Sarcolectin: an interferon antagonist extracted from hamster sarcomas and normal muscles. Isolation, characterization, and purification

In the present work we show that sarcoma and normal hamster tissues contain a protein which agglutinates normal and transformed cells. The inhibition of agglutination by galacturonic acid and occasionally by fucose suggests a resemblance of this protein with vegetal lectins. When added 5 h after interferon, the crude semipurified and electrophoretically homogeneous preparations reduce within 20 h the antiviral state pre-established by interferon. These two biological tests have enabled us to monitor the subsequent purification steps. The isolation of the biologically active protein is greatly facilitated by its resistance to pepsin and nucleases, whereas its sensitivity to trypsin and Pronase suggests its proteinaceous character. Furthermore, the molecule is stable when heated 1-2 min at 100 degrees C in the presence or absence of sodium dodecyl sulfate. After pepsin treatment, Sephacryl G-200 gel filtration, and ion exchange chromatography on DEAE-cellulose, 25-40-fold purification can be obtained. When controlled by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a double band (DEAE-cellulose sample) or single band (octyl-agarose sample) is detected in the 65,000-dalton region and no other contaminator is present. The eluted protein retains full biological activity when assayed by the degradation of the interferon-induced antiviral protection in the cell or titrated by cell agglutination.     

Characterization of alloimmunization-induced T lymphocytes reactive against AKR leukemia in vitro and correlation with graft-vs-leukemia activity in vivo

We have reported that immunization of H-2k mice with lymphoid cells from various allogeneic strains induced a population of cells that could eliminate first-passage spontaneous AKR leukemia from the spleens of immuno-suppressed AKR (H-2k) hosts. In the present study, we examined the nature of the cells responsible for this graft-vs-leukemia (GVL) reaction and compared them to cytolytic cells detected in vitro. Spleen cells from alloimmunized CBA/J (H-2k) mice were selectively depleted of various subpopulations by treatment with antibody and complement (C), then tested in vivo for GVL reactivity. Cell suspensions depleted of Thy-1.2+, Lyt-1+, or Lyt-2+ lymphocytes had no significant GVL reactivity, whereas suspensions depleted of NK-1.2+ cells retained GVL reactivity. The GVL-reactive cells persisted in H-2-compatible donor mice for up to 56 days. Lyt-1+2+ lymphocytes that were cytotoxic for cultured AKR leukemia cells in vitro could be detected in the spleens of alloimmunized H-2-compatible mice after expansion of the cells in T cell growth factor. Using quantitative limiting dilution cytotoxicity assays, we found that the frequency of leukemia-reactive cytotoxic lymphocytes (CL) in the spleen showed a direct correlation with the GVL efficacy of the cells in vivo. Alloimmunization was essential for induction of the GVL-reactive cell population. CL in alloimmunized mice consisted of heterogeneous cytotoxic specificities; i.e., some CL were leukemia-specific, others lysed only nonleukemic AKR target cells, and a third group mediated killing of both leukemic and nonleukemic target cells. The CL appeared to be H-2 restricted and specific for non-H-2 antigens shared by the AKR leukemia and the alloimmunizing cells.     

Acyl-CoA oxidase from Candida tropicalis

Acyl coenzyme A oxidase (acyl-CoA oxidase) has been isolated in good yield from Candida tropicalis pK 233 grown on n-alkanes. Gel filtration, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and measurement of flavin content suggest that the oxidase is an octamer of Mr 75 000 subunits each containing one flavin. The oxidase yields the red semiquinone form on dithionite or photochemical reduction, slowly forms an N-5 adduct with 0.16 M sulfite at pH 7.4, and is rapidly reduced by borohydride, forming the 3,4-dihydroflavin isomer. The red flavosemiquinone is only kinetically stabilized with respect to disproportionation in the free enzyme but is thermodynamically stabilized on binding enoyl-CoA derivatives. The enzyme is reduced by butyryl-, octanoyl-, and palmitoyl-CoA without formation of prominent long-wavelength bands. Acyl-CoA oxidase and the acyl-CoA dehydrogenases share many similarities in their interaction with CoA derivatives. For example, both enzymes stabilize the anionic radical on binding enoyl-CoA derivatives, both dehydrogenate 2-oxoheptadecyldethio-CoA but cannot utilize S-heptadecyl-CoA, both form long-wavelength bands with CoA persulfide species, and both enzymes are attacked by the suicide substrates 3,4-pentadienoyl-CoA and (methylene-cyclopropyl)acetyl-CoA at the flavin prosthetic group.     

Effect of Oxygen on Heme and Porphyrin Accumulation from δ-Aminolevulinic Acid by Suspensions of Anaerobically Grown Staphylococcus epidermidis

The effect of various conditions on the accumulation of porphyrins and heme by resting suspensions of anaerobically grown cells of Staphylococcus epidermidis was examined. Anaerobically grown cells contain 10 to 15% of the amount of protoheme found in cells grown aerobically. Resting suspensions of anaerobically grown cells, when incubated aerobically in buffer with delta-aminolevulinic acid and glucose for 60 min, exhibited a fourfold increase in protoheme content. At high levels of delta-aminolevulinic acid, there was also a significant accumulation of porphyrins with the solubility and chromatographic properties of coproporphyrin and uroporphyrin. Protoporphyrin was not accumulated. When oxygen was excluded from the incubation mixture, accumulation of protoheme was prevented, but accumulation of coproporphyrin and total porphyrin was enhanced. Nitrate served as an electon acceptor as indicated by its reduction to nitrite; however, nitrate did not substitute for oxygen in causing the accumulation of protoheme. These results suggested that oxygen is required for one of the late steps of heme synthesis in S. epidermidis, possibly for the conversion of coproporphyrinogen to protoporphyrin. The inability of nitrate to substitute for oxygen suggests a role for molecular oxygen as a substrate rather than as an electron acceptor for heme synthesis.     

SIEVE TUBES AND CALLOSE IN ELODEA LEAVES

Detachment and incubation of Elodea leaves promoted callose synthesis in all cells, especially in epidermal pits and in sieve tubes. Phloem was detected in the midrib by fluorescent staining of callose induced to form on sieve plates. In EM views of mature sieve elements nucleus and tonoplast were lacking, mictoplasm replaced cytoplasm, mitochondria were fewer in number, and large plastids contained crystalline inclusion bodies. Slime was present as compact aggregates and as individual fibrils in mictoplasm and sieve pores. Deposition of callose is considered in relation to the blockage concept of callose function.     

溴氰菊酯对鼠脑蛋白质磷酸化作用的影响

以小鼠大脑碎片与[γ-~(32)P]ATP一起保温,观察到溴氰菊酯对蛋白1—3磷酸化的刺激作用和对4、5磷酸化的抑制作用,表明溴氰菊酯对大脑蛋白质磷酸化产生了影响。从鼠脑分离了C、D、S三个组分,分别进行的蛋白质磷酸化试验结果表明,C、D组分可能是重要的磷酸化部位。 蛋白1、2、3的磷酸化明显地受到溴氰菊酯的刺激,这三个蛋白质可能是“蛋白Ⅲb”的几种形式。溴氰菊酯对“蛋白Ⅲb”磷酸化的刺激,可能会影响神经末梢的神经激素释放,从而影响到与其相关的某些神经功能。