Search

The evolutionarily ancient Aquificales bacterium Sulfurihydrogenibium spp. dominates filamentous microbial mat communities in shallow, fast-flowing, and dysoxic hot-spring drainage systems around the world. In the present study, field observations of these fettuccini-like microbial mats at Mammoth Hot Springs in Yellowstone National Park are integrated with geology, geochemistry, hydrology, microscopy, and multi-omic molecular biology analyses. Strategic sampling of living filamentous mats along with the hot-spring CaCO3 (travertine) in which they are actively being entombed and fossilized has permitted the first direct linkage of Sulfurihydrogenibium spp. physiology and metabolism with the formation of distinct travertine streamer microbial biomarkers. Results indicate that, during chemoautotrophy and CO2 carbon fixation, the 87–98% Sulfurihydrogenibium-dominated mats utilize chaperons to facilitate enzyme stability and function. High-abundance transcripts and proteins for type IV pili and extracellular polymeric substances (EPSs) are consistent with their strong mucus-rich filaments tens of centimeters long that withstand hydrodynamic shear as they become encrusted by more than 5 mm of travertine per day. Their primary energy source is the oxidation of reduced sulfur (e.g., sulfide, sulfur, or thiosulfate) and the simultaneous uptake of extremely low concentrations of dissolved O2 facilitated by bd-type cytochromes. The formation of elevated travertine ridges permits the Sulfurihydrogenibium-dominated mats to create a shallow platform from which to access low levels of dissolved oxygen at the virtual exclusion of other microorganisms. These ridged travertine streamer microbial biomarkers are well preserved and create a robust fossil record of microbial physiological and metabolic activities in modern and ancient hot-spring ecosystems.

Summary Objective To assess the functional changes of Transient receptor potential vanilloid 1 (TRPV1) receptor and to clarify its mechanism in a rat mono-iodoacetate (MIA)-induced joint pain model (MIA rats), which has joint degeneration with cartilage loss similar to osteoarthritis. Methods Sensitization of TRPV1 in MIA rats was assessed by transient spontaneous pain behavior induced by capsaicin injection in knee joints and electrophysiological changes of dorsal root ganglion (DRG) neurons innervating knee joints in response to capsaicin. Mechanisms of TRPV1 sensitization were analyzed by a newly developed sandwich enzyme-linked immunosorbent assay that detects phosphorylated TRPV1, followed by functional and expression analyses of protein kinase C (PKC) in vivo and in vitro , which involves TRPV1 phosphorylation. Results Pain-related behavior induced by intra-articular injection of capsaicin was significantly increased in MIA rats compared with sham rats. In addition, capsaicin sensitivity, evaluated by capsaicin-induced inward currents, was significantly increased in DRG neurons of MIA rats. Protein levels of TRPV1 remained unchanged, but phosphorylated TRPV1 at Ser800 increased in DRG neurons of MIA rats. Phosphorylated-PKCɛ (p-PKCɛ) increased and co-localized with TRPV1 in DRG neurons of MIA rats. Capsaicin-induced pain-related behavior in MIA rats was inhibited by intra-articular pretreatment of the PKC inhibitor bisindolylmaleimide I. In addition, intra-articular injection of the PKC activator phorbol 12-myristate 13-acetate increased capsaicin-induced pain-related behavior in normal rats. Conclusion TRPV1 was sensitized at the knee joint and at DRG neurons of MIA rats through PKC activation. Thus, TRPV1 sensitization might be involved in chronic pain caused by osteoarthritis.

We have fabricated thin-film transistors (TFTs) and liquid-crystal displays (LCDs) with monolithic drivers on glass substrates and achieved ultralow power consumption by operating with a low power-supply voltage at 3 V. The gate insulator of the TFTs has a stack structure with an ultrathin interfacial SiO2 layer formed by nitric acid oxidation of silicon and a 40-nm-thick SiO2 layer formed by plasma-enhanced chemical vapor deposition. Owing to the TFTs with the thin gate insulator, the driving circuits of the LCDs can be operated at a supply voltage of 1.5 V, which is much lower than that of conventional LCDs of 10-15 V.

東京都内の水道水について，有機フッ素化合物（PFCs）である11種のペルフルオロアルキルカルボン酸類および5種のペルフルオロアルキルスルホン酸類を測定した。40地点のPFCsの総濃度は0.72～95 ng・L-1の範囲で，平均値は19 ng・L-1であった。最多検出地点では12種のPFCsが検出され，PFCs組成比を用いたクラスター分析の結果，都内の水道水は多摩地域と区部の二種類にほぼ大別できた。区部の水道水のPFCs組成比および濃度は類似しているのに対して，多摩地域の水道水の組成比および濃度にはばらつきがみられた。これは区部の水道水が表流水を原水としているのに対して，多摩地域では各浄水所において表流水に地下水を混合して原水としていることによるものと考えられた。諸外国のPFCsの指針値等と測定値を比較した結果，都内の水道水中の個々のPFC濃度は指針値等を下回った。

