Application of 7-amino-actinomycin D for the fluorescence microscopical analysis of DNA in cells and polytene chromosomes

Summary The cytochemical properties of a guanine-specific synthetic fluorescent analogue of actinomycin D, 7-amino-actinomycin D, have been studied in fixed and living preparations of L cells and polytene chromosomes of salivary glands ofChironomus thummi thummi andDrosophila lummei (Hackman).7-Amino-actinomycin D has been shown to bind to DNA-containing structures, thereby inducing in them a bright red fluorescence. No specific fluorescence has been found in RNA-containing structures treated with this fluorescent probe.The fluorescence pattern of some regions of polytene chromosomes with a known nucleotide composition was analysed. It has been established that 7-amino-actinomycin D induces a very weak fluorescence in GC-poor chromosome regions of theDrosophila lummei toromere structure. Data indicating a nonlinear dependence between the fluorescence intensity of a stained chromosome region and the GC content in its DNA have been obtained. The influence of DNA nucleotide composition in a chromosome region on the fluorescence of 7-amino-actinomycin D is discussed. In combination with quinacrine staining and the Feulgen fluorescence reaction, treatment with 7-amino-actinomycin D provides useful information about the distribution of GC base pairs in the chromosome region under study.     

Comparative study of antibody formation in ontogenetically determined and experimentally shaped populations of cooperating mouse cells

When studying the levels of immune response to the sheep erythrocytes in the adoptive transfer system in a population of cooperating bone marrow and spleen cells of the intact mice formed under the effect of hydrocortisone, differences have been found in their capacity to produce antibody-forming cells. Unlike the population of spleen cells formed during ontogenesis, the population of cooperating bone marrow cells acquires the capacity to develop an intensive enough immune response only after twofold immunization of the recipient and remains insensitive to the priming treatment of the donors with the sheep erythrocytes. These behavioral peculiarities of the population of cooperating bone marrow cells are due to relative deficiency of mature T-B helpers; the primed spleen T-lymphocytes are not involved in the formation of the pool of the latter.     

Hybrid plasmid with bacterial and fungal markers carrying the denV gene of T4 phage and restoring the UV-resistance of E. coli uvrA

The hybrid plasmid pYBP2 with bacterial (ampR), yeast (LEU2) and bacteriophage T4 (denV) genes has been constructed. The plasmid transformed Escherichia coli CSR603 uvrA recA ampS leuA phr- to ampicillin resistance, leucine independence, UV-resistance similar to the one of uvrA+ recA strain. Cell-free extracts of transformed Escherichia coli cells contain low level of ultraviolet-endonuclease activity in contrast to nontransformed cells containing no enzyme.     

Isolation and characteristics of the thermolabile enterotoxin from Escherichia coli strain H74-114

The modified method for isolation and purification of LT-enterotoxin from Escherichia coli strain H74-114 has been proposed. Modification is based on exclusion of concentration of bacterial cell lysates by 3-5 fold dilution and changing PH during chromatography. The proposed modification permitted to increase the yield of purified and active product 8-10 fold. The obtained preparations are homogenic due to PAAG-SDS data and immunochemical analysis. B-component of TL-enterotoxin is shown to consist of 4 subunits B in contrast to B-component of choleragen, consisting of 5 subunits. The mol mass of A component of both toxins is identical but in contrast to choleragen the component of LT-enterotoxin is not subject to proteolysis.     

Vivid molecular divergence over volcanic remnants: the phylogeography of Megadromus guerinii on Banks Peninsula,New Zealand

Megadromus guerinii, an endemic carabid beetle (Carabidae), is the most common carabid throughout its restricted range on Banks Peninsula, a formation of extinct volcanoes in Canterbury, New Zealand. This study characterises the small-scale phylogeographic patterns of M. guerinii across the formerly volcanically active Banks Peninsula using mitochondrial and ribosomal genes. Between the eastern and western areas of the peninsula, the mitochondrial, but not nuclear, DNA has a well-defined geographic distribution. Specifically, mitochondrial cytochrome c oxidase subunit I (CO1) identifies two distinct groups (> 6% divergence between eastern and western beetles) while ribosomal genes show no discernible pattern. Whether such a pattern represents male-biased dispersal, Wolbachia infection, a recent range expansion of a divergent lineage, or a deeper historic separation is explored. There is potential that male-biased dispersal could have occurred. Wolbachia infection was not detected. We conclude that historical processes have likely separated taxa in the eastern and western peninsula.     

