Metagenomic analysis of the microbial communities and associated network of nitrogen metabolism genes in the Ryukyu limestone aquifer.

While microbial biogeochemical activities such as those involving denitrification and sulfate reduction have been considered to play important roles in material cycling in various aquatic ecosystems, our current understanding of the microbial community in groundwater ecosystems is remarkably insufficient. To assess the groundwater in the Ryukyu limestone aquifer of Okinawa Island, which is located in the southernmost region of Japan, we performed metagenomic analysis on the microbial communities at the three sites and screened for functional genes associated with nitrogen metabolism. 16S rRNA amplicon analysis showed that bacteria accounted for 94-98% of the microbial communities, which included archaea at all three sites. The bacterial communities associated with nitrogen metabolism shifted by month at each site, indicating that this metabolism was accomplished by the bacterial community as a whole. Interestingly, site 3 contained much higher levels of the denitrification genes such as narG and napA than the other two sites. This site was thought to have undergone denitrification that was driven by high quantities of dissolved organic carbon (DOC). In contrast, site 2 was characterized by a high nitrate-nitrogen (NO3-N) content and a low amount of DOC, and this site yielded a moderate amount of denitrification genes. Site 1 showed markedly low amounts of all nitrogen metabolism genes. Overall, nitrogen metabolism in the Ryukyu limestone aquifer was found to change based on environmental factors.

Mechanism of denitrification in subsurface-dammed Ryukyu limestone aquifer, southern Okinawa Island, Japan.

Denitrification crucially regulates the attenuation of groundwater nitrate and is unlikely to occur in a fast-flowing aquifer such as the Ryukyu limestone aquifer in southern Okinawa Island, Japan. However, evidences of denitrification have been observed in several wells within this region. This study analyzed environmental isotopes (δ15NNO3 and ẟ18ONO3) to derive the rationale for denitrification at this site. Additionally, the presence of two subsurface dams in the study area may influence the processes involved in nitrate attenuation. Herein, we analyzed 150 groundwater samples collected spatially and seasonally to characterize the variations in the groundwater chemistry and stable isotopes during denitrification. The values of δ15NNO3 and δ18ONO3 displayed a progressive trend up to +59.7 ‰ and + 21 ‰, respectively, whereas the concentrations of NO3--N decreased to 0.1 mg L-1. In several wells, the enrichment factors of δ15NNO3 ranged from -6.6 to -2.1, indicating rapid denitrification, and the δ15NNO3 to δ18ONO3 ratios varied from 1.3:1 to 2:1, confirming the occurrence of denitrification. Denitrification intensively proceeds under conditions of depleted dissolved oxygen concentrations (<2 mg L-1), sluggish groundwater flow with longer residence times, high concentrations of dissolved organic carbon (>1.2 mg L-1), and low groundwater levels during the dry season with precipitation rates of <100 mm per month (Jun-Sep). SF6 analysis indicated the exclusive occurrence of denitrification in specific wells with groundwater residence times exceeding 30 years. These wells are located in close proximity to the major NE-SW fault system in the Komesu area, where the hydraulic gradient was below 0.005. Detailed geological and lithological investigations based on borehole data revealed that subsurface dams did not cause denitrification while the major NE-SW fault system uplifted the impermeable basement rock of the Shimajiri Group, creating a lithological gap at an equivalent depth that ultimately formed a sluggish groundwater area, promoting denitrification.

Effect of groundwater residence time on geogenic fluoride release into groundwater in the Mt. Meru slope area, Tanzania, the Great Rift Valley, East Africa.

