Enhancement of cytotoxic T-lymphocyte responses in patients with gastrointestinal malignancies following vaccination with CEA peptide–pulsed dendritic cells

Carcinoembryonic antigen (CEA) is strongly expressed in a vast majority of gastrointestinal carcinomas. Recently, epitope peptides of CEA were identified. We have demonstrated HLA-A24–restricted peptide, CEA652[9] (TYACFVSNL), was capable of eliciting specific cytotoxic T lymphocytes (CTLs) which could lyse tumor cells expressing HLA-A24 and CEA. HLA-A24 is the most applicable MHC class I allele in the Japanese population. In this pilot study, we have used the peptide-pulsed dendritic cells (DCs) generated from peripheral blood mononuclear cells (PBMCs) supplemented with GM-CSF and IL-4 as the source of the vaccine. Eight patients with advanced CEA-expressing gastrointestinal malignancies received subcutaneous injections every 2 or 3 weeks. Immunomonitoring was performed by ELISpot (enzyme-linked immunosorbent spot) assay to measure the precursor frequency of CTLs and their capacity to elicit antitumor CTLs in vitro. Four of seven patients have developed their CTL response after vaccinations. DTH reaction was observed in one of eight patients at the DC-injected site. Skin biopsy at the injected site showed the infiltration of the lymphocytes. Furthermore, A24/CEA peptide tetramer assay revealed an increase in peptide-specific T-cell precursor frequency in vaccinated patients. No significant toxic adverse effects were observed, except for mild diarrhea in one case after three vaccinations. Three patients have shown stabilization of the disease after vaccinations. In conclusion, our results clearly demonstrated that our vaccination protocol was safe and might develop a CEA-specific CTL response in cancer patients.     

Effects of a novel gaseous antioxidative system containing a rosemary extract on the oxidation induced by nitrogen dioxide and ultraviolet radiation

Rosemary is commonly used as a spice and a flavoring agent in food processing. Although the antioxidative properties of its extracts have been investigated, there have been few reports on the volatile components of rosemary. We designed a novel antioxidative system which can generate the volatile constituents in the gaseous phase from a rosemary extract and evaluated the gaseous antioxidative activities against both lipid peroxidation and cell death induced by nitrogen dioxide and ultraviolet radiation. The antioxidative effects of the major volatile components on the oxidation of linoleic acid induced by azo compounds were also investigated in a solution. The volatile components in the novel antioxidative system suppressed the Jurkat cell death induced by nitrogen dioxide and the intracellular formation of reactive oxygen species in fibroblast cells induced by ultraviolet radiation. 1,8-Cineole among the volatile components exerted an antioxidative effect against the oxidation of linoleic acid in a solution induced by azo compounds and ultraviolet radiation. These data suggest that the volatile constituents of a rosemary extract had antioxidative properties and that gaseous exposure antioxidant is a promising method for promoting health.     

Detection of lipid peroxidation in vivo: total hydroxyoctadecadienoic acid and 7-hydroxycholesterol as oxidative stress marker

It is important to assess the oxidative injury in vivo accurately and inclusively, as the oxidative stress induced by various oxidants in a random and destructive fashion is considered to play an important role in the pathophysiology of a number of human disorders and diseases. We have developed an improved method for the measurement of lipid peroxidation in vivo, where total hydroxyoctadecadienoic acids (HODE) and 7-hydroxycholesterol (FCOH) were determined by GC/MS analysis from physiological samples after reduction with sodium borohydride and saponification by potassium hydroxide. In this method, both free and ester forms of hydroperoxides and ketones as well as hydroxides of linoleate and cholesterol are measured as HODE and FCOH, respectively. The ratio of stereo-isomers, (E,E)-HODE/(E,Z)-HODE, could be also measured. The plasma concentrations of total HODE were obtained as 76.5, 666 and 2225 nM for human, rat and mouse, respectively. It was found that HODE and FCOH could be measured satisfactorily by the present method from plasma, erythrocyte and urine of humans and experimental animals. It was also found that HODE in urine arose from both free and ester forms, while 8-iso-prostaglandin F2alpha was present primarily as a free acid form. As the concentrations of HODE were much higher than 8-iso-prostaglandin F2alpha, HODE may well be used as a good oxidative marker in vivo.     

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.     

Coumarins and gamma-pyrone derivatives from Prangos pabularia: antibacterial activity and inhibition of cytokine release

The n-hexane and ethyl acetate extracts of the stems and the ethyl acetate extracts of roots from Prangos pabularia afforded an gamma-pyrone derivative and furanocoumarin derivatives with three glucose and gamma-pyrone (pabularin A, B and C), along with 26 previously known compounds (18 coumarins, six terpenoids and two glycosides). Their structures were established on the basis of spectroscopic studies. Of these, 16 coumarin derivatives isolated from P. pabularia were tested for antibacterial activity and inhibition of cytokine release.     