We have fabricated submicrometer ultralow-power thin-film transistors (TFTs) with stack gate dielectric structure formed by the nitric acid oxidation of Si (NAOS) method. A 1.8 nm NAOS SiO2 layer effectively blocks the leakage current, and consequently, the thickness of a gate oxide layer deposited on the NAOS SiO2 layer can be made as thin as 20 nm. Because of the thin gate oxide layer, submicrometer TFTs with gate length in the range of 0.6-0.9 μm can be fabricated. The operation voltage of the TFTs can be set as low as 1.5 V because of the low threshold voltages (i.e., -0.6 V for P-ch TFT and 0.6 V for N-ch TFT). The drain current versus source-drain voltage curves possess an ideal feature with sufficiently high saturation currents even at 1.5 V operation voltage. The drain current versus gate voltage curves show a sharp current increase, and the subthreshold swing value is ~80 mV/dec for both P-ch and N-ch TFTs. The on/off ratio is ~109 for both P-ch and N-ch TFTs, and the channel mobility is ~100 cm2/V·s for P-ch TFT and ~200 cm2/V·s for N-ch TFT.

Plasma metabolite and immunoreactive insulin concentrations and activities of enzymes related to energy metabolism in peripheral leukocytes were measured in growing Holstein calves. A ratio of girth of abdomen divided by girth of thorax (A/T ratio) of calves was significantly elevated after weaning, and the A/T ratio maybe a good indicator to evaluate rumen development. Plasma glucose and free fatty acid concentrations were changed in calves accompanying change in feeding. Activities of lactate dehydrogenase with pyruvate as substrate (LDH-P) and hexokinase (HK) in cytosolic fractions of peripheral leukocytes decreased significantly after weaning the calves reflecting the change of energy source from milk replacer with high percentages of fat and glucose and lactose as absorbable carbohydrate to pelleted feed containing starch as less absorbable carbohydrate and roughage. Some peripheral leukocyte enzymes such as LDH and HK may be good indicators to evaluate changes in energy metabolism of growing calves.

A problem faced in proteomics studies is the recovery of tagged protein complexes in their native and active form. Here we describe a peptide, Bio-Ox, that mimics the immunoglobulin G (IgG) binding interface of Staphylococcus aureus Protein A, and competitively displaces affinity-purified Protein A fusion proteins and protein complexes from IgG-Sepharose. We show that Bio-Ox elution is a robust method for the efficient and rapid recovery of native tagged proteins, and can be applied to a variety of structural genomics and proteomics studies.

To extend the application range of epitaxial NbN devices, we developed epitaxial TiN thin-film resistors. TiN has approximately the same lattice constant and crystalline structure as NbN and MgO. However, high quality TiN thin films show superconductivity at the liquid helium temperature. We attempted to remove this superconductivity using TiO impurity. The ratio of Ar and N/sub 2/ was fixed at 5:1 and the O/sub 2/ flow ratio varied from 0 to 1.6% in relation to the total gas flow. When O/sub 2/ ranged from 0.5 to 1.6%, superconductivity vanished and the resistivity obtained was about 50-145 /spl mu//spl Omega/cm at 4.2 K. X-ray analysis showed that the TiN films grew epitaxially on the MgO substrate as did the NbN films on the TiN films. Using the TiN resistor, a fully epitaxial NbN Josephson array oscillator was fabricated and tested. The oscillator was formed with 10 TiN-shunted NbN/MgO/NbN tunnel junctions and NbN microstrip resonators. At around 1.0THz, Shapiro steps induced by the Josephson oscillation were observed in the detector I-V characteristics.

Histone acetylation and methylation play a critical role in transcription and gene regulation. Identification of sites of lysine acetylation and methylation in histones or other proteins by mass spectrometry (MS) is of increasing interest. In this paper, we report the use of MS to differentiate between peptides containing acetylated or tri-methylated lysines. High accuracy matrix-assisted laser desorption/ionization-time of flight MS gives better than five parts per million measurement accuracy, which is sufficient to verify acetylation and/or methylation. Electrospray ionization tandem mass spectrometry was used to assign modification sites and to differentiate acetylation from methylation. Typically, an immonium ion at m/z 98 corresponds to a mono-methylated lysine and an immonium ion at m/z 126 corresponds to an acetylated lysine. The neutral loss ion (MH(+)-59) is unique for a tri-methylated lysine. For a peptide with two or more modification sites of acetylation or tri-methylation or one site containing partial acetylation and tri-methylation, the a(2)-, b(2)-type ion is the characteristic index for an acetylated lysine whereas the b(2)-59 ion is indicative of a tri-methylated lysine in the N-terminus. The y-type ions and y-59 ions are characteristic of an acetylated lysine and a tri-methylated lysine at the C-terminus, respectively. We demonstrated that a lysine in a peptide modified by methylation or acetylation can be differentiated by MS using our method. Even if more then one lysine is present in a peptide and different modifications of this amino acid occur, they can be distinguished. This method was successful for the determination of the acetylation and methylation status of lysine 9 of histone H3 in chicken erythrocytes and human HeLa cell lines.

