PubMed Health⌕ Search

Biomedical subjects

E Severin

Publications and source records attributed to E Severin.

At least 37 records · Page 2Linked to original sources
PubMed1988

[Synchronization of tumor cells with 5-fluorouracil plus uracil and with vinblastine and irradiation of the synchronized cultures. A contribution to combined radio-chemotherapy].

In this article, some arguments are put forward which support the conception of a combined radio-chemotherapy acting by a reversible inhibition of tumor cells with cytostatic drugs in a not cytocidal dose and the following selective killing by irradiation of the cells blocked in a radiosensitive phase. The two cytostatic drugs 5-fluorouracil (FU) and vinblastine (VLB), as inhibitors of DNA synthesis and mitosis, respectively, are tested in vitro both separately and combined in two tumor cell lines of the mouse, i.e. the Ehrlich ascites tumor and the sarcoma S 180. A cell-proliferative and, as far as possible, not cytocidal dose is used because of the inevitable side effects exerted by these drugs on normal tissues. A reversible synchronization of the ascites tumor is achieved even in the young mouse by FU in a dose of 15 ng to 500 ng (applied seven times every two hours), if the synchronization is controlled by applying the antimetabolite together with uracil in an equimolar concentration and then stimulating the growth of the cells inhibited during DNA synthesis by the administration of thymidine. The statistical analysis of dose-effect curves after X-ray irradiation shows an increased radiosensitivity of the synchronized cell population, provided that the optimum moment had been chosen for the irradiation.

Animals↗
PubMed1987

A fluorescent redox dye. Influence of several substrates and electron carriers on the tetrazolium salt-formazan reaction of Ehrlich ascites tumour cells.

This study was performed to elaborate the best conditions for measuring the redox activity of Ehrlich ascites tumour cells by using a new tetrazolium salt, cyantolyl tetrazolium chloride (CTC). This tetrazolium salt forms a fluorescent water-insoluble formazan on reduction on the surface of intact vital cells. The influences of fixation and of various substrates and electron carriers on the cellular reduction of CTC were investigated quantitatively using an elution technique. The amount of formazan obtained after incubating vital cells with Meldola Blue as electron carrier was greater than that obtained with Methylene Blue, menadione, 2,6-dichloroindophenol, 1-methoxyphenazine methosulphate or phenazine methosulphate. Using flow cytometry, the formazan production per cell and, after staining the nuclear DNA, the distribution of the redox activity in the cell population can be visualized with satisfactory resolution. We conclude from our findings that dehydrogenases are only partially involved in the reduction of tetrazolium salts by intact cells and that a redox activity, probably related to a cell membrane-bound NAD(P)H-oxidase system, is mainly measured.

Animals↗
PubMed1985

Genetic characterization and partial sequence determination of a Treponema pallidum operon expressing two immunogenic membrane proteins in Escherichia coli.

A detailed physical and genetic map of a previously cloned 5.5-kilobase segment of Treponema pallidum DNA is described. This segment expressed two proteins that are cell membrane associated in Escherichia coli. The structural genes of these treponemal membrane proteins, tmpA and tmpB, are coordinately expressed, and transcription in E. coli can start from at least two different treponemal promoters. The tmpA and tmpB proteins are the products of in vivo proteolytic cleavage from precursor proteins which are 2 and 4 kilodaltons larger, respectively, than the mature proteins. Because the sizes of the corresponding proteins produced in T. pallidum were identical to those of the mature membrane proteins in E. coli, we concluded that a similar proteolytic processing takes place in both E. coli and T. pallidum. Although tmpA and tmpB were controlled by the same transcription signals, tmpB was expressed to a higher extent than tmpA, and only the tmpB product could be overproduced by placing the left lambda promoter in front of the structural genes. The nucleotide sequence of the T. pallidum tmpA gene was established. This is the first T. pallidum gene sequenced. Codon usage and the nature of transcriptional and translational signals are discussed. The deduced amino acid sequence indicated the presence of a sequence that was characteristic for a signal peptide. This sequence information allowed the construction of hybrid genes coding for proteins having beta-galactosidase enzyme activity as well as TmpA epitopes. The enzyme-linked antigen was expressed at a high level in E. coli when transcriptional and translational signals from coliphage lambda were used. In this case the protein produced was a sandwich protein consisting of 21 amino acids of the lambda cro protein, 204 amino acids of the T. pallidum TmpA protein, and 1,020 amino acids of the E. coli lambda-galactosidase. The potential use of this enzyme-linked antigen for the serodiagnosis of syphilis is discussed.