People living in the Great Rift Valley in East Africa suffer from fluorosis resulting from their consumption of groundwater. This paper shows that geogenic fluoride contamination in a natural water system has changed in the last two decades in the Mt. Meru slope area of northern Tanzania based on water quality, dating of the residence time, and stable isotopes of groundwater. The results demonstrate that 1) the average recharge altitude of groundwater with a high geogenic fluoride concentration is estimated to range from 1900 m to 3000 m on the southern slope of Mt. Meru, and the fluoride concentration tends to increase with an increase in the recharge altitude, 2) the fluoride concentration increases with increasing groundwater residence time for groundwater with a residence time of 20 years or longer, suggesting that water-rock interaction processes (weathering, dissolution, and ion exchange), which depend on the contact time between the volcanic aquifer and groundwater, have predominated for approximately 20 years or longer, and 3) the mixing of aerobic young water and old groundwater has been active for approximately 20 years, and the fluoride concentration is increasing in some shallower well waters. The mixing of fluoride-contaminated groundwater with aerobic water infiltrating the aquifer through pumping groundwater in the last two decades may increase the spread of groundwater contaminated with fluoride due to increased water demand caused by rapid population growth, and urbanization, industrial growth, and the expansion of irrigated agriculture.

Visualisation of Phosphate in Subcalicoblastic Extracellular Calcifying Medium and on a Skeleton of Coral by Using a Novel Probe, Fluorescein-4-Isothiocyanate-Labelled Alendronic Acid.

The overload of nutrients of anthropogenic origin, including phosphate, onto coastal waters has been reported to have detrimental effects on corals. However, to the best of our knowledge, the phosphate concentration threshold for inhibiting coral calcification is unclear owing to a lack of information on the molecular mechanisms involved in the inhibitory effect of phosphate. Therefore, in this study, we prepared a new phosphate analogue, fluorescein-4-isothiocyanate (FITC)-labelled alendronic acid (FITC-AA), from commercially available reagents and used it as a novel probe to demonstrate its transfer pathway from ambient seawater into Acropora digitifera. When the juveniles at 1 d post-settlement were treated with FITC-AA in a laboratory tank, this phosphate analogue was found in the subcalicoblastic extracellular calcifying medium (SCM) and was absorbed on the basal plate in the juveniles within a few minutes. When the juveniles bear zooxanthellae at 3 months post-settlement, FITC-AA was observed on the corallite walls within a few minutes after adding ambient seawater. We concluded that FITC-AA in ambient seawater was transferred via a paracellular pathway to SCM and then absorbed on the coral CaCO3 skeletons because FITC-AA with a high polarity group cannot permeate through cell membranes.

Phosphate bound to calcareous sediments hampers skeletal development of juvenile coral.

To test the hypothesis that terrestrial runoff affects the functions of calcareous sediments in coral reefs and hampers the development of corals, we analysed calcareous sediments with different levels of bound phosphate, collected from reef areas of Okinawajima, Japan. We confirmed that phosphate bound to calcareous sediments was readily released into ambient seawater, resulting in much higher concentrations of phosphorous in seawater from heavily polluted areas (4.3-19.0 µM as compared with less than 0.096 µM in natural ambient seawater). Additionally, we examined the effect of phosphate released from calcareous sediments on the development of Acropora digitifera coral juveniles. We found that high phosphate concentrations in seawater clearly inhibit the skeletal formation of coral juveniles. Our results demonstrate that calcareous sediments in reef areas play a crucial role in mediating the impact of terrestrial runoff on corals by storing and releasing phosphate in seawater.

Phosphate Enrichment Hampers Development of Juvenile Acropora digitifera Coral by Inhibiting Skeleton Formation.

Coral reef degradation due to various local stresses, such as nutrient enrichment and terrestrial run-off into coastal waters, is an increasing global concern. Inorganic phosphates have been considered to possibly inhibit skeleton formation in corals. Despite many studies available on the effects of nutrients on corals, a clear consensus on how nutrients exert deteriorative effects on corals has not been established satisfactorily. In this study, we examined the effects of phosphates and nitrates on in vitro aragonite CaCO3 formation by using biogenic polyamines and in vivo aragonite formation in the skeleton of juvenile Acropora digitifera corals. We showed that the phosphates at similar concentrations clearly inhibited both in vitro and in vivo CaCO3 formation. In contrast, nitrates inhibited neither in vitro aragonite CaCO3 formation nor in vivo aragonite formation in juvenile coral skeleton. Furthermore, our findings showed that inhibition of coral skeleton formation was due to absorption of phosphate on the skeleton, which inorganically inhibited normal development of juvenile coral skeleton.