Mutation spectrum of the <Emphasis Type="Italic">PAH</Emphasis> gene in phenylketonuria patients in the Karachay-Cherkess Republic (Russia)

A comprehensive population and medical-genetic study was carried out in ten districts and two cities in the Karachay-Cherkess Republic (Russia). As a result, 57 patients with phenylketonuria were revealed. PAH gene genotypes for 40 probands and their diseased and healthy relatives were determined. The mutation spectrum of the PAH gene in the Karachay-Cherkess Republic was investigated. The major mutation in this region is R261X with allelic frequency of 68.4%. We elaborated a convenient system for detection of six PAH gene mutations common in the Karachay-Cherkess Republic, with the total information content of the system being 89.9%. As a result of processing the clinical data, association of the diet and phenylalanine levels in the blood was verified. Genophenotypic analysis confirms the association of the residual activity of phenylalanine hydroxylase and the severity of the disease. It is shown that common mutation R261X is severe and that patients who are homozygous for this mutation have classical phenylketonuria (PKU).     

Therapeutic form of doxycycline hydrochloride for intravenous administration in the treatment of suppurative-septic diseases]

Efficacy of doxycycline hydrochloride administered intravenously was studied in treatment of severe purulent inflammatory diseases such as pneumonia, lung abscesses, pyothorax, skin and soft tissue infections, peritonitis, purulent cholangitis, etc.. Doxycycline showed significant advantages over tetracyclines: prolonged action, higher efficacy and good tolerance. Favourable results were observed in 85 per cent of the cases.     

Membranotropic effects of ω-hydroxypalmitic acid and Ca<Superscript>2+</Superscript> on rat liver mitochondria and lecithin liposomes. Aggregation and membrane permeabilization

The paper examines membranotropic Ca²⁺-dependent effects of ω-hydroxypalmitic acid (HPA), a product of ω-oxidation of fatty acids, on the isolated rat liver mitochondria and artificial membrane systems (liposomes). It was established that in the presence of Ca²⁺, HPA induced aggregation of liver mitochondria, which was accompanied by the release of cytochrome c from the organelles. It was further demonstrated that the addition of Ca²⁺ to HPA-containing liposomes induced their aggregation and/or fusion. Ca²⁺ also caused the release of the fluorescent dye sulforhodamine B from liposomes, indicating their permeabilization. HPA was shown to induce a high-amplitude swelling of Ca²⁺-loaded mitochondria, to decrease their membrane potential, to induce the release of Ca²⁺ from the organelles and to result in the oxidation of the mitochondrial NAD(P)H pool. Those effects of HPA were not blocked by the MPT pore inhibitor CsA, but were suppressed by the mitochondrial calcium uniporter inhibitor ruthenium red. The effects of HPA were also observed when Ca²⁺ was replaced with Sr²⁺ (but not with Ba²⁺ or Mg²⁺). A supposition is made that HPA can induce a Ca²⁺-dependent aggregation of mitochondria, as well as Ca²⁺dependent CsA-insensitive permeabilization of the inner mitochondrial membrane – with the subsequent lysis of the organelles.     

7-Amino-actinomycin D as a specific fluorophore for DNA content analysis by laser flow cytometry

A technique for DNA amount determination by flow cytometry based on the use of 7-amino-actinomycin D (7-amino-AMD), a fluorescent analogue of antibiotic actinomycin has been investigated, and a particular staining procedure has been developed. The procedure includes short fixation in 70% ethanol and staining for 20 min in 10(-5)M solution of 7-amino-AMD at pH7. The results of DNA content measurements are very reproducible. The histograms obtained have a coefficient of variation less than 3%. The absorption maximum of the complex of 7-amino-AMD with DNA is situated in the green spectrum region, making this stain particularly suitable for argon laser flow cytometry.     

Temperature-induced radioresistance of Escherichia coli cells

A marked increase in radioresistance of E. coli cells, of the wild-type repair genotype, was observed when they were exposed to gamma-radiation at 40-45 degrees C. The effect of the thermoinduced radioresistance did not depend on the growth medium and the pre-incubation temperature but disappeared completely after treatment of cells by chloramphenicol or CaCl2 or after modification of cell membranes by exogenous cholesterol. This phenomenon was not observed with UV-irradiation. It is suggested that the thermoinduced radioresistance is connected with the activation of the membrane-associated repair complex.