p53 alteration, proliferating cell nuclear antigen, and nucleolar organizer regions in thymic epithelial tumors

We examined p53 protein expression, p53 gene mutation, proliferating cell nuclear antigen (PCNA), and argyrophilic nuclear organizer regions (AgNOR), in 30 patients with surgically-treated thymic tumors (26 thymoma and 4 thymic carcinoma cases). p53 expression ratio with DO-1 was divided as p53 negative (0% positivity), low expressor (<10% positivity), high expressor (>10% positivity). The incidence of p53 low and high expressor in thymoma were 19% (5/26) and 8% (2/26), respectively. p53 immunopositivity in thymoma was significantly correlated with PCNA labeling index (LI). p53 expression ratio in invasive thymoma (33%) tended to be higher than that in non-invasive thymoma (18%). p53 expression was detected in one of the thymic carcinoma. There were no p53 gene mutations in 15 invasive thymoma, although one of four (25%) thymic carcinomas showed two point mutations. p53 gene alterations seem to be associated with malignant activity of tumor cells, and therefore detection of p53 gene mutations is useful as a diagnostic factor.     

Circularly polarized dielectric-loaded planar antenna excited bythe parallel feeding waveguide network

A new receiving planar array antenna for DBS (direct broadcasting satellite) is proposed. The element antenna is a short waveguide aperture mounted in the ground plane, loaded with a dielectric and polarizers, and excited through its side wall by another feeding rectangular waveguide. The gain of the element antenna loaded with a dielectric is so high that the grating lobes can be reduced sufficiently even if the element spacing in the array is wider than the wavelength in free space. Therefore we can reduce the number of the array elements, and parallel feeding by the low loss waveguide network can be feasible to provide a planar array antenna. This paper describes the experimental results of several kinds of the circularly polarized dielectric-loaded element antennas and the planar antennas fed by the waveguide network. In the 12 GHz band the planar antenna with 64-element radiators has a maximum gain of 31.9 dBi with an aperture efficiency of 94.7%, the 1 dB-down frequency bandwidth of the gain is about 800 MHz (6.7% for a center frequency of 11.85 GHz), and the frequency bandwidth of the axial ratio is less than 1 dB of 850 MHz (7.2%)     

Carbonization of adamantane to a graphitizable carbon

Adamantane was carbonized in sealed tubes to 1073 K. Despite the non-planar molecular structure of adamantane, the resulting carbon was graphitizable. Using i.r. and u.v. spectrometry, mass spectrometry and X-ray diffraction, it was found that adamantane begins to decompose at ≈ 823 K to yield such planar molecules as phenanthrene, methyl-phenanthrene and pyrene. These molecules lead to a graphitizing carbon through a carbonization process which is similar to that of a pitch.     

Noncompetitive inhibition by L-cysteine and activation by L-glutamate of the iron-oxidizing activity of a mixotrophic iron-oxidizing bacterium strain OKM-9

A mesophilic, mixotrophic iron-oxidizing bacterium strain OKM-9 uses ferrous iron as a sole source of energy and L-glutamate as a sole source of cellular carbon. Uptake of L-glutamate into OKM-9 cells is absolutely dependent on ferrous iron oxidation. Thus, the Fe(2+)-dependent L-glutamate uptake system of strain OKM-9 is crucial for the bacterium to grow mixotrophically in iron medium with L-glutamate. The relationship between iron oxidation and L-glutamate transport activities was studied. Iron oxidase containing cytochrome a was purified 9-fold from the plasma membrane of OKM-9. A purified iron oxidase showed one rust-colored band following disc gel electrophoresis after incubation with Fe(2+). The Fe(2+)-dependent L-glutamate transport system was also purified 14.5-fold from the plasma membrane using the same purification steps as for iron oxidase. Fe(2+)-dependent L-glutamate and L-cysteine uptake activities of OKM-9 were 0.36 and 0.24 nmol/mg/min, respectively, when a concentration of 18 mM of these amino acids was used as a substrate. Both uptake activities were completely inhibited by potassium cyanide (KCN), suggesting that cytochrome a in the iron oxidase is involved in the transport process. The iron-oxidizing activity of strain OKM-9 was activated 1.7-fold by 80 mM L-glutamate. In contrast, the activity was noncompetitively inhibited by L-cysteine. The Michaelis constant of iron oxidase for Fe(2+) was 12.6 mM and the inhibition constant for L-cysteine was 41.6 mM. A marked inhibition of iron oxidase by 50 mM L-cysteine was completely reversed by the addition of 60 mM L-glutamate. The results suggest the possibility that iron oxidase has a binding site for L-cysteine and the cysteine first bound to the iron oxidase was replaced by the added L-glutamate.     