Atmospheric CO2 captured by biogenic polyamines is transferred as a possible substrate to Rubisco for the carboxylation reaction.

Biogenic polyamines are involved in a wide range of plant cellular processes, including cell division, morphogenesis and stress responses. However, the exact roles of biogenic polyamines are not well understood. We recently reported that biogenic polyamines that have multiple amino groups can react with CO2 and accelerate calcium carbonate formation in seawater. The ability of biogenic polyamines to capture atmospheric CO2 prompted us to examine their roles in photosynthesis. Here, we demonstrated that atmospheric CO2 captured by biogenic polyamines is a candidate substrate for the carboxylation reaction of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), which is an enzyme involved in the first major step of carbon fixation during photosynthesis, and that biogenic polyamines can accelerate the carboxylation reaction of this enzyme because of their specific affinity for CO2. Moreover, the results of our nuclear magnetic resonance (NMR) analysis showed that putrescine, which is the most common biogenic polyamine, reacts with atmospheric CO2 and promotes the formation of carbamate derivatives and bicarbonate in aqueous environments. A sufficient amount of CO2 is well known to be produced by carbonic anhydrase from bicarbonate in vivo. The present study indicates that CO2 would be also produced by the equilibrium reaction from carbonate produced by biogenic polyamines and would be used as a substrate of Rubisco, too. Our results may suggest a new photosynthetic research strategy that involves CO2-concentrating mechanisms and also possibly constitutes a potential tool for reducing atmospheric CO2 levels and, consequently, global warming.

Hepatitis B virus prevents excessive viral production via reduction of cell death-inducing DFF45-like effectors.

The relationship between hepatitis B virus (HBV) infection and lipid accumulation remains largely unknown. In this study, we investigated the effect of HBV propagation on lipid droplet growth in HBV-infected cells and HBV-producing cell lines, HepG2.2.15 and HBV-inducible Hep38.7-Tet. The amount of intracellular triglycerides was significantly reduced in HBV-infected and HBV-producing cells compared with HBV-lacking control cells. Electron and immunofluorescent microscopic analyses showed that the average size of a single lipid droplet (LD) was significantly less in the HBV-infected and HBV-producing cells than in the HBV-lacking control cells. Cell death-inducing DFF45-like effectors (CIDEs) B and C (CIDEB and CIDEC), which are involved in LD expansion for the improvement of lipid storage, were expressed at a significantly lower level in HBV-infected or HBV-producing cells than in HBV-lacking control cells, while CIDEA was not detected in those cells regardless of HBV production. The activity of the CIDEB and CIDEC gene promoters was impaired in HBV-infected or HBV-producing cells compared to HBV-lacking control cells, while CIDEs potentiated HBV core promoter activity. The amount of HNF4α, that can promote the transcription of CIDEB was significantly lower in HBV-producing cells than in HBV-lacking control cells. Knockout of CIDEB or CIDEC significantly reduced the amount of supernatant HBV DNA, intracellular viral RNA and nucleocapsid-associated viral DNA, while the expression of CIDEB or CIDEC recovered HBV production in CIDEB- or CIDEC-knockout cells. These results suggest that HBV regulates its own viral replication via CIDEB and CIDEC.

Involvement of FKBP6 in hepatitis C virus replication.

The chaperone system is known to be exploited by viruses for their replication. In the present study, we identified the cochaperone FKBP6 as a host factor required for hepatitis C virus (HCV) replication. FKBP6 is a peptidyl prolyl cis-trans isomerase with three domains of the tetratricopeptide repeat (TPR), but lacks FK-506 binding ability. FKBP6 interacted with HCV nonstructural protein 5A (NS5A) and also formed a complex with FKBP6 itself or FKBP8, which is known to be critical for HCV replication. The Val(121) of NS5A and TPR domains of FKBP6 were responsible for the interaction between NS5A and FKBP6. FKBP6 was colocalized with NS5A, FKBP8, and double-stranded RNA in HCV-infected cells. HCV replication was completely suppressed in FKBP6-knockout hepatoma cell lines, while the expression of FKBP6 restored HCV replication in FKBP6-knockout cells. A treatment with the FKBP8 inhibitor N-(N', N'-dimethylcarboxamidomethyl)cycloheximide impaired the formation of a homo- or hetero-complex consisting of FKBP6 and/or FKBP8, and suppressed HCV replication. HCV infection promoted the expression of FKBP6, but not that of FKBP8, in cultured cells and human liver tissue. These results indicate that FKBP6 is an HCV-induced host factor that supports viral replication in cooperation with NS5A.

