Pigmented squamous cell carcinoma of the skin. Report of a case with epiluminescence microscopic observation.
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Aged↗
Biomedical subjects
T Ohnishi
Publications and source records attributed to T Ohnishi.
Hepatocyte growth factor in gingival crevicular fluid and the distribution of hepatocyte growth factor-activator in gingival tissue from adult periodontitis.
Hepatocyte growth factor (HGF), also known as scatter factor, is a broad-spectrum and multifunctional cytokine required for the development, growth and regeneration of various organs and tissues. The expression of HGF in human gingival fibroblasts is induced by inflammatory cytokines such as interleukin 1. Thus, although it is possible that content of HGF in gingival crevicular fluid (GCF) in periodontitis is increased, this has not so far been reported because the volume of GCF is too small to determine HGF by the available enzyme-linked immunosorbent assay (ELISA). A recently developed, highly sensitive ELISA for HGF, with a detection limit of 1 pg/ml sample, has now enabled HGF to be measured in GCF.The mean HGF content in GCF from sites with clinically healthy gingiva, defined by the absence of overt signs of gingival inflammation and a probing depth (PD) <3 mm, was 1.7 ng/ml, and that of periodontitis, defined by obvious alveolar bone loss detected by radiographic examination and a PD> or =3 mm, was 3.23 ng/ml. Although treating the periodontitis did not significantly decrease the HGF concentration despite significantly improved clinical scores such as PD and Gingival Index, the total amount of HGF in GCF did decrease significantly after treatment. HGF was expressed by gingival fibroblasts and inflammatory cells as determined by in situ hybridization. HGF-activator (HGFA), which converts inactive pro-HGF to active mature HGF, was detected in gingival epithelial cells by immunostaining. The expression of HGFA was also confirmed in gingival tissue by reverse transcription-polymerase chain reaction (RT-PCR). These findings indicate that HGF is synthesized and activated in gingiva that is clinically healthy or associated with periodontitis.
Adult↗
Radiobiological characteristics of solid tumours depending on the p53 status of the tumour cells, with emphasis on the response of intratumour quiescent cells.
Human head and neck squamous cell carcinoma cells transfected with mutant TP53 (SAS/mTP53) or with a neo vector as a control (SAS/neo) were inoculated subcutaneously (s.c.) into both hind legs of Balb/cA nude mice. Mice bearing tumours received 5-bromo-2'-deoxyuridine (BrdU) continuously to label all proliferating (P) cells in the tumours. The mice then received gamma-ray irradiation. Another group of mice received a series of test doses of gamma-rays while alive or after tumour clamping to obtain hypoxic fractions (HFs) in the tumours. Right after irradiation, the tumour cells were isolated and incubated with a cytokinesis blocker. The micronucleus (MN) frequency in the cells without BrdU labelling (=quiescent (Q) cells) was determined using immunofluorescence staining for BrdU. Meanwhile, 6 h after irradiation, tumour cell suspensions obtained in the same manner were used for determining the frequency of apoptosis in the Q cells. The MN frequency and apoptosis frequency in total (P+Q) tumour cells were determined from the tumours that were not pretreated with BrdU. In total cell populations, SAS/mTP53 cells were more radioresistant than SAS/neo cells in clonogenic survival. Q tumour cells exhibited a significantly lower apoptosis and MN frequency, probably due to their much larger HF, than total cells. In both total and Q cell fractions, SAS/mTP53 cells were less susceptible to apoptosis and more susceptible to micronucleation than SAS/neo cells. Obviously, TP53 status had the potential to influence the radiosensitivity of not only the total cells, but also the Q cells. However, irrespective of the TP53 status, significant differences in radiosensitivity between total and Q tumour cells were consistently observed. From the viewpoint of tumour control as a whole, including intratumour Q tumour cell control, a treatment modality for enhancing the Q cell response has to be considered.
Animals↗
Transfection of mutant p53 gene depresses X-ray- or CDDP-induced apoptosis in a human squamous cell carcinoma of the head and neck.
