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Radiation research has its foundation on the target and hit theories, which assume that the initial stochastic deposition of energy on a sensitive target in a cell determines the final biological outcome. This assumption is rather static in nature but forms the foundation of the linear no-threshold (LNT) model of radiation carcinogenesis. The stochastic treatment of radiation carcinogenesis by the LNT model enables easy calculation of radiation risk, and this has made the LNT model an indispensable tool for radiation protection. However, the LNT model sometimes fails to explain some of the biological and epidemiological data, and this suggests the need for insight into the mechanisms of radiation carcinogenesis. Recent studies have identified unique characteristics of the tissue stem cells and their roles in tissue turnover. In the present report, some important issues of radiation protection such as the risk of low-dose-rate exposures and in utero exposures are discussed in light of the recent advances of stem cell biology.

We describe the direct electrochemical detection of DNA methylation in relatively long sequences by using a nanocarbon film electrode. The film was formed by employing the electron cyclotron resonance sputtering method and had a nanocrystalline sp(2) and sp(3) mixed bond structure. Our methylation detection technique measures the differences between the oxidation currents of both 5-methylcytosine and cytosine without a bisulfite reaction or labeling. This was possible because this film electrode has a wide potential window while maintaining the high electrode activity needed to quantitatively detect both bases by direct oxidation. By optimizing the electrode surface conditions using electrochemical pretreatment, we used this film to quantitatively detect single cytosine methylation regardless of the methylation position in the sequence including retinoblastoma gene fragments (approximately 24 mers). This was probably due to the high stability of this film electrode, which we achieved by controlling the surface hydrophilicity to suppress the fouling, and by maintaining electrode activity against all the bases. The pH optimization of the oligonucleotide measurements was also useful for distinguishing both bases separately. Under the optimized conditions, this film electrode allowed us to realize the quantitative detection of DNA methylation ratios solely by measuring methylated 5'-cytosine-phosphoguanosine (CpG) repetition oligonucleotides (60 mers) with different methylation ratios., (Copyright 2010 Elsevier Inc. All rights reserved.)

This paper reports the development and characterization of a highly sensitive enzyme linked immunosorbent assay realized by the electrogenerated chemiluminescence (ECL) detection of a thiol monolayer formed by an enzyme labeled antibody. We used two monoclonal anti tumor necrosis factor-alpha (TNF-alpha) antibodies for a sandwich immunoassay. One was a capture antibody, and the other was a detection antibody labeled with an enzyme via an avidin-biotin interaction. Acetylcholinesterase was used as the labeling enzyme to convert acetylthiocholine to thiocholine. Then the thiocholine was collected on a gold electrode surface by gold-thiol binding. A bright and distinctive emission was observed at 1150 mV (vs Ag-AgCl) on the gold electrode with a thiocholine monolayer as a coreactant in the presence of tris(2,2'-bipyridyl)ruthenium complex. This method can greatly enhance the immunoassay signal since a large number of coreactant molecules can be generated by the enzymatic reaction, which is advantageous compared with a previously reported ECL based immunoassay that directly labels the detection antibody with a coreactant or luminophore. In addition, a surface accumulated coreactant is superior to the previously reported coreactant system in a bulk solution, because ECL emission occurs only very close to an electrode surface. As a result, high sensitivity and a low detection limit of 0.2 pM (3.4 pg/mL) TNF-alpha were achieved with excellent reproducibility by optimizing the conditions for the immuno-reaction, thiocholine accumulation, and ECL generation.

Inhibition of heat shock protein 90 (Hsp90) is an attractive modality for cancer therapy. Recent studies presented that an Hsp90 inhibitor, 17AAG (17-allylamino-17-demethoxygeldanamycin), enhanced tumor radio-sensitivity, while this was not observed in normal cells. One of the studies reported that the effect of this drug was only observed in tumor cells carrying the wild-type p53 gene, thus demonstrating p53-dependent tumor radio-sensitization by 17AAG. We have now tested the effects of 17AAG on two human lymphoblastoid cell lines from the same donor, TK6 cells with the wild-type p53 gene and WTK1 cells with the mutated p53 gene. The effects of 17AAG were tested at concentrations of 10 and 100 nM on various parameters, including growth inhibition of the cells, enhancement of radio-sensitivity by colony formation assay, apoptosis and chromosomal radio-sensitivity and abrogation of radiation induced G2/M checkpoint. When 100 nM 17AAG was applied, all of these parameters were enhanced in a similar fashion in both cell lines, indicating that the drug effect is p53-independent. Our results suggest that 17AAG is likely to be an effective sensitizer for radiotherapy, even on tumors with mutated p53.

Tri(ethylene glycol) terminated short alkylchain thiols (TEGCnSHs) offer good potential for constructing ultra-thin protein-resistant monolayers because they have an alkylchain for forming a densely packed monolayer and a flexible-hydrophilic oligo ethylene glycol arm for avoiding non-specific adsorption. Hybrid monolayers consisting of TEGCnSH and a maltoside ligand (MalC12SH, for capturing lectin) were effective in detecting concanavalin A (Con A). This hybrid monolayer was more suitable for Con A detection than that modified with 100% ligands in terms of the detection limit and time. The anti-fouling properties, packing densities, interaction and homogeneity of TEGCnSH monolayers were confirmed in detail by surface plasmon resonance (SPR) measurements and electrochemical methods. SPR measurements revealed their excellent repellency to proteins and peptides of various sizes (M(W) 400-104000). The electrochemical results indicated that the lower defects in the TEGCnSH monolayers suppressed the permeation of small peptides. The stability, homogeneity and packing density of the TEGCnSH monolayers were gradually improved as their alkylchain length increased.

We demonstrated the imaging of local electron transfer-rate differences on a flat conductive carbon substrate, attributed to only surface functional groups, by using a scanning electrochemical microscopy (SECM) technique. These differences were clearly imaged by using a redox mediator with surface state sensitive electron transfer rates, even if the conductivity of each imaging area were almost identical. The carbon electrode surface was masked with a patterned photoresist, and selectively introduced oxygen functional groups using an oxygen plasma treatment. This patterned surface exhibited hardly any topographical features when observed by scanning electron microscopy (SEM), and a height difference of only 1.0 nm was observed with atomic force microscopy (AFM). However, the SECM feedback mode and substrate generation-tip collection (SG-TC) mode are able to distinguish these interfaces with an almost micrometer order resolution by utilizing the difference in the electron transfer rate for the Fe(2+/3+) mediator, and the current ratios for regions rich and poor in oxygen containing groups were 1.5 and 2.0, respectively. This technique could be employed for imaging and monitoring the electron transfer rates on various electrode surfaces, including fluorine and nitrogen terminated surfaces and a monolayer film patterned with micro or nano contact printing techniques.

