intravenous injection of ETX were obtained as follows: prekallikrein (18.0 +/- 4.8%), HMW kininogen (36.2 +/- 1.9 %), factor XII (54.0 +/- 0.7%), factor VIII (86.1 +/- 1.8%), factor VII (35.6 +/- 7.7%), factor V (90.6 +/- 0.8%), and factor I (fibrinogen) (>89.6 +/- 0.0%). Thus, coagulation factor I (fibrinogen) and factors V and VIII (cofactors) were consumed preferentially. The extrinsic coagulation pathway was dominantly activated, whereas the intrinsic coagulation pathway, including plasma kallikrein-kinin system, played less important role in the ETX-induced consumption coagulopathy in rat.
Animals↗
New techniques enable comparative analysis of microtubule orientation, wall texture, and growth rate in intact roots of Arabidopsis.
This article explores root epidermal cell elongation and its dependence on two structural elements of cells, cortical microtubules and cellulose microfibrils. The recent identification of Arabidopsis morphology mutants with putative cell wall or cytoskeletal defects demands a procedure for examining and comparing wall architecture and microtubule organization patterns in this species. We developed methods to examine cellulose microfibrils by field emission scanning electron microscopy and microtubules by immunofluorescence in essentially intact roots. We were able to compare cellulose microfibril and microtubule alignment patterns at equivalent stages of cell expansion. Field emission scanning electron microscopy revealed that Arabidopsis root epidermal cells have typical dicot primary cell wall structure with prominent transverse cellulose microfibrils embedded in pectic substances. Our analysis showed that microtubules and microfibrils have similar orientation only during the initial phase of elongation growth. Microtubule patterns deviate from a predominantly transverse orientation while cells are still expanding, whereas cellulose microfibrils remain transverse until well after expansion finishes. We also observed microtubule-microfibril alignment discord before cells enter their elongation phase. This study and the new technology it presents provide a starting point for further investigations on the physical properties of cell walls and their mechanisms of assembly.
Arabidopsis↗
MYB-related transcription factor NtMYB2 induced by wounding and elicitors is a regulator of the tobacco retrotransposon Tto1 and defense-related genes.
Transposition of the tobacco retrotransposon Tto1 is regulated mainly by transcription from the long terminal repeat (LTR). Functional analysis of the LTR showed that the 13-bp motif is a cis-regulatory element involved in activation by tissue culture, wounding, and treatment with elicitors. The 13-bp motif contains a conserved motif (L box) that has been implicated in the expression of phenylpropanoid synthetic genes in response to defense-related stresses. To gain further insight into the regulatory mechanism of the retrotransposon and defense-related genes, cDNAs encoding four different proteins binding to the 13-bp motif have been isolated and characterized. One protein is identical to the previously reported NtMYB1, the RNA for which is induced by virus infection; the others are also MYB-related factors. One of these factors, NtMYB2, was analyzed in detail. NtMYB2 mRNA was induced by wounding and by treatment with elicitors. NtMYB2 activated expression from the promoter with the 13-bp motif and from the promoter of the phenylalanine ammonia lyase gene (Pv-PAL2) in tobacco protoplasts. Overexpression of NtMYB2 cDNA in transgenic tobacco plants induced expression of Tto1 and a PAL gene. Together, these results indicate that NtMYB2 is involved in the stress response of the retrotransposon and defense-related genes.
Amino Acid Sequence↗
A novel trinuclear palladium cluster compound: di-mu 3-chloro-tris
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Journal Article↗
CD56 and terminal deoxynucleotidyl transferase positive cutaneous lymphoblastic lymphoma.
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Adult↗
Rfc5, in cooperation with rad24, controls DNA damage checkpoints throughout the cell cycle in Saccharomyces cerevisiae.
RAD24 and RFC5 are required for DNA damage checkpoint control in the budding yeast Saccharomyces cerevisiae. Rad24 is structurally related to replication factor C (RFC) subunits and associates with RFC subunits Rfc2, Rfc3, Rfc4, and Rfc5. rad24Delta mutants are defective in all the G(1)-, S-, and G(2)/M-phase DNA damage checkpoints, whereas the rfc5-1 mutant is impaired only in the S-phase DNA damage checkpoint. Both the RFC subunits and Rad24 contain a consensus sequence for nucleoside triphosphate (NTP) binding. To determine whether the NTP-binding motif is important for Rad24 function, we mutated the conserved lysine(115) residue in this motif. The rad24-K115E mutation, which changes lysine to glutamate, confers a complete loss-of-function phenotype, while the rad24-K115R mutation, which changes lysine to arginine, shows no apparent phenotype. Although neither rfc5-1 nor rad24-K115R single mutants are defective in the G(1)- and G(2)/M-phase DNA damage checkpoints, rfc5-1 rad24-K115R double mutants become defective in these checkpoints. Coimmunoprecipitation experiments revealed that Rad24(K115R) fails to interact with the RFC proteins in rfc5-1 mutants. Together, these results indicate that RFC5, like RAD24, functions in all the G(1)-, S- and G(2)/M-phase DNA damage checkpoints and suggest that the interaction of Rad24 with the RFC proteins is essential for DNA damage checkpoint control.
Cell Cycle↗