The present study examined whether X-ray- and CDDP-sensitivities depend on p53 gene status in human squamous cell carcinoma of the head and neck (SAS cells) showing dominant negative nature of mutant p53 protein. SAS cells were transfected with a vector carrying a mutant p53 gene (SAS/Trp248 cells) or neomycin resistant gene control vector (SAS/neo cells). Sensitivities of the transfected cells to X-ray or CDDP were measured with colony formation assay. The incidence of apoptosis by X-ray or CDDP was analyzed with Hoechst staining or DNA ladder formation assay. The activation of caspase-3 was estimated as an indicator of apoptosis by the detection of fragmentation of caspase-3 or poly (ADP ribose) polymerase (PARP) with Western blot. SAS/Trp248 cells showed X-ray- and CDDP-resistance due to the dominant negative nature of mutant p53, compared with SAS/neo cells. The incidence of DNA ladders and apoptotic bodies increased markedly in SAS/neo cells after X-ray irradiation or CDDP treatment, but increased only slightly in SAS/Trp248 cells. Fragmentation of caspase-3 and PARP was observed in SAS/neo cells, but almost no such fragmentation was observed in SAS/Trp248 cells after X-ray irradiation or CDDP treatment. The present results strongly suggest that the X-ray- and CDDP-sensitivities of human squamous cell carcinomas are p53-dependent, and that the sensitivities are tightly correlated with the induction of apoptosis through caspase-3 activation. The p53-dependent X-ray- or CDDP-sensitivity was supported by results from p53-null human lung cancer H1299 cells which were transfected with wild-type or mutant p53 gene.
Apoptosis↗
Apocrine poroma with follicular differentiation: a case report and immunohistochemical study.
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Acrospiroma↗
Radiation-induced apoptosis in the scid mouse spleen after low dose-rate irradiation.
PURPOSE: To elucidate the process of radioadaptation, the role of DNA-PK activity was examined using the scid mouse defective in DNA-PKcs. MATERIALS AND METHODS: The induction of apoptosis in the spleens of the C.B-17 Icr scid mouse and the parental mouse was studied after chronic irradiation with gamma-rays at 1.5 Gy (0.001 Gy min(-1) for 25 h) followed by challenge irradiation with X-rays at 3.0 Gy (1.0 Gy min(-1) for 3 min). RESULTS: When the wild-type mouse was previously exposed to chronic irradiation (1.5 Gy) at a low dose-rate (0.001 Gy min(-1)), apoptosis induced by acute irradiation (3.0 Gy, 1.0 Gy min(-1)) was significantly suppressed, especially in the splenic white pulp. There was no change by acute irradiation after chronic irradiation in the scid mouse, although an effect was detected in the spleen after acute irradiation alone. CONCLUSIONS: These data suggest that DNA-PK activity might play a major role in the radioadaptive response following pre-irradiation at a low dose-rate.
Animals↗
Inducible nitric oxide synthase inhibition of weibel-palade body release in cardiac transplant rejection.
BACKGROUND: Inducible nitric oxide synthase (iNOS, or NOS2) reduces the severity of accelerated graft arteriosclerosis (AGA) in transplanted organs, although the precise mechanism is unclear. METHODS AND RESULTS: We transplanted wild-type murine hearts into either wild-type or NOS2-null recipient mice; we then measured cardiac allograft survival and analyzed tissue sections by immunohistochemistry. We have confirmed that NOS2 increases cardiac allograft survival. We now show that there is less inflammation of cardiac allografts in wild-type hosts than in NOS2-null hosts. Furthermore, staining for von Willebrand factor reveals that the presence of NOS2 is correlated with the presence of Weibel-Palade bodies inside endothelial cells, whereas the absence of NOS2 is correlated with the release of Weibel-Palade bodies. CONCLUSIONS: Weibel-Palade bodies contain mediators that promote thrombosis and inflammation. Therefore, nitric oxide (NO) may stabilize the vessel wall and prevent endothelial activation in part by inhibiting the release of the contents of Weibel-Palade bodies. Prevention of Weibel-Palade body release might be a mechanism by which NO protects the vessel wall from inflammatory disorders such as atherosclerosis or graft arteriosclerosis.
Animals↗
Chromosome 6p21.2, 18q21.2 and human papilloma virus (HPV) DNA can predict prognosis of cervical cancer after radiotherapy.