Densely packed co-adsorbed ultrathin mono molecular layers of short tri(ethylene glycol)-alkanethiolate (for repelling proteins) and maltoside terminated alkanethiolate (for capturing lectin) provided an extremely high signal to noise ratio surface: the repelling molecules, which had two different functions (highly flexible-hydrophilic arm and rigid packing tail group), worked as "nano barriers" in the recognition monolayer.

Ionizing radiation is known to induce delayed chromosome and gene mutations in the descendants of the irradiated tissue culture cells. Molecular mechanisms of such delayed mutations are yet to be elucidated, since high genomic complexity of mammalian cells makes it difficult to analyze. We now tested radiation induction of delayed recombination in the fission yeast Schizosaccharomyces pombe by monitoring the frequency of homologous recombination after X-irradiation. A reporter with 200 bp tandem repeats went through spontaneous recombination at a frequency of 1.0 x 10(-4), and the frequency increased dose-dependently to around 10 x 10(-4) at 500 Gy of X-irradiation. Although the repair of initial DNA damage was thought to be completed before the restart of cell division cycle, the elevation of the recombination frequency persisted for 8-10 cell generations after irradiation (delayed recombination). The delayed recombination suggests that descendants of the irradiated cells keep a memory of the initial DNA damage which upregulates recombination machinery for 8-10 generations even in the absence of DNA double-strand breaks (DSBs). Since radical scavengers were ineffective in inhibiting the delayed recombination, a memory by continuous production of DNA damaging agents such as reactive oxygen species (ROS) was excluded. Recombination was induced in trans in a reporter on chromosome III by a DNA DSB at a site on chromosome I, suggesting the untargeted nature of delayed recombination. Interestingly, Rad22 foci persisted in the X-irradiated population in parallel with the elevation of the recombination frequency. These results suggest that the epigenetic damage memory induced by DNA DSB upregulates untargeted and delayed recombination in S. pombe.

A simple and highly specific protein detection system using glycoconjugated gold nanoparticles was investigated. This system was based on the aggregation of gold nanoparticles coated with carbohydrate alkanethiols in the presence of corresponding proteins (lectins) that had specific recognition for certain carbohydrates. In order to construct an efficient specific recognition system, maltoside alkanethiol was adopted as an effective sensing modifier having a disaccharide group and a flexible long alkyl chain. The surface modification of gold nanoparticles with maltoside alkanethiol resulted in a shift and broadening (from 520 to 610 nm) of the absorption peak. Monodispersed maltoside-adsorbed gold nanoparticles aggregated with the specific lectin, concanavalin A (Con A). This phenomenon was used to detect the presence of Con A and to estimate concentrations of Con A in sample solutions. The precipitate of the maltoside-gold nanoparticle-Con A mixture was redispersed by addition of methyl alpha-D-mannopyranoside whose adsorption coefficient is larger than that of maltoside with Con A.

The anti-CD20 chimeric monoclonal antibody (mAb) rituximab is the most widely used therapeutic antibody for B-cell malignancies. However, approximately 50% of non-Hodgkin's lymphoma (B-NHL) patients respond to treatment with this antibody. Novel humanized antibodies target membrane CD20 with enhanced effector properties should improve treatment for a broader patient population with relapsed and refractory disease. A novel chimerized form of the murine anti-CD20 1K1791 exerts more potent antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) activities and induces cell death by a non-caspase dependent process. Humanized mAbs derived from 1K1791 were designed using four different humanization techniques and characterized. In contrast to rituximab or 2F2 (human anti-CD20 mAb), several of these exhibited superior ADCC, CDC, inhibition of cell growth and cell death. There was a wide range of functional differences among the humanized forms of 1K1791 despite a modest replacement of amino acid residues in the CDRs. To determine whether the superior activities exhibited by parental murine mAb 1K1791 were due to differences in VH and VL rearrangement, we analyzed its germline and compared it to other anti-CD20 mAbs. A remarkable conservation of VH and Vk (VL kappa) gene usage was observed in the murine anti-CD20 mAbs. 18/23 used the same germline gene J558.42 and 4/23 used closely related genes of the 'J558' group. Thus, 22/23 belonged to VH1 family. One exception was the mAb 1K1791, which was derived from the VH9.12 germline gene. 1K1791 was also unique in its use of a Vk19/28 family gene whereas most other mAbs (21/23) used Vk4/5 family genes. A formal relationship between the particular germline gene recruitment and antibody functionality has not been established, however, the present findings identified humanized mAbs with functional activities that were superior to rituximab and 2F2. These in vitro results support future in vivo animal testing and subsequent clinical trials.

The core proteins of the spindle assembly checkpoint (SAC), Mads, Bubs, and Mps1, first identified in the budding yeast, are thought to be functionally and structurally conserved through evolution. We found that fission yeast Bub3 is dispensable for SAC, as bub3 null mutants blocked mitotic progression when spindle formation was disrupted. Consistently, the bub3 mutation only weakly affected the stability of minichromosome Ch16 compared with other SAC mutants. Fission yeast Rae1 has sequence homology with Bub3. The bub3 rae1 double mutant and rae1 single mutant did not have defective SAC, suggesting that these genes do not have overlapping roles for SAC. Observations of living cells revealed that the duration of the mitotic prometaphase/metaphase was longer in the bub3 mutant and was Mad2 dependent. Further, the bub3 mutant was defective in sister centromere association during metaphase. Together, these findings suggest that fission yeast Bub3 is required for normal spindle dynamics, but not for SAC.

Dikaryons, cells with two haploid nuclei contributed by the members of a mating pair, are part of the life cycle of many filamentous fungi, but the molecular mechanisms underlying the division of dikaryons are largely unknown. We found that the fission yeast Schizosaccharomyces pombe has a latent ability to divide as a dikaryon. Cells capable of restarting the mitotic cycle with two nuclei were prepared by transient inactivation of the septation initiation network. Close pairing of the two nuclei before mitosis was dependent on minus-end-directed kinesin Klp2p and was essential for propagation as a dikaryon. The two spindles extended in opposite directions, keeping their old spindle pole bodies at the prospective site of cell division until the mid-anaphase. The spindles then overlapped, exchanging the inner nuclei. Finally, twin mitosis was followed by a single cytokinesis, producing two daughter dikaryons carrying copies of the original pair of nuclei.

