Enhanced Material Assimilation in a Toroidal Plasma Using Mixed H_{2}+Ne Pellet Injection and Implications to ITER.

The ablation and assimilation of cryogenic pure H_{2} and mixed H_{2}+Ne pellets, which are foreseen to be used by the ITER tokamak for mitigating thermal and electromagnetic loads of major disruptions, are observed by spatially and temporally resolved measurements. It is experimentally demonstrated that a small fraction (here ≈5%) of neon added to hydrogenic pellets enhances the core density assimilation with reduced outward transport for the low magnetic-field side injection. This is consistent with theoretical expectations that line radiation increased by doped neon in dense plasmoids suppresses the plasmoid pressure and reduces the E[over →]×B[over →] transport of the ablated material.

Universal GFP reporter for the study of vascular development.

We report the generation and characterization of transgenic mouse and zebrafish expressing green fluorescent protein (GFP) specifically in vascular endothelial cells in a relatively uniform fashion. These reporter lines exhibit fluorescent vessels in developing embryos and throughout adulthood, allowing visualization of the general vascular patterns with single cell resolution. Furthermore, we show the ability to purify endothelial cells from whole embryos and adult organs by a single step fluorescence activated cell sorting. We expect that these transgenic reporters will be useful tools for imaging vascular morphogenesis, global gene expression profile analysis of endothelial cells, and high throughput screening for vascular mutations.

Crystallographic and functional studies of very short patch repair endonuclease.

Vsr endonuclease plays a crucial role in the repair of TG mismatched base pairs, which are generated by the spontaneous degradation of methylated cytidines; Vsr recognizes the mismatched base pair and cleaves the phosphate backbone 5' to the thymidine. We have determined the crystal structure of a truncated form of this endonuclease at 1.8 A resolution. The protein contains one structural zinc-binding module. Unexpectedly, its overall topology resembles members of the type II restriction endonuclease family. Subsequent mutational and biochemical analyses showed that certain elements in the catalytic site are also conserved. However, the identification of a critical histidine and evidence of an active site metal-binding coordination that is novel to endonucleases indicate a distinct catalytic mechanism.

Investigation of the mechanisms of DNA binding of the human G/T glycosylase using designed inhibitors.

Deamination of 5-methylcytosine residues in DNA gives rise to the G/T mismatched base pair. In humans this lesion is repaired by a mismatch-specific thymine DNA glycosylase (TDG or G/T glycosylase), which catalyzes specific excision of the thymine base through N-glycosidic bond hydrolysis. Unlike other DNA glycosylases, TDG recognizes an aberrant pairing of two normal bases rather than a damaged base per se. An important structural issue is thus to understand how the enzyme specifically targets the T (or U) residue of the mismatched base pair. Our approach toward the study of substrate recognition and processing by catalytic DNA binding proteins has been to modify the substrate so as to preserve recognition of the base but to prevent its excision. Here we report that replacement of 2'-hydrogen atoms with fluorine in the substrate 2'-deoxyguridine (dU) residue abrogates glycosidic bond cleavage, thereby leading to the formation of a tight, specific glycosylase-DNA complex. Biochemical characterization of these complexes reveals that the enzyme protects an approximately 20-bp stretch of the substrate from DNase I cleavage, and directly contacts a G residue on the 3' side of the mismatched U derivative. These studies provide a mechanistic rationale for the preferential repair of deaminated CpG sites and pave the way for future high-resolution studies of TDG bound to DNA.

Cloning of a yeast 8-oxoguanine DNA glycosylase reveals the existence of a base-excision DNA-repair protein superfamily.

BACKGROUND: Reactive oxygen species, ionizing radiation, and other free radical generators initiate the conversion of guanine (G) residues in DNA to 8-oxoguanine (OG), which is highly mutagenic as it preferentially mispairs with adenine (A) during replication. Bacteria counter this threat with a multicomponent system that excises the lesion, corrects OG:A mispairs and cleanses the nucleotide precursor pool of dOGTP. Although biochemical evidence has suggested the existence of base-excision DNA repair proteins specific for OG in eukaryotes, little is known about these proteins. RESULTS: Using substrate-mimetic affinity chromatography followed by a mechanism-based covalent trapping procedure, we have isolated a base-excision DNA repair protein from Saccharomyces cerevisiae that processes OG opposite cytosine (OG:C) but acts only weakly on OG:A. A search of the yeast genome database using peptide sequences from the protein identified a gene, OGG1, encoding a predicted 43 kDa (376 amino acid) protein, identical to one identified independently by complementation cloning. Ogg1 has OG:C-specific base-excision DNA repair activity and also intrinsic beta-lyase activity, which proceeds through a Schiff base intermediate. Targeted disruption of the OGG1 gene in yeast revealed a second OG glycosylase/lyase protein, tentatively named Ogg2, which differs from Ogg1 in that it preferentially acts on OG:G. CONCLUSIONS: S. cerevisiae has two OG-specific glycosylase/lyases, which differ significantly in their preference for the base opposite the lesion. We suggest that one of these, Ogg1, is closely related in overall three-dimensional structure to Escherichia coli endonuclease III (endo III), a glycosylase/lyase that acts on fragmented and oxidatively damaged pyrimidines. We have recently shown that AlkA, a monofunctional DNA glycosylase that acts on alkylated bases, is structurally homologous to endo III. We have now identified a shared active site motif amongst these three proteins. Using this motif as a protein database searching tool, we find that it is present in a number of other base-excision DNA repair proteins that process diverse lesions. Thus, we propose the existence of a DNA glycosylase superfamily, members of which possess a common fold yet act upon remarkably diverse lesions, ranging from UV photoadducts to mismatches to alkylated or oxidized bases.