Loss of heterozygosity (LOH) is one of the most important mechanisms for inactivation of tumor-suppressor genes. Studies of LOH in patients with cervical carcinoma have reported a high frequency of LOH on 3p21.3, 6p21.2, 17p13.1, and 18q21.2. Our study explored whether p53 status, human papilloma virus (HPV), and LOH on chromosome 3p21.3, 6p21.2, 17p13.1, and 18q21.2 are associated with treatment outcome in 65 patients with cervical cancer after radiotherapy. Tumors and normal DNA were analyzed by polymerase chain reaction (PCR) for genetic losses at 10 polymorphic microsatellite loci. The presence of HPV and its type were analyzed by PCR-based assay using the consensus primers for E6, E7, and L1 region. Mutations of the p53 gene were identified by a single-strand conformation polymorphism analysis. Chromosomes 3p21.3, 6p21.2, 17p13.1, and 18q21.2 were involved in the LOH in 23.1%, 41.5%%, 33.8%, and 23.1% of the tumors in our study, respectively. HPV-positive tumors were found in 73.8% of the patients and p53 mutation in 10.8%. The patients with LOH on chromosome 6p21.2 and 18q21.2 survived significantly shorter compared with those without LOH on chromosome 6p21.2 and 18q21.2 in both the overall survival (P = 0.006 and P = 0.007) and the disease-free survival (P = 0.005 and P = 0.008). The HPV-negative patients survived significantly shorter compared with the HPV-positive patients in both the overall survival (P = 0.01) and the disease-free survival (P = 0.04). According to multivariate analysis, HPV status (P = 0.0004, P = 0.01), LOH on 6p21.2 (P = 0.006, P = 0.02), and LOH on 18q21.2 (in both P = 0.01) is a significant predictor of both overall and disease-free survival time. The results of our study suggest that absence of HPV infection, LOH on 6p21.2, and LOH on 18q21.2 are the most important determinants of outcome of patients with cervical carcinoma after radiotherapy.
Adult↗
N-linked glycosylations at Asn(26) and Asn(114) of human MD-2 are required for toll-like receptor 4-mediated activation of NF-kappaB by lipopolysaccharide.
MD-2 is physically associated with Toll-like receptor 4 (TLR4) and is required for TLR4-mediated LPS signaling. Western blotting analysis revealed the presence of three forms of human (h)MD-2 with different electrophoretic mobilities. After N-glycosidase treatment of the cellular extract prepared from cells expressing hMD-2, only a single form with the fastest mobility was detected. Mutation of either one of two potential glycosylation sites (Asn(26) and Asn(114)) of MD-2 resulted in the disappearance of the slowest mobility form, and only the fastest form was detected in hMD-2 carrying mutations at both Asn(26) and Asn(114). Although these mutants were expressed on the cell surface and maintained its ability to associate with human TLR4, these mutations or tunicamycin treatment substantially impaired the ability of MD-2 to complement TLR4-mediated activation of NF-kappaB by LPS. LPS binding to cells expressing CD14, TLR4, and MD-2 was unaffected by these mutations. These observations demonstrate that hMD-2 undergoes N-linked glycosylation at Asn(26) and Asn(114), and that these glycosylations are crucial for TLR4-mediated signal transduction of LPS.
Acute-Phase Proteins↗
Human SMG-1, a novel phosphatidylinositol 3-kinase-related protein kinase, associates with components of the mRNA surveillance complex and is involved in the regulation of nonsense-mediated mRNA decay.
Nonsense-mediated mRNA decay (NMD) is a conserved surveillance mechanism that eliminates imperfect mRNAs that contain premature translation termination codons (PTCs) and code for nonfunctional or potentially harmful polypeptides. We show that a novel phosphatidylinositol 3-kinase-related protein kinase, hSMG-1, is a human ortholog of a product of Caenorhabditis elegans smg-1, one of seven smg genes involved in NMD. hSMG-1 phosphorylates hUPF1/SMG-2 in vivo and in vitro at specific serine residues in SQ motifs. hSMG-1 can associate with hUPF1/SMG-2 and other components of the surveillance complex. In particular, overexpression of a kinase-deficient point mutant of hSMG-1, hSMG-1-DA, results in a marked suppression of the PTC-dependent beta-globin mRNA degradation; whereas that of wild-type hSMG-1 enhances it. We also show that inhibitors of hSMG-1 induce the accumulation of truncated p53 proteins in human cancer cell lines with p53 PTC mutation. Taken together, we conclude that hSMG-1 plays a critical role in NMD through the direct phosphorylation of hUPF1/SMG-2 in the evolutionally conserved mRNA surveillance complex.
Amino Acid Sequence↗
AMY-1, a c-Myc-binding protein, is localized in the mitochondria of sperm by association with S-AKAP84, an anchor protein of cAMP-dependent protein kinase.
We have reported that a novel c-Myc-binding protein, AMY-1 (associate of Myc-1), stimulated the transcription activity of c-Myc. To access the molecular function of AMY-1, a two-hybrid screening of cDNAs encoding AMY-1-binding proteins was carried out with AMY-1 as a bait using a human HeLa cDNA library, and a clone encoding cAMP-dependent protein kinase anchor protein 149 (AKAP149), was obtained. AMY-1 was found to bind in vitro and in vivo to the regulatory subunit II binding region of AKAP149 and S-AKAP84, a splicing variant of AKAP149 expressed in the testis. AMY-1 was expressed postmeiotically in the testis, as S-AKAP84 was expressed. Furthermore, S-AKAP84 and regulatory subunit II, a regulatory subunit of cAMP-dependent protein kinase, made a ternary complex in cells, and AMY-1 was localized in the mitochondria of HeLa and sperm in association with AKAP149 and S-AKAP84, respectively. These results suggest that AMY-1 plays a role in spermatogenesis.