Several redox enzymes were examined for enzymatic/electrochemical-recycling systems in order to measure p-aminophenol (PAP) with high sensitivity. Glucose oxidase (GOD) and diaphorase (DI) worked well as catalysts for recycling electrode systems: these enzymes effectively reduced p-iminoquinone (PIQ), the electrochemically-oxidized form of PAP, and caused an enhancement in the electrochemical signals (anodic currents in the voltammogram and amperogram) by approximately 100 fold. The lower detection limits for PAP were estimated to be 50 nM with the GOD system and 2 nM with the DI system. We combined the enzymatic-recycling electrode using DI with an enzyme immunoassay system to measure atrial natriuretic peptide (ANP), an important marker peptide hormone involved in heart diseases. ANPs from serum samples at ppt-levels were determined appropriately using the present assay system.

We describe the quantitative nonlabel electrochemical detection of both cytosine (C) and methylcytosine (mC) in oligonucleotides using newly developed nanocarbon film electrodes. The film consists of nanocrystalline sp2 and sp3 mixed bonds formed by employing the electron cyclotron resonance (ECR) sputtering method. We successfully used this film to develop a simple electrochemical DNA methylation analysis technique based on the measurement of the differences between the oxidation currents of C and mC since our ECR nanocarbon film electrode can directly measure all DNA bases more quantitatively than conventional glassy carbon or boron-doped diamond electrodes. The excellent properties of ECR nanocarbon film electrodes result from the fact that they have a wide potential window while maintaining the high electrode activity needed to oxidize oligonucleotides electrochemically. Proof-of-concept experiments were performed with synthetic oligonucleotides including different numbers of C and mC. This film allowed us to perform both C- and mC-positive assays solely by using the electrochemical oxidation of oligonucleotides without bisulfite or labeling processes.

A cellular analyzing system including a "real-time cellular imaging system" and a "comprehensive analyzing system for cellular responses" was developed. A "real-time cellular imaging system" is a system used to measure real-time imaging of multiple phenomena of a single cell with high special and temporal resolutions for the purpose to understand the pathology and physiology in a single cell and realize to single cell level diagnosis. A "real-time cellular imaging system" includes multi-probe imaging with AFM (atomic force microscopy), optical and SECM (scanning electrochemical microscopy) modes, which provides us with topological information and biochemical reactions at the local area of the interior and exterior of a cell. Scanning electrochemical/optical microscopy was applied to image PC12 cells. On the other hand, cells respond to their specific substances via their ligands. Therefore, the comprehensive analysis of protein-protein interaction is the important issue to determine the functions of cells. For this purpose, a "comprehensive analysis system for cellular responses" was developed. This system is based on SPR (surface plasmon resonance) and MS (mass spectrometry) using a nano-fabricated substrate. The interaction between IL-1 beta and anti-IL-1 beta antibodies was detected.

We developed an electrochemical surface plasmon resonance flow cell for the simultaneous measurement of the binding affinity and catalytic activity of bifunctional biomolecules. These measurements will be useful for evaluating the performance of such biomolecules as ribozyme and abzyme. The simultaneous measurements were performed on a gold surface modified with a multilayer consisting of poly-l-lysine and poly(styrene sulfonate) assembled with the layer-by-layer method using an enzyme-labeled monoclonal antibody as a model compound. We obtained the amount of immunocomplex formation from the surface plasmon resonance angle shift value by injecting the compound into the flow cell containing the multilayer modified with tumor necrosis factor-alpha. Then we compared this surface plasmon resonance result with that for the in situ electrochemical oxidation of p-aminophenol formed by the catalytic reaction of labeled enzyme on the same gold film. We were able to obtain a high correlation coefficient of 0.999 between the two responses. This is because the compound could be captured with high stability with a less than 3% coulometric response decrease in the catalyzed product in the multilayer whose thickness was easily controllable. In addition, we were able to measure the catalytic activity by coulometry and thus avoid the effect of peak broadening. We also report that the dephosphorylation activity of a bound compound could be estimated from the measurement results and an equation.

The early stage embryogenesis of higher eukaryotes lacks some of the damage response pathways such as G1/S checkpoint, G2/M checkpoint and apoptosis. We examined here the damage response of preimplantation stage embryos after fertilization with 6 Gy irradiated sperm. Sperm-irradiated embryos developed normally for the first 2.5 days, but started to exhibit a developmental delay at day 3.5. p21 was activated in the delayed embryos, which carried numerous micronuclei owing to delayed chromosome instability. Apoptosis was observed predominantly in the inner cell mass of the day 4.0 embryos. Sperm-irradiated p21-/- embryos lacked the delay, but chromosome instability and apoptosis were more pronounced than the corresponding p21 wild-type embryos. We conclude from the result that damage responses come in a stage-specific manner during preimplantation stage development; p53-dependent S checkpoint at the zygote stage, p21-mediated cell cycle arrest at the morula/blastocyst stages and apoptosis after the blastocyst stage in the inner cell mass.

This article describes the use of nanobiologic techniques in diagnosis and preventive medicine. It discusses the engineering of fluorescent and bioluminescent proteins to visualize biologic functions; single-molecule measurements of protein structure and function; two-photon microscopy for molecular imaging of the structure and function in living cells and tissues; and imaging ligand-induced protein conformations and interactions by fluorescence in single living cells.

We prepared an amorphous indium tin oxide (ITO) film and studied it with respect to its surface characterization and the effect of phosphate adsorption on its electrochemical properties. The film was deposited using RF sputtering under ambient low-oxygen conditions at room temperature. The XPS results revealed that the amount of phosphate adsorbed on the amorphous ITO film was more than 4.6 times greater than that adsorbed on commercially available polycrystalline ITO film in spite of the smaller microscopic surface area of the former. Electrochemical responses for anionic species such as L-ascorbic acid (AA) and 3,4-dihydroxyphenylacetic acid (DOPAC) on the phosphate-adsorbed ITO film electrodes were more effectively suppressed at the amorphous ITO film electrode than at the polycrystalline ITO film electrode when a phosphate-containing electrolyte was used. Such suppression could be attributed to the electrostatic repulsion between the anionic species and more heavily adsorbed phosphate on our amorphous ITO film electrode surface. This effect is made more pronounced by increasing the phosphate concentration to 1 mM. With 1 mM phosphate, the amorphous ITO film electrode showed the highest selectivity for dopamine (DA) against the anionic species, namely, 880 for DA/AA and 330 for DA/DOPAC, respectively. In contrast, the selectivity was 120 for DA/AA and 20 for DA/DOPAC with the polycrystalline ITO film electrode.

