Biomedical subjects

J Smarda

Publications and source records attributed to J Smarda.

At least 37 records · Page 2Linked to original sources

PubMed1998

Colicins--exocellular lethal proteins of Escherichia coli.

Colicins are toxic exoproteins produced by bacteria of colicinogenic strains of Escherichia coli and some related species of Enterobacteriaceae, during the growth of their cultures. They inhibit sensitive bacteria of the same family. About 35% E. coli strains appearing in human intestinal tract are colicinogenic. Synthesis of colicins is coded by genes located on Col plasmids. Until now more than 34 types of colicins have been described, 21 of them in greater detail, viz. colicins A, B, D, E1-E9, Ia, Ib, JS, K, M, N, U, 5, 10. In general, their interaction with sensitive bacteria includes three steps: (1) binding of the colicin molecule to a specific receptor in the bacterial outer membrane; (2) its translocation through the cell envelope; and (3) its lethal interaction with the specific molecular target in the cell. The classification of colicins is based on differences in the molecular events of these three steps.

Colicins↗

PubMed1998

A possible cross-talk of retinoic acid- and TPA-driven myeloid differentiation pathways.

Retinoic acid (RA) and phorbol esters are important regulators of cellular proliferation and differentiation processes which are capable of arresting growth and inducing differentiation of various leukemic cells. We have studied and compared the effects of these agents on the line of v-myb oncogene-transformed chicken monoblasts BM2. We found that although there are differences in the differentiation kinetics and cell cycle stop points, the differentiation pathways induced by RA and phorbol esters seem to be significantly interconnected.

Animals↗

PubMed1998

[The effect of low-frequency electromagnetic fields on living organisms].

This report studies effect of alternating low-frequency electromagnetic fields (Bm = 5-21.5 mT, f = 50 Hz, duration of exposure t = 0-24 min) on viability of bacteria Escherichia coli. We have shown that the growth of bacteria is impaired the electromagnetic field. Their ability to form colonies on a solid medium decreases in dependence on magnitude of magnetic field and on duration of exposure. The growth curve is influenced by the electromagnetic field as well. Effects of electromagnetic fields are independent of biological age in first four hours of their growth. We have found no morphological changes in bacterial systems in electromagnetic field by optical microscope. Viability of bacteria is bigger in a liquid medium and less in a solid medium. Bacteriophage BF 23 attach less to bacteria influenced by electromagnetic field. And finally, magnetic field did not make induction of production of bacteriophage. This effect indicates, that magnetic field did not damage DNA of exposed bacteria.

Electromagnetic Fields↗

PubMed1995

By-pass of TPA-induced differentiation and cell cycle arrest by the c-Myb DNA-binding domain.

Monoblasts transformed by v-Myb can be induced to differentiate into macrophages by treatment with phorbol ester (TPA). This differentiation occurs during both the G1 and the G2 phases of the cell cycle and is accompanied by cell cycle arrest. The introduction of a protein consisting of the three repeats (3R) of the c-Myb DNA-binding domain permits the by-pass of this phorbol ester-induced differentiation and cell cycle arrest. In particular, monoblasts which express the 3R protein progress through both the G1/S and G2/M transitions in the presence of phorbol ester. However, the 3R protein contains no detectable transcriptional activation domain. These results demonstrate that the c-Myb DNA-binding domain can regulate the cell cycle without functioning as a direct transcriptional activator.

Animals↗

PubMed1995

[From the information on a structural gene to the disease].

Clinical genetics know more than 4000 hereditary diseases: 7.9% of live born children are born with one of them. For more than 80% of them mutations of structural genes are responsible: either of one or most frequently of several genes. The structural gene is a nucleotide sequence in DNA bearing genetic information for a primary structure of the peptide (protein) chain. A mutation of the structural gene most frequently implies a pathological alteration or loss of biological function of the protein coded by it--which leads to the development of symptoms of disease. Impaired DNA and impaired expression of its information may be caused also by aberration of the chromosome where it is located. Chains of causal relationships between the altered information of the structural gene and the clinical picture of the disease can be followed up in both directions. so far the most frequently used pathway is from a known syndrome to an unknown gene. As an example, we may quote the elucidation of the genetic background of Down's syndrome. Symptoms of Down's syndrome--trisomy of a 21 autochromosome or at least of strip 22 of its q arm in its translocation form--ensue from the abnormally enhanced formation of products of genes located there as a result of an increased gene dose; so far five are known.(ABSTRACT TRUNCATED AT 250 WORDS)

Chromosome Aberrations↗

PubMed1995

Retinoic acid receptor alpha suppresses transformation by v-myb.