Hepatitis B virus efficiently infects non-adherent hepatoma cells via human sodium taurocholate cotransporting polypeptide.

Sodium taurocholate cotransporting polypeptide (NTCP) has been reported as a functional receptor for hepatitis B virus (HBV) infection. However, HBV could not efficiently infect HepG2 cells expressing NTCP (NTCP-HepG2 cells) under adherent monolayer-cell conditions. In this study, NTCP was mainly detected in the basolateral membrane region, but not the apical site, of monolayer NTCP-HepG2 cells. We hypothesized that non-adherent cell conditions of infection would enhance HBV infectivity. Non-adherent NTCP-HepG2 cells were prepared by treatment with trypsin and EDTA, which did not degrade NTCP in the membrane fraction. HBV successfully infected NTCP-HepG2 cells at a viral dose 10 times lower in non-adherent phase than in adherent phase. Efficient infection of non-adherent NTCP-HepG2 cells with blood-borne or cell-culture-derived HBV was observed and was remarkably impaired in the presence of the myristoylated preS1 peptide. HBV could also efficiently infect HepaRG cells under non-adherent cell conditions. We screened several compounds using our culture system and identified proscillaridin A as a potent anti-HBV agent with an IC50 value of 7.2 nM. In conclusion, non-adherent host cell conditions of infection augmented HBV infectivity in an NTCP-dependent manner, thus providing a novel strategy to identify anti-HBV drugs and investigate the mechanism of HBV infection.

Hallmarks of hepatitis C virus in equine hepacivirus.

UNLABELLED: Equine hepacivirus (EHcV) has been identified as a closely related homologue of hepatitis C virus (HCV) in the United States, the United Kingdom, and Germany, but not in Asian countries. In this study, we genetically and serologically screened 31 serum samples obtained from Japanese-born domestic horses for EHcV infection and subsequently identified 11 PCR-positive and 7 seropositive serum samples. We determined the full sequence of the EHcV genome, including the 3' untranslated region (UTR), which had previously not been completely revealed. The polyprotein of a Japanese EHcV strain showed approximately 95% homology to those of the reported strains. HCV-like cis-acting RNA elements, including the stem-loop structures of the 3' UTR and kissing-loop interaction were deduced from regions around both UTRs of the EHcV genome. A comparison of the EHcV and HCV core proteins revealed that Ile(190) and Phe(191) of the EHcV core protein could be important for cleavage of the core protein by signal peptide peptidase (SPP) and were replaced with Ala and Leu, respectively, which inhibited intramembrane cleavage of the EHcV core protein. The loss-of-function mutant of SPP abrogated intramembrane cleavage of the EHcV core protein and bound EHcV core protein, suggesting that the EHcV core protein may be cleaved by SPP to become a mature form. The wild-type EHcV core protein, but not the SPP-resistant mutant, was localized on lipid droplets and partially on the lipid raft-like membrane in a manner similar to that of the HCV core protein. These results suggest that EHcV may conserve the genetic and biological properties of HCV. IMPORTANCE: EHcV, which shows the highest amino acid or nucleotide homology to HCV among hepaciviruses, was previously reported to infect horses from Western, but not Asian, countries. We herein report EHcV infection in Japanese-born horses. In this study, HCV-like RNA secondary structures around both UTRs were predicted by determining the whole-genome sequence of EHcV. Our results also suggest that the EHcV core protein is cleaved by SPP to become a mature form and then is localized on lipid droplets and partially on lipid raft-like membranes in a manner similar to that of the HCV core protein. Hence, EHcV was identified as a closely related homologue of HCV based on its genetic structure as well as its biological properties. A clearer understanding of the epidemiology, genetic structure, and infection mechanism of EHcV will assist in elucidating the evolution of hepaciviruses as well as the development of surrogate models for the study of HCV.