A Kinase Anchor Proteins↗
Atypical protein kinase C is involved in the evolutionarily conserved par protein complex and plays a critical role in establishing epithelia-specific junctional structures.
We have previously shown that during early Caenorhabditis elegans embryogenesis PKC-3, a C. elegans atypical PKC (aPKC), plays critical roles in the establishment of cell polarity required for subsequent asymmetric cleavage by interacting with PAR-3 [Tabuse, Y., Y. Izumi, F. Piano, K.J. Kemphues, J. Miwa, and S. Ohno. 1998. Development (Camb.). 125:3607--3614]. Together with the fact that aPKC and a mammalian PAR-3 homologue, aPKC-specific interacting protein (ASIP), colocalize at the tight junctions of polarized epithelial cells (Izumi, Y., H. Hirose, Y. Tamai, S.-I. Hirai, Y. Nagashima, T. Fujimoto, Y. Tabuse, K.J. Kemphues, and S. Ohno. 1998. J. Cell Biol. 143:95--106), this suggests a ubiquitous role for aPKC in establishing cell polarity in multicellular organisms. Here, we show that the overexpression of a dominant-negative mutant of aPKC (aPKCkn) in MDCK II cells causes mislocalization of ASIP/PAR-3. Immunocytochemical analyses, as well as measurements of paracellular diffusion of ions or nonionic solutes, demonstrate that the biogenesis of the tight junction structure itself is severely affected in aPKCkn-expressing cells. Furthermore, these cells show increased interdomain diffusion of fluorescent lipid and disruption of the polarized distribution of Na(+),K(+)-ATPase, suggesting that epithelial cell surface polarity is severely impaired in these cells. On the other hand, we also found that aPKC associates not only with ASIP/PAR-3, but also with a mammalian homologue of C. elegans PAR-6 (mPAR-6), and thereby mediates the formation of an aPKC-ASIP/PAR-3-PAR-6 ternary complex that localizes to the apical junctional region of MDCK cells. These results indicate that aPKC is involved in the evolutionarily conserved PAR protein complex, and plays critical roles in the development of the junctional structures and apico-basal polarization of mammalian epithelial cells.
Actins↗
Preparation of anhydrothrombin and characterization of its interaction with natural thrombin substrates.
Thrombin is a serine proteinase that plays a key role in thrombosis and haemostasis through its interaction with several coagulation factors. Anhydrothrombin was prepared from PMSF-inactivated thrombin under alkaline conditions, and the folded anhydrothrombin was successfully recovered after dialysis in the presence of glycerol. Anhydro-derivatives of factor Xa, factor VIIa and activated protein C could also be prepared essentially by the same procedure. Anhydrothrombin retained affinity for various natural substrates of thrombin, including fibrinogen, factor VIII, factor XIII and protein C. In addition, these proteins were bound to anhydrothrombin-agarose in a reversible manner. The K(d) values for factor VIII, fibrinogen, factor XIII and protein C were 1.2x10(-8), 4.4x10(-8), 2.8x10(-7) and 8.1x10(-5) M, respectively. Thus thrombin substrates known to interact with the exosite I of thrombin demonstrated high affinity for anhydrothrombin. Furthermore, in the presence of Na+, substantial enhancement of the association rate constant (k(ass)) was observed for interactions of fibrinogen and factor VIII with anhydrothrombin. These results suggest that anhydrothrombin is useful in the purification of thrombin substrate proteins as well as in the investigation of detailed interactions between thrombin and these substrates in their activation or degradation processes.
Chromatography, Affinity↗
Metabolic activation of mitomycin C by NADPH-ferredoxin reductase in vitro.
