Anti-prion activity of protein-bound polysaccharide K in prion-infected cells and animals

Protein-bound polysaccharide K (PSK) is a clinical immunotherapeutic agent that exhibits various biological activities, including anti-tumor and anti-microbial effects. In the present study, we report on the anti-prion activity of PSK. It inhibited the formation of protease-resistant abnormal prion protein in prion-infected cells without any apparent alterations in either the normal prion protein turnover or the autophagic function in the cells. Its anti-prion activity was predominantly composed of the high molecular weight component(s) of the protein portion of PSK. A single subcutaneous dose of PSK slightly but significantly prolonged the survival time of peritoneally prion-infected mice, but PSK-treated mice produced neutralizing antibodies against the anti-prion activity of PSK. These findings suggest that PSK is a new anti-prion substance that may be useful in elucidating the mechanism of prion replication, although the structure of the anti-prion component(s) of PSK requires further evaluation.     

The effect of oxycholesterols on thermo-induced membrane dynamics

The effect of temperature change(s) on the dynamics of giant unilamellar vesicles containing oxidized and non-oxidized cholesterol was investigated and characterized. We have demonstrated that (i) major cholesterol auto-oxidation products, 7β-hydroxycholesterol (7β) and 7-ketocholesterol (7keto), rendered vesicles more responsive to temperature changes; (ii) 7keto imparted greater thermo-induced membrane dynamics than 7β; (iii) 7β and 7keto vesicles synergistically were more thermo-responsive than the individual oxysterols; (iv) the thermo-responsiveness of 7keto-containing vesicles was equivalent to that of 25 hydroxycholesterol (25OH)-containing vesicles; and (v) we have characterized the observed membrane dynamics. The results provide a new plausible mechanism: oxidative-stressed membranes in conjunction with temperature change induce membrane dynamics. These findings improve the mechanisms reported previously that attributed the induced dynamics solely to membrane oxidation.     

Cellular expression and localization of DGKζ-interacting NAP1-like proteins in the brain and functional implications under hypoxic stress

Diacylglycerol kinase (DGK) catalyzes conversion of a lipid second messenger diacylglycerol to another messenger molecule phosphatidic acid. Consequently, DGK plays a pivotal role in cellular pathophysiology by regulating the levels of these two messengers. We reported previously that DGKζ translocates from the nucleus to cytoplasm in hippocampal neurons under ischemic/hypoxic stress. In addition, we also identified nucleosome assembly protein 1 (NAP1)-like proteins NAP1L1 and NAP1L4 as novel DGKζ-interacting partners using a proteomic approach and revealed that these NAP1-like proteins induce cytoplasmic translocation of DGKζ in overexpressed cells because NAP1-like proteins associate with the nuclear localization signal of DGKζ and block its nuclear import via importin α. In the present study, we examined whether NAP1-like proteins are expressed in the brain and whether the molecular interaction of DGKζ and NAP1-like proteins would be changed in the brain after hypoxic stress. Immunohistochemistry revealed that NAP1L1 and NAP1L4 are widely expressed in neurons and glial cells in the brain with some differences. After 3 days of transient whole-body hypoxic stress, DGKζ translocated from the nucleus to cytoplasm in hippocampal pyramidal neurons, whereas NAP1-like proteins remained in the cytoplasm. Contrary to our expectations, NAP1-like proteins showed no change in their expression levels. The molecular interaction between DGKζ and NAP1-like proteins was attenuated after hypoxic stress. These results suggest that DGKζ cytoplasmic translocation in neurons under hypoxic stress is regulated by some mechanism which differs from that mediated by NAP1-like proteins.     

Cytoprotective effects of the lipoidic‐liquiform pro‐vitamin C tetra‐isopalmitoyl‐ascorbate (VC‐IP) against ultraviolet‐A ray‐induced injuries in human skin cells together with collagen retention,MMP inhibition and p53 gene repression

Irradiation with ultraviolet‐A (UVA) ray at doses of 20–100 J/cm² diminished the cell viability of human keratinocytes HaCaT and human melanoma cells HMV‐II, both of which were protected by pre‐irradiational administration with the ascorbic acid (Asc) derivative, VC‐IP (2,3,5,6‐O‐tetra‐2′‐hexyldecanoyl‐L‐ascorbic acid; vitamin C‐isopalmityl tetraester), which is the first lipoidic‐liquiform pro‐vitamin C by itself that is materialized by esterization of all four intramolecular hydroxyl groups of an Asc molecule with branched chain fatty groups, resulting in molecular fluidity higher than that of the corresponding straight chains. Irradiation with UVA to HaCaT keratinocytes was shown to cause the formation of 8‐hydroxydeoxyguanosine (8‐OHdG), translocation of phosphatidylserine in the inner layer into the outer layer of cell membrane, and lowering of a mitochondrial membrane potential, all of which were repressed by pre‐irradiational administration with VC‐IP. Expression of p53 gene, another hallmark of UV‐induced DNA damages, was promoted by UVA irradiation to the keratinocytes but also repressed by VC‐IP. Administration with VC‐IP of 10–50 µM to human fibroblasts NHDF achieved the enhancement of collagen synthesis, repression of matrix metalloprotease‐2/9 activity, and increasing of intracellular Asc contents more markedly than that with Asc itself of the same concentrations. Thus UVA‐induced diverse harmful effects could be prevented by VC‐IP, which was suggested to ensue intrinsically from the persistent enrichment of intracellular Asc, through esterolytic conversion of VC‐IP to a free‐form Asc molecule, resulting in relief to UVA‐caused oxidative stress. J. Cell. Biochem. 106: 589–598, 2009. © 2009 Wiley‐Liss, Inc.     