Progressive renal dysfunction in 5/6 nephrectomized (NX) rats can be physiologically divided into three stages, coinciding with morphological stages, after definition of physiological parameters for identification of stage. Now, for the establishment of a toxicity screening approach using 5/6 NX rats, our concept, “Differential toxicity synchronized with renal dysfunction process could be identified using 5/6 NX rats” was examined by dosing gentamicin. Firstly, electrophoretic fractional changes of urinary proteins during gentamicin treatment were clarified with determination of amino acid sequences and the three differential features were proven, revealing the unpredictable depression of urinary albumin with progression of the stages in NX rats. Secondly, marked elevation of urinary lactate dehydrogenase (LDH) and glucose (GLU) was evident, indicating the intensified hypoxic conditions and glycolysis in tubular cells synchronized with increased tubular damage. Thirdly, these transit metabolic changes were proven as intensive cause for the advancement of renal dysfunction by the reduction of FRelectrolytes and water at the end of each dosing period. These results indicate that toxicity studies of newly developed drugs using 5/6 NX rats have potentiality prior to clinical dosing to the patients.

High-temperature ion implantation of arsenic (As+) into the 4H-silicon carbide (SiC) substrates with high dose of 7×1015 cm−2 has been investigated as an effective doping method of n-type dopant for SiC power electron devices fabrication. Regardless of the ion implantation temperature, the sheet resistances (Rs) decrease below 1600°C post-annealing and increase above 1700°C as the post-annealing temperature increases. The low Rs value (213 Ω/□) is achieved in the sample implanted at 500°C and annealed at 1600°C, an order of magnitude smaller than that implanted at room temperature (RT). Atomic force microscopy (AFM) images reveal that the surface roughness of ion-implanted SiC increases with the increase of post-annealing temperature. Secondary ion mass spectroscopy (SIMS) results show that As+ dopant depth profiles of the sample implanted at 500°C do not change before and after the post-annealing. On the other hand, for the sample implanted at RT, the As+ concentration in the ion-implanted layer decreases due to the outer-diffusion. These results indicate that high-temperature ion implantation is an effective method to prevent the outer-diffusion of As+ dopants during high-temperature post-annealing. It is considered that these post-annealing temperature dependences are caused by the evaporation of SiC surface layer.

Mass spectrometry is a powerful technique for the identification of proteins at nanogram quantities. However, some degree of sample preparation prior to mass spectrometry is required, and silver-stained protein gel samples are most problematic. Here we report our strategy to obtain peptide mass profiles from silver-stained protein gel samples from one- or two-dimensional gels by destaining prior to enzymatic digestion. This study demonstrates that by using the destaining method, the sensitivity and quality of mass spectra is increased for matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometric analysis, permitting more proteins to be identified by peptide mass database analysis.

Water soluble pollutants in the atmosphere are incorporated into fog and their concentrations in the fog generated in the polluted air may be very high. Waldman et al. (1982) have reported that the pH of the polluted fog water is very low and the concentrations of ions in fog water are usually much higher than those in rain water. Inorganic ions in fog water have been measured to clarify the acidification mechanism of fog water (Weathers, et al., 1988; Hosono et al., 1994; Gundel et al., 1994). However, incorporation of many trace organic contaminants into fog has been ignored. Only a few measurements for carboxylic acids (Kawamura and Kaplan, 1984; Winiwarter and Puxbaum, 1988; Suzuki, 1997) and carbonyls (Grosjean and Wright, 1983; Hosono et al., 1994) in fog water have been reported.

Undoped epitaxial channel n-MOSFET with high transconductance was developed. In order to obtain a good crystal quality of the epitaxial layer and, thus, to achieve high performance, it is important to reduce the oxygen concentration at the epitaxial Si/Si substrate interface. In this paper, we describe the relationship between the electrical characteristics and the surface density of oxygen at the epitaxial Si/Si substrate. We also describe the dependence of the electrical characteristics on epitaxial Si thickness. The g/sub m/ of n-MOSFET with 40-nm epitaxial Si for 0.10-/spl mu/m gate length was 630 mS/mm at V/sub d/-1.5 V, and the drain current was 0.77 mA//spl mu/m. This g/sub m/ value in the case of the epitaxial Si channel is about 20% larger than that of bulk the MOSFET. These results show that epitaxial Si channel MOSFET's are useful for future high-speed ULSI devices.

The use of samarium-153 in the context of radioimmunotherapy of cancers has been limited by the instability of antibody labelling, which produces high uptake concentrations in liver and bone. This study compares the pharmacokinetics and biodistribution of 153Sm-labelled OC125 monoclonal antibody, in whole or F(ab')2 fragment form and with diethylene triamine penta-acetic acid (DTPA) or 6-p-isothiocyanatobenzyl diethylene triamine penta-acetic acid (CITCDTPA) coupling, in nude mice grafted subcutaneously with an ovarian adenocarcinoma line (SHIN-3) expressing CA125 antigen. The specific activity of the immunoconjugates was 18.5-55.5 MBq/mg, and their immunoreactivity exceeded 65%. With 153Sm-DTPA-OC125F(ab')2, the stability study in serum indicated that 50% of the metal remained bound to the antibody. The pharmacokinetic study showed a retention half-life of 25.1 h and blood clearance of 0.72 ml/h. The biodistribution study indicated tumour uptake of 4.53%+/-0.49% of injected activity per gram (%ID/g) at 24 h and tumour-to-liver and tumour-to-bone ratios of 0.23+/-0.02 and 1.54+/-0.49 respectively at 24 h. With 153Sm-CITCDTPA-OC125F(ab')2, serum stability was greater (87% of the metal remaining bound to the antibody), retention half-life was 22.25 h and blood clearance was 2.23 ml/h. Tumour was better targeted (8.30%+/-3.56%ID/g at 24 h), and tumour-to-liver and tumour-to-bone ratios were 1.17+/-0.36 and 7.08+/-3.09 respectively at 24 h. However, renal retention remained elevated (29.76%+/-9. 41%ID/g at 24 h). With intact IgG, renal uptake decreased (1.41%+/-0. 49%ID/g at 24 h), but tumour uptake was lower than with fragments (1. 46%+/-0.58%ID/g at 24 h). Liver uptake was higher (tumour-to-liver ratio 0.10+/-0.05), and blood clearance was slower. The stability and distribution of 153Sm-CITCDTPA were more favourable than those of 153Sm-DTPA for application in radioimmunotherapy. Quantitative analysis performed using digitized images obtained by conventional autoradiography and the imaging plate system indicated that the latter system is suitable for biodistribution studies of immunoconjugates.

