Induction of competence and progression signals in human T lymphocytes by phorbol esters and calcium ionophores

We have investigated the induction of competence (IL-2 responsiveness) and progression in human T lymphocyte proliferation triggered by phorbol ester and calcium ionophore. The degree of proliferation induced with the phorbol ester, phorbol 12,13-dibutyrate (PDB) and the calcium ionophore ionomycin was dependent on the duration of exposure to these agents, with more than 6 h required for obtaining maximum proliferation. Following brief exposure to both agents for 30 min, which did not cause significant proliferation, T cells became competent to proliferate in response to exogenous interleukin 2 (IL-2). These competent T cells also progressed to DNA synthesis following incubation with PDB in the absence of ionomycin. Induction of competence to proliferate in response to either PDB or IL-2 was blocked by EGTA, suggesting that transmembrane Ca2+ flux was obligatory at this stage. Since other phorbol esters and synthetic diacylglycerols also stimulated DNA synthesis in competent cells, it is likely that progression was triggered by activation of protein kinase C. Following a brief exposure to PDB and ionomycin, subsequent incubation with PDB induced gene expression and secretion of IL-2 and augmented the expression of IL-2 receptors in the competent cells. Thus, we have demonstrated that Ca2+ mobilization is required for rendering T cells competent to express functional IL-2 receptors, to produce IL-2 in response to subsequent incubation with PDB, and that sustained activation of protein kinase C seems necessary for IL-2 production and subsequent progression of competent T cells to DNA synthesis.     

Production of daunorubicin by immobilized growing Streptomyces peucetius cells

Summary Streptomyces peucetius cells were immobilized by entrapment in calcium alginate and a photosensitive synthetic polymer, and used for the production of daunorubicin (daunomycin), which is known to be an antitumour reagent. The use of cultivation media removed insoluble components in a natural medium prevented rapid decrease in daunorubicin titer after maximum production. These entrapped cells could be used at least five times for repeated daunorubicin production; the total cultivation period was 45 days.     

Molecular cloning and sequencing of cDNA encoding goat pre alpha-lactalbumin

In poly(A)+RNA extracted from a lactating goat mammary gland, mRNA of about 750 nucleotides was shown to encode pre alpha-lactalbumin by using in vitro translation and immunoprecipitation. From the total poly(A)+RNA, the cDNA library was constructed using the Escherichia coli plasmid pUC18; it was screened with the oligodeoxyribonucleotide probe corresponding to the amino acid sequence of Trp60-Gln65 of goat alpha-lactalbumin. A plasmid containing almost full-length cDNA of goat pre alpha-lactalbumin, pGLA-1, was identified. The cDNA insert of pGLA-1 comprises 727 base pairs and contains the signal peptide and mature protein sequence.     

Expression of goat alpha-lactalbumin in Escherichia coli and its refolding to biologically active protein

A cDNA encoding the mature region of goat alpha-lactalbumin and the 3'-non-coding region was fused to cDNA of the N-terminal half of porcine adenylate kinase which had been placed under the control of the tac promoter in an expression vector in Escherichia coli. In addition, a methionine codon was inserted between the two cDNAs. When the plasmid carried the full-length 3'-non-coding region, little accumulation of the fused protein was observed. However, the deletion of two-thirds of the 3'-non-coding region produced significant expression of the fused protein in E. coli strain JM105. Since goat alpha-lactalbumin contains no methionine residue, the mature goat alpha-lactalbumin was isolated by CNBr digestion of the fused insoluble protein and refolded using thioredoxin. The homogeneous and biologically active goat alpha-lactalbumin was purified by Ca2+ ion-dependent hydrophobic chromatography.     

Functional and phenotypic analyses of interleukin 2-activated tumor-infiltrating lymphocytes

The tumor-infiltrating lymphocytes (TILs) were cultured with interleukin 2 (IL-2) to induce the activated killer cells possessing autologous tumor-killing activity, and analysed their cell surface phenotypes and assessed anti-tumor killing activity. Furthermore, the activated TILs were transferred into 7 patients adoptively resulting in complete remission in a patient with pancreatic cancer and partial remission in another patient with gastric cancer.The cytotoxic activities of activated TILs at 3 weeks-incubation was 72 ± 15, 42 ± 26, 27 ± 21 and 25 ± 15% against K562, Daudi, KATO-III and autologous tumor, respectively. The negative selection method, indicated that the killer cells recognizing autologous tumor cells consisted of CD4- or CD8-positive T lymphocytes and CD16- or CD56-positive natural killer cells. The activated TILs could not only lyse cultured tumor cell lines, but also autologous tumor cells.     

Purification and characterization of immunoglobulin production stimulating factor derived from human B lymphoblastoid HO-323 cells

An immunoglobulin (Ig) production stimulating factor (IPSF) for hybridomas was found in spent medium of the human B lymphoblastoid cell line, HO-323. The IPSF was purified by serial use of DEAE chromatography, ultrafiltration, gel filtration and HPLC-DEAE chromatography. Purified IPSF was estimated to be a 410 k macro molecule by gel filtration, and contained three types of isomers which were separated from each other by native polyacrylamide gel electrophoresis. All of the isomers were, however, assumed to have the same protein components by SDS polyacrylamide gel electrophoresis.The IPSF was effective for human-human and mouse-mouse hybridomas producing IgM, but not for IgG producers in the experimental condition used here. Human-human hybridoma HF10B4, cultured in IPSF-containing medium, produced 20 times more IgM than in IPSF-free medium under serum-free conditions. The IPSF showed very little proliferation stimulating activity on HF10B4 cells.     

Determination of base composition of DNA by high performance liquid chromatography of its nuclease P1 hydrolysate

The base composition of DNA was directly determined by high performance liquid chromatography (HPLC) of its nuclease P1 hydrolysate. This method can satisfactorily be employed even if the sample of DNA contains RNA or the amount of the sample is very small.