Rituximab is the first anti-cancer antibody approved by the FDA for the treatment of B-cell non-Hodgkin lymphoma (B-NHL), alone or in combination with chemotherapeutic drugs. Further, rituximab is now being examined in a variety of CD20+ neoplastic diseases as well as B-cell-induced autoimmune diseases. The clinical response to rituximab is significant, resulting not only in tumor regression but also prolongation of survival. However, a subset of patients does not initially respond to rituximab or develops resistance to its further treatment. Therefore, alternative therapies for these patients are strongly desired. Rituximab activity has been thought to be by antibody-dependent cellular cytotoxicity, complement-dependent cytotoxicity and apoptosis, and studies in model systems established the role of rituximab in cell signaling-induced perturbation of anti-apoptotic survival pathways, suggesting that the patients unresponsive to rituximab may be overcome with other CD20 antibodies with different activities. This study investigated eight novel murine antibodies directed against CD20 for their physical and biological properties in comparison with 2B8 and c2B8 (rituximab). These antibodies were derived by various antigenic and immunization procedures and selected for CD20 activity. Analysis of these antibodies revealed that they all bound to various B-cell lines and CD20-transfected CHO cells. Six of the eight antibodies shared similar variable-region amino acid sequences that were also shared by 2B8 while two monoclonal antibodies did not. Of them, 1K1791 has a distinct heavy chain and both 1K1791 and 1K1782 have distinct light chains. Not all of the antibodies inhibited cell growth and only two antibodies reacted with fixed GST-CD20 recombinant fusion protein. Noteworthy, 1K1791 was found to inhibit cell proliferation and also induced caspase-independent apoptosis in the absence of cross-linker. These findings identified new antibodies with properties and epitope specificities different from 2B8. The potential clinical application of such antibodies in the treatment of B-NHL and rituximab-resistant B-NHL is discussed.

Mice were exposed at various ages to 1 Gy or 2 Gy of X rays, and translocation frequencies in peripheral blood T cells, spleen cells, and bone marrow cells were determined with FISH painting of chromosomes 1 and 3 when the animals were 20 weeks old. It was found that the mean translocation frequencies were very low (< or =0.8%) in mice exposed in the fetal or early postnatal stages. However, with the increase in animal age at the time of irradiation, the frequency observed at 20 weeks old became progressively higher then reached a plateau (about 5%) when mice were irradiated when > or =6 weeks old. A major role of p53 (Trp53)-dependent apoptosis for elimination of aberrant cells was not suggested because irradiated fetuses, regardless of the p53 gene status, showed low translocation frequencies (1.8% in p53(-/-) mice and 1.4% in p53(+/-) mice) compared to the frequency in the p53(-/-) mother (7.4%). In contrast, various types of aberrations were seen in spleen and liver cells when neonates were examined shortly after irradiation, similar to what was observed in bone marrow cells after irradiation in adults. We interpreted the results as indicating that fetal cells are generally sensitive to induction of chromosome aberrations but that the aberrant cells do not persist because fetal stem cells tend to be free of aberrations and their progeny replace the pre-existing cell populations during the postnatal growth of the animals.

A previously isolated fission yeast gamma-tubulin mutant containing apparently stabilized microtubules proliferated at an approximately identical rate as wild type, yet the mutant mitosis spindle dynamics were aberrant, particularly the kinetochore microtubule dynamics. Progression through mitosis in the mutant, however, resulted in mostly accurate chromosome segregation. In the absence of the spindle assembly checkpoint gene, mad2+, the spindle dynamics in the gamma-tubulin mutant were greatly compromised, leading to a high incidence of chromosome missegregation. Unlike in wild-type cells, green fluorescent protein (GFP)-tagged Mad2 protein often accumulated near one of the poles of an elongating spindle in the gamma-tubulin mutant. We isolated novel mad2 mutants that were defective in arresting mitotic progression upon gross perturbation of the spindle formation but remained functional for the viability of the gamma-tubulin mutant. Further, the mad2 mutations did not appreciably destabilize minichromosomes in unperturbed mitoses. When overexpressed ectopically, these mutant Mad2 proteins sequestered wild-type Mad2, preventing its function in mitotic checkpoint arrest, but not in minichromosome stability. These results indicated that the Mad2 functions required for checkpoint arrest and chromosome stability in unperturbed mitosis are genetically discernible. Immunoprecipitation studies demonstrated that GFP-fused mutant Mad2 proteins formed a Mad1-containing complex with altered stability compared to that formed with wild-type Mad2, providing clues to the novel mad2 mutant phenotype.

p53 is one of the most frequently mutated genes in mammary carcinomas (MCs). To detect tumor suppressor genes cooperating with a hetero-deficient p53 gene in mammary carcinogenesis, we first examined allelotypes in MCs from (BALB/cHeA x MSM/Ms) F(1)- p53(+/-) and (BALB/cHeA x 129/SvEv) F(1)- p53(+/-) female mice, and then surveyed down-regulated genes in the allelic loss regions. Genome-wide screening at 42 loci identified frequent (more than 30%) loss of heterozygosity (LOH) on chromosomes 5, 8, 11, 12, 14 and 18 in the MCs from either of the F(1) mice. The MCs in the p53(+/- )mice indicated highly frequent LOH, especially on chromosomes 8, 11 and 12, distinct from other mouse tumors. More than 60% of the 38 MCs from (BALB/cHeA x MSM/Ms) F(1)- p53 (+/-) mice showed LOH in a region ranging from D8Mit85 (105.0 Mb from centromere) to D8Mit113 (111.8 Mb) on chromosome 8, a region syntenic to human chromosome 16q22.1, on which LOH has been found in breast cancers. RT-PCR analyses revealed that the LOH of chromosome 8 was associated with the reduced and/or complete loss of expression of Cdh1 and Cdh5 genes in 15 (58%) and 8 (31%) of 26 MCs derived from the F(1) mice, respectively. Thus, inactivation of Cdh1 and Cdh5 is likely to cooperate with the loss of p53, suggesting a possible tumor suppressive function of these genes in mammary carcinogenesis.