Mammalian NADPH-ferredoxin reductase (EC 1.18.1.2) functions in the mitochondrial electron transport chain for cytochrome P-450-dependent steroid hydroxylation. Significant homology of three-dimensional structure exists in the surroundings of FAD between NADPH-ferredoxin reductase and NADH-cytochrome b5 reductase. The latter is involved in the bioreduction of mitomycin C (MC), a prototype antitumor agent. In this study, we assessed the capacity of NADPH-ferredoxin reductase to activate MC. Mitomycin C increased the NADPH oxidase activity of NADPH-ferredoxin reductase. In the absence of ferredoxin, the Km value of NADPH-ferredoxin reductase for MC was 73.5 +/- 2.3 microM. While in the presence of 500 nM ferredoxin, a Lineweaver-Burk plot exhibited a biphasic curve. NADPH-ferredoxin reductase-mediated reduction of MC resulted in the formation of an alkylated complex of 4-(p-nitrobenzyl) pyridine and an increase in plasmide DNA single-strand breaks under hypoxic conditions. With the addition of 500 nM ferredoxin, the amount of the alkylated complex of 4-(p-nitrobenzyl) pyridine and the plasmide DNA single-strand breaks increased by 40% and 37%, respectively. However, neither alkylated complex of 4-(p-nitrobenzyl) pyridine nor DNA strand breaks was observed in the presence of SOD and catalase under aerobic conditions. These findings demonstrate that NADPH-ferredoxin reductase is capable of catalyzing the bioactivation of mitomycin C under hypoxic conditions in vitro.
Alkylation↗
Crystal structure of the Holliday junction migration motor protein RuvB from Thermus thermophilus HB8.
We report here the crystal structure of the RuvB motor protein from Thermus thermophilus HB8, which drives branch migration of the Holliday junction during homologous recombination. RuvB has a crescent-like architecture consisting of three consecutive domains, the first two of which are involved in ATP binding and hydrolysis. DNA is likely to interact with a large basic cleft, which encompasses the ATP-binding pocket and domain boundaries, whereas the junction-recognition protein RuvA may bind a flexible beta-hairpin protruding from the N-terminal domain. The structures of two subunits, related by a noncrystallographic pseudo-2-fold axis, imply that conformational changes of motor protein coupled with ATP hydrolysis may reflect motility essential for its translocation around double-stranded DNA.
Adenosine Triphosphatases↗
Differentiation of Dictyostelium discoideum vegetative cells into spores during Earth orbit in space.
We reported previously that emerged amoebae of Dictyostelium (D.) discoideum grew, aggregated and differentiated to fruiting bodies with normal morphology in space. Here, we investigated the effects of space radiation and/or microgravity on the number, viability, kinetics of germination, growth rate and mutation frequency of spores formed in space in a radiation-sensitive strain, gamma s13, and the parental strain, NC4. In gamma s13, there were hardly spores in the fruiting bodies formed in space. In NC4, we found a decrease in the number of spores, a delay in germination of the spores and delayed start of cell growth of the spores formed in space when compared to the ground control. However, the mutation frequency of the NC4 spores formed in space was similar to that of the ground control. We conclude that the depression of spore formation might be induced by microgravity and/or space radiation through the depression of some stage(s) of DNA repair during cell differentiation in the slime mold.
Animals↗
The effects of microgravity on induced mutation in Escherichia coli and Saccharomyces cerevisiae.
We examined whether microgravity influences the induced-mutation frequencies through in vivo experiments during space flight aboard the space shuttle Discovery (STS-91). We prepared dried samples of repair-deficient strains and parental strains of Escherichia (E.) coli and Saccharomyces (S.) cerevisiae given DNA damage treatment. After culture in space, we measured the induced-mutation frequencies and SOS-responses under microgravity. The experimental findings indicate that almost the same induced-mutation frequencies and SOS-responses of space samples were observed in both strains compared with the ground control samples. It is suggested that microgravity might not influence induced-mutation frequencies and SOS-responses at the stages of DNA replication and/or DNA repair. In addition, we developed a new experimental apparatus for space experiments to culture and freeze stocks of E. coli and S. cerevisiae cells.
Alkylating Agents↗
Alkylating agent (MNU)-induced mutation in space environment.
In recent years, some contradictory data about the effects of microgravity on radiation-induced biological responses in space experiments have been reported. We prepared a damaged template DNA produced with an alkylating agent (N-methyl-N-nitroso urea; MNU) to measure incorrect base-incorporation during DNA replication in microgravity. We examined whether mutation frequency is affected by microgravity during DNA replication for a DNA template damaged by an alkylating agent. Using an in vitro enzymatic reaction system, DNA synthesis by Taq polymerase or polymerase III was done during a US space shuttle mission (Discovery, STS-91). After the flight, DNA replication and mutation frequencies were measured. We found that there was almost no effect of microgravity on DNA replication and mutation frequency. It is suggested that microgravity might not affect at the stage of substrate incorporation in induced-mutation frequency.
Alkylating Agents↗