Changes of immunological functions after acute exacerbation in schizophrenia.

We investigated the changes of immunological functions in 14 schizophrenic patients (DSM-III-R; six men and eight women) who were hospitalized due to acute exacerbation of schizophrenia. The following immunological functions were studied on admission, 4 and 8 weeks after admission: serum immunoglobulins (Ig)G, A, and M; serum complement CH50; lymphocyte responses to mitogens (phytohemagglutinin, concanavalin A, and pokeweed mitogen); lymphocyte subpopulations (CD3%, 4%, 8%, 16%, 20%, 25%, and 56%); and natural killer cell (NK) activity. Psychological status of the patients, which was assessed by using Brief Psychiatric Rating Scale, improved gradually after admission. Changes in immune functions were analyzed using one-way analysis of variance and a randomized block analysis of variance with multiple comparison. NK activity on admission was significantly lower than those at 4 and 8 weeks after admission (p < .03). Serum IgG levels on admission and at 4 weeks after admission were significantly decreased as compared with those at 8 weeks after admission (p < .05); they were also lower than those in controls (p < .05). CD56% on admission and CD25% 4 weeks after admission were significantly increased as compared with controls (p < .05). These results indicate that several immunological functions might change related to time course after acute exacerbation. It is suggested that clinical conditions be carefully taken into consideration to evaluate immunological studies in schizophrenia.

Pericentric region of chromosome 9 is a possible candidate region for linkage study of schizophrenia.

We have undertaken a systematic G-banding survey to find structural chromosomal abnormalities among patients with schizophrenia. Of 120 patients with DSM-III-R schizophrenia, four (3.3%) had a pericentric inversion of chromosome 9 and three (2.5%) had a X/XX mosaicism. The frequency of pericentric inversion of chromosome 9 among patients with schizophrenia was statistically higher than those among newborns and Asian populations. Our results indicate that the pericentric region of chromosome 9 might be one of the potential regions of interest for linkage analysis of schizophrenia.

Neuronal pp60c-src(+) in the developing chick spinal cord as revealed with anti-hexapeptide antibody.

Polyclonal antibody was raised in rabbits against a synthetic hexapeptide R-K-V-D-V-R corresponding to a unique amino acid sequence of the neuron-specific c-src gene product pp60c-src(+). The antibody was purified by affinity chromatography. A single band with an apparent molecular mass of 60 kDa was recognized when the supernatant of homogenates of brain and spinal cord from chick embryos and chicks was probed with the affinity purified anti-hexapeptide antibody after SDS-polyacrylamide gel electrophoresis followed by Western blotting. Specificity of the antibody was further characterized by autophosphorylation assay of immunoprecipitate in comparison with the monoclonal antibody 327. Immunocytochemical studies by light microscopy revealed that pp60c-src(+) was localized in flake-like aggregates in neuronal cell bodies of the spinal cord in 7-15-day-incubated chick embryos and newly hatched chicks. Developing spinal ganglia and muscle cells were also immunoreactive at early developmental stages. By electron microscopy, the reaction product was observed mainly in two regions. One region was at polysomes and along the membranes of the rough endoplasmic reticulum. The other region was along the neuronal plasma membrane--at subsurface cisterns and at synapses. At synapses, the postsynaptic density, presynaptic membrane and synaptic vesicle membranes were immunostained. Immunoreactivity at synapses were more frequently observed at earlier stages than at later stages of development. These findings suggest that pp60c-src(+) is actively produced in developing neurons and has some important roles in synaptogenesis. In mature synapses, pp60c-src(+) may be involved in the interaction of synaptic vesicles with the presynaptic membrane.

Interaction of cellular hydrogenase, cytochrome c3, and desulfoviridin in Desulfovibrio vulgaris Miyazaki with their antibodies.

Anti-sera for hydrogenase, cytochrome c3, and desulfoviridin (abbreviated as anti-hyd, anti-c3, and anti-dvn, respectively) were raised in mice, and used to locate these antigens in cells of Desulfovibrio vulgaris Miyazaki. The activity of the intact cells to absorb H2 with methyl viologen or sulfite as an electron acceptor was cumulatively inhibited by treating the cells with anti-hyd and anti-c3 but unaffected by anti-dvn treatment. The activity of the intact cells to produce H2 from formate was also inhibited by anti-c3 treatment, but the inhibition by anti-hyd treatment was not significant. The fluorescent antibody technique applied to intact cells of D. vulgaris Miyazaki indicated that both hydrogenase and cytochrome c3 are localized on the surface of the cell. These results are not exactly in conformity with the hydrogen-cycling hypothesis for proton gradient formation in the energy metabolism in Desulfovibrio. The procedure described in the present paper provides a new technique to elucidate the roles of proteins by applying anti-sera to intact cells without destroying the cellular structure.