Pattern modification of a neuronal network for individual-cell-based electrophysiological measurement using photothermal etching of an agarose architecture with a multielectrode array

A new type of individual-cell-based on-chip multielectrode array (MEA) cell-cultivation system with an agarose microchamber (AMC) array for topographical control of the network patterns of a living neuronal network has been developed. The advantages of this system are that it allows control of the cell positions and numbers for cultivation using AMCs, as well as easy and flexible control of the pattern of connections between the AMCs through photothermal etching where a portion of the agarose layer is melted with a 1480 nm infrared laser beam. With adequate laser power, narrow micrometer-order grooves (microchannels) can easily be fabricated that can be used to combine neighbouring AMCs to enable topographical control of the neural network pattern. Using this system, an individual-cell-based neural network pattern was formed of rat hippocampal cells within the AMC array without cells escaping from the electrode positions in the microchamber during an eight-day cultivation, and could record cell firing in response to 1.5 V, 500 kHz stimulation through an electrode. This demonstrated the potential of the on-chip AMCMEA cell cultivation system for long-term single-cell-based electrophysiological measurement of a neural network system.     

Volatilization and recovery of mercury from mercury wastewater produced in the course of laboratory work using Acidithiobacillus ferrooxidans SUG 2-2 cells

The iron-oxidizing bacterium Acidithiobacillus ferrooxidans SUG 2-2 is markedly resistant to mercuric chloride and can volatilize mercury (Hg0) from mercuric ion (Hg2+) under acidic conditions. To develop a microbial technique to volatilize and recover mercury from acidic and organic compound-containing mercury wastewater, which is usually produced in the course of everyday laboratory work in Okayama University, the effects of organic and inorganic chemicals on the mercury volatilization activity of A. ferrooxidans cells were studied. Among 55 chemicals tested, the mercury volatilization from a reaction mixture (pH 2.5) containing resting cells of SUG 2-2 (1 mg of protein) and mercury chloride (14 nmol) was strongly inhibited by AgNO3 (0.05 mM), K2CrO7 (1.0 mM), cysteine (1.0 mM), trichloroethylene (1 microM), and commercially produced detergents (0.05%). However, the strong inhibition by trichloroethylene and detergents was not observed when these organic compounds were chemically decomposed using Fenton's method before the treatment of the wastewater with SUG 2-2 cells. When 20 ml of water acidified with sulfuric acid (pH 2.5) containing ferrous sulfate (3%), diluted mercury wastewater (17.5 nmol of Hg2+) and SUG 2-2 cells (0.05 mg of protein) were incubated for 10 d at 30 degrees C, 47% of the total mercury in the wastewater was volatilized and recovered into a trapping reagent for metal mercury. However, when the organic compounds in the mercury wastewater were decomposed using Fenton's method and then treated with A. ferrooxidans cells, approximately 100% of the total mercury in the wastewater was volatilized and recovered.     

Conformational properties of the guanine-binding site of ribonuclease T1 inferred from the X-ray structure and protein engineering

Recognition by ribonuclease T₁ of guanine bases via multidentatehydrogen bonding and stacking interactions appears to be mediatedmainly by a short peptide segment formed by one stretch of aheptapeptide, Tyr⁴²-Asn⁴³-Asn⁴⁴-Tyr⁴⁵-Gly⁴⁶-Gly⁴⁷-Phe⁴⁸. Thesegment displays a unique folding of the polypeptide chain—consistingof a reverse turn, Asn⁴⁴-Tyr⁴⁵-Glu⁴⁶-Gly⁴⁷, stabilized by ahydrogen-bond network involving the side chain of Asn⁴⁴, themain-chain atoms of Asn⁴⁴, Gly⁴⁷ and Phe⁴⁸ and one water molecule.The segment is connected to the C terminus of a ß-strandand expands into a loop region between Asn⁴³ and Ser⁵⁴. Lowvalues for the crystallographic thermal parameters of the segmentindicate that the structure has a rigidity comparable to thatof a ß-pleated sheet. Replacement of Asn⁴⁴ with alanineleads to a far lower enzymatic activity and demonstrates thatthe side chain of Asn⁴⁴ plays a key role in polypeptide foldingin addition to a role in maintaining the segment structure.Substitution of Asn⁴³ by alanine to remove a weak hydrogen bondto the guanine base destabilized the transition state of thecomplex by 6.3 kJ/mol at 37°C. In contrast, mutation ofGlu⁴⁶ to alanine to remove a strong hydrogen bond to the guaninebase caused a destabilization of the complex by 14.0 kJ/mol.A double-mutant enzyme with substitutions of Asn⁴³ by a histidineand Asn⁴⁴ by an aspartic acid, to reproduce the natural substitutionsfound in ribonuclease M_s, showed an activity and base specificitysimilar to that of the wild-type ribonuclease M_s. The segmenttherefore appears to be well conserved in several fungal ribonucleases.     

