In vivo monitoring of hydroxyl radical generation caused by x-ray irradiation of rats using the spin trapping/EPR technique

Measurement of hydroxyl radical (*OH) in living animals irradiated with ionizing radiation should be required to clarify the mechanisms of radiation injury and the in vivo assessment of radiation protectors, because generation of *OH is believed to be one of the major triggers of radiation injury. In this study, *OH generation was monitored by spin trapping the secondary methyl radical formed by the reaction of *OH with dimethyl sulfoxide (DMSO). Rats were injected intraperitoneally with a DMSO solution of alpha-phenyl-N-tert-butylnitrone (PBN). X-irradiation of the rats remarkedly increased the six-line EPR signal in the bile. The strengthened signal was detectable above 40 Gy. Use of 13C-substituted DMSO revealed that the signal included the methyl radical adduct of PBN as a major component. The EPR signal of the PBN-methyl radical adduct was completely suppressed by preadministration of methyl gallate, a scavenger of *OH but not of methyl radical. Methyl gallate did not reduce the spin adducts to EPR-silent forms. These observations indicate that what we were measuring was *OH generated in vivo by x-irradiation. This is the first report of the in vivo monitoring of *OH generation at a radiation dose close to what people might receive in the case of radiological accident or radiation therapy.     

Evaluation of a high-affinity QCM immunosensor using antibody fragmentation and 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer

This study evaluated construction of a highly affinitive quartz crystal microbalance (QCM) immunosensor using anti-C-reactive protein (CRP) antibody and its fragments for CRP detection. Three types of antibody were immobilized on the surface of a QCM via covalent-bounding. Then affinity was evaluated through antigen-antibody binding between CRP and its antibody. Affinity between antigen-antibody was shown to be highest when anti-CRP F(ab')2-IgG antibody (70 microg/mL) was immobilized on the QCM. In case of anti-CRP F(ab')2-IgG antibody, affinity which was attributable to antigen-antibody binding was almost twice that of anti-CRP IgG antibody, which is used conventionally for QCM immunosensors. In addition, when it was treated with 2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate, so-called MPC polymer, highly affinitive and selective immunosensing for CRP was achieved without non-specific binding from plasma proteins in human serum. When anti-CRP F(ab')2-IgG antibody was immobilized on the QCM, the detection limit and the linearity of CRP calibration curve were achieved at concentrations from 0.001 to 100 microg/dL even during investigation in serum samples. Experimental results verified the successful construction of a highly affinitive and selective QCM-immunosensor which was modified with anti-CRP F(ab')2-IgG antibody and MPC polymer.     

Characterization of multiple members of the HSP70 family in platyfish culture cells: molecular evolution of stress protein HSP70 in vertebrates

A shift from 28 to 37 degrees C in the incubation temperature of a culture of the platyfish fibroblast cell line, EHS cells (platyfish fibroblast cell line), induced a set of stress proteins. A two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) analysis showed that the cells expressed three genetically distinct forms of heat-shock protein 70 (HSP70) family proteins: heat-inducible forms of HSP70, the constitutively expressed heat-shock cognate protein 70 (HSC70) and its phosphorylated isoform, and the glucose-regulated protein 78 (GRP78). Three different clones encoding two major isoforms of heat-inducible HSP70, platyfish HSP70-1 and HSP70-2, and of the HSC70 were isolated from a platyfish cDNA library. We compared the deduced amino acid sequences of the platyfish HSP70 and HSC70 proteins with those of other vertebrates. Phylogenetic analysis showed that vertebrate HSP70 could be classified into four cluster groups: (a) fish HSP70, with two isoforms of heat-inducible HSP70 in fish, fish HSP70-1 and HSP70-2; (b) the mammalian testis-specific HSP70-related protein HST70; (c) the mammalian heat-inducible HSP70B'; and (d) the mammalian major histocompatibility complex (MHC)-linked HSP70, including the MHC-linked heat-inducible HSP70 and the testis-specific HSP70-related protein. These findings suggest that vertebrate HSP70 was derived from a single ancestral HSP70 gene during vertebrate evolution and that multiple copies of heat-inducible HSP70 were probably evolved during genetic divergence in fish and higher vertebrates.     

Plant-specific microtubule-associated protein SPIRAL2 is required for anisotropic growth in Arabidopsis

In diffusely growing plant cells, cortical microtubules play an important role in regulating the direction of cell expansion. Arabidopsis (Arabidopsis thaliana) spiral2 (spr2) mutant is defective in directional cell elongation and exhibits right-handed helical growth in longitudinally expanding organs such as root, hypocotyl, stem, petiole, and petal. The growth of spr2 roots is more sensitive to microtubule-interacting drugs than is wild-type root growth. The SPR2 gene encodes a plant-specific 94-kD protein containing HEAT-repeat motifs that are implicated in protein-protein interaction. When expressed constitutively, SPR2-green fluorescent protein fusion protein complemented the spr2 mutant phenotype and was localized to cortical microtubules as well as other mitotic microtubule arrays in transgenic plants. Recombinant SPR2 protein directly bound to taxol-stabilized microtubules in vitro. Furthermore, SPR2-specific antibody and mass spectrometry identified a tobacco (Nicotiana tabacum) SPR2 homolog in highly purified microtubule-associated protein fractions from tobacco BY-2 cell cultures. These results suggest that SPR2 is a novel microtubule-associated protein and is required for proper microtubule function involved in anisotropic growth.     