A novel electrochemical technique for lipopolysaccharide (LPS) detection has been developed using a combination of ferrocenylboronic acid derivatives and an enzyme-modified electrode. The enzyme-modified electrode was constructed from a gold electrode modified with a bovine serum albumin membrane containing diaphorase. Ferrocenylboronic acid derivatives are oxidized on the electrode, and then regenerated by a diaphorase-catalyzed reaction in the presence of NADH. The consumption/regeneration cycle for ferrocenylboronic acid derivatives resulted in a chemically amplified current response. The current response for ferrocenylboronic acid derivatives decreased in association with its complexation with glycosyl units of LPS, and this current decrease caused by LPS was also amplified by the recycling process. On the other hand, the addition of a monosaccharide such as D-mannose or D-galactose induced no response at the same LPS concentration. The enzyme membrane immobilized on the electrode plays an important role in selectivity as well as chemical amplification. In addition, the enzyme-modified electrode exhibited a rapid response of 5 min for LPS, which is much faster than the currently used method. The detection limit of LPS from Escherichia coli O127:B8 was as low as 50 ng ml-1.

Within minutes of the induction of DNA double-strand breaks in somatic cells, histone H2AX becomes phosphorylated in the serine 139 residue at the damage site. The phosphorylated H2AX, designated as gamma-H2AX, is visible as nuclear foci in the irradiated cells which are thought to serve as a platform for the assembly of proteins involved in checkpoint response and DNA repair. It is known that early stage mammalian embryos are highly sensitive to radiation but the mechanism of radiosensitivity is not well understood. Thus, we investigated the damage response of the preimplantation stage development by analyzing focus formation of gamma-H2AX in mouse embryos gamma-irradiated in utero. Our analysis revealed that although H2AX is present in early preimplantation embryos, its phosphorylation after 3 Gy gamma-irradiation is hindered up to the two cell stage of development. When left in utero for another 24-64 h, however, these irradiated embryos showed delayed phosphorylation of H2AX. In contrast, phosphorylation of H2AX was readily induced by radiation in post-compaction stage embryos. It is possible that phosphorylation of H2AX is inefficient in early stage embryos. It is also possible that the phosphorylated H2AX exists in the dispersed chromatin structure of early stage embryonic pronuclei, so that it cannot readily be detected by conventional immunostaining method. In either case, this phenomenon is likely to correlate with the lack of cell cycle arrest, apoptosis and high radiosensitivity of these developmental stages.

Adlayers of cobalt(II) 5,10,15,20-tetrakis(alpha,alpha,alpha,alpha-2-pivalamidophenyl)porphyrin (CoTpivPP) were prepared by immersing either Au(111) or Au(100) substrate in a benzene solution containing CoTpivPP molecules, and they were investigated in 0.1 M HClO4 and 0.1 M H2SO4 by cyclic voltammetry and in situ scanning tunneling microscopy (STM). The adlayer structure and electrochemical properties of CoTpivPP are compared to those of 5,10,15,20-tetraphenyl-21H,23H-porphine cobalt(II) (CoTPP). Characteristic nanobelt arrays consisting of CoTpivPP molecules were produced on both Au(111) and Au(100) surfaces. The stability of the nanobelt arrays was controlled by manipulating the electrode potential. On the other hand, the formation of nanobelt arrays consisting of O2-adducted CoTpivPP molecules depended upon the crystallographic orientation of Au. The state of O2 trapped in the cavity of CoTpivPP was distinctly observed in STM images as a bright spot in the nanobelt array formed on reconstructed Au(100)-(hex) surface, but not on Au(111) surface. This result suggests that the arrangement of underlying Au atoms plays an important role in the formation of nanobelt arrays with the sixth ligand coordination.

This paper describes the characterization, electrochemical properties, and applications of carbon films prepared by the electron cyclotron resonance (ECR) sputtering method. The ECR-sputtered carbon film was deposited within several minutes at room temperature. The optimized sputtering conditions significantly change the film structure, which includes many more sp3 bonds (sp3/sp2 = 0.702) than previously reported film (sp3/sp2 = 0.274)1 with an extremely flat surface (0.7 A). The ECR-sputtered carbon films exhibit excellent electrochemical properties. For example, they have nearly the same potential window in the positive direction as that of high-quality, boron-doped diamond (moderately doped, 10(19)-10(20) boron atoms/cm3)2 and an even wider potential window in the negative direction with a low background current, high stability, and suppression of fouling by electroactive species without pretreatment. The electron-transfer rates at ECR-sputtered carbon films are similar to those of glassy carbon (GC) for Ru(NH3)(6)(2+)/(3+) and Fe(CN)(6)(3-)/(4-), whereas they are much slower than those of GC for Fe2+/3+, dopamine oxidation, and O2 reduction due to weak interactions between electroactive species and the ECR-sputtered carbon film surface. Such a response can be attributed to the ultraflat surface and low surface O/C ratios of ECR-sputtered carbon films. ECR-sputtered carbon film is advantageous for measuring biochemicals with high oxidation potentials because of its wide potential window and high stability. Highly reproducible and well-defined cyclic voltammograms were obtained for histamine and azide ions with a peak potential at 1.25 and 1.12 V vs Ag/AgCl, respectively. The film is very stable for continuous voltammetry measurements in 10 microM bisphenol A, which usually fouls the electrode surface with oxidation products.

Imbalances of gene expression in aneuploids, which contain an abnormal number of chromosomes, cause a variety of growth and developmental defects. Aneuploid cells of the fission yeast Schizosaccharomyces pombe are inviable, or very unstable, during mitotic growth. However, S. pombe haploid cells bearing minichromosomes derived from the chromosome 3 can grow stably as a partial aneuploid. To address biological consequences of aneuploidy, we examined the gene expression profiles of partial aneuploid strains using DNA microarray analysis. The expression of genes in disomic or trisomic cells was found to increase approximately in proportion to their copy number. We also found that some genes in the monosomic regions of partial aneuploid strains increased their expression level despite there being no change in copy number. This change in gene expression can be attributed to increased expression of the genes in the disomic or trisomic regions. However, even in an aneuploid strain that bears a minichromosome containing no protein coding genes, genes located within about 50 kb of the telomere showed similar increases in expression, indicating that these changes are not a secondary effect of the increased gene dosage. Examining the distribution of the heterochromoatin protein Swi6 using DNA microarray analysis, we found that binding of Swi6 within ~50 kb from the telomere occurred less in partial aneuploid strains compared to euploid strains. These results suggest that additional chromosomes in aneuploids could lead to imbalances in gene expression through changes in distribution of heterochromatin as well as in gene dosage.