We have already reported that anti-erythrocyte antibody induced by Babesia gibsoni infection (aEAb) showed higher reactivity against aged and/or oxidized erythrocytes than intact one in vitro (Morita et al., 1995). To clarify the meaning of such a binding character of aEAb in vivo, changes in erythrocyte oxidation were observed in artificially infected dogs. The ratio of methemoglobin concentration against total hemoglobin concentration (metHb%) as the indicator of erythrocyte oxidation was increased by the artificial infection in the intact dogs, suggesting that aEAb played a more important role in erythrocyte destruction in infected dogs. Though parasitemia approximately three times higher was observed in splenectomized dogs than that in intact dogs, metHb% did not increase in the splenectomized dogs. This suggests that the spleen plays a very important role in erythrocyte oxidation in dogs infected with B. gibsoni.

Effects of ageing treatment of erythrocytes to binding activity of the anti-erythrocyte antibody(s) in the serum of dogs infected artificially with Babesia gibsoni were examined by enzyme-linked immunosorbent assay (ELISA) using fixed cell antigen. When erythrocytes made senescent by two artificial ageing treatments, one of which was storing erythrocytes at 37 degrees C in medium for some days and the other was oxidation of erythrocytes with phenylhydrazine, reactivity of anti-erythrocyte antibody(s) in the infected serum to both types of treated erythrocytes was increased. These results suggest that such antibody(s) could play a role as one of the causes of anemia in canine babesiosis.

Segregation of arsenic and suppression of decomposition of source molecules during the growth of silicon by ultra-high-vacuum chemical vapor deposition is investigated. At the surface, the incidence and the desorption of arsenic balances with a small amount of incorporation. Almost all dopant atoms segregate on the surface during the growth and the rest are incorporated. Thus, the doping concentration is governed by the competition between the growth rate of Si crystal and the segregation speed of the dopant. The high doping concentration was obtained at high growth temperature, because the increment of the growth rate is larger than that of the segregation speed in accordance with the temperature. The dependence of doping concentration on AsH 3 pressure is explained by this model.

Protein kinase specificity is of fundamental importance to pathway regulation and signal transduction. Here, we report a convenient system to monitor the activity and specificity of recombinant protein kinases expressed in E. coli. We apply this to the study of the cytoplasmic domain of the plant receptor kinase BRASSINOSTEROID-INSENSITIVE 1 (BRI1), which functions in brassinosteroid (BR) signaling. Recombinant BRI1 is catalytically active and both autophosphorylates and transphosphorylates E. coli proteins in situ. Using enrichment approaches followed by LC-MS/MS, phosphosites were identified allowing motifs associated with auto- and transphosphorylation to be characterized. Four lines of evidence suggest that transphosphorylation of E. coli proteins by BRI1 is specific and therefore provides meaningful results: (1) phosphorylation is not correlated with bacterial protein abundance; (2) phosphosite stoichiometry, estimated by spectral counting, is also not related to protein abundance; (3) a transphosphorylation motif emerged with strong preference for basic residues both N- and C-terminal to the phosphosites; and (4) other protein kinases (BAK1, PEPR1, FLS2, and CDPKβ) phosphorylated a distinct set of E. coli proteins and phosphosites. The E. coli transphosphorylation assay can be applied broadly to protein kinases and provides a convenient and powerful system to elucidate kinase specificity.

Mycoplasma parasites escape host immune responses via mechanisms that depend on remarkable phenotypic plasticity. Identification of these mechanisms is of great current interest. The aminoacyl-tRNA synthetases (AARSs) attach amino acids to their cognate tRNAs, but occasionally make errors that substitute closely similar amino acids. AARS editing pathways clear errors to avoid mistranslation during protein synthesis. We show here that AARSs in Mycoplasma parasites have point mutations and deletions in their respective editing domains. The deleterious effect on editing was confirmed with a specific example studied in vitro. In vivo mistranslation was determined by mass spectrometric analysis of proteins produced in the parasite. These mistranslations are uniform cases where the predicted closely similar amino acid replaced the correct one. Thus, natural AARS editing-domain mutations in Mycoplasma parasites cause mistranslation. We raise the possibility that these mutations evolved as a mechanism for antigen diversity to escape host defense systems.