Effect of recent changes in atomic bomb survivor dosimetry on cancer mortality risk estimates

The Radiation Effects Research Foundation has recently implemented a new dosimetry system, DS02, to replace the previous system, DS86. This paper assesses the effect of the change on risk estimates for radiation-related solid cancer and leukemia mortality. The changes in dose estimates were smaller than many had anticipated, with the primary systematic change being an increase of about 10% in gamma-ray estimates for both cities. In particular, an anticipated large increase of the neutron component in Hiroshima for low-dose survivors did not materialize. However, DS02 improves on DS86 in many details, including the specifics of the radiation released by the bombs and the effects of shielding by structures and terrain. The data used here extend the last reported follow-up for solid cancers by 3 years, with a total of 10,085 deaths, and extends the follow-up for leukemia by 10 years, with a total of 296 deaths. For both solid cancer and leukemia, estimated age-time patterns and sex difference are virtually unchanged by the dosimetry revision. The estimates of solid-cancer radiation risk per sievert and the curvilinear dose response for leukemia are both decreased by about 8% by the dosimetry revision, due to the increase in the gamma-ray dose estimates. The apparent shape of the dose response is virtually unchanged by the dosimetry revision, but for solid cancers, the additional 3 years of follow-up has some effect. In particular, there is for the first time a statistically significant upward curvature for solid cancer on the restricted dose range 0-2 Sv. However, the low-dose slope of a linear-quadratic fit to that dose range should probably not be relied on for risk estimation, since that is substantially smaller than the linear slopes on ranges 0-1 Sv, 0-0.5 Sv, and 0- 0.25 Sv. Although it was anticipated that the new dosimetry system might reduce some apparent dose overestimates for Nagasaki factory workers, this did not materialize, and factory workers have significantly lower risk estimates. Whether or not one makes allowance for this, there is no statistically significant city difference in the estimated cancer risk.     

Non-destructive on-chip cell sorting system with real-time microscopic image processing

Studying cell functions for cellomics studies often requires the use of purified individual cells from mixtures of various kinds of cells. We have developed a new non-destructive on-chip cell sorting system for single cell based cultivation, by exploiting the advantage of microfluidics and electrostatic force. The system consists of the following two parts: a cell sorting chip made of poly-dimethylsiloxane (PDMS) on a 0.2-mm-thick glass slide, and an image analysis system with a phase-contrast/fluorescence microscope. The unique features of our system include (i) identification of a target from sample cells is achieved by comparison of the 0.2-μm-resolution phase-contrast and fluorescence images of cells in the microchannel every 1/30 s; (ii) non-destructive sorting of target cells in a laminar flow by application of electrostatic repulsion force for removing unrequited cells from the one laminar flow to the other; (iii) the use of agar gel for electrodes in order to minimize the effect on cells by electrochemical reactions of electrodes, and (iv) pre-filter, which was fabricated within the channel for removal of dust contained in a sample solution from tissue extracts. The sorting chip is capable of continuous operation and we have purified more than ten thousand cells for cultivation without damaging them. Our design has proved to be very efficient and suitable for the routine use in cell purification experiments.     

Improved synthesis with high yield and increased molecular weight of poly(alpha,beta-malic acid) by direct polycondensation

The development of synthetic biodegradable polymers, such as poly(alpha-hydroxy acid), is particularly important for constructing medical devices, including scaffolds and sutures, and has attracted growing interest in the biomedical field. Here, we report a novel approach to preparing high molecular weight poly(malic acid) (HMW--PMA) as a biodegradable and bioabsorbable water-soluble polymer. We investigated in detail the reaction conditions for the simple direct polycondensation of l-malic acid, including the reaction times, temperatures, and catalysts. The molecular weight of synthesized alpha,beta-PMA is dependent on both the reaction temperature and time. The optimum reaction condition to obtain alpha,beta-PMA by direct polycondensation using tin(II) chloride as a catalyst was thus determined to be 110 degrees C for 45 h with a molecular weight of 5300. The method for alpha,beta-PMA synthesis established here will facilitate production of alpha,beta-PMA of various molecular weights, which may have a potential utility as biomaterials.     

Fourteen morphotypes of Entodinium ovumrajae (Ophryoscolecidae, Entodiniomorphida) found in the Dromedary camel of Egypt

During a survey of the ciliate protozoal composition of the stomach contents of nine dromedary camels of Egypt, fourteen morphotypes of Entodinium ovumrajae, which has been considered as a species peculiar to camels, were found in six camels. Except for five morphotypes including one originally described as an independent species and its forms, these were newly detected. These morphotypes, divided into three groups, can be identified mainly by the morphology of their ectoplasmic processes. Each camel had on average, about five morphotypes of this species.     

Fission yeast Rhp51 is required for the maintenance of telomere structure in the absence of the Ku heterodimer

The Schizosaccharomyces pombe Ku70–Ku80 heterodimer is required for telomere length regulation. Lack of pku70⁺ results in telomere shortening and striking rearrangements of telomere-associated sequences. We found that the rearrangements of telomere-associated sequences in pku80⁺ mutants are Rhp51 dependent, but not Rad50 dependent. Rhp51 bound to telomere ends when the Ku heterodimer was not present at telomere ends. We also found that the single-stranded G-rich tails increased in S phase in wild-type strains, while deletion of pku70⁺ increased the single-stranded overhang in both G₂ and S phase. Based on these observations, we propose that Rhp51 binds to the G-rich overhang and promotes homologous pairing between two different telomere ends in the absence of Ku heterodimer. Moreover, pku80 rhp51 double mutants showed a significantly reduced telomere hybridization signal. Our results suggest that, although Ku heterodimer sequesters Rhp51 from telomere ends to inhibit homologous recombination activity, Rhp51 plays important roles for the maintenance of telomere ends in the absence of the Ku heterodimer.