Comparative study on the action of tocopherols and tocotrienols as antioxidant: chemical and physical effects

alpha-Tocopherol is known as the most abundant and active form of vitamin E homologues in vivo, but recently the role of other forms of vitamin E has received renewed attention. The antioxidant properties were compared for alpha-, beta-, gamma- and delta-tocopherols and tocotrienols. The following results were obtained: (1). the corresponding tocopherols and tocotrienols exerted the same reactivities toward radicals and the same antioxidant activities against lipid peroxidation in solution and liposomal membranes; (2). tocopherols gave more significant physical effect than tocotrienols on the increase in rigidity at the membrane interior; (3). tocopherols and tocotrienols showed similar mobilities within the membranes, but tocotrienols were more readily transferred between the membranes and incorporated into the membranes than tocopherols; (4). alpha-tocopherol and alpha-tocotrienol, but not the other forms, reduced Cu(II) to give Cu(I) together with alpha-tocopheryl and alpha-tocotrienyl quinones, respectively and exerted prooxidant effect in the oxidation of methyl linoleate in SDS micelles.     

Seasonal Variations in the Presence of Antibodies against Coxiella burnetii in Dairy Cattle in Hokkaido,Japan

The prevalence and seasonal variations of infection by Coxiella burnetii in cattle were investigated seroepidemiologically on a farm in Hokkaido, Japan, by an immunofluorescent antibody test. A total of 364 serum samples from 28 cows were collected from August 1993 to October 1995 in two barns on the farm. It was found that the number of antibody-positive cows and their antibody titers were significantly elevated in winter and decreased in summer. In addition, antibodies were detectable in seroconverted cows for about five months.     

Transient generation of hydrogen peroxide is responsible for carcinostatic effects of hydrogen combined with platinum nanocolloid,together with increases intracellular ROS,DNA cleavages,and proportion of G2/M-phase

In our previous study, we demonstrated that combined treatment with hydrogen (H₂) and platinum nanocolloid (Pt-nc) exerted markedly antiproliferative effects on cancer cells compared with each treatment alone. However, because the related mechanisms remain unclear, we investigated carcinostatic mechanisms of the combined treatment with H₂ + Pt-nc. Significant suppression of cell proliferation was confirmed at 52?h following combined treatment, and the similar effect was also observed by the 30- or 40-min transient treatment with H₂?+?Pt-nc. The transient treatments led to changes in cell size and morphology, loss of microvilli, and apoptosis-like cell death at 120 h after treatment. Moreover, transient combined treatment with H₂?+?Pt-nc induced cell-cycle arrest, as reflected by decreased proportions of G1-phase cells and accumulation of G2/M-phase cells. In contrast, intracellular peroxide levels were temporarily and significantly increased immediately after H₂?+?Pt-nc treatment but not after treatment with H₂ or Pt-nc alone. Additionally, combined treatment-induced carcinostatic effects were significantly diminished in the presence of catalase, and marked hydrogen peroxide (H₂O₂) generation was confirmed after mixing Pt-nc into cell culture media containing a high concentration of H₂. These changes are in agreement with the results that carcinostatic effects were induced after only 40 min of treatment with H₂?+?Pt-nc. Thus, transient and marked generation of H₂O₂ is responsible for the carcinostatic effects of combined treatment with H₂?+?Pt-nc.     

Javanicinosides D-H, quassinoid glucosides from Picrasma javanica

From Picrasma javanica, five new quassinoid glucosides, javanicinosides D-H, together with known quassinoids, neoquassin and picrasin A and triterpenoids, hispidol A and lanosta-7,24-dien-3-one were isolated. The structures have been determined by spectral analysis and chemical evidence.     

Bi-phenotypic t(9;22)-positive leukemia cell lines from a patient with acute leukemia: NALM-20, established at the onset; and NALM-21, NALM-22 and NALM-23, established after relapse.

Four leukemia cell lines; NALM-20, established at the onset of leukemia and NALM-21, -22 and -23 established at the relapse of the disease were found to be t(9;22)-positive leukemia lines having the biphenotypic feature of B cell and myeloid cell characteristics. In addition, a polyclonal Epstein-Barr virus-transformed normal B cell line, B250, was established from the peripheral blood at the onset of the disease.     

Renaturation, purification, and characterization of human truncated macrophage colony-stimulating factor expressed in Escherichia coli

A human truncated macrophage colony-stimulating factor (M-CSF) encoding the amino acid residues from 3 to 153 of the native M-CSF was expressed by using a two-cistron expression system in Escherichia coli. The truncated M-CSF found in inclusion bodies was renatured and had CSF activity. Purification, which included a QAE-ZeTa preparative cartridge concentration step followed sequentially by HPLC on TSK-gel Phenyl-5PW and TSK-gel DEAE-5PW columns, gave an overall yield of 63.8%. The purified truncated M-CSF had a specific activity of 4 x 10(7) units/mg of protein. Peptide mapping of a lysylendopeptidase digest by reversed-phase HPLC confirmed the amino acid sequence predicted from the cDNA sequence. SDS-PAGE of the purified truncated M-CSF gave a single band at 17 kDa under reducing conditions and at 32 kDa under non-reducing conditions. Activated Thiol-Sepharose 6B column chromatography and other experiments failed to detect any free cysteine residue in spite of the existence of 7 cysteine residues in the truncated M-CSF subunit. These results indicate that it is a dimeric structure linked by one or more intermolecular disulfide bonds.