Aneuploid generation and stability are biologically important. In the present study, we investigated fission yeast aneuploids, focusing on the process through which aneuploidy is resolved into stable euploidy. The viability and growth patterns of aneuploid spores were greatly influenced by culture conditions, including nutrition and temperature. Germ tube formation and DNA synthesis in a major portion of aneuploids were greatly delayed or arrested. Observation of individual spores and their growth profiles revealed that a certain type(s) of aneuploid resolved its aneuploidy into normal euploids through anomalous cell divisions, which in many cases produced dead cells. Another type of aneuploid, disomy of chromosome 3, the only maintainable aneuploid between n and 2n, showed a peculiar cell division arrest phenotype under a certain growth condition. Microcolonies that formed from this type of aneuploid often contained a population of cells that became incompetent for cell division. This cell division arrest was not due to a nutritional limitation. During this peculiar process of colony formation, stable haploids or diploids were frequently produced. All other types of aneuploids are usually inviable, at least under our experimental conditions. To examine the aneuploid issue more systematically, we constructed a system to select for disomy of chromosome 1 or 2 using intragenic complementation of ade6-M210 and -M216 alleles. This genetic selection system revealed that fission yeast aneuploids can be stabilized through structural chromosome changes, including partial duplication and circular mini-chromosomes., (Copyright 2006 John Wiley & Sons, Ltd.)

The S-phase DNA damage checkpoint is activated by DNA damage to delay DNA synthesis allowing time to resolve the replication block. We previously discovered the p53-dependent S-phase DNA damage checkpoint in mouse zygotes fertilized with irradiated sperm. Here, we report that the same p53 dependency holds in mouse embryonic fibroblasts (MEFs) at low doses of irradiation. DNA synthesis in p53 wild-type (WT) MEFs was suppressed in a biphasic manner in which a sharp decrease below 2.5 Gy was followed by a more moderate decrease up to 10 Gy. In contrast, p53-/- MEFs exhibited radioresistant DNA synthesis below 2.5 Gy whereas the cells retained the moderate suppression above 5 Gy. DNA fiber analysis revealed that 1 Gy irradiation suppressed replication fork progression in p53 WT MEFs, but not in p53-/- MEFs. Proliferating cell nuclear antigen (PCNA), clamp loader of DNA polymerase, was phosphorylated in WT MEFs after 1 Gy irradiation and redistributed to form foci in the nuclei. In contrast, PCNA was not phosphorylated and dissociated from chromatin in 1 Gy-irradiated p53-/- MEFs. These results demonstrate that the novel low-dose-specific p53-dependent S-phase DNA damage checkpoint is likely to regulate the replication fork movement through phosphorylation of PCNA.

This paper reports a miniaturized immunosensor designed to determine a trace level cardiac marker, B-type natriuretic peptide (BNP), using a microfluidic device combined with a portable surface plasmon resonance (SPR) sensor system. Sample BNP solution was introduced into the microchannel after an immunoreaction with acetylcholine esterase-labeled antibody (conjugate), and only unbound conjugate was trapped on the BNP-immobilized surface in the flow channel. Then, the thiol compound generated by the enzymatic reaction with the trapped conjugate was accumulated on a gold thin film located downstream in the microchannel to monitor the real-time SPR angle shift. We achieved a detectable concentration range of 5 pg/mL-100 ng/mL by monitoring the SPR angle shift, which covers the required detection range for the BNP concentrations found in blood. This success resulted from the use of a T-shaped microfluidic device structure, which prevents the sample solution from flowing over the gold film used for SPR detection. We were able to measure trace levels of BNP peptide (15 fg) within 30 min since the procedure with our immunosensor is simpler than a multistep immunoassay through the simultaneous use of a labeled enzymatic reaction and the real-time monitoring of enzymatic product accumulation in the microfluidic device. We employed the procedure to detect serum BNP by using spiked samples in human serum and achieved satisfactory recovery for heat-treated samples to denature the esterase in the serum before the immunoreaction.

Ionizing radiation is a well-known carcinogen for various human tissues and a complete carcinogen that is able to initiate and promote neoplastic progression. Studies of radiation-induced mouse thymic lymphomas, one of the classic models in radiation carcinogenesis, demonstrated that even the unirradiated thymus is capable of developing into full malignancy when transplanted into the kidney capsule or subcutaneous tissue of irradiated mice. This suggests that radiation targets tissues other than thymocytes to allow expansion of cells with tumorigenic potential in the thymus. The idea is regarded as the 'indirect mechanism' for tumor development. This paper reviews the indirect mechanism and genes affecting the development of thymic lymphomas that we have analyzed. One is the Bcl11b/Rit1 tumor suppressor gene and the other is Mtf-1 gene affecting tumor susceptibility.

Radiation induction of genomic instability has two features: induction of untargeted mutation and delayed mutation. These phenomena have been studied mostly in tissue culture cells, but analyses have also been conducted in whole body systems. The study of response in whole body systems frequently applies repeat sequences as markers to detect mutations. These studies have generated conflicting findings. In addition, lack of knowledge of the mechanisms involved in repeat mutation confounds the interpretation of the biological significance of increased rates of repeat mutation. In this review, some of the existing controversies of genomic instability are discussed in relation to the mechanism of repeat mutation. Analyses of published and unpublished studies indicate a mechanistic similarity between radiation-induced genomic instability at repeat loci and dynamic mutations of triplet repeats. Because of their repetitive nature, repeat sequences frequently block progression of replication forks and are consequently resolved by slippage and/or recombination. Irradiation of cells induces S checkpoints and promotes slippage/recombination mediated repeat mutations. Thus, genomic instability at repeat loci might be viewed as a consequence of cellular attempts to restore the stability of replication in the face of the stalled replication fork; this process can occur both spontaneously as well as after exposure to radiation.

We have developed ultra-flat carbon film electrodes with a wide potential window and a low capacitive current by the electron cyclotron resonance (ECR) sputtering method. The film consists of sp2 and sp3 bonds (sp3/sp2 ratio = 0.702) and is sufficiently conductive for electrochemical measurements without doping. The film has average roughness of 0.7 A, which is much flatter than that of nanocrystalline diamond film. The potential limit of ECR sputtered carbon (current limit < +/-500 muA/cm2) in the positive direction is 2.0 V vs Ag/AgCl, which is slightly lower than that of boron-doped diamond (2.1 V) and much wider than that of a glassy carbon (GC) electrode (1.7 V). In contrast, a much wider potential window can be obtained in the negative direction. The capacitive current is also much lower than that of a GC electrode due to the ultra-flat surface and the low number of oxygen-containing groups at the film surface. ECR sputtered carbon film can be used to measure each base of oligonucleotides by electrochemical oxidation without any pretreatment. The ultra-flat surface and low surface oxygen concentration suppress fouling with electroactive species, such as oligonucleotides, NADH, and bisphenol A.

