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.     

Primary structures of human protein kinase C beta I and beta II differ only in their C-terminal sequences

Two types of cDNA clones encoding human protein kinase C (PKC) were isolated from a spleen cDNA library using rabbit protein kinase C beta I/beta II cDNA as a hybridization probe. Nucleotide sequence analyses of these cDNA inserts revealed complete primary structures of two distinct types of human protein kinase C beta I and beta II which differ only in their C-terminal 50 or 52 amino acid residues. It was concluded that there exist four distinct types of PKC, PKC alpha, beta I, beta II and gamma, in human as well as rabbit, and that the corresponding sequences are strictly conserved among mammalian species.     

Immunocytochemical study of the intracellular localization of M protein of vesicular stomatitis virus

Summary The purpose of this paper is to describe the immunocytochemical localization of M protein of vesicular stomatitis virus (VSV) in infected cells. Vero cells, MDBK cells, Swiss 3T3 cells, and BHK cells were examined at various times after infection. For immunofluorescent staining, the cells were fixed with PLP fixative and then treated with 0.05% Triton X-100 before incubation with antibodies. Three hours after infection, M protein exhibited diffuse immunostaining throughout the cytoplasm and later accumulated along the cell membrane. The localization of M protein differed from the granular localization of the nucleocapsid N protein of VSV in the cytoplasm. For electron microscopy, the cells were fixed first in a mixture of 2% paraformaldehyde and 0.05% glutaraldehyde and then with PLP fixative, this being followed by treatment with 0.05% saponin. They were then immunostained using the immunoperoxidase method. The M protein was found to be distributed throughout the cytoplasm and later under the cell membrane, especially at virus budding sites. We also used postembedding immunostaining and freeze-fracture immunostaining to avoid the translocation of M protein caused by the detergent treatment. These techniques confirmed our previous results. Our findings are consistent with the view that the M protein of VSV is synthesized on free ribosomes and is then associated with the cell membrane where viral assembly may occur.S. Ohno was a visiting fellow from the Fogarty International Center at the National Institutes of Health, USA, from September 1981 to August 1983, while some parts of this work were in progress.     

In vitro splicing of a chicken delta-crystallin pre-mRNA in a mammalian nuclear extract

An in vitro splicing system was constructed using portions of chicken delta-crystallin pre-mRNA synthesized in vitro and a HeLa nuclear extract. Analysis of the reaction products revealed that about 25% of the pre-mRNA was precisely spliced at 30 degrees C in 2 h under the standard conditions. The other major products of the reaction detected were a 5'-exon fragment and three RNA species showing unusual electrophoretic mobilities on polyacrylamide gels. Structural analyses showed that these three RNAs contain a branch (lariat) structure as seen in the in vitro splicing reactions of human beta-globin, adenovirus, and yeast pre-mRNAs. In addition, methylation at the N-7 position of the blocking guanosine of the 5'-terminal cap structure of pre-mRNA has been suggested to play an important role in the splicing reaction.     

Cell type-specific expression of the genes for the protein kinase C family: down regulation of mRNAs for PKC alpha and nPKC epsilon upon in vitro differentiation of a mouse neuroblastoma cell line neuro 2a

By the use of cloned cDNAs for protein kinase C isozymes alpha, beta I, beta II, gamma, and those for novel protein kinase C, epsilon and zeta, the expression of the corresponding mRNA species was examined in various mouse tissues, human lymphoid cell lines, and mouse cell lines of neuronal origin. In adult brain, mRNAs for all the isozymes of PKC family are expressed. However, the expression of these mRNA species in brain is low at birth. A similar pattern of expression was also observed for beta I/beta II mRNAs in spleen. These expression patterns are in clear contrast to that for beta I/beta II mRNAs in thymus where the mRNAs are expressed at birth and the levels of expression decrease with age. Human lymphoid cell lines express large amounts of PKC beta mRNAs in addition to PKC alpha. Further, nPKC epsilon mRNA is expressed in some of these cell lines. On the other hand, all the mouse cell lines of neuronal origin tested express nPKC epsilon and zeta in addition to PKC alpha. In a mouse neuroblast cell line, Neuro 2a, down modulation of mRNAs for both PKC alpha and nPKC epsilon was observed in association with in vitro differentiation.     

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.