Amino Acid Sequence↗
PubMed1985

Longterm effect of neonatal whole-body X-ray exposure on ploidy development in mouse liver cells.

Mice of the strains C3H, DBA, NZB, and NMRI were X-irradiated with 0.475 Gy and 2.85 Gy (or 3.80 Gy) at newborn stage or at the age of six months. Their livers were prepared at the age of one to maximum 18 months and the hepatocytes isolated. The DNA content of both whole cells and separated nuclei was measured by flow cytometry and the liver ploidy pattern was determined applying a correcting calculation. Irradiation with 0.475 Gy of newborns of strain C3H results in a relative decrease of octoploid cells and an increase of tetraploid nuclei during lifetime (significance level of 0.001). After irradiation with 3.80 Gy a reversed reaction was observed. However, if the animals were irradiated as adults with a dose of 3.80 Gy the 8c cells decreased significantly three months later. Similarly, newborns of the outbred strain NMRI irradiated with 2.85 Gy, showed an increase of octoploid cells from the third month on, but an oscillating pattern of the 4c nuclear DNA content. In strains DBA and NZB, significantly altered ploidy values could not be found, perhaps due to a too great variance of experimental values.

Age Factors↗
PubMed1984

Flow cytometric analysis of mouse hepatocyte ploidy. I. Preparative and mathematical protocol.

Preparative and mathematical procedures are presented for the investigation of the ploidy pattern of liver cells. The DNA content of enzymatically-isolated liver cells and of nuclei was measured by flow cytometry. The true DNA content could not be measured directly due to super-position of statistical coincidences (demanding "first mode correction") and incomplete separation of the nuclei in binucleate hepatocytes (demanding "second mode correction"). The statistical coincidences (caused by simultaneous measurement of two or more particles or subsequent reaggregation of particles) were corrected by splitting the "unnatural" i.e., aneuploid DNA content, and classifying it with the normal ploidy classes. In addition, the higher normal ploidy classes were reduced by the proportion of the measured coincidences in favour of the lower ones. The second mode correction applied to nuclear distributions only. It is a probability calculation based on counting nuclear pairs on microscope slides, and resulted in a 10% increase of diploid nuclei and a larger standard deviation between the age groups. 8c and 16c values were reduced. The tetraploid values were unchanged.

Animals↗
PubMed1984

Flow cytometric analysis of mouse hepatocyte ploidy. II. The development of polyploidy pattern in four mice strains with different life spans.

The development of liver ploidy in mice aged up to 24 months was investigated by flow cytometry in four mouse strains. A mathematical procedure was applied for correction of flow cytometry histograms. In two of the mouse strains, C3H and DBA, both cellular and nuclear ploidy proceed in the same way. The octoploid cell with two tetraploid nuclei is the most numerous cell type in adulthood. On the other hand, strain NZB and the out-bred strain NMRI show at the corresponding age a higher proportion of diploid cells with strikingly low proportions of 4c cells. In addition, high values of 16c cells and nuclei are present in NMRI. In all strains the proportion of binucleate hepatocytes is in the same range (60%). However, the strains differ in ploidy classes of binucleate cells. Development of liver polyploidization does not depend on life span of the specific strain.

Aging↗
PubMed1984

[Impulse cytofluorometry of the redox activity of single cells using fluorescent formazan].

The first application of a newly developed fluorescent formazan in flow cytometry is described. The cell surface redox activity of isolated mouse hepatocytes after incubation with the tetrazolium salt forming the new fluorescent formazan and the DNA content after Hoechst staining have been measured simultaneously. 2 parametric distribution patterns have been obtained. This new and sensitive fluorometric technique can be used for automatic measurements of single cells correlating redox activity with other cell parameters.

Animals↗
PubMed1983

A new flow chamber and processing electronics for combined laser and mercury arc illumination in an impulse cytophotometer flow cytometer.

A flow cytometer of the impulse cytophotometer (ICP)-type has been developed for two-parameter flow cytometry. It uses two different methods of excitation simultaneously: an argon laser beam focused onto the cell stream within a modified flow chamber, and the light of a high pressure mercury (Hg) lamp using Köhler illumination. The two fluorescence signals are collected sequentially by the same photomultiplier tube. The signals are processed on a cell-by-cell basis using new hardwired analog signal processing electronics enabling their accumulation and display as two-parameter frequency distribution histograms.

Computers↗
PubMed1982

Flow cytometric analysis of chromosomes and cells using a modified BrdU-Hoechst method.

