Subject(s)
Fatty Acid Desaturases/chemistry , Fatty Acid Desaturases/genetics , Animals , Bacterial Proteins/chemistry , Cell Membrane/metabolism , Cytochromes/metabolism , Fungal Proteins/chemistry , Histidine/chemistry , Humans , Mixed Function Oxygenases/chemistry , Models, Chemical , Models, Molecular , Phylogeny , Plant Proteins/chemistry , Protein Structure, Tertiary , Sphingolipids/metabolismABSTRACT
In the genome of Arabidopsis thaliana, two genes were identified encoding isoenzymes for C4-hydroxylation of long chain bases (LCB) in plant sphingolipids. Both predicted proteins consist of 258 amino acid residues (77% identity) which show sequence similarity to di-iron-binding enzymes, such as Sur2p and Erg3p from yeast, involved in oxygen-dependent lipid modifications. Heterologous expression of these genes in a yeast sur2Delta-null mutant lacking C4-LCB hydroxylation resulted in the formation of D-ribo-C(18)- and -C(20)-phytosphinganine. The identity and stereochemical configuration of the isolated trihydroxybases was confirmed by electrospray ionization-mass spectroscopy, gas-liquid chromatography-mass spectrometry and 1H-nuclear magnetic resonance spectroscopy. These results represent the first functional identification of SUR2 genes from plants as well as from any organism other than yeast.
Subject(s)
Arabidopsis/enzymology , Genes, Plant/physiology , Mixed Function Oxygenases/genetics , Saccharomyces cerevisiae Proteins , Sphingosine/biosynthesis , Arabidopsis/genetics , Base Sequence , DNA, Plant , Fatty Acid Desaturases/genetics , Gas Chromatography-Mass Spectrometry/methods , Molecular Sequence Data , Nuclear Magnetic Resonance, Biomolecular/methods , Saccharomyces cerevisiae/metabolism , Sphingosine/analogs & derivativesABSTRACT
Interleukin 10 (IL-10) is a strong B-cell-activating factor. Since murine Ly1-positive peritoneal or lymphoma B cells strongly express IL-10, we examined malignant cells from patients with acute and chronic leukemias by reverse transcriptase polymerase chain reaction (RT-PCR) for the expression of IL-10 mRNA. High expression was found in three out of four samples from CD10+, CD5- common acute lymphoblastic leukemia (cALL) cells, whereas T-ALL samples generally did not contain IL-10 mRNA. In contrast to the murine system, only low levels of IL-10 expression were seen in 10 out of 11 samples from patients with CD5+ CLL. Myeloid derived cell lines were negative for IL-10. Epstein Barr virus (EBV) positive and negative Burkitt's lymphoma (BL) cell lines showed heterogenous IL-10 expression: BL cell lines with a nonactivated germinal center phenotype (CD10+, CD77+, CD23-, CD30-, CDw70-) expressed little or no IL-10, whereas EBV-positive and negative BL cell lines with an activated phenotype (CD77-, CD23+, CD30+, CDw70+) expressed easily detectable amounts of IL-10 mRNA. The highest expression of IL-10 was found in EBV-immortalized lymphoblastoid cell lines (LCL). Upon superinfection with non-defective EBV, the EBV-negative cell line BL 41 up-regulated IL-10 expression. Thus, in vivo IL-10 expression is not restricted to cells showing a specific (CD5+) phenotype. IL-10 may play an important role in c-ALL. The expression of IL-10 in BL cell lines is correlated to an activated phenotype in vitro and is independent of the EBV carrier status. EBV can induce IL-10 expression.
Subject(s)
B-Lymphocytes/metabolism , Interleukin-10/metabolism , Antigens, CD/analysis , B-Lymphocytes/immunology , Burkitt Lymphoma/metabolism , Burkitt Lymphoma/microbiology , CD5 Antigens , Cell Transformation, Viral , Herpesvirus 4, Human/isolation & purification , Humans , Immunophenotyping , Interleukin-10/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/immunology , Leukemia, Lymphocytic, Chronic, B-Cell/metabolism , Lymphocyte Activation , Lymphoma, Non-Hodgkin/metabolism , Neprilysin/analysis , Polymerase Chain Reaction , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/immunology , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/metabolism , RNA, Messenger/analysis , Tumor Cells, Cultured/immunology , Tumor Cells, Cultured/metabolismABSTRACT
Specific amplification of nucleic acid sequences by PCR has been extensively used for the detection of gene rearrangements and gene expression. Although successful amplification of DNA sequences has been carried out with DNA prepared from formalin-fixed, paraffin-embedded (FFPE) tissues, there are only a few reports regarding RNA analysis in this kind of material. We describe a procedure for RNA extraction from different types of FFPE tissues, involving digestion with proteinase K followed by guanidinium-thiocyanate acid phenol extraction and DNase I digestion. These RNA preparations are suitable for PCR analysis of mRNA and even of intronless genes. Furthermore, the universally expressed porphobilinogen deaminase mRNA proved to be useful as a positive control because of the lack of pseudogenes.
Subject(s)
Polymerase Chain Reaction/methods , RNA/genetics , Base Sequence , Cells, Cultured , Cytological Techniques , DNA/genetics , Formaldehyde , Humans , Molecular Sequence Data , Paraffin , RNA/isolation & purification , Sequence Analysis, RNA/methodsABSTRACT
The bcl-2 oncogene blocks programmed cell death (apoptosis). Epstein-Barr virus (EBV) can immortalize B lymphocytes into continuously growing lymphoblastoid cell lines (LCL) by the coordinate expression of at least 9 latent genes (EBV nuclear antigen [EBNA] 1-6, latent membrane protein [LMP], and terminal proteins [TP] 1 and 2). We analyzed transcription and expression of bcl-2 and latent EBV genes in Burkitt's lymphoma (BL) cell lines with a germinal center phenotype (group I) as well as activated BL cell lines (group III) and LCLs. We found high expression of bcl-2 as well as the full spectrum of latent EBV genes in LCLs and activated group III BL cell lines. Group I BL cells expressed little or no bcl-2, EBNA-2, and LMP. Superinfection with nondefective EBV or an EBNA-2-defective virus as well as transfection with EBNA-2- or LMP-carrying vectors into the EBV-negative cell lines RAMOS, DG75, U698, or BJAB induced upregulation of bcl-2 expression. The strongest effect on bcl-2 was obtained by transfection with LMP, or infection with the nondefective virus. No change of bcl-2 expression was observed with EBNA-1. Our data indicate that the immortalization capacity of EBV and the growth advantage of EBV-positive compared with EBV-negative BL cells in vitro may predominantly be mediated via induction of bcl-2 and the main effectors are EBNA-2 and LMP.