The structural basis for pseudoreversion of the H95N lesion by the secondary S96P mutation in triosephosphate isomerase

The structural basis for the 3000-fold decrease in catalytic efficiency of the H95N mutant chicken triosephosphate isomerase and the 60-fold regain of catalytic efficiency in the double mutant, H95N.S96P, have been analyzed. The results from a combination of X-ray crystallography and Fourier transform infrared spectroscopy experiments indicate that the predominant defect in the H95N mutant isomerase appears to be its inability to bind the substrate in a coplanar, cis conformation. The structures of each mutant isomerase were determined from X-ray crystallography of the complex of phosphoglycolohydroxamate (PGH), an intermediate analog with the isomerase, and each was solved to a resolution of 1.9 A. The PGH appeared to be in two different conformations in which the enediol-mimicking atoms, O2-N2-C1-O1, of the PGH were not coplanar. No density was observed that would correspond to the coplanar conformation. Two bands are observed for the dihydroxyacetone phosphate carbonyl in the H95N mutant FTIR spectrum, and these can be explained if the O1 of DHAP, like the O1 of PGH in the crystal structure, is in two different positions. Two ordered water molecules are located between O1 of PGH and N delta of N95. Comparison of the structure of the pseudorevertant, H95N.S96P with that for the H95N single mutant, shows that S96P mutation causes the double mutant to regain the ability to bind PGH predominantly in the coplanar, cis conformation. Electron density for a single ordered water molecule bridging the N95 amide side chain and the O2 of PGH is observed, but the density was weak, perhaps indicating that the water molecule is somewhat disordered. Whether or not a water molecule is hydrogen bonded to O2 of PGH may explain the two carbonyl stretching frequencies observed for the GAP carbonyl. Together, the crystal structures and the FTIR data allow a complete explanation of the catalytic properties of these two mutant isomerases.     

Diversity of 16S ribosomal DNA-defined bacterial population in acid rock drainage from Japanese pyrite mine

Four acidophilic bacteria (YARDs1-4) were isolated from an acid rock drainage (ARD) from Yanahara mine, Okayama prefecture, Japan. The physiological and 16S rDNA sequence analyses revealed that YARD1 was closely affiliated with Acidithiobacillus ferrooxidans, YARD2 was an Acidiphilium-like bacterium, and YARD3 and YARD4 were sulfur-oxidizing bacteria with a relatively close relationship to A. ferrooxidans in the phylogenetic analysis. A molecular approach based on the construction of a 16S rDNA clone library was used to investigate the microbial population of the ARD. Small-subunit rRNA genes were PCR amplified, subsequently cloned and screened for variation by a restriction fragment length polymorphism (RFLP) analysis. A total of 284 clones were grouped into 133 operational taxonomic units (OTUs) by the RFLP analysis. Among them, an OTU showing the same RFLP pattern as those of the isolates from the ARD was not detected. The phylogenetic analysis based on the 16S rDNA sequences from 10 major OTUs and their close relatives revealed that 4 OTUs containing 32.1% of the total clones were loosely affiliated with Verrucomicrobia, 2 OTUs containing 6.6% of the total clones were loosely affiliated with Chloribi, and other OTUs were affiliated with Actinobacteria, Nitrospirae, and beta-Proteobacteria.     

Preparation and properties of carbon dental implant with fine rahmen surface layer

Blade- and tooth-root-form carbon dental implants were prepared by fixing fine Rahmen surface (FRS) layers of carbon fabric on a carbon fiber-reinforced carbon (CFRC) core. The fixing was successfully performed through the low-temperature deposition of pyrolytic carbon using cis-1,2-dichloroethylene as the raw material. The resultant implants showed flexural and compressive strength more than 150 and 100 MPa, respectively. The outer part of this FRS layer contained many pores ranging from 30 to 300 μm in diameter and 40–75% in volume fraction.New bone was generated in the open pores after being embedded in the femur of a monkey for 6 months. The shear strength of the embedded implant vs bone interface was 15 MPa. On the other hand, the blade-form FRS implant in the mandible of a monkey gave a locking system consisting of “bone-collagen fiber-calcified layer” after two years under a masticating pressure. The latter is very similar to the natural tooth system.