Monoclonal Antibodies Specific for NADH-Ferricyanide Reductase Domain of Spinach Leaf Nitrate Reductase

Two monoclonal antibodies, 17(3)9 and 36(79)4, were preparedagainst nitrate reductase from Spinacia oleracea L. leaves.An enzyme-linked immunosorbent assay showed that 17(3)9, butnot 36(79)4, reacted more strongly to heat-denatured than nativeantigen. These antibodies inhibited NADH-nitrate reductase aswell as its various partial activities including reduced methylvilogen-nitrate reductase, reduced flavin mononucleotide-nitratereductase and NADH-cytochrome c reductase activities, but notNADH-ferricyanide reductase activity. Immunoblotting after electrophoreticseparation of nitrate reductase fragments obtained by Staphyrococcusaureus V8 protease digestion of native enzyme revealed thatthe two monoclonal antibodies bind to different epitopes locatedon the 28 kDa of the NADH-ferricyanide reductase domain. (Received October 2, 1987; Accepted June 9, 1988)     

The regions of α-neurotoxin binding on the extracellular part of the α-subunit of human acetylcholine receptor

A set of 18 synthetic uniform overlapping peptides spanning the entire extracellular part (residues 1–210) of the α-subunit of human acetylcholine receptor were studied for their binding activity of¹²⁵I-labeled α-bungarotoxin and cobratoxin. A major toxin-binding region was found to reside within peptide α122–138. In addition, low-binding activities were obtained with peptides α34–49 and α194–210. It is concluded that the region within residues α122–138 constitutes a universal major toxin-binding region for acetylcholine receptor of various species.     

Increase in nucleoside diphosphatase in rat brain striatum lesioned with kainic acid

The activity of ammoniagenesis from guanine nucleotides was found to increase significantly in rat brain after infusion of kainic acid into the striatum. Among the enzymes involved in degrading guanine nucleotides, nucleoside diphosphatase was markedly increased in the lesioned striatum. The enzyme activity began to increase 2 days after the infusion, and reached the maximum on the 13th day, the level being 4 times as high as that of the intact contralateral region. The increased activity was due to Type L enzyme, judging from its substrate specificity. Puromycin and cycloheximide inhibited this increase, indicating that the increased activity resulted from an increase in the net synthesis of the enzyme. These findings suggest that Type L NDPase might play some important roles in gliosis after neuronal lesion.     

Isolation and characterization of a new Ca2+/calmodulin-dependent protein kinase from isoproterenol-stimulated proliferating rat parotid acinar cells.

A new Ca2+/calmodulin-dependent serine kinase was isolated from rat parotid gland acinar cells following chronic treatment with the beta-agonist isoproterenol. A single-step purification was performed on a calmodulin-agarose affinity column, following solubilization with Triton X-100. Among various substrates tested, bovine galactosyltransferase was the preferred substrate of the kinase, followed by glycogen synthetase greater than histone greater than phosphodiesterase greater than phenylalanine hydroxylase greater than phosphorylase b greater than bovine serum albumin. In comparison, a spleen preparation of Ca2+/calmodulin-dependent kinase did not show galactosyltransferase to be the preferred substrate. Thus, the enzyme would appear to be similar to the human galactosyltransferase-associated kinase. The kinase activity was saturable with 100 microM Ca2+ and 2 microM calmodulin. The molecular mass determined by nondenaturing and sodium dodecyl sulfate polyacrylamide gel electrophoreses was 75 kDa with a pI of 4.3. The Vmax was 3500 mumol/(min.mg protein) with a Km of 1.6 microM for the transferase substrate. Leukotriene C and prostaglandin E2 were found to be specific noncompetitive inhibitors of the rat galactosyltransferase-associated kinase.     