Biogenic polyamines capture CO2 and accelerate extracellular bacterial CaCO3 formation.

Bacteria, including cyanobacteria, as well as some fungi, are known to deposit calcium carbonate (CaCO(3)) extracellularly in calcium-containing artificial medium. Despite extensive investigation, the mechanisms involved in extracellular formation of CaCO(3) by bacteria have remained unclear. The ability of synthetic amines to remove carbon dioxide (CO(2)) from natural gas led us to examine the role of biogenic polyamines in CaCO(3) deposition by bacteria. Here, we demonstrated that biogenic polyamines such as putrescine, spermidine, and spermine were able to react with atmospheric CO(2) and the resultant carbamate anion was characterized by using nuclear magnetic resonance (NMR) analysis. Biogenic polyamines accelerated the formation of CaCO(3), and we artificially synthesized the dumbbell-shaped calcites, which had the same form as observed with bacterial CaCO3 precipitates, under nonbacterial conditions by using polyamines. The reaction rate of calcification increased with temperature with an optimum of around 40 °C. Our observation suggests a novel scheme for CO(2) dissipation that could be a potential tool in reducing atmospheric CO(2) levels and, therefore, global warming.

Anticancer effects of phenoxazine derivatives revealed by inhibition of cell growth and viability, disregulation of cell cycle, and apoptosis induction in HTLV-1-positive leukemia cells.

Adult T-cell leukemia (ATL) is a malignant tumor of human CD4(+) T cells infected with a human retrovirus, T lymphotropic virus type-1 (HTLV-1). The aim of the present study was to investigate the apoptotic effects of phenoxazines, 2-amino-4,4alpha-dihydro-4alpha,7-dimethyl-3H-phenoxazine-3-one (Phx-1), 3-amino-1,4alpha-dihydro-4alpha,8-dimethyl-2H-phenoxazine-2-one (Phx-2), and 2-aminophenoxazine-3-one (Phx-3) on a T cell leukemia cell line from ATL patients, MT-1 cells; HTLV-1 transformed T-cell lines, HUT-102 cells and MT-2 cells; and an HTLV-1-negative rat sarcoma cell line, XC cells. Among these phenoxazines, Phx-3 at concentrations of less than 10 microg/ml extensively inhibited growth and cell viability; arrested cell cycles at sub G(0)/G(1) phase; and augmented apoptosis of MT-1, HUT-102, and MT-2 cells. However, these phenoxazines did not affect the cell viability of an HTLV-1-negative rat sarcoma cell line, XC cells, and phytohemaggutinin-activated human peripheral blood mononuclear cells, although they markedly inhibited the growth of these cells. The transmission of HTLV-1 from HTLV-1-positive cells (MT-2 cells) to HTLV-1-negative cells (XC cells) was considered to be prevented by Phx-1, Phx-2, or Phx-3 because the syncytium formation between these cells was inhibited markedly in the presence of these phenoxazines. The present results suggest that Phx-1, Phx-2, and, in particular, Phx-3 may be useful as therapeutic agents against ATL, which is extremely refractory to current therapies.

Protein microarray analysis of apoptosis-related protein expression following heat shock in human tongue squamous cell carcinomas containing different p53 phenotypes.

PURPOSE: Hyperthermia is useful in the treatment of human head and neck cancers, because it is relatively easy to regulate temperatures when compared to tumors located in deep organs. In this study, attention was focused on p53 as a possible predictive indicator for the efficacy of hyperthermic cancer therapy. METHODS: Two kinds of cell lines were used. These were derived from a human squamous cell carcinoma (SAS) and had identical genetic backgrounds except for their p53 gene status. It was previously reported that the heat sensitivity and frequency of apoptosis in wild-type p53 cells (SAS/neo) were clearly elevated when compared with mutated p53 cells (SAS/mp53). In order to study the expression of apoptosis related proteins after heat treatment, protein microarray analysis was used. RESULTS: The expression of apoptosis inhibitory proteins such as Bcl-2, Bcl-xL, NF-kappaB, COX2, STAT3, IL-6, and IKKalpha/1 was seen to increase after heat treatment in SAS/mp53 cells, but not in SAS/neo cells. CONCLUSION: The result of these observations indicates that apoptosis inhibitory proteins (such as Bcl-2, Bcl-xL, IL-6, etc.) were highly induced in SAS/mp53 cells after heat treatment when compared to control SAS/neo cells.