We present our data on the cytotoxicity of microtubule inhibitors (paclitaxel and taxotere) and their mode of action against cisplatin-sensitive or -resistant ovarian cancer cell lines in vitro. To try to establish an optimal administration schedule in clinical use, the cytotoxicity of microtubule inhibitors alone and in sequential combination with cisplatin was investigated. With microtubules alone, a marked dose- and schedule-dependent growth inhibition was demonstrated in lower concentrations and only schedule-dependency in higher concentrations. The cytotoxicity of taxotere was approximately 0.8- to 132-fold that of paclitaxel. No cross resistance to cisplatin was observed. In the relation between assay AUC (area under the concentration curve) and growth inhibition, increasing AUC by dose escalation seemed not to be advantageous. In a combination with cisplatin, the treatment of microtubules over 48 h followed by cisplatin administration demonstrated the most effective cytotoxicity in cisplatin-resistant cell line.

Four hybrid cell lines producing monoclonal antibodies (designated AC2, AH12, DB10 and DD11) were derived from mice immunized with the large chondroitin sulphate proteoglycan isolated and purified from the bovine temporomandibular joint disc. The epitopes were partially characterized by enzyme-linked immunosorbent assays and staining patterns on immunoblots of intact proteoglycans and digests made with glycosidases and proteinases. All four monoclonal antibodies appeared to recognize some form of keratan sulphate although the epitopes for two (AC2 and DD11) were probably identical. One antibody (AH12) showed almost no reactivity with corneal keratan sulphate but stained a small keratan sulphate proteoglycan extracted from the disc, in addition to the large chondroitin sulphate proteoglycan. These antibodies were used for immunohistochemical staining of sections of the disc and showed that keratan sulphate associated with the large chondroitin sulphate proteoglycan was concentrated inside and away from the periphery of the structure but close to the inferior and superior surfaces, in a pattern which may reflect the adaptation of the extracellular matrix to the mechanical stresses placed on it by mastication.

In order to develop a highly reliable method of routine charged particle activation analysis on a commercial basis by request of an outside company, (1) a new target holder was developed for brittle or low melting-point samples, (2) some modifications and improvements were introduced in the chemical separation, and (3) the reliability of the results was further examined. Oxygen, nitrogen, carbon, and boron in a greater variety of matrices have thus become analyzable with a higher reliability and efficiency.

We have developed a novel direct conversion detector for digital radiography by using a fullerene (C 60 )-doped polymer layer added on a thick amorphous selenium (a-Se) layer coupled to an amorphous silicon thin-film transistor (a-Si TFT) array. This detector exhibits considerable improvement in the lag characteristics and durability in high ambient temperatures. The C 60 -doped polymer layer, which is directly and uniformly solution cast on the a-Se layer and followed by an inorganic electron-transporting layer, smoothly changes the electronic junction between the a-Se layer and the inorganic layer. It lubricates the emission of photocurrents from the a-Se photo-conversion layer and leads to the improved lag characteristics. Another merit of using a C 60 -doped polymer is that it is stabile in high-temperature ambient conditions and is not degraded by humidity or a large amount of X-ray exposure. The polymer layer prevents the crystallization of a-Se, which otherwise occurs on exposure of a-Se to high temperature not only during the deposition of the inorganic layer or the metal electrode layer in the manufacturing process but also in actual use. A prototype detector, with a size of 17 in × 17 in and a pixel pitch of 150 μm, exhibited a good resolution; its DQE is approximately 48% at 1 cy/mm in 258 μC/kg (RQA5). This new development can simplify cooling apparatus and detector modules and also make a wide range of operational environments available. In addition, the improved lag characteristics make it possible to reduce the exposure intervals for static imaging, tomosynthesis, and other various exposure techniques.

The structure of the nucleosome has been under intense investigation using neutron crystallography, x-ray crystallography, and neutron solution scattering. However the dimension of the histone octamer inside the nucleosome is still a subject of controversy. The radius of gyration (Rg) of the octamer obtained from solution neutron scattering of core particles at 63% 2H2O, 37% 1H2O is 33 A, and x-ray crystallography study of isolated histone octamer gives a Rg of 32.5 A, while the reported values using x-ray crystallography of core particles from two individual studies are 29.7 and 30.4 A, respectively. We report here studies of isolated histone octamer and trypsin-limited digested octamer using both neutron solution scattering and small angle x-ray scattering. The Rg of the octamer obtained is 33 A, whereas that of the trimmed octamer is 29.8 A, similar to the structure obtained from the crystals of the core particles. The N-terminal domains of the core histones in the octamer have been shown by high resolution nuclear magnetic resonance (Schroth, G.P., Yau, P., Imai, B.S., Gatewood, J.M., and Bradbury, E.M. (1990) FEBS Lett. 268, 117-120) to be mobile and flexible; it is likely that these regions are disordered and "not seen" by x-ray crystallography.

An enzyme catalyzing the formation of an unusual C-P bond that is involved in the biosynthesis of the antibiotic bialaphos (BA) was isolated from the cell extract of a mutant (NP71) of Streptomyces hygroscopicus SF1293. This enzyme, carboxyphosphonoenolpyruvate (CPEP) phosphonomutase, was first identified as a protein lacking in a mutant (NP213) defective in one of the steps in the pathway to BA. The first 30 residues of the amino terminus of this protein were identical to those predicted by the nucleotide sequence of the gene that restored BA production to NP213. The substrate of the enzyme, a P-carboxylated derivative of phosphoenolpyruvate named CPEP, was also isolated from the broth filtrate of NP213 as a new biosynthetic intermediate of BA. CPEP phosphonomutase catalyzes the rearrangement of the carboxyphosphono group of CPEP to form the C-P bond of phosphinopyruvate.

