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At least 19 recordsLinked to original sources
PubMed1975

The effect of bleomycin on rapidly proliferating epidermis. A comparative investigation using micro-flow fluorometry, H3Tdr incorporation and a stathmokinetic method (colcemid).

At different time intervals after injection of Bleomycin (BLM) th effect on several kinetic parameters of the hairless mouse epidermis stimulated to proliferate by previous adhesive tape stripping was measured. Micro-flow fluorometry was used to determine the relative number of cells in the various phases of the cell cycle (G1, S and G2). Tritiated thymidine was used to determine labelling indices and grain counts. Colcemid was used to observe the mitotic rate. An initial decrease followed by a subsequent significant increase compared to the non-BLM-treated controls was observed in all parameters studied except the mitotic rate, which remained lower than in the control animals during all 48 hours. The transit time of the cells through the S-phase was initially slightly prolonged, but thereafter it seemed to be shorter than that of the controls. BLM seems to provoke a partial blocking of cells in the G1 phase. When the block is released, a greater number of cells pass through the S phase in partial synchrony at a higher than normal speed. BLM induced a low mitotic rate which remained below the level of that of the normal animals after stripping, even though there obviously was a considerably higher influx of cells from the S phase to the G2 phase. This resulted in a subsequent accumulation of cells in the G2-phase. Thus, BLM has also a blocking effect on the G2-M boundary of the cell cycle. This inhibitory effect of BLM on the mitotic rate was shown to be independent of the effect of BLM on the DNA synthesis. BLM therefore seems to have complex influence on epidermal cell kinetics in vivo. Cells in G1-phase are partially and transiently blocked, but this block is soon released. These cells thereafter pass through the S-phase and pile up in the G2-phase, because BLM also blocks the passage of cells from the G2-phase to mitosis. The overall reduction in cell proliferation seen after BLM in vivo seems mainly to be due to the effect on the G2-M boundray of the cell cycle.

Animals↗
PubMed1977

Fluorometric detection of deoxyribonucleic acid synthesis; possibilities for interfacing bromodeoