Roumen Tsanev

Direct spectrophotometric analysis of ribonucleic acid fractionation by agar-gel electrophoresis.

Abstract The fractionation of rat-liver RNA was studied by direct ultraviolet densitometry of dry agar electrophoregrams. Highly reproducible electrophoretic patterns of phenol extracted RNA and residual RNA were obtained. Phenol-extracted RNA was resolved into 3 main and 4–6 minor fractions. The main fractions accounted in most cases for 90 % of the absorbancy area and corresponded to the two components of ribosomal RNA and soluble RNA. Residual RNA extracted with phenol at 65° in the presence of sodium dodecylsulphate was resolved into 11–13 fractions, 3 of them being components of ribosomal RNA and low-molecular-weight RNA. A slow-moving region was also present in which the two slowest components are extracted only in the presence of sodium dodecylsulphate.

Mycoplasma contamination of cell cultures: interference with 32P-labelling pattern of RNA.

Abstract 32P-labelled RNA was isolated from stable cell lines by a SDS -hot phenol procedure and fractionated by electrophoresis on agar gel. Apart from the RNA components typical of mammalian cells, two additional RNA fractions were detected with approximate S values of 16S and 23S. These fractions represented only a very small portion of the total cellular RNA, but had a high incorporation rate and a G+C A+U ratio of about 1. Microbiological, electron microscopical and biochemical studies proved that these fractions originated from mycoplasma contaminants. After treatment of the cultures with Tylan the microbiological tests for detection of mycoplasma became negative but the labelling of the 16S and 23S RNA fractions still persisted. Thus the 32P-labelling combined with fractionation of RNA on agar gel may be used as a very sensitive, although unspecific method for detection of mycoplasma in cell cultures. These studies emphasize the serious danger which the mycoplasma contamination represents when cell cultures are used for studies of RNA metabolism.

Ribonucleic acids fractionation by density-gradient centrifugation and by agar gel electrophoresis: A comparison

Abstract The pattern of RNA fractionation by agar gel electrophoresis is analogous to that obtained by sucrose density-gradient centrifugation. An almost linear relationship between electrophoretic mobility and sedimentation coefficient is established with different RNA preparations. As revealed by agar gel electrophoresis, RNA fractions obtained by singlerun sucrose density-gradient centrifugation are contaminated to a varying extent by the lighter fraction.

Nuclear matrix and transcriptional activity of the mouse α-globin gene

Abstract The association of the mouse α-globin gene with the nuclear matrix was studied when the gene was permanently repressed in Ehrlich ascites cells, potentially active in uninduced Friend cells or actively transcribed in induced Friend cells. Matrix-associated DNA was obtained by two methods, differing in the order of treatment of the nuclei with high salt and DNase I. By using a cloned α-globin probe, no enrichment in α-globin sequences was found in the matrix-associated DNA after DNase I digestion of high-salt treated nuclei from Ehrlich ascites and uninduced Friend cells. In induced Friend cells, a high enrichment (up to 20 times) of α-globin sequences was detected in the DNA left with the nuclear matrix structures. The size of the DNA fragments obtained by this procedure indicated a random attack and did not correspond to a progressive top-to-bottom cleavage model. No enrichment in α-globin sequences was found in induced Friend cells if nuclear matrices were obtained by DNase I digestion of the nuclei before the treatment with high salt. Our data suggest that the enrichment in actively transcribed genes of matrices from nuclei treated with high salt does not reflect a localization of these genes close to the attachment sites of the chromatin loops but rather their artefactual association with some high salt-insoluble proteins of the transcriptional complexes.

Immunofluorescent localization of histone H10 in the nuclei of proliferating and differentiating friend cells

By using affinity-purified antibodies to H10 and to H1AB the localization of these histones was studied by indirect immunofluorescence in the nuclei of proliferating (EAT and uninduced Friend cells) and of differentiating (induced Friend cells) cell populations. While with H1AB antibodies a bright fluorescence all over the chromatin was obtained, the localization of H10 varied depending on the state of the cell population. In the proliferating EAT cells it was localized strictly in the nucleoli. The Friend cell population revealed a heterogeneous picture with two types of H10 localization-nucleolar predominating in uninduced cell populations and peripheral predominating in induced cells. A comparison with literature data suggests that H10 seems to be associated with chromatin regions containing active genes.

Stable DNA-protein complexes in eukaryotic chromatin

Demembranized sperm and somatic nuclei of mammalian origin were extracted with high salt/urea/2-mercaptoethanol, treated with detergents and purified in CsCl density gradients to isolate DNA. Under these conditions a protein component still remained bound to DNA. This stable DNA-protein complex could be reduced to an oligodeoxynucleotide-peptide complex by extensive sequential digestions with DNase I and Pronase E. Chemical and enzymatic treatments of this complex indicated the presence of a phosphoester bond between DNA and a hydroxyamino acid. Two-dimensional tryptic peptide mapping revealed a remarkable similarity among the covalently linked protein components in all types of chromatin studied. These maps differed from the maps of mammalian topoisomerases I and II.