In this paper, a synchronous high-speed logic standard cells using bootstrapped technology for low temperature poly silicon thin film transistor -liquid crystal display (LTPS TFT-LCD) panel is proposed. The proposed TFT standard cells operate at high frequency region owing to the deep non-saturation operation by using the bootstrapped technology (BST). The proposed TFT standard cells can be reduced the pattern size by using the TFT capacitors for the bootstrapped capacitors. To confirm some characteristics of the proposed TFT standard cells, a full adder (FA) are simulated. As a result, the power delay products obtain about 1/5-9/10 times less value than that of the conventional standard cells using LTPS TFT.

Polyethylene (PE) is widely used as an insulating material for power cable. Recently, the simultaneous observation system for electroluminescence (EL) and dissipation current waveform has been developed by authors. By using this system, the simultaneous observation in low-density polyethylene (LDPE) film was carried out at 50, 100 and 200 Hz, respectively. From the results of experiments, the relation between the time and spatial distributions of EL and the dissipation current waveforms were carefully examined. At the 1st 50 Hz electric field application, the plenty number of EL pulses have occurred. Also, at the consecutive experiments at 100 and 200 Hz, the number of EL pulses shows the positive frequency dependence. However, at the 2nd 50 Hz electric field application just after that at 200 Hz, the number of EL pulses extremely decreased compared with 1st application. On the other hand, the nonlinearity of dissipation current at the 1st 50 Hz application seems to small compare with that at the 2nd application. The fundamental component itself at 1st one is smaller than that at 2nd one. The EL images showed that EL occurred at the multiple weak points within the circular observation area. The spatial distributions of EL are almost same for all frequencies. It is considered that a lot of recombination center in material will exist in initial. But the long period ac high field application makes the number of recombination center decrease. This decrement due to occupation by carrier leads the carrier density increase and it may cause the nonlinearity of dissipation current waveform.

Plasma metabolite concentrations and activities of enzymes related to energy metabolism in plasma, peripheral leukocytes and liver of pregnant Romney ewes with restricted feeding were measured to assess those metabolites and enzymes as indicators for evaluating metabolic conditions in the ewes. The body weights and plasma lactate concentrations of the low-feeding ewes (about 1.0 times maintenance) were significantly lower than those of the high-feeding ewes (about 1.5 times maintenance). There were no significant differences in plasma protein, glucose, triglyceride, cholesterol, pyruvate and immunoreactive insulin concentrations and plasma and leukocyte enzyme activities between both groups. Hepatic malate dehydrogenase activities were significantly higher and hexokinase activities were significantly lower in the low-feeding ewes than in the high-feeding ewes. Restricted feeding could maintain pregnancy and some plasma metabolites and peripheral leukocyte enzymes may be useful indicators for evaluating metabolic changes in ewes.

To investigate the effect of Agaricus mushroom meal on the energy metabolism in animal tissues; plasma glucose, triglyceride, cholesterol and immunoreactive insulin (IRI) concentrations and activities of enzymes related to energy metabolism in plasma and peripheral leukocytes were measured in Japanese Black Wagyu × Holstein F1 calves supplemented with Agaricus blazei Murill ( A. blazei ) extract in milk-replacer at the dose of 60 g/head/day for 4 weeks. Activities of malate dehydrogenase and aspartate aminotransferase in cytosol and glutamate dehydrogenase in mitochondria, and the malate dehydrogenase/lactate dehydrogenase ratio in cytosol in peripheral leukocytes of calves with A. blazei were significantly higher than those in control calves without A. blazei . It was concluded that supplementation of Agaricus mushroom meal extract was effective in activation of enzymes related to energy metabolism in peripheral leukocytes of calves.

Pits appearing on selectively grown Si(100) epitaxial film were examined by scanning electron microscopy (SEM), cross-sectional transmittance electron microscopy (TEM) and energy dispersive X-ray spectroscopy (EDX). The pits were only observed on selectively grown Si films on SiO 2 patterned Si wafers, but not on mirror Si wafers. Amorphous nuclei consisting of silicon and oxygen existed at the bottom of the pits. The amorphous nuclei were about 50–200 nm in width and 5–10 nm in height. It was found that the Si film grew in such a manner as to avoid the amorphous Si-O nuclei and finally facets were formed, which appeared as the pits.

Activities of enzymes related to energy metabolism and isoenzyme patterns of lactate dehydrogenase (LDH) were determined in peripheral leukocytes and livers of Holstein dairy cows and Institute of Cancer Research (ICR) mice. In dairy cow liver, activities of enzymes in glycolysis, malate-aspartate shuttle and lipogenesis were lower, but activities of glucose-6-phosphatase in gluconeogenesis were higher than those in mouse liver. Glucokinase activities were below detection limit in leukocytes and liver of the cows. Dairy cow leukocytes and liver showed the isoenzyme patterns with dominance of LDH-1, -2 and-3, whereas mouse leukocytes and liver showed that LDH-5 was dominant. The LDH isoenzyme patterns were very similar between leukocytes and liver in each animal species. Some enzymes in leukocytes may reflect those enzymes activities in liver and be a useful indicator for energy metabolism in animals.

