Fragmentation of the large subunit of ribulose-1,5-bisphosphate carboxylase by reactive oxygen species occurs near Gly-329

The large subunit (LSU) of ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) in the illuminated lysates of wheat (Triticum aestivum L.) chloroplasts is broken down by reactive oxygen radicals into 37- and 16-kDa polypeptides. Analysis of the terminal amino acid residues of both fragments revealed that the C terminus of the 37-kDa fragment was Ser-328 and the N terminus of the 16-kDa fragment was Thr-330. Gly-329, which links the two fragments, was missing, suggesting that the fragmentation of the LSU in the lysates driven by oxygen-free radicals occurs at Gly-329. Purified rubisco, exposed to a hydroxyl radical-generating system, was also cleaved at the same site of the LSU. The cleavage site was positioned at the N-terminal end of the flexible loop (loop 6) within the beta/alpha-barrel domain, constituting the catalytic site of rubisco. The binding of a reaction intermediate analogue, 2-carboxyarabinitol 1,5-bisphosphate, to the active form of rubisco completely protected the enzyme from the fragmentation. The fragmentation was differentially affected by CO2, Mg2+, ribulose 1, 5-bisphosphate, or 2-carboxyarabinitol 1,5-bisphosphate. All these results indicate that the conformation of the catalytic site of the enzyme is involved as an important factor determining the breakdown of rubisco by reactive oxygen species. Reactive oxygen species generated at its catalytic site by a Fenton-type reaction may trigger the site-specific degradation of the LSU in the lysates of chloroplasts in the light.     

A New Slip Frequncy Detector of an Induction Motor Utilizing Rotor Slot Harmonics

A method to detect slip frequency from rotor slot harmonics of a three-phase squirrel-cage induction motor is proposed. The rotor slot harmonic voltage Is obtained by summing the three phase voltages, and after being sampled with a multiple of the stator frequency, it is changed into slip frequency waves, from which a voltage proportional to the slip frequency is obtained. Sophisticated sampling techniques allow elimination of the third harmonic voltage induced in the sum of the three-phase voltages and also allow reduction of time constant of the slip frequency detector. Expenmental results show that the slip frequency detector has a good linearity in the range of slip frequency of about -50 to +30 percent of the stator frequency.     

Effects of α-tocopherol level in raw venison on lipid oxidation and volatiles during storage

Relationships between α-tocopherol concentration in the muscle and development of lipid oxidation or volatiles in raw venison were studied. Fourteen Japanese Shika Deer (Cervus nippon) were fed various amounts (0–3.0 g of α-tocopheryl acetate per animal) during the different periods (0–37 days) and then M. longissimus thoracium et lumborum (LD muscles) with a range of α-tocopherol concentrations (4.1–15.1 mg/kg tissue) were obtained. For stabilizing the lipid during storage for 11 days under air, over ca. 9 mg of α-tocopherol per kg tissue were required based on levels of 2-thiobarbituric acid reacting substances (TBARS) numbers. Nine compounds were identified in headspace volatiles, and one of the volatiles was hexanal which has been recognized as off-flavour component. For depressing the hexanal evolution, at least ca. 9 mg of α-tocopherol per kg tissue were also required for 11 days’ storage. This value was much higher than other species. The reasons for higher requirement of α-tocopherol were possibly due to the higher concentration of unsaturated fatty acid and myoglobin in venison.     

A pneumatically actuated full in-channel microvalve with MOSFET-like function in fluid channel networks

A pneumatically actuated silicon microvalve applicable to integrated microfluidic systems is presented. All the ports of this microvalve are in-channel, and connectable to any surface fluid channels in microfluidic systems. This microvalve controls fluid flow by means of the controlled gap between glass and silicon diaphragm actuated by a control pressure. In addition, the diaphragm is also deformed by the outlet pressure of the microvalve. Due to the feature, this microvalve shows saturation of flow rate like MOSFETs operated at saturation region. The fabricated microvalve device was evaluated focusing on analogous relationship between MOSFET and the microvalve. Fluids such as air and DI-water were well controlled by the control pressure. Fluid starts to flow in the microvalve when the control pressure exceeds its "threshold pressure." Hysteresis due to sticking of diaphragm was not observed in the characteristics. Air flow rate of the microvalve was gradually saturated with the increasing of the outlet pressure as expected. Through the evaluation, analogous relationship between this microvalve and MOSFET has been experimentally demonstrated.     

Highly selective fluorometric determination of polyamines based on intramolecular excimer-forming derivatization with a pyrene-labeling reagent

We introduce a novel approach in highly selective and sensitive fluorescence derivatization of polyamines. This method is based on an intramolecular excimer-forming fluorescence derivatization with a pyrene reagent, 4-(1-pyrene)butyric acid N-hydroxysuccinimide ester (PSE), followed by reversed-phase high-performance liquid chromatography (HPLC). Polyamines, having two to four amino moieties in a molecule, were converted to the corresponding dipyrene- to tetrapyrene-labeled derivatives by reaction (100 degrees C, 20 min) with PSE. The derivatives afforded intramolecular excimer fluorescence (450-520 nm), which can clearly be discriminated from the monomer (normal) fluorescence (360-420 nm) emitted from PSE, its hydrolysate and monopyrene-labeled derivatives of monoamines. The structures of the derivatives were confirmed by HPLC with mass spectrometry, and the emission of excimer fluorescence could be proved by spectrofluorometry and time-resolved fluorometry. The PSE derivatives of four polyamines [putrescine (Put), cadaverine (Cad), spermidine (Spd), and spermine (Spm)] could be separated by reversed-phase HPLC on a C8 column with linear gradient elution. The detection limits (signal-to-noise ratio of 3) for the polyamines were 1 (Put), 1 (Cad), 5 (Spd), and 8 (Spm) fmol on the column. Furthermore, the present method was so selective that biogenic monoamines gave no peak in the chromatogram.     