Nucleosome segregation in chromatin replicated in the presence of cycloheximide

Abstract Ehrlich ascites tumour cells and L cells were grown in the presence of [14C]thymidine to label DNA replicated under normal conditions and were then cultured in the presence of cycloheximide and [3H]thymidine to label DNA replicated in the absence of histone synthesis, Chromatin from these cells was digested with micrococcal nuclease and with restriction endonuclease BspRI (an isoschizomer of HaeIII). The rates of digestion of the 14C-labelled and of the 3H-labelled DNA, and the size and buoyant density of the BspRI-generated chromatin fragments showed that: (1) chromatin replicated in the presence of cycloheximide contained half the normal amount of histones; (2) it did not contain long stretches of naked DNA; and (3) it was organized in nucleosomes distributed along DNA in groups of several particles separated by relatively short stretches of histone-free DNA. Control experiments showed that this could not be the result of a long-distance sliding of nucleosomes. These data suggest a bilateral mode of nucleosome segregation during DNA replication.

Distribution of high mobility group and other acid-soluble proteins in fractionated chromatin

Chromatin was fractionated by digestion with deoxyribonuclease II and precipitation with MgCl2. The Mg2+-soluble fraction, known to be enriched in transcribed DNA sequences, was enriched also in high mobility group proteins 1 and 2 and contained almost all other acid-soluble nonhistone proteins.

A comparison of histone variants in different rat tissues.

The histone patterns of rat liver, spleen, kidney and brain were compared by means of a two-dimensional electrophoresis in the presence of Triton X-100. In addition, a two-step procedure was introduced for quantitative estimation of different histone variants. Tissue differences wee found in the relative content of the different variants of H2a, H3 and the subfraction H1O. It was also found that the high salt induced H2a specific proteolytic activity was very high in spleen, very low in liver and kidney and absent in brain.

Immune interferon : properties and clinical applications

FORWARD ABBREVIATIONS THE CYTOKINE FAMILY INTERFERON GAMMA(IFNg) Differences Between IFNg and IFN a/b Interferon-Producing Cells. Inducers and Inhibitors. Effect of Aging Structure and Physico-Chemical Properties of IFNg Interferon Gamma Receptors BIOLOGICAL ACTIVITIES OF INTERFERON GAMMA Antiviral Activity Antiproliferative Activity Immunostimulatory Activity Modulation of Gene Activity Antibacterial Activity Antitumor Activity MOLECULAR MECHANISMS OF IFNg ACTIVITIES. TRANSMEMBRANE SIGNAL TRANSDUCTION PRODUCTION OF INTERFERON GAMMA GAMMA-INTERFERON PREPARATIONS Interferon Gamma-1b Gammaferon Others INDICATIONS AND CONTRAINDICTIONS ROUTES OF ADMINISTRATION, PHARMACOKINETICS, DOSAGE SIDE EFFECTS INFECTIOUS DISEASES Viral Infections Herpes Simplex Type I and Type II Herpes Zoster Herpetic and Adenoviral Eye Infections Respiratory Viral Infections Human Papilloma Viruses (HPVs) HIV Infection Hepatitis Non-Viral Intracellular Pathogens Fungi Non-Viral Extracellular Pathogens NON-INFECTIOUS NON-MALIGNANT DISEASES Fibroproliferative Diseases Atherosclerosis Multiple Sclerosis (MS) Psoriasis Chronic Granulomatous Disease (CGD) Skin Ulcers in Autoimmune Vasculitis Rheumatoid Arthritis (RA) Osteopetrosis Allergic Disorders Lupus Erythematosus (SLE) Heavy Traumas, Burns and Surgical Operations MALIGNANT DISEASES Background for Using IFNg in Malignancies Basocellular Skin Cancer (Basalioma) Cancer of the Larynx Lung Cancer Colorectal Cancer Malignant Melanoma Renal Cell Carcinoma (RCC) Breast Cancer Cervical Cancer Gastric Cancer Prostate Cancer Ovarian Cancer Cancer of the Bladder Hepatocellular Carcinoma (HCC) Sarcomas Kaposis Sarcoma Neuroblastoma Gliomas/Glyoblastomas Hematologic Malignancies Chronic Myelogenic Leukemia (CML) B and T Chronic Lymphatic Leukemias (B-CLL and T-CLL) Acute Leukemias Lymphomas Myelodisplastic Syndrome (MDS) Multiple Myeloma (MM) Conclusions on the Use of IFN-g In Malignant Diseases GENERAL CONCLUSIONS REFERENCES