It has been proposed that phosphoinositide 3-kinase (PI3K), one of the phosphatidylinositol kinases, can be regulated by G-protein-coupled receptor as well as nerve growth factor-associated receptors. The aim of the present study was to investigate whether in vivo treatment with morphine, a mu-opioid receptor (MOR) agonist, could directly regulate PI3Kgamma isoform in the mouse periaqueductal gray matter (PAG). Using the polyclonal antibody recognizing a p110gamma catalytic subunit of PI3Kgamma, PI3Kgamma-like immunoreactivity (IR) was mostly seen in the membrane of the cell labeled by anti-neuron-specific nuclear protein. A single s.c. injection of morphine caused a marked increase in the number of PI3Kgamma-IR expressing cells in the PAG. Double immunolabeling assay showed that MOR-IR was mostly overlapped with PI3Kgamma-IR on the cell surface in the PAG section. Additionally, phosphorylated-phospholipase Cgamma1 (PLCgamma1-IR) was highly detected in the membrane compartment of the increased PI3Kgamma-IR-positive cells of this region. Further pharmacological evidence for the critical role of PI3Kgamma in MOR-mediated antinociceptive response was provided by the warm-plate test. The dose-response lines for antinociceptive effects of morphine were significantly shifted to the right following i.c.v. treatment with PI3K inhibitors. These findings suggested that acute treatment with morphine may directly activate the PI3Kgamma/PLCgamma1 pathway in the PAG. This effect may, at least in part, result in the expression of morphine-induced pharmacological actions including antinociception in mice.

The structure and crystallinity of a segregation on a borophosphosilicate glass (BPSG) film have been clarified by scanning electron microscopy (SEM), transmission electron microscopy (TEM), transmission electron diffraction (TED), and electron energy loss spectroscopy (EELS). The segregation arises on the BPSG film surface after annealing in nitrogen ambient. Its size is below 0.5 μm. The segregation is identified as crystalline BPO4 from the results of TED and EELS. In this letter, TED pattern of crystalline BPO4 is indicated in detail. The growth mechanism of crystalline BPO4 is proposed on the basis of a series of the results.

1. Both activities of NADH- and NADPH-linked aquacobalamin reductases were found in some human tissues, liver, kidney pancreas, heart, spleen, lung, cerebrum, cerebellum, adrenal glands, stomach, duodenum, jejunum, ileum, colon and bone marrow. 2. Human liver contained both enzymes with higher specific activities than any other tissues. 3. The liver NADH-linked enzyme was distributed in both mitochondrial (approx. 60%) and microsomal (40%) fractions; similar to the distribution of the NADPH-linked enzyme, but of which 40% activity was found in the mitochondria and the remaining activity was recovered in the microsomes. 4. The results suggest that the synthetic systems of the cobalamin coenzymes occur in both mitochondria and microsomes of human liver.

We constructed a scanning tunneling spectroscopy (STS) system which uses ac voltage as a tunneling bias. In this system, for a measurement of a constant current image, time‐averaged value of rectified ac tunneling current was maintained to be constant by a feedback circuit, while, for an STS measurement, differential conductance data were obtained using a circuit for analog differentiaion. Therefore, this system enables us on line monitoring of differential conductance images at various voltages as well as a constant current image. As a preliminary result, atomic resolution of a graphite sample in air and tunneling bias voltage dependence of differential conductance images were obtained.

Six year-old Japanese pear (Pyrus serotina Reheder cv. Kosui) grafted on P. serotina cv. Nihonyamanashi was subjected to CO2 concentration (0.35 and 1.40 kPa) for 2 to 79 consecutive days at the fruit growing stage under controlled environmental conditions. Diurnal changes in stem and fruit diameters, apparent photosynthetic rate (Po), transpiration rate and fruit quality were measured. CO2 enrichment increased average weight of matured fruit, however, it had no significant effect on fruit quality such as size, acidity, and hardness. Po of leaves on spur and vegetative lateral branches was increased by CO2 enrichment, however, transpiration rate was reversely affected mainly due to stomatal closure. The fruit diameter increased with elevated CO2 concentration. The stem diameter started to increase 4 h after elevating CO2 concentration. The stem diameter sharply decreased in the morning and increased in the evening, however CO2 enrichment alleviated the reduction in the stem diameter during daytime largely due to suppression of shrinkage in the inner part (including xylem) of the stem. These results suggest that response of stem and fruit growth to CO2 enrichment could be detected by a micromorphometric technique within a short period.

Using ion exchange HPLC and ammonium sulfate precipitation, we have purified a 70-kDa protein (P70) specific to the cobalt-induced epileptogenic cortex of rat cerebrum and determined certain of its biochemical properties. P70 has a similar isoelectric point (pI; 4.6–4.8), amino acid composition and N-terminal amino acid sequence to rat serum albumin (RSA). Intracortical application of purified P70 to the motor area of normal rat cerebrum induces both ECoG seizure discharges and behavioral seizures. The data suggest that P70 is a novel albumin-like protein linked to the generation of seizure activities. However, it can be clearly distinguished from RSA, since it is able to produce seizure, is a glycoprotein and can be readily separated from RSA by 2-dimensional electrophoresis.

