Controlled direct electron transfer kinetics of fructose dehydrogenase at cup-stacked carbon nanofibers.

Graphene edge sites not only facilitate heterogeneous electron transfer reactions of redox species because of localization of electrons, but also allow sensitivities and selectivities to be tuned by controlling the atomic oxygen/carbon (O/C) ratio. Here, we immobilized fructose dehydrogenase (FDH) onto the surface of cup-stacked carbon nanofibers (CSCNFs), which provide highly ordered graphene edges with a controlled O/C ratio, and investigated the direct electron communication with FDH. As the O/C ratio decreased at the CSCNF surface, the negative zeta potential was mitigated and the electrochemical communication with FDH was facilitated. This is likely due to improved orientation of FDH molecules on the CSCNF surface. CSCNFs with a controlled O/C ratio could be applied to FDH-based d-fructose biosensors with tunable dynamic range and fructose biofuel cells with a controlled maximum current.

Degradation of bisphenol A in water by TiO2 photocatalyst.

The photocatalytic degradation of bisphenol A (BPA), a representative endocrine disruptor, was carried out in TiO2 aqueous suspension. The main purposes were to confirm the total mineralization of BPA and to evaluate the estrogenic activity in the treated water during the photocatalytic reaction. An initial BPA concentration of 175 microM in water was totally degraded to carbon dioxide by TiO2-photocatalyzed reactions under UV irradiation of 10 mW cm-2 for 20 h. Four HPLC peaks indicating intermediate products appeared in chromatograms monitored at 275 nm, but the heights relative to that of the initial BPA were very low, at most 0.04 in the time period 5-10 h after the start of UV irradiation. All of the peaks finally disappeared after 20 h. For the treated water, the transcriptional estrogenic activity in response to human estrogen receptor in a yeast hybrid assay decreased drastically to less than 1% of the initial BPA's activity within 4 h. On the basis of these results, we conclude that TiO2 photocatalysis could be a useful technology for the purification of water containing BPA without generating any serious secondary pollution.

Self-sterilizing and self-cleaning of silicone catheters coated with TiO(2) photocatalyst thin films: a preclinical work.

TiO(2) photocatalysts were successfully coated on silicone catheters or medical tubes by pretreatment of the silicone surface with a sulfuric acid solution (5 M) for 3 h. The TiO(2) film adhered to the silicone substrate strongly against tensile and bending stresses. On the TiO(2)-coated silicone-catheters under UV illumination, both the bleaching of methylene blue dye and the photocatalytic bactericidal effect on Escherichia coli (E. coli) cells were confirmed. Thus, this type of catheter can be sterilized and cleaned simply by irradiation with low-intensity UV light and can, therefore, be useful in the protection from catheter-related bacterial infections.

Telomerase activity in peripheral blood for diagnosis of hepatoma.

BACKGROUND: Telomerase activity may be used as a molecular marker for the detection of circulating hepatoma cells in blood of patients with hepatoma. METHODS: Telomerase activity in peripheral blood from hepatocellular carcinoma (HCC) patients was assessed by using a highly sensitive and non-radioisotope telomerase polymerase chain reaction (PCR) ELISA. Initially, tissue telomerase activity was measured in the hepatoma and non-tumour portions by using PCR ELISA within the same specimen, to compare its sensitivity with the conventional telomeric repeat amplification protocol (TRAP) method. Second, telomerase activity was measured in the peripheral blood obtained from patients with HCC, patients with chronic liver disease and in healthy controls. RESULTS: Of the 17 HCC patients, telomerase activity was found to be positive in 14 (82%) by using TRAP and 15 (88%) by using PCR ELISA, indicating that PCR ELISA is a reliable tool for the measurement of telomerase activity. By using the Telomerase PCR ELISA assay, telomerase activities in the peripheral blood of 20 HCC patients was 1.65 +/- 0.78 units. This was significantly greater than the results obtained for 20 chronic liver disease patients (0.43 +/- 0.36 units) and 20 healthy controls (0.39 +/- 0.14 units; P < 0.0001).When the arbitrary cut-off level was set at 0.7 units (maximum value of healthy controls + 0.1), the positive frequency of telomerase activity was 25% for chronic liver disease and 80% for HCC patients (sensitivity 80%, specificity 75%). Among the HCC patients, high telomerase activity in the peripheral blood was shown at stage III HCC with vascular invasion (2.10 +/- 0.62 units, n = 9). This was significantly higher than patients at stage II of HCC (1.28 +/- 0.72 units, n = 11, without vascular invasion; P = 0.012). CONCLUSION: These results suggest that peripheral blood telomerase activity, which may reflect haematogenous micrometastasis, is potentially a practical diagnostic/predictive marker of HCC.

