Calcium-independent induction of cytocidal activity of polymorphonuclear leukocytes by phorbol myristate acetate-like tumor promoters

Tumor promoters were tested for the ability to induce cytocidal activity of polymorphonuclear leukocytes (PMNs), and the extracellular calcium-dependency of their PMN cytotoxicities were examined in comparison with that by some immunomodulators. Immunomodulators such as linear beta-1, 3-D-glucan (TAK) induced potent cytocidal activity of PMNs. The induction was dependent on extracellular Ca2+. Tumor promoters such as phorbol 12-myristate 13-acetate (PMA) and its derivatives, teleocidin which is structurally unrelated to PMA, and croton oil as an example of mixture also induced potent PMN cytotoxicities. In the latter cases, however, the induction was not dependent on extracellular Ca2+. The ability of these tumor promoters to induce PMN cytotoxicity correlated well with their skin-tumor promoting activities. These results indicate that inductions by PMA-like tumor promoters are distinguishable from those by TAK-like immunomodulators in not being Ca2+-dependent. The application of Ca2+-independent PMN cytotoxicity to detect PMA-like tumor promoters is discussed.     

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.     

cis-Elements involved in expression of unspliced RNA in Moloney murine leukemia virus.

Murine leukemia virus (MLV) produces the unspliced RNA and the singly spliced RNA at a proper ratio at a time. To identify cis-elements involved in the production of the unspliced RNA, we examined the level of unspliced RNA in a series of mutants derived from a prototype Moloney MLV mutant gag-encoding G3.6. Our present data indicated that nt 1560-1906 region in the CA-encoding region in gag was the negative cis-element and nt 5119-5355 region, which was immediately upstream of the splice acceptor site, was the positive cis-element for expression of the unspliced RNA. It was found that the former element made expression of the unspliced RNA dependent upon the latter. These two elements were functional as discrete elements and their activities were relatively position-independent.     

Bacterial expression and characterization of molluscan IDO-like myoglobin

The indoleamine 2,3-dioxygenase (IDO)-like myoglobin (Mb) is a unique type of Mb isolated from the buccal mass of several archgastropod species. Here, we expressed Sulculus diversicolor IDO-like Mb as a GST-fusion protein in bacteria. The visible spectrum of GST-fusion IDO-like Mb shows characteristic α- and β-peaks, indicating that it binds oxygen. To identify residues important in heme and oxygen binding, we constructed site-directed mutants. We initially replaced each of the 7 histidines of S. diversicolor IDO-like Mb with alanine. The spectra of three mutants (H74A, H288A, and H332A) revealed a remarkable loss of absorbance around 414 nm, indicating that they cannot bind heme. His⁷⁴, His²⁸⁸, and His³³² were also replaced by arginine or tyrosine. Neither H332R nor H332Y contains heme, suggesting that His³³² is the proximal ligand of IDO-like Mb. In contrast, both H74R and H288Y mutants were isolated in the heme-binding oxy-form. The autoxidation rates of these two mutants showed that they can bind oxygen as stably as wild-type. His⁷⁴ and His²⁸⁸ might be partially associated with heme-binding, but do not act as the distal ligand. The S. diversicolor IDO-like Mb seems to stably bind oxygen in a different manner from normal myoglobins.     

Structural requirements of flavonoids for inhibition of antigen-Induced degranulation,TNF-alpha and IL-4 production from RBL-2H3 cells