LY294002, an inhibitor of PI-3K, enhances heat sensitivity independently of p53 status in human lung cancer cells.

The aim of this study was to ascertain whether LY294002, an inhibitor of PI-3K, enhances heat sensitivity in human cancer cells regardless of their p53 status. Colony formation assays showed that LY294002 enhanced heat sensitivity in two human lung cancer cell lines; H1299/wild-type p53 (wtp53) and H1299/mutated p53 (mp53) cells. These cell lines have identical genetic backgrounds except for their p53 status. LY294002 suppressed the heat-induced accumulation of heat shock protein 27 (hsp27) and heat shock protein 72 (hsp72) in these cell lines. Heat-induced apoptosis was observed more frequently in H1299/wtp53 cells than in H1299/mp53 cells, and was enhanced by LY294002 in both cell lines. In addition, both the heat-induced phosphorylation of Akt and the accumulation of survivin were suppressed by LY294002. These results suggest that LY294002 inhibits anti-apoptosis signaling through hsp27 and hsp72 as well as cell survival signaling through Akt and survivin. LY294002 appears to be an attractive candidate for a p53-independent heat sensitizer in hyperthermic cancer therapy.

Glycerol enhances radiosensitivity in a human oral squamous cell carcinoma cell line (Ca9-22) bearing a mutant p53 gene via Bax-mediated induction of apoptosis.

Radiotherapy for oral squamous cell carcinomas is limited in its efficacy and in its ability to improve the survival rate in patients at an advanced stage. A protocol is described here which may elevate the therapeutic efficacy of radiation for these cells. The addition of glycerol to the culture medium prior to irradiation of an oral squamous cell carcinoma cell line (Ca9-22) bearing a mutant p53 (mp53) gene was found to increase the radiosensitivity of these cells. A colony formation assay was used to evaluate the effect of glycerol on the radiation sensitivity of Ca9-22 cells. Apoptosis was analyzed using Hoechst 33342 staining, Western blotting, and a DNA ladder formation assay. Glycerol, when present in the culture medium, enhanced the radiation sensitivity and extent of apoptosis following X-irradiation in the Ca9-22 cells, although neither X-rays or glycerol alone increased the extent of apoptosis. Bax protein was accumulated after treatment with X-rays plus glycerol, but not after exposure to X-rays or glycerol alone. A gel mobility-shift assay showed that glycerol restored the DNA-binding activity of mp53 for a p53-consensus sequence to levels similar to that of wild-type p53. These findings suggest that pre-treatment with glycerol may enhance the effectiveness of radiotherapy against oral squamous cell carcinomas bearing an mp53 gene mutation.

Evidence for the involvement of double-strand breaks in heat-induced cell killing.

To identify critical events associated with heat-induced cell killing, we examined foci formation of gammaH2AX (histone H2AX phosphorylated at serine 139) in heat-treated cells. This assay is known to be quite sensitive and a specific indicator for the presence of double-strand breaks. We found that the number of gammaH2AX foci increased rapidly and reached a maximum 30 minutes after heat treatment, as well as after X-ray irradiation. When cells were heated at 41.5 degrees C to 45.5 degrees C, we observed a linear increase with time in the number of gammaH2AX foci. An inflection point at 42.5 degrees C and the thermal activation energies above and below the inflection point were almost the same for cell killing and foci formation according to Arrhenius plot analysis. From these results, it is suggested that the number of gammaH2AX foci is correlated with the temperature dependence of cell killing. During periods when cells were exposed to heat, the cell cycle-dependent pattern of cell killing was the same as the cell cycle pattern of gammaH2AX foci formation. We also found that thermotolerance was due to a depression in the number of gammaH2AX foci formed after heating when the cells were pre-treated by heat. These findings suggest that cell killing might be associated with double-strand break formation via protein denaturation.