Electron transfer from diamond electrodes to heme peptide and peroxidase.

Direct electron transfer from boron-doped diamond electrodes to heme undecapeptide and horseradish peroxidase (HRP) was examined and evaluated for the application to H2O2 biosensors. As-grown and oxygen plasma-treated diamond electrodes on which heme peptide is adsorbed exhibited cathodic current responses to H2O2 on the basis of the direct electron transfer. In a comparative study of carbon electrodes on which heme peptide was adsorbed, an oxygen plasma-treated diamond electrode exhibited responses comparable with those of an edge-oriented pyrolytic graphite (EOPG) electrode, despite much smaller roughness. However, electron transfer to compounds I and II of HRP from the diamond electrodes was much slower than that from EOPG or glassy carbon, suggesting that the pi electrons of an sp2 carbon may play an important role in the direct electron transfer to the heme moiety of HRP. To examine the applicability of heme peptide-modified diamond electrodes to oxidase-based biosensors, anodic current responses of the oxygen plasma-treated diamond electrode to possible interfering agents, ascorbic acid and uric acid, were examined and compared with those of EOPG. Since the diamond electrode exhibited much less sensitivity to those interfering agents, the heme peptide-modified diamond electrode should be a promising H2O2 biosensor for the application to oxidase-based biosensors.

Amelioration of tumor necrosis factor release by cyclosporine in warm ischemia/reperfusion injury of the rat liver: with special reference to hepatic ultrastructure.

Mechanisms by which an immunosuppressant (cyclosporine, CsA) ameliorates warm ischemic injury of the liver were studied. Female Sprague-Dawley rats were subjected to 60-min normothermic liver ischemia. Animals were assigned to one of two groups: group I, controls with vehicle treatment; group II, treatment with CsA (10 mg/kg). CsA was given orally for 4 consecutive days prior to the induction of hepatic ischemia. In addition to a survival study, plasma levels of endotoxin, serum activity of tumor necrosis factor-alpha (TNF), and serum levels of aminotransferases were measured in blood samples collected from the suprahepatic vena cava, and hepatic ultrastructural alterations were examined under an electron microscope. The 7-day survival rate was significantly higher in the CsA-treated animals. In the control group, serum TNF levels were elevated following reperfusion and peaked at 3 h. When the values at 3 h post reflow were compared, the animals given CsA had significantly lower levels of TNF (170.0 +/- 30.5 pg/ml for group I, 67.6 +/- 13.7 for group II, mean +/- SEM; P < 0.05). The sinusoidal lining cells and hepatocytes were drastically destroyed at 6 h post reflow in the control group, although the degree of injury at 1-3 h was less severe. On the other hand, the endothelium and parenchymal liver cells in the CsA-treated group were well preserved at 6 h in comparison with those in the control group. Our data suggest that modulation of TNF production is one of the mechanisms through which CsA prevents the exacerbation of ischemia/reperfusion injury of the liver.

The fas and fas ligand pathways in liver allograft tolerance.

The Fas and Fas ligand (Fas/FasL) pathways may play a central role in cytotoxicity or immunoregulation in liver transplantation. Here, in an attempt to examine the role of Fas/FasL on drug-free tolerance, we measured mRNA levels of Fas/FasL in livers by reverse transcriptase-polymerase chain reaction (RT-PCR), and also protein levels of Fas/FasL in livers by immunohistochemistry and in serum by dot blot assay. PVG recipients bearing DA livers showed serious rejection between post-operative (POD) days 7 and 14, but this rejection was naturally overcome without any immunosuppression. Fas gene and protein products were expressed on almost every cell in livers taken from naive rats, and at any time point in both syngeneic and allogeneic orthotopic liver transplantation (OLT) rats. In contrast, FasL mRNA in DA livers was detectable at POD 2, peaked at POD 14, and declined at POD 63 in allogeneic OLT (DA-PVG). Although the FasL gene was detectable in isografts at POD 14, its expression was much lower than in allografts. The time course and localization of FasL expression indicated that the expression of FasL gradually switched from infiltrating cells to hepatocytes when the rejection was naturally overcome and tolerance was induced in this OLT model. Soluble Fas could constitutively be detected at any time point in the serum of the tolerogenic OLT (DA-PVG) rats and was not diminished during the rejection phase. Soluble FasL peaked at POD 14 in allogeneic OLT, while sFasL was significantly lower in the serum of normal and syngeneic OLT rats. These findings suggest that the Fas and FasL pathways, including soluble forms, may contribute to the control of the immune response in this drug-free tolerance OLT model.