Retinoic acid (RA) is capable of inducing the differentiation of various myelomonocytic cell lines. During this differentiation process, the levels of c-myb expression decline, suggesting that the RA receptor (RAR) may act in part by down-regulating this proto-oncogene. We have now investigated whether the RAR can also inhibit the function of Myb proteins themselves. We have found that transcriptional activation of a Myb-responsive reporter gene can be inhibited by RA in a human monocytic cell line. This inhibition could not be overcome by the expression of exogenous Myb. The RAR did not interfere with DNA binding by Myb proteins in vitro, suggesting that the functional inhibition occurs at the level of transcriptional activation. To determine the biological relevance of the inhibition of Myb proteins by the RAR, we have used v-myb-transformed monoblasts. These cells differentiate into macrophages in the presence of phorbol ester (tetradecanoyl phorbol acetate [TPA]) but are normally unresponsive to RA treatment. The introduction of an inducible, exogenous RAR alpha into v-myb-transformed monoblasts permitted an RA-dependent differentiation into macrophage-like cells similar to those induced by TPA. These results demonstrate that transformation by v-myb is recessive to RAR alpha and imply that many types of non-RA-responsive leukemia cells may become responsive following the introduction of the RAR.

Animals↗

PubMed1994

c-Myb prevents TPA-induced differentiation and cell death in v-Myb transformed monoblasts.

Myelomonocytic cells transformed by v-Myb or altered forms of c-Myb do not contain the full-length c-Myb protein found in most immature hematopoietic cells. To determine if c-Myb was a dominant inhibitor of v-Myb, we have induced the synthesis of full-length c-Myb in monoblasts transformed by v-Myb. We found that although some morphological changes occurred, the presence of both c-Myb and v-Myb was compatible with cell growth. However, the response to phorbol ester (TPA) was significantly altered by c-Myb. Monoblasts transformed by v-Myb can be induced to differentiate into macrophages by treatment with TPA. This process is accompanied by a significant amount of cell death. However, when c-Myb was made TPA-inducible in these cells, TPA-induced differentiation into macrophages was blocked and cell death was prevented. These results demonstrate a significant difference in the biological effects of v-Myb and c-Myb in transformed myelomonocytic cells.

Animals↗

PubMed1993

Dicistronic selection for nuclear proteins in living animal cells.

We present an expression/selection system designed for the purification of cell lines inducibly expressing genes coding for unselectable proteins by using dicistronic selection for the cell surface marker CD4. This system enabled us to establish and purify c-myb expressing variants of the v-myb transformed chicken monoblast cell line BM2 with high efficiency.

Animals↗

PubMed1991

Isolation of DNA-dependent RNA polymerase from Streptomyces granaticolor and its binding to phage phi 29 DNA.

Partially purified DNA-dependent RNA polymerase of Streptomyces granaticolor was further separated on phosphocellulose in 50% glycerol and a single activity peak was obtained. The enzyme isolated in this way consisted of 4 main proteins with molar mass of 145, 132, 50 and 46 kg/mol. These four subunits represented 93% proteins of the active fraction. To test the ability of RNA polymerase to recognize specific sites on DNA, binding sites for RNA polymerase on phage phi 29 DNA were mapped by electron microscopy. The specific binding sites detected were compared with those for RNA polymerases from Escherichia coli and Bacillus subtilis.

Bacteriophages↗

PubMed1990

Colicins E7 and E8 degrade DNA in sensitive bacteria.

The primary target of colicin E7 in sensitive bacteria are their DNA molecules. In agarose gel electrophoresis of lysates of cells treated with colicin E7, both chromosomal and plasmid DNA bands disappear, in direct relation to E7 concentration and to the duration of treatment. DNA degradation is followed by a cessation of DNA synthesis. In E7-immune bacteria, no damage to DNA due to colicin E7 occurs. The mode of action of colicin E7 thus appears to be equal to that of colicin E2. Also, colicin E8 causes a distinct damage to chromosomal and plasmid DNA in sensitive, but not in immune bacteria. None of the colicins E1, E3, E4, E5, E6 or E9 has any influence on bacterial DNA.