The combined effect of lymphokine activated killer cell and radiation therapy on rat brain tumorin vitro

Thein vitro effect of a combined treatment with lymphokine activated killer (LAK) cell and radiation therapy on rat brain tumor was examined using⁵¹Cr release assay. The tumor cell-line used in this experiment was 9L rat brain tumor derived from a Fischer 344 rat. LAK cells were obtained by culturing rat lymphocytes with recombinant human interleukin 2 for at least 3 days. The cytotoxic activity of the LAK cells was examined by⁵¹Cr release assay. Irradiation was done by exposing the microtiter plate in which the¹⁵Cr labeled 9L cells and LAK cells were cultured to a¹³⁷Cs gamma cell unit. Without irradiation, there was 18% cytotoxicity in the 1:100 tumor-to-LAK cell ratio specimen after 24 hrs cocultivation. However, if 5 Gy of irradiation was given, followed by 12 hrs incubation, the cytotoxicity was enhanced significantly at the same cell ratio (30%). This enhancement effect was the most prominent when the cell ratio was 1:100 and the irradiation dose was 5 Gy. To generate the enhancement effect, an incubation time of over 8 hrs both before and after irradiation was required. The supernatant of the LAK cells showed 19.8% and 11.4% cytotoxicity with and without irradiation, respectively. This result indicates the participation of a cytotoxic factor released from LAK cells.This work is supported in part by grant from Univeristy of Tsukuba Project Research.     

Influence of stress on the maturity of T-cells

Stress is known to influence the immune function via an effect on the central nervous system. We previously presented data showing that stress alters the population of T-cell subsets in mice. The variations of T-cell subsets in the thymus, peripheral blood, and spleen in mice similarly stressed by immobilization or by unavoidable and opioid-dependent stress were measured by flow cytometry using the monoclonal antibodies anti-L3T4, anti-Lyt 1, anti-Lyt 2 and anti-Thy 1, 2. Immobilization stress was applied for three days and T-cell subsets were measured on the days 1, 2 and 3, as well as on day 7 after release from immobilization. Lyt 2-positive cells in the thymus were the most sensitive to stress, showing significant variations. The proportion of immature T-cells increased in the thymus, blood and spleen of the stressed mice. When diazepam or naloxone were administered 30 min before the initiation of stress, these variations tended to decrease. Thus, the ratio of T-cell subsets varied with the duration of immobilization stress. This appeared to be partly mediated by the opioid system and the central nervous system.     

Distribution and possible intergradation betweenMacaca tonkeana andM. ochreata at the borderland of the species in Sulawesi

Morphological observations of pet and wild monkeys were made in the area that was inferred to be the borderland betweenMacaca tonkeana andM. ochreata in Sulawesi. Almost all individual monkeys could be classified into one of two species by their external characteristics. The possible borderland was estimated to extend from the La River in the east and to around Karaena River in the west. These two species may make contact in the forest in the western area of the borderland. Some external characteristics exhibited wide individual variations in the two species. Some monkeys originating from the borderland showed external characteristics that were intermediate between those of the two species. The possible intergradation between these two species is discussed in terms of the morphological variations found in the two species.     

Type B nucleoside-diphosphatase of rat brain. Purification and properties of an enzyme with high thiamin pyrophosphatase activity

Type B nucleoside-diphosphatase was purified from membranes of rat brain by solubilization with a non-ionic detergent and successive column chromatographies on DEAE-cellulose DE-52, concanavalin-A-Sepharose, Bio-Gel HT, blue-Sepharose CL-6B, chelating Sepharose 6B, Ultrogel AcA44 and TSK gel G3000 SW. The purified enzyme gave a single protein band on SDS/polyacrylamide gel electrophoresis and its molecular mass was estimated to be 75 kDa. It hydrolyzed thiamin diphosphate as well as GDP, IDP and UDP. Thiamin diphosphate (TPP) was hydrolyzed twice as efficiently as nucleoside diphosphates in the presence of Mn2+ at pH 7.4. The Km values for TPP, GDP, IDP and UDP were 0.66, 0.40, 0.54 and 1.06 mM respectively. ATP, ADP and pyridoxal 5'-phosphate inhibited thiamin-pyrophosphatase activity competitively and their Ki values were 2.3 mM, 1.0 mM and 0.59 mM respectively. The optimum pH of thiamin-pyrophosphatase activity was 7.4 in the presence of Mn2+ and that of GDP-hydrolytic activity was 6.5 in the presence of Mg2+.