Chromosomes and interphase cells were harvested from cultures of the Chinese hamster line B14 F28 grown in medium containing BrdU up to four cell cycles and stained with the fluorescent dye 33342 Hoechst for flow cytometry. The newly synthetized BrdU-DNA is not stainable by the Hoechst dye which is highly specific for thymidine. The temporal development of the DNA fluorescence after addition of BrdU to the growth medium has been investigated. The chromosomal fluorescence intensity is reduced one step per generation. The extent of the intensity decrease by BrdU incorporation is proportional to the amount of new DNA and it is realized by repeated measurement following an UV-exposure. This UV-illumination stops the quenching by BrdU of the Hoechst stain induced DNA fluorescence. Therefore, the entire DNA content of these chromosomes now becomes measurable. The obtained intensity gain serves as a measure of the extent of the previous BrdU caused intensity shift. In this way we could establish 3 successive mitoses. Principally, this method is suitable also for measurement of whole cells in order to obtain both the number of generations in the experimental period and the phase distribution of the cell cycle.

Animals↗
PubMed1982

UV dose-dependent increase in the Hoechst fluorescence intensity of both normal and BrdU-DNA.

If the DNA nucleoside thymidine is replaced by bromodeoxyuridine, the fluorescence of the nuclei of Hoechst-stained cells is quenched. The decrease of fluorescence intensity determined by flow cytometry and fluorometry is neutralized independent of the degree of BrdU substitution by an UV-exposure with a dose of 5-10 kJ/m2 to the unfiltered spectrum of a 100 W mercury high-pressure lamp. This dose is equivalent to that obtained in fluorescence microscopy after exposure for about 1 s. We suppose that this approximate matching of the intensities both of normal and BrdU in the DNA resulting in no further quenching. However, the fluorescence intensity of normal Hoechst-stained DNA also is increased by a previous exposure to UV light. We explain the time pattern of the Hoechst fluorescence in the course of an exposure with constant dose rate, by the superimposition of the well-known bleaching by an additional increase of the fluorescence intensity. Our results suggest that the UV-exposure of Hoechst dye creates a brightly fluorescing photoproduct which differs spectroscopically from the original dye. This product is stable in the dark and seems to fluorochrome DNA only if it is formed when the Hoechst dye is bound to DNA, thus increasing the nuclear fluorescence. Phosphorescence was not found.

Animals↗
PubMed1980

Role of fatty acid oxidation in mechanism of action of gastric secretagogues.

The role of beta-oxidation in the mechanism of stimulation of acid secretion was examined in toad gastric mucosa in vitro. The incubation with 4-pentenoate selectively inhibited in a dose-dependent manner the rate of 14CO2 formation from [1-14C]octanoate. Pretreatment with 20 mM 4-pentenoate sharply reduced the respiratory and secretory responses to theophylline and histamine. Tracer studies showed a major utilization of exogenous octanoate over glucose and pyruvate by the in vitro toad gastric mucosa. Theophylline and histamine stimulated by 69% the rate of octanoate oxidation. Over 60% of the increments in oxygen uptake produced by theophylline and histamine accounted for the increments in octanoate oxidation, whereas glucose and pyruvate together accounted for less than 25%. Octanoate-dependent respiration was shown to correlate with octanoate oxidation under both inhibition with 4-pentenoate and stimulation with theophylline. Theophylline stimulated by 25% the rate of octanoate oxidation in Cl--free glucuronate-nutrient solutions. The present work provides further evidence for the primary role of fatty acid oxidation in the mechanism of acid secretion in amphibian.

Animals↗
PubMed1979

Intermembrane inclusions induced by anoxia in heart and skeletal muscle mitochondria.

Heart and skeletal muscle from rats of different ages were incubated in vitro in an oxygen-free medium supplied with substrates in order to investigate the effect of anoxia on muscle fine structure, particulary on the mitochondria. In skeletal muscle fibers anoxia has been found to induce changes similar to those previously described in ischemic muscles in vivo namely giant mitochondria, apparently derived by mitochondrial fusion, and intermembrane inclusions with a paracrystalline structure. The plate-like inclusions are mostly located in the intracristal spaces and are closely associated to cristal membranes even in markedly swollen mitochondria. Identical inclusions have been observed in cardiac muscle cells following anoxic injury, whereas they are never found in non-muscle cells such as endothelia, fibroblasts and nerve fibers. Cardiac and skeletal muscle fibers from newborn rats maintained in an oxygen-free medium show mitochondrial swelling but no intermembrane inclusions. The different response of mitochondria from developing vs adult striated muscle to anoxia may be due to changes during postnatal development in the quality or quantity of the protein component(s) involved in paracrystal formation.

Animals↗