We have already reported a method for fabricating ultramicroelectrodes (Suzuki, K. JP Patent, 2004-45394, 2004). This method is based on the selective chemical etching of optical fibers. In this work, we undertake a detailed investigation involving a combination of etched optical fibers with various types of tapered tip (protruding-shape, double- (or pencil-) shape and triple-tapered electrode) and insulation with electrophoretic paint. Our goal is to establish a method for fabricating nanometer-sized optical fiber electrodes with high reproducibility. As a result, we realized pencil-shaped and triple-tapered electrodes that had radii in the nanometer range with high reproducibility. These nanometer-sized electrodes showed well-defined sigmoidal curves and stable diffusion-limited responses with cyclic voltammetry. The pencil-shaped optical fiber, which has a conical tip with a cone angle of 20 degrees , was effective for controlling the electrode radius. The pencil-shaped electrodes had higher reproducibility and smaller electrode radii (r(app) < 1.0 nm) than those of other etched optical fiber electrodes. By using a pencil-shaped electrode with a 105-nm radius as a probe, we obtained simultaneous electrochemical and optical images of an implantable interdigitated array electrode. We achieved nanometer-scale resolution with a combination of scanning electrochemical microscopy SECM and optical microscopy. The resolution of the electrochemical and optical images indicated sizes of 300 and 930 nm, respectively. The neurites of living PC12 cells were also successfully imaged on a 1.6-microm scale by using the negative feedback mode of an SECM.

We have developed a microfluidic device consisting of a gold film working electrode modified with lactate oxidase and Os-poly(vinylpyridine) mediator containing horseradish peroxide, and reference and counter electrodes in a microflow detection channel separated by a microdialysis membrane from another microflow channel used for sample injection. The dialysis membrane is cellulose with a molecular weight cut off of 10 kDa. We achieved control over a wide recovery rate range of 3-94% because the device is capable of controlling both flow rates in the dual thin-layer channels. We were able to measure the lactate concentration in blood samples within a few minutes without any pretreatment because biomolecules are simultaneously separated by molecular weight and detected in the device. We achieved quantitative and reproducible measurements of the lactate concentration in blood samples, and obtained a relative standard deviation of 1.5% (n = 8). With our device, the lactate concentration in dog whole blood was measured with high stability without any pretreatment.

Many studies have confirmed that radiation can induce genomic instability in whole body systems. Although the molecular mechanisms underlying induced genomic instability are not known at present, this interesting phenomenon could be the manifestation of a cellular fail-safe system in which fidelity of repair and replication is down-regulated to tolerate DNA damage. Two features of genomic instability namely, delayed mutation and untargeted mutation, require two mechanisms of ;damage memory' and ;damage sensing, signal transduction and execution' to induce mutations at a non damaged-site. In this report, the phenomenon of transgenerational genomic instability and possible mechanisms are discussed using mouse data collected in our laboratory as the main bases.

A novel electrochemical enzyme immunoassay system with a 10 ng L(-1) level detection limit was developed for the determination of B-type natriuretic peptide (BNP), an important marker for the diagnosis of heart failure. Sample BNP was added to a solution containing a certain concentration of acetylcholinesterase(AChE)-labeled anti-BNP antibody to undergo an immunological reaction. After the immunological reaction, we proposed two assay schemes. One involves measuring the amount of antibody-enzyme conjugate that reacted with two BNP molecules (reacted conjugate). The other involves measuring the amount of antibody-enzyme conjugate with at least one free binding site (unreacted conjugate). Then the amount of reacted or unreacted conjugate was determined by measuring the AChE activity after the recovery of each conjugate from the immunological reaction mixture. To determine the trace level of the recovered antibody-enzyme conjugate, the AChE activity was determined with high sensitivity on the basis of the chemisorption/electrochemical desorption process of thiocholine, which was produced through the enzymatic reaction, on a silver surface. The thiocholine chemisorption (i.e., accumulation) on the silver electrode surface resulted in a sensitivity for the electrochemical determination of the AChE activity that was 2 orders of magnitude greater than that obtained when using direct measurement without accumulation. The procedure for determining the AChE activity of unreacted conjugate after its recovery on a BNP-modified disk was applied to the determination of BNP in serum samples. This procedure involves the removal of the immunological reaction mixture before the enzymatic reaction process, which allows the AChE activity to be measured without any interference from endogenous pseudocholinesterase, which exists with high activity in serum. With both procedures, the BNP could be measured within an hour. The detection limits were 20 and 40 ng L(-1) using the reacted and unreacted conjugate measuring procedures, respectively.

We demonstrated the discrimination of volatile sulfur compound mixtures with different mixing ratios by using an array of the plasma-polymerized film (PPF)-coated quartz crystal resonators. The PPF sensor array, which contains PPFs prepared from amino acids and synthetic polymers, exhibited different response patterns to mono or mixed volatile sulfur compounds (VSCs) (hydrogen sulfide and methanethiol) under a dry environment. The sensor array was installed in a desktop-size relative humidity controller. The relative humidity and temperature conditions of the sample flow to the sensor cell were equalized to those of the inner atmosphere of the sensor cell based on the concept of the two-separate-temperatures method. In this way, the baseline drift of PPF sensor response caused by the introduction of a highly humid sample was successfully suppressed. We compared the sensor array responses under the controlled humidity conditions. Presorption of water molecules by PPFs caused a decrease of sensor sensitivity, but the films still had the ability to discriminate sub-ppmv VSC mixtures having 6:1, 1:1, and 1:6 mixture ratios of hydrogen sulfide and methanethiol.

The gas selectivities of highly ordered mesoporous silicates and commercially-obtained porous silicates with respect to benzene, toluene and xylene were studied. After studying the porosities, pore uniformities, and surface silanol structures of the silicates and their relationships to gas selectivity in detail, we found that we could achieve high benzene selectivity by controlling the micropore size (less than 1 nm). Concluding that mesoporous silicate has a suitable micropore size and structure for benzene selectivity, we also observed that mesoporous silicate SBA-16 exhibited a high (>6) benzene selectivity from toluene and xylene even in a pseudo-atmospheric environment. A benzene detection limit of about 100 ppb was achieved by introducing SBA-16 into a microfluidic device originally developed for the separate detection of benzene, toluene, and xylene gases.