Chromosomes, Bacterial↗

PubMed1990

Sensitivity of Shigella flexneri and Escherichia coli bacteria to bacteriophages and to colicins, lost or established by the acquisition of R plasmids.

Lac+ recombinant of Shigella flexneri 2a carrying R-factors of the standard set have been constructed. The R+ transconjugants obtained have been compared with the original strain with respect to their susceptibility to a set of bacteriophages recommended for phagetyping of Shigella strains and to a set of colicins. It have been found that as a result of acquiring some R-factors the susceptibility to bacteriophages changes; presence of certain R-factors causes susceptibility, that of other R-factors--resistance. Similar changes have been observed in the case of susceptibility to colicins. Propable mechanisms of the changes of susceptibility to these factors are discussed.

Bacteriophages↗

PubMed1988

The effects of some protein-modifying reagents on the interaction of colicins A, E2, E3, and K with their respective Escherichia coli cell receptors.

Colicins attach themselves--through specific protein-protein interactions--onto receptors in the outer membrane of sensitive bacterial cells. An attempt was made to analyze amino-acid groups and attractive forces involved in this interaction, following treatment of either colicins A, E2, E3 and K or sensitive bacteria with various physico-chemical factors and several protein-modifying reagents. The amounts of colicin bound were checked by a quantitative biological assay. Ionic conditions and specific spatial conformation of both colicin and its receptor molecules are crucial in their interaction and, hence, in the biological effect of colicin. Formaldehyde, naphthalene-diisocyanate and osmium tetroxide strongly inhibit the binding ability of all colicins tested. The results suggest that NH2 and SH groups are involved in their binding onto receptors; also, CH3S groups seem to be engaged in the attachment of colicins E2 and E3 and phenol-OH groups in that of colicin K. The possible involvement of further groups (NH, SH etc.) should be checked using more specific reagents. The attitude of colicins E2 and E3 to their common receptor Btu B protein is nearly, but not completely the same. Receptors for all colicins tested should be oxidized to achieve optimal interactions; obviously, carbonyl groups are produced and newly formed anions increase the negative load of bacterial surface. In agreement, reduction of at least colicins A and E3 enhances their receptor binding reactivity. The binding capacity of each receptor can be modulated by a set of amino acid reagents in a specific manner.

Ammonia↗

PubMed1988

Mutation analysis of the receptor for colicins E1-E7. A pilot study.

Thirty eight mutant clones of the colicin indicator strain Escherichia coli K 12 ROW, selected by their insensitivity to any of the colicins E1-E7, were isolated. Comparison of their sensitivity-resistance patterns to colicins E1-E7 enabled us to draw a rough preliminary map of the receptor for E colicins. In this receptor, the highly specific binding site for colicin E1 partially overlaps with the domain shared by all colicins E2 through E7. A specific binding site of this domain appears to be common for colicins E3 and E6; a part of the E3 and E6 binding site is also common for colicins E4 and E5 and a small, least specific, part also for colicins E2 and E7. Using colicin assay experiments, the binding capacity of colicin E receptor mutants could be estimated. A decreased, but not completely lost ability of certain mutants to bind colicins E, correlated to their lowered sensitivity to them, was found. Thus the phenomenon of partial colicin resistance was established, showing that colicin sensitivity--resistance is not a qualitative but a quantitative marker.

Colicins↗

PubMed1988

Modes of action of colicins E4-E7: rates of basic biosyntheses inhibition.

The progress of the inhibition of prominent biopolymer syntheses (namely of DNA, RNA and protein synthesis) in cultures of susceptible bacteria Escherichia coli treated with colicins E4, E5, E6 or E7 was followed. The method used was the measurement of incorporation of specific radioactive precursors into an instantly precipitable fraction. All the three syntheses being inhibited successively, progressively and with different urgency during unfolding of the lethal effect of each colicin, emphasis was put on a comparative mathematical analysis of the development of the biosyntheses inhibition rates. In bacteria treated with colicins E4, E5 or E6, protein synthesis was blocked preferentially and most heavily, followed by cessation of DNA synthesis and finally also by a rather slowly proceeding impairment of RNA synthesis. (In E6-treated cells, damage to DNA synthesis started prior to that to protein synthesis.) Thus, the effect of colicins E4, E5 and E6 follows the general pattern of colicin E3 action. Bacteria treated with colicin E7 developed an immediate block of DNA synthesis, soon followed by protein and (again after a significant delay) by RNA synthesis switch-off. Thus the action of colicin E7 is closely related to that of colicin E2. The presumptive direct targets of these colicins remain to be elucidated.