Multichannel quartz crystal microbalance.

Arrays of quartz crystal resonators are fabricated on a single quartz wafer as a multichannel quartz crystal microbalance (MQCM). Three types of four-channel array of 10-MHz resonators are prepared and tested. Mechanical oscillation of each channel is entrapped within the channel almost completely, so that the interference between the channels via the quartz crystal plate is almost negligible. A mass change on each channel is quantitatively evaluated on the basis of Sauerbrey's law. Thus, each channel of a MQCM device can be used as an independent QCM. Influence from a longitudinal wave generated from another channel is also negligible compared to the influence from the wave from the monitored channel itself. The simultaneous oscillation of channels is also possible. The potential applicability of MQCM to the two-dimensional mapping of mass changes is demonstrated.

A transient increase in endogeneous erythropoietin levels after paediatric liver transplantation.

We investigated whether liver transplantation affects endogeneous erythropoietin (EPO) synthesis. Serum EPO levels were measured before transplantation and during the peri-transplant period in ten consecutive paediatric patients who had received a liver allograft without recombinant EPO therapy. All patients were anaemic on post-operative day 1 (POD 1); however, the haemoglobin levels of three patients gradually increased and required phlebotomy on POD 5-9. The serum levels of EPO in all patients were within the normal range before surgery, but six of the ten patients had a transient increased level of EPO at 1248 h after transplantation. A transient increase of endogeneous EPO following paediatric liver transplantation may be associated with extramedullary erythropoiesis in human liver grafts.

Electrochemical/Piezoelectric dual-response biosensor for heme ligands.

A heme peptide-modified quartz crystal oscillator is fabricated as a dual-response biosensor, giving electrochemical and piezoelectric responses simultaneously. Binding of a ligand to heme peptide causes inhibition of its catalytic activity, which is observed as the electrochemical response, and a mass increase is monitored as the piezoelectric response. The dual-response sensor can give two independent pieces of information at the same time. That is, qualitative (identification of the species) and quantitative (determination of its concentration) analyses can be made simultaneously. Simultaneous quantitative analysis for two components is also possible. These two modes are theoretically explained and then experimentally demonstrated employing imidazole and histidine as interferants.

H(2)O(2)-generating peroxidase electrodes as reagentless cyanide sensors.

Inexpensive, reagentless, and simple (single-electrode) cyanide biosensors are developed using a pyrolytic graphite (PG) electrode on which horseradish peroxidase (HRP) is adsorbed. The electrode is poised at -300 mV vs Ag/AgCl for 40 s to reduce dissolved O(2) to H(2)O(2) at the PG surface. The generated H(2)O(2) accumulates in the diffusion layer. The potential is then stepped to 0 mV, at which the accumulated H(2)O(2) is reduced, though the O(2) reduction does not proceed. Since the H(2)O(2) reduction is catalyzed by HRP, the transient cathodic current is inhibited by cyanide. Therefore, the transient current is a function of the cyanide concentration. A HRP/PG electrode with saturated HRP coverage is reliable, and it can determine 10(-)(5)-10(-)(3) M cyanide. On the other hand, the electrode with lower HRP coverage is less reliable, though it is so sensitive as to detect 2 × 10(-)(7) M cyanide because the system is under kinetic control.

Linamarin sensors: interference-based sensing of linamarin using linamarase and peroxidase.