To clarify the structure-activity relationships of flavonoids for antiallergic activity, the inhibitory effects of various flavonoids on the release of beta-hexosaminidase, as a marker of degranulation of RBL-2H3 cells, were examined. Among them, luteolin (IC(50)=3.0 microM), diosmetin (2.1 microM), and fisetin (3.0 microM) were found to show potent inhibitory activity, and the results suggested the following structural requirements of flavonoids: (1) the 2-3 double bond of flavones and flavonols is essential for the activity; (2) the 3- or 7-glycoside moiety reduced the activity; (3) as the hydroxyl groups at the 3'-, 4'-, 5-, 6-, and 7-positions increased in number, the inhibitory activities become stronger; (4) the flavonols with a pyrogallol type moiety (the 3',4',5'-trihydroxyl groups) at the B ring exhibited less activity than those with a phenol type moiety (the 4'-hydroxyl group) or catechol type moiety (the 3',4'-dihydroxyl groups) at the B ring; (5) the activities of flavones were stronger than those of flavonols; and (6) methylation of flavonols at the 3-position reduced the activity. However, (7) several flavones and flavonols with the 4'- and/or 7-methoxyl groups did not obey rules (3), (4), and (5). In addition, several flavonoids, that is apigenin, luteolin, diosmetin, fisetin, and quercetin, inhibited the antigen-IgE-mediated TNF-alpha and IL-4 production from RBL-2H3 cells, both of which participate in the late phase of type I allergic reactions.     

Fusion with maltose-binding protein (MBP) affects neither RNA N-glycosidase activity nor immunogenicity of karasurin-A, a ribosome-inactivating protein from Trichosanthes kirilowii var. japonica

A genomic clone encoding mature karasurin-A (KRNA), a ribosome-inactivating protein from Trichosanthes kirilowii var. japonica, was efficiently expressed in E. coli using an expression cassette vector pMAL-c2. The resultant recombinant KRNA fused with maltose-binding protein (MBP) was recovered from the soluble fraction of the bacterial cells and purified to near homogeneity after one round of the affinity chromatography. Neither the karasurin precursor retaining both N- and C-terminal peptides, nor the protein with the N-terminal peptide was successufully produced even as a MBP-fusion. The protein with its C-terminal peptide was over-produced but was recovered in an insoluble fraction. Both the recombinant MBP-KRNA fusion protein and recombinant KRNA with MBP removed were as active as the native KRNA from root tubers. The immunogenicity of the recombinant KRNA was also unaffected by fusion with MBP.     

Rosmarinic acid formation and differential expression of tyrosine aminotransferase isoforms in Anchusa officinalis cell suspension cultures

Time-course changes in rosmarinic acid (RA) formation and activities of tyrosine aminotransferase (TAT) isoforms were examined in Anchusa officinalis suspension cultures. Three TAT isoforms (TAT-1, TAT-3, TAT-4) were resolved by Mono-Q anion-exchange column chromatography. The proportion of the TAT-3 activity within the total TAT activity remained high regardless of the growth stage of the cultured cells. TAT-1 activity was positively correlated with the rate of RA biosynthesis during linear growth stage of the culture cycle, while TAT-4 activity was rapidly induced in conjunction with transfer to fresh medium coincident with a transient increase in RA synthesis. Based on these results, as well as the substrate specificity of each TAT isoform, it was concluded that both TAT-1 and TAT-4 are closely involved in RA biosynthesis. TAT-1 controls conversion of tyrosine to 4-hydroxyphenyl pyruvate, and TAT-4 acts by participating in the formation of tyrosine and phenylalanine via prephenate.Abbreviations PAL phenylalanine ammonia-lyase - TAT tyrosine aminotransferase - RA rosmarinic acid     

Induction of rosmarinic acid biosynthesis in Lithospermum erythrorhizon cell suspension cultures by yeast extract

Summary A transient increase in rosmarinic acid (RA) content in cultured cells of Lithospermum erythrorhizon was observed after addition of yeast extract (YE) to the suspension cultures, reaching a maximum at 24 hr. The highest increase of the RA content (2.5-fold) was obtained when 6-day-old cells in the exponential growth phase were treated with YE. Preceding the induced RA accumulation, phenylalanine ammonia-lyase (PAL) activity increased rapidly, whereas tyrosine aminotransferase (TAT) activity was largely unaffected by the treatment. The incorporation of both ¹⁴C-phenylalanine and ¹⁴C-tyrosine into RA was enhanced in the YE-treated cells, consistent with increased synthesis of the ester.Abbreviations 2,4-D 2,4 dichlorophenoxyacetic acid - PAL phenylalanine ammonia-lyase - TAT tyrosine aminotransferase - RA rosmarinic acid - YE yeast extract