Bacterial Proteins↗

PubMed1988

Colicin K decreases the density of intramembrane particles (IMP) in the cell membrane of Escherichia coli.

Toxic effects of both main colicin types, i.e. of porin and nuclease types, involve the direct contact of their molecules with the plasma membrane of sensitive cells. In the present study, it was tested whether this contact provokes a lateral or vertical movement of intramembrane protein particles (IMP) or a direct cleavage of the proteins. IMP were visualized by freeze-fracturing and electron microscopy on the protoplasmic fracture face (PF) of colicin-treated cells of Escherichia coli. Possible changes in distribution and in density of IMP due to treatment with colicins E1-E7 and K were followed. As a control, the bacteria were equilibrated at 0 degrees C before quenching, which caused a reversible formation of smooth areas and a decrease in the mean density of IMP on the PF. Colicins E1-E7 had no clear-cut effect on the disposition of IMP. Only colicin K decreased the IMP density, by 10% in E. coli strain 58-161 and by 17% in strain C6; the distribution of IMP remained homogeneous. Trypsin reactivation of colicin-K-inactivated bacteria was not reflected by restoration of the original density of IMP; on the contrary, it led to a further decrease, of 1-13%, in IMP density, presumably by proteolytic cleavage. Varying densities of IMP in different strains of the same bacterial species (under standard conditions) were confirmed.

Cell Membrane↗

PubMed1987

Cytotoxic effects of colicins E1-E5 and K on hamster fibroblasts.

As a further model of mammalian tissue cells, Chinese hamster fibroblasts of the stable line V79 were used to check the cytotoxic effects of colicins. The efficiency of plating of cells treated with colicins E1-E5 and with colicin K was followed. The V79 cells were, in general, poorly sensitive to colicins; only colicins E1, E3 and E5 lowered the number of colony-forming cells to some degree. Again, a different action of colicins E1 and K (which is the same in bacteria) was found in eukaryotic cells.

Animals↗

PubMed1987

Tolerated variations in a genome: the case of closely related Bacillus phages PZA, phi 29 and phi 15--a review.

Restriction-site analysis was used to estimate the relationship of bacteriophages PZA, phi 29 and phi 15. Complete nucleotide sequences of PZA and luminal diameter 29 genomes were compared and tolerated variations were assessed. Most of the base-pair changes are silent nucleotide substitutions in the third position of codons but amino acid changing substitutions are also observed. The terminal portions of the phage genomes diverged faster than their central parts. Gene mutations in phage PZA were induced by hydroxylamine and their frequency was compared with the evolutionary mutability.

Bacillus↗

PubMed1987

Colicin Js of Shigella sonnei: classification of type colicin "7".

Colicinotype 7 of Shigella sonnei, introduced by Abbott and Graham, is one of those most frequently found in epidemiological screenings. It is represented by the production of a single colicin (acidic protein of m. w. 46 kD) endowed with some striking features. It is unstable at pH 8.0 and at the temperature of 70 degrees C. Its inhibitive activity is specific for the species Shigella sonnei, most strains of which seem to be sensitive. It is the only colicin known so far inactive towards Escherichia coli, including the indicator strains of broad-spectrum sensitivity, such as K12 Row. It shows no cross-resistance with any other colicin type. Its adsorption on sensitive bacteria and inhibitive effect on them proceed extremely quickly. As indicated by indirect fluorimetry, its mode of action is extremely quickly. As indicated by indirect fluorimetry, its mode of action is probably different from those known for other colicins: it does not concern the plasma membrane of sensitive bacteria under aerobiosis, but it interferes with it under anaerobic conditions. These data show clearly that it is a unique colicin type. It is proposed to include it into the current system of classified colicins as "colicin Js".

Bacterial Proteins↗