Cell cycle arrest in response to DNA damage is important for the maintenance of genomic integrity in higher eukaryotes. We have previously reported the novel p53-dependent S-phase checkpoint operating in mouse zygotes fertilized with irradiated sperm. In the present study, we analysed the detail of the p53 function required for this S-phase checkpoint in mouse zygotes. The results indicate that ATM kinase is likely to be indispensable for the p53-dependent S-phase checkpoint since the suppression was abrogated by inhibitors such as caffeine and wortmannin. However, ATM phosphorylation site mutant proteins were still capable of suppressing DNA synthesis when microinjected into sperm-irradiated zygotes lacking the functional p53, suggesting that the target of the phosphorylation is not p53. In addition, the suppression was not affected by alpha-amanitin, and p53 protein mutated at the transcriptional activation domain was also functional in the suppression of DNA synthesis. However, p53 proteins mutated at the DNA-binding domain were devoid of the suppressing activity. Taken together, the transcription-independent function of p53 associated with the DNA-binding domain is involved in the S-phase checkpoint in collaboration with yet another unidentified target protein(s).

Mouse thymic lymphomas are readily induced by radiation and also arise without irradiation when the mice are null in Trp53 functions. In the present study, spontaneous thymic lymphomas in Trp53-/- mice were compared to those arising in irradiated Trp53+/- mice, revealing three features characteristic of the spontaneous lymphomas. (1) Mp53D2, a Trp53 modifier that affects the latent period of radiogenic thymic lymphomas in Trp53+/- mice, had no effect on the development of spontaneous lymphomas. (2) A sex difference in the latency was found. (3) A marked difference was noted in the frequency of allelic loss at the Ikaros gene on chromosome 11, encoding a transcription factor required for normal lymphocyte development and differentiation; 2% in the lymphomas of Trp53-/- mice and 78% in the radiogenic lymphomas of Trp53+/- mice, suggesting that loss of Trp53 may reduce the requirement for the loss of Ikaros for lymphomagenesis. Furthermore, allelic loss analysis on chromosome 19 localized a region that may harbor an unknown tumor suppressor gene. These results suggest intricate steps of lymphomagenesis influenced by the presence or absence of Trp53.

Genetic predisposition to cancers is significant to public health because a high proportion of cancers probably arise in a susceptible human subpopulation. Using a mouse model of gamma-ray-induced thymic lymphomas, we performed linkage analysis and haplotype mapping that suggested Mtf-1, metal-responsive transcription factor-1 (Mtf-1), as a candidate lymphoma susceptibility gene. Sequence analysis revealed a polymorphism of Mtf-1 that alters the corresponding amino acid at position 424 in the proline-rich domain from a serine in susceptibility strains to proline in resistant strains. The transcriptional activity of Mtf-1 encoding serine and proline was compared by transfecting the DNA to Mtf-1-null cells, and the change to proline conferred a higher metal responsiveness in transfections. Furthermore, the resistant congenic strains possessing the Mtf-1 allele of proline type exhibited higher radiation inducibility of target genes than susceptible background strains having the Mtf-1 allele of serine type. Since products of the targets such as metallothionein are able to suppress cellular stresses generated by irradiation, these results suggest that highly inducible strains having Mtf-1 of proline type are refractory to radiation effects and hence are resistant to lymphoma development.

Introduction: Ataxia-telangiectasia is an autosomal-recessive disease that affects neuro-immunological functions, associated with increased susceptibility to malignancy, chromosomal instability and hypersensitivity to ionizing radiation. Although ataxia-telangiectasia mutated (ATM) heterozygous deficiency has been proposed to increase susceptibility to breast cancer, some studies have not found excess risk. In experimental animals, increased susceptibility to breast cancer is not observed in the Atm heterozygous deficient mice (Atm+/-) carrying a knockout null allele. In order to determine the effect of Atm heterozygous deficiency on mammary tumourigenesis, we generated a series of Atm+/- mice on the p53+/- background with a certain predisposition to spontaneous development of mammary carcinomas, and we examined the development of the tumours after X-irradiation., Methods: BALB/cHeA-p53+/- mice were crossed with MSM/Ms-Atm+/- mice, and females of the F1 progeny ([BALB/cHeA x MSM/Ms]F1) with four genotypes were used in the experiments. The mice were exposed to X-rays (5 Gy; 0.5 Gy/min) at age 5 weeks., Results: We tested the effect of haploinsufficiency of the Atm gene on mammary tumourigenesis after X-irradiation in the p53+/- mice of the BALB/cHeA x MSM/Ms background. The singly heterozygous p53+/- mice subjected to X-irradiation developed mammary carcinomas at around 25 weeks of age, and the final incidence of mammary carcinomas at 39 weeks was 31% (19 out of 61). The introduction of the heterozygous Atm knockout alleles into the background of the p53+/- genotype significantly increased the incidence of mammary carcinoma to 58% (32 out of 55) and increased the average number of mammary carcinomas per mouse. However, introduction of Atm alleles did not change the latency of development of mammary carcinoma., Conclusion: Our results indicate a strong enhancement in mammary carcinogenesis by Atm heterozygous deficiency in p53+/- mice. Thus, doubly heterozygous mice represent a useful model system with which to analyze the interaction of heterozygous genotypes for p53, Atm and other genes, and their effects on mammary carcinogenesis.

Although germline cells can form multipotential embryonic stem (ES)/embryonic germ (EG) cells, these cells can be derived only from embryonic tissues, and such multipotent cells have not been available from neonatal gonads. Here we report the successful establishment of ES-like cells from neonatal mouse testis. These ES-like cells were phenotypically similar to ES/EG cells except in their genomic imprinting pattern. They differentiated into various types of somatic cells in vitro under conditions used to induce the differentiation of ES cells and produced teratomas after inoculation into mice. Furthermore, these ES-like cells formed germline chimeras when injected into blastocysts. Thus, the capacity to form multipotent cells persists in neonatal testis. The ability to derive multipotential stem cells from the neonatal testis has important implications for germ cell biology and opens the possibility of using these cells for biotechnology and medicine.

This paper proposes a very simple procedure for preparing a biocompatible sensor based on a protein (bovine serum albumin, BSA), enzyme and vinylferrocene (VF) composite membrane modified electrode. The membrane was prepared simply by first casting vinylferrocene and then coating it with BSA and glucose oxidase immobilised with glutaraldehyde. The sensor response was independent of dissolved oxygen concentration from 3 to 10 ppm and showed good stability for serum sample measurement, unlike the commonly used BSA/enzyme modified electrode. The sensor response was almost unchanged over the measurement time (>10 h) whereas the responses of a BSA and glucose oxidase modified platinum electrode and an osmium-polyvinylpyridine wired horseradish peroxidase modified electrode (Ohara et al., 1993) fell to 68% of their initial value in a serum sample containing 10mM glucose.