An interference-based linamarin sensor is developed. Horseradish peroxidase (HRP) is adsorbed on a pyrolytic graphite (PG) electrode, and then linamarase from cassava is cross-linked with glutaraldehyde on the electrode surface. The prepared bienzyme electrode is poised at -300 mV vs Ag/AgCl for 40 s to reduce dissolved O(2) to H(2)O(2) at the PG surface. The potential is then stepped to 0 mV, at which point the accumulated H(2)O(2) is reduced, though the O(2) reduction does not proceed. Since the H(2)O(2) reduction is catalyzed by HRP, the transient cathodic current is inhibited by cyanide, which is liberated from linamarin by linamarase. Therefore, the transient current is a function of the linamarin concentration. This sensor responds to 1 × 10(-5)-5 × 10(-3) M linamarin and can estimate a linamarin concentration of a cassava extract.

Importance of hepatovenous back-perfusion for maintenance of liver viability during the Pringle manoeuvre.

The role of hepatovenous back-perfusion in maintaining hepatic viability was investigated during inflow occlusion (Pringle manoeuvre) of the pig liver. The study compared two ischaemia procedures of 60 min duration: hepatic inflow occlusion and inflow plus outflow occlusion (vascular exclusion). Each procedure was carried out in six pigs and liver tissue perfusion, energy metabolism, lipid peroxidation and 7-day survival were assessed. Although all pigs survived after inflow occlusion, five of six died after vascular exclusion (P < 0.01). Exclusion induced a significant decrease in perfusion to 15.3 per cent of the value before ischaemia compared with 32.4 per cent after hepatic inflow occlusion alone (P < 0.01). Although cellular adenosine 5'-triphosphate levels were significantly decreased by ischaemia in both groups, the fall was less in pigs with inflow occlusion alone (to 55 per cent of the preclamp value) than in those with exclusion (to 24 per cent of the preclamp value) (P < 0.01). The plasma phosphatidylcholine hydroperoxide level rose immediately after reperfusion in pigs with exclusion, while the level remained constant after inflow occlusion alone. There is a fundamental difference between the two ischaemia procedures: back-perfusion from the vena cava contributes to the maintenance of liver function during inflow occlusion.

Hepatoprotection by a PGI2 analogue in complete warm ischemia of the pig liver. Prostanoid release from the reperfused liver.

We examined the hepatoprotective effect of a prostaglandin (PG)I2 analogue by analyzing the endogenous release of prostanoid from the pig liver. Fourteen female pigs underwent 1 hr complete hepatic vascular exclusion (HVE); the portal and vena caval circulation was actively decompressed. The animals were divided into one of two groups (n = 7, each) according to pretreatment with the prostacyclin analogue (OP 2507, OP) administered via a mesenteric vein branch for 30 min at a rate of 2 micrograms/kg/min immediately prior to HVE. The plasma levels of prostaglandin E2 (PGE2), 6-keto-prostaglandin F1-alpha (6-keto-PGF1 alpha), and thromboxane B2 (TXB2), from the blood samples from the aorta, the hepatic vein, and the portal vein were serially compared for 60 min after the restoration of blood flow. Other parameters included 7-day survival rate, serum biochemistry, and endotoxin assay. A significant improvement in 7-day survival rate (6/7 vs. 1/7 for the control, P < 0.02) was observed in the OP-treated animals, associated with amelioration of serum transaminase activities but with no differences in plasma endotoxin levels. The reperfused liver progressively and substantially released PGE2 but did not generate other prostanoids (TXB2 and 6-keto-PGF1 alpha). OP pretherapy substantially suppressed hepatic generation of the PGE2 postreflow, correlating with serum transaminase levels (rs = 0.80; P < 0.01, at 60 min). We conclude that the PGI2 analogue ameliorates hepatic ischemia/reperfusion injury by down-regulating PGE2 production from the reperfused liver.

Substrate-purging enzyme electrodes. Peroxidase/catalase electrodes for H2O2 with an improved upper sensing limit.

A substrate-purging enzyme electrode carrying a sensing enzyme and a substrate-purging catalyst on its surface is proposed and was examined theoretically and experimentally. Since the substrate-purging catalyst partially consumes the substrate without consuming a cosubstrate, the sensitivity of the enzyme electrode is lowered while the maximum response remains unchanged leading to a raising of the upper sensing limit. Thus, by coating it with a cross-linked film of catalase and albumin, the upper sensing limit of a peroxidase-incorporated polypyrrole membrane electrode as an H2O2 sensor was raised by approximately 2 orders of magnitude.