High-LET radiation enhanced apoptosis but not necrosis regardless of p53 status.

PURPOSE: We analyzed the death pattern of human lung cancer cells harboring different p53 statuses after irradiation with different levels of linear energy transfer (LET). METHODS AND MATERIALS: We used three kinds of human lung cancer cell lines with identical genotypes, except for the p53 gene. These cells were exposed to X-rays or accelerated carbon-ion beams. The cellular sensitivities were determined by a colony-forming assay. The detection and quantification of cell death (apoptosis and necrosis) were evaluated and compared by acridine orange/ethidium bromide double staining for fluorescence microscopy. RESULTS: We found that (1) there was no significant difference in cellular sensitivity to LET radiation >70 KeV/microm, although wild-type p53 cell sensitivity to X-rays was higher than that of mutated p53 or p53-null cells; (2) low-LET radiation effectively induced apoptosis in wild-type p53 cells as compared with mutated p53 and p53-null cells; and (3) high-LET radiation induced p53-independent apoptosis. CONCLUSIONS: Our findings suggest that high-LET radiotherapy is expected to be a valid application for patients carrying mutated p53 cancer cells. We proposed that the elucidation of the p53-independent apoptosis-related genes might provide new insights into radiotherapy for cancer.

Grit-blasted and hydroxyapatite-coated total hip arthroplasty: an 11- to 14-year follow-up study.

We report long-term results of the first clinical trial of hydroxyapatite-coated total hip arthroplasty conducted in Japan. The hemispherical cup and the straight-tapered stem were made of titanium alloy with a grit-blasted, hydroxyapatite-coated surface. The surface roughness before and after hydroxyapatite coating was 1.4 microm and 3.4 microm, respectively. Thirty-three patients (35 hips) were followed prospectively; of these, 1 patient was lost to follow-up, 5 were deceased at the latest follow-up, and 27 were followed for 11 to 14 years. Two cups and one stem (two patients) were revised. Survivorship, with radiological acetabular loosening as the endpoint, was 62.3% at 14 years. At the latest radiological follow-up, stable fixation with bone ongrowth was achieved in 46% of the acetabular cups and 89% of the femoral stems. Acetabular cups with host bone coverage of less than 60% had a high rate of failure. The suboptimal result of the hydroxyapatite-coated smooth cup indicates that porous coatings under the hydroxyapatite coating would be beneficial for hydroxyapatite-coated total hip implants, especially for the acetabular components.

Glycerol enhances CDDP-induced growth inhibition of thyroid anaplastic carcinoma tumor carrying mutated p53 gene.

To increase the chemo-sensitivity of anaplastic thyroid carcinoma, we examined the effects of glycerol on the tumor growth after CDDP treatment. The cultured cells of an anaplastic thyroid carcinoma cell line (8305c) carrying a mutated p53 gene (mp53) were transplanted into the thighs of nude mice. Tumor growth was evaluated until 24 days after intraperitoneal injection of CDDP and/or pre-injection of glycerol to the tumor. We treated the mice with half the tumor volume of glycerol (1.2 M) and/or CDDP at 6 mg/kg (BW) either of which hardly inhibited tumor growth by itself. When we treated the mice with the combination of glycerol and CDDP at these concentrations, however, a clear delay of the tumor growth was observed. We also immunohistochemically analyzed the effects of glycerol on the induction of caspase-3 activity and apoptosis. Cells positive for cleavage to active caspase-3 and 85 kDa PARP, and apoptosis were hardly observed in the tumors when they were treated with glycerol or CDDP alone. In contrast, when they were treated with CDDP combined with glycerol, such positive cells were significantly increased. It has been shown that glycerol synergistically enhanced the effects of CDDP as a tumor suppressive agent through the induction of caspase-3-mediated apoptosis in 8305c tumors. Therefore, glycerol might be useful for chemotherapy in patients with mp53 cancer cells.