Bcl-2 prevents hypoxia/reoxygenation-induced cell death through suppressed generation of reactive oxygen species and upregulation of Bcl-2 proteins

The function of bcl-2 in preventing cell death is well known, but the mechanisms whereby bcl-2 functions are not well characterized. One mechanism whereby bcl-2 is thought to function is by alleviating the effects of oxidative stress upon the cell. To examine whether Bcl-2 can protect cells against oxidative injury resulting from post-hypoxic reoxygenation (H/R), we subjected rat fibroblasts Rat-1 and their bcl-2 transfectants b5 to hypoxia (5% CO2, 95% N2) followed by reoxygenation (5% CO2, 95% air). The bcl-2 transfectants exhibited the cell viability superior to that of their parent non-transfectants upon treatment with reoxygenation after 24-, 48-, or 72-h hypoxia, but not upon normoxic serum-deprivation or upon serum-supplied hypoxic treatment alone. Thus bcl-2 transfection can prevent cell death of some types, which occurred during H/R but yet not appreciably until termination of hypoxia. The time-sequential events of H/R-induced cell death were shown to be executed via (1) reactive oxygen species (ROS) production at 1-12 h after H/R, (2) activation of caspases-1 and -3, at 1-3 h and 3-6 h after H/R, respectively, and (3) loss of mitochondrial membrane potential (DeltaPsi) at 3-12 h after H/R. These cell death-associated events were prevented entirely except caspase-1 activation by bcl-2 transfection, and were preceded by Bcl-2 upregulation which was executed as early as at 0-1 h after H/R for the bcl-2 transfectants but not their non-transfected counterpart cells. Thus upregulation of Bcl-2 proteins may play a role in prevention of H/R-induced diminishment of cell viability, but may be executed not yet during hypoxia itself and be actually operated as promptly as ready to go immediately after beginning of H/R, resulting in cytoprotection through blockage of either ROS generation, caspase-3 activation, or DeltaPsi decline.     

Role of endo-1,4-beta-glucanases from neisseria sicca SB in synergistic degradation of cellulose acetate

An enzyme hydrolyzing beta-1,4 bonds in cellulose acetate was purified 10.5-fold to electrophoretic homogeneity from a culture supernatant of Neisseria sicca SB, which assimilate cellulose acetate as the sole carbon and energy source. The enzyme was an endo-1,4-beta-glucanase, to judge from the substrate specificity and hydrolysis products of cellooligosaccharides, we named it endo-1,4-beta-glucanase I (EG I). Its molecular mass was 50 kDa, 9 kDa larger than EG II from this strain, and its isoelectric point was 5.0. Results of N-terminal and inner-peptide sequences of both enzymes, and a similarity search, suggested that EG I contained a carbohydrate-binding module at the N-terminus and that EG II lacked this module. The pH and temperature optima of EG I were 5.0-6.0 and 45 degrees C. It hydrolyzed water-soluble cellulose acetate (degree of substitution, 0.88) and carboxymethyl cellulose. The Km and Vmax for these compounds were 0.296% and 1.29 micromol min(-1) mg(-1), and 0.448% and 13.6 micromol min(-1) mg(-1), respectively. Both glucanases and cellulose acetate esterase from this strain degraded water-insoluble cellulose acetate synergistically.     

Ascorbic acid stimulation of production of a highly branched ,beta-1,3-glucan by Aureobasidium pullulans K-1--oxalic acid, a metabolite of ascorbic acid as the stimulating substance

The production of a highly branched beta-1,3-glucan by Aureobasidium pullulans K-1 in Czapek's medium has been found to be stimulated by ascorbic acid. When the culture supernatant, after removal of polysaccharide from the culture filtrate by ethanol precipitation, was concentrated, then added to a new medium and this strain was cultured in the medium, the polysaccharide production was stimulated the same as when L-ascorbic acid was added to the medium. The stimulating substance was partially purified from the supernatant, and was found to be oxalic acid; 0.03% oxalic acid was the most effective concentration for the stimulation of polysaccharide production. The stimulating substance, oxalic acid, was proved to be derived from ascorbic acid added to a medium in an experiment using L-[1-14C]ascorbic acid. We suggest that oxalic acid generated from the metabolism of ascorbic acid in cells of Aureobasidium pullulans K-1 participated in the stimulation of the polysaccharide production by ascorbic acid.     

Presence of two trans-o-hydroxybenzylidenepyruvate hydratase-aldolases in naphthalenesulfonate-assimilating Sphingomonas paucimobilis TA-2: comparison of some properties

Two trans-o-hydroxybenzylidenepyruvate hydratase-aldolases named tHBP HA A and tHBP HA B were purified from a cell-free extract of naphthalenesulfonate-assimilating Sphingomonas paucimobilis (formerly Pseudomonas sp.) TA-2 to an electrophoretically homogeneous state by successive column chromatographies on DEAE-cellulose, DEAE-Toyopearl 650M, Sephacryl S-100, Hydroxyapatite, and Mono Q. These enzymes were similar to each other in molecular mass (ca. 37 kDa on SDS-PAGE, ca. 110 kDa on ultracentrifugation), thermal stability (<50 degrees C) and optimum pH (pH 9.0). However, they differed from each other in N-terminal amino acid sequences, pH stability, K(m) values for trans-o-hydroxybenzylidenepyruvate (tHBP), and inhibition by p-chloromercuribenzoic acid (PCMB). tHBP HA B had a homologous N-terminal amino acid sequence with tHBP HAs from Pseudomonas vesicularis DSM 6383 (strain BN6) and Sphingomonas aromaticivorans F119, and tHBP HA A had a homologous sequence with tHBP HAs of Pseudomonas putida strain OUS82, Pseudomonas sp. strain C18 and NAH7 plasmid. tHBP HA B was inhibited by PCMB, but tHBP HA A was not. Their K(m) values for tHBP were 9 and 3 M, respectively. tHBP HA B was stable in the range of pH 7.1 to pH 10.7, and tHBP HA A was stable in the range of pH 6.0 to 9.3.