Characterization of natural resin shellac by reactive pyrolysis-gas chromatography in the presence of organic alkali

A method to determine chemical composition of natural resin shellac was developed on the basis of reactive pyrolysis-gas chromatography (Py-GC) in the presence of an organic alkali, tetramethylammonium hydroxide ((CH(3))(4)NOH, TMAH). Py-GC using 25% TMAH aqueous solution enabled the highly sensitive determination of terpenic acids, aleuritic acid, several minor fatty acids, and the wax components of shellac as their methyl derivatives on the resulting pyrograms with less than 2.0% relative standard deviations without using any cumbersome pretreatment. The observed average distributions of each resin acid component determined by reactive Py-GC for shellac samples from India and Thailand showed that the average ratios among terpenic acids, aleuritic acid, and the other fatty acids were about 53:34:14 for Indian shellac and 51:35:14 for Thailand shellac, respectively, suggesting a slightly significant difference. However, clearer discrimination of the shellac samples from the two different growing places was attainable by applying principal component analysis for the mole percent distributions of all the acidic components determined by reactive Py-GC.     

17O Knight shift study in the superconducting state of Sr2RuO4

¹⁷O Knight shift measurements in Sr₂RuO₄ were performed over the wide range of magnetic field 3.2-11.4kOe parallel to the basal RuO₂ planes. The spin susceptibility is totally unchanged through its T_c, evidencing that the spin-triplet superconducting state is realized in Sr₂RuO₄. The result indicates that the Cooper pairs consist of the parallel spin pairs | > and | > with their quantization axis perpendicular to the c-axis direction. The in-plane 2D nearly ferromagnetic spin fluctuations may play a role for the stabilization of this state among various representations of spin-triplet order parameter.     

Fas-independent and nonapoptotic cytotoxicity mediated by a human CD4(+) T-cell clone directed against an acute myelogenous leukemia-associated DEK-CAN fusion peptide

The mechanism underlying the cytotoxicity mediated by a human CD4(+) cytotoxic T-lymphocyte (CTL) clone directed against a peptide derived from the acute myelogenous leukemia-associated fusion protein, DEK-CAN, was investigated. A DEK-CAN fusion peptide-specific CD4(+) Th0 CTL clone, designated HO-1, was established from the peripheral blood lymphocytes of a healthy individual. HO-1 exerted direct but not "innocent bystander" cytotoxicity within 2 hours. The cytotoxicity mediated by HO-1 was completely Ca2+-dependent. Because HO-1 lysed peptide-loaded Fas-deficient target cells derived from a patient with a homozygous Fas gene mutation, its cytotoxicity appeared to be mediated by a Fas-independent pathway. In addition, its cytotoxicity was only partially inhibited by treatment with concanamycin A and strontium ions, which are inhibitors of the perforin-based cytotoxic pathway. Although membrane-bound type of tumor necrosis factor-alpha (TNF-alpha) was expressed on HO-1, an anti-TNF-alpha antibody had no effect on HO-1-mediated cytotoxicity. HO-1 expressed mRNA for apoptosis-inducing mediators, including perforin, granzyme B, Fas ligand, TNF-alpha, and lymphotoxin; however, no DNA fragmentation was detected in target cells incubated with HO-1 by 5-[125I]Iodo-2'-deoxyuridine release assay and agarose gel electrophoresis of DNA. Although it has been suggested that the Fas/Fas ligand system is the main pathway by which CD4(+) CTL-mediated cytotoxicity is exerted in murine systems, HO-1 produced peptide-specific and HLA-restricted cytotoxicity via a Fas-independent and nonapoptotic pathway. The present study thus describes a novel mechanism of cytotoxicity mediated by CD4(+) CTL.     

Regulation of selectin binding activity by cyclization of sialic acid moiety of carbohydrate ligands on human leukocytes

We provide here evidence that supports the occurrence of a biologically dormant form of selectin ligand carbohydrate, the sialyl 6-sulfo Lewis X containing modified sialic acid, in human leukocytes. The modification of sialic acid involves first de-N-acetylation of sialic acid moiety through ubiquitous de-N-acetylation/re-N-acetylation cycle, followed by the dehydrative cyclization of de-N-acetyl sialic acid to form "cyclic sialic acid." The enzyme involved in the dehydration of de-N-acetyl sialic acid is a calcium-dependent enzyme having neutral-alkaline pH optimum. De-N-acetyl sialyl 6-sulfo Lewis X retained selectin binding activity as well as parental sialyl 6-sulfo Lewis X, but cyclic sialyl 6-sulfo Lewis X was devoid of selectin binding activity. Sialyl 6-sulfo Lewis X carrying the cyclic sialic acid is specifically recognized by the newly generated mAb, G159. The determinant was distributed widely among normal human leukocytes, especially on monocytes and subsets of lymphocytes including NK cells, helper memory T cells, Tcr-gammadelta T cells, and a part of B cells. The determinant was detected also on several cultured lymphocytic leukemia cell lines and O-tetradecanoylphorbol 13-acetate-activated lymphoid cells. Cyclic sialyl 6-sulfo Lewis X is efficiently formed by the action of the partly membrane-bound calcium-dependent enzyme, tentatively called "sialic acid cyclase," and a possible physiological significance of this reaction could be a rapid inactivation of selectin binding activity at the cell surface. Conversely, the accumulated intracellular cyclic sialyl 6-sulfo Lewis X determinant may function as a dormant pool of selectin ligands, which, on appropriate stimulation, is hydrolyzed and becomes active in selectin-dependent cell adhesion.