SUMMARY In order to investigate the proposed involvement of neuropeptides in musculoskeletal inflammation we pretreated rats, in an adjuvant spondylitis model, with capsaicin, a neurotoxin. Immunohistochemistry showed that administration of capsaicin to newborn rats depicted irreversibly the neuropeptide substance P. Elimination of capsaicin-sensitive fibres by the neonatal injection of capsaicin did not suppress the peridiscitis of rats in which adjuvant spondylitis was induced at 7 weeks of age. However, elimination of capsaicin-sensitive fibres did suppress the inflammation usually seen in the bone marrow. We speculate that this intramedullary inflammation is normally induced or sustained by capsaicin-sensitive fibres.

Characteristics of keratins of five carcinomas of sweat gland origin were immunohistochemically investigated with several antikeratin monoclonal antibodies with differing specificities. Specimens were obtained from two cases of mucinous carcinoma of the skin, two cases of classic type of eccrine adenocarcinoma, and a case of eccrine porocarcinoma. The tumor cells of mucinous carcinoma expressed only simple epithelial keratins. In a case of eccrine adenocarcinoma, simple epithelial keratin 19 was diffusely expressed. The expression of the other simple epithelial keratins was confined to the luminal cells, whereas the remaining tumor cells further expressed stratified epithelial keratins. Eccrine porocarcinoma and a second case of eccrine adenocarcinoma did not express simple epithelial keratins, although stratified epithelial keratins were diffusely expressed. These data suggest that carcinomas of sweat glands express various combinations of simple and stratified epithelial keratins. Development of additional data along these lines may help to further define their classification.

First, we will briefly describe how our study on the mechanisms of relaxant effects of nitroglycerin and related compounds (NG) on the vascular smooth muscle developed to a study on the sarcolemmal (SL) Ca(2+)-ATPase. These compounds were found to have no effects on the voltage-dependent Ca2+ channel and Ca(2+)-induced Ca2+ release from the sarcoplasmic reticulum (SR) and Na(+)-Ca2+ exchange mechanism has been shown not to play an important role in the vascular smooth muscle. Furthermore, when we initiated our study, the idea of receptor-operated Ca2+ channels and Ca2+ release from the SR by inositol triphosphates were not known. A major part of this review is devoted to a description of the characteristic features of SL Ca(2+)-ATPase and its regulation by various protein kinases. References to SR enzyme are made when necessary. Brief mention is made of the putative molecular structure and its possible variations as envisaged by genetic engineering techniques. However, particular attention is directed to the regulation by cyclic GMP-dependent protein kinase as this seems to be most important as regards the mechanisms of vascular smooth muscle relaxation by NG.

An incomplete proviral genome of endogenous mammary tumour virus (MMTV) was found in DNA of several strains of mice. This MMTV-related sequence was assigned to the Y chromosome since it was clearly observed in male mice only. This MMTV provirus contained a sequence related to LTR (long terminal repeat), but not to gag-pol and env genes. NFS, NIH Swiss/S, STS/A, and DD/Tbr mice have this sequence but BALB/cHeA, SHN, SLN, C57BL/6NJcl, C3H/HeNJcl and CBA/JJcl mice are negative. In the strains containing this sequence, a DNA test for the sequence makes it possible to easily distinguish the DNAs of male or female mice.

In an attempt to clarify the mechanism of sarcoplasmic reticulum (SR) dysfunction during the genesis of irreversible damage in the ischemic-reperfused myocardium, the changes in SR Ca2(+)-pumping ATPase (Ca2(+)-activated, Mg2(+)-dependent ATPase; Ca2(+)-ATPase) activity were studied during ischemia and subsequent reperfusion in the isolated perfused guinea pig heart preparation and correlated with the accumulation of calcium in the myocardium. Although the SR Ca2(+)-ATPase activity was not affected by ischemia of 40 min, reperfusion of the ischemic myocardium resulted in a definite time-dependent decrease in the enzyme activity. The reduction of SR Ca2(+)-ATPase activity was associated with a concomitant decrease in the enzyme concentration in the isolated SR and was in a good correlation with a substantial accumulation within the myocardium. As the results indicated the possibility that proteolytic degradation by a calcium-activated protease(s) was responsible for the reduction of enzyme activity, we examined for the possible involvement of calcium-activated neutral protease (CANP). However, SR Ca2(+)-ATPase obtained either from the normal hearts or from the hearts after 40-min ischemia was found to be quite resistant to proteolytic actions of the two forms of CANP, i.e., microCANP and mCANP partially purified from the guinea pig heart. These results suggest that a destructive process leading to the degradation of SR Ca2(+)-ATPase is activated by reperfusion, but not by ischemia per se, and that CANP is not implicated in the degradation of SR Ca2(+)-ATPase.

The histone octamer from chicken erythrocytes was studied in 2 M NaCI using 500 mHz 1H NMR spectroscopy. We compared the spectrum of control octamers with that of octamers isolated from trypsinized nucleosome core particles. We observe that the sharp resonances found in the spectrum of the native octamer disappear completely after trypsinization. Therefore, within the time frame of the NMR experiment, all of the mobile amino acid residues in the histone octamer are found in the well defined trypsin sensitive domains. These results indicate that there is a very clear structural demarcation between the random coil N- and C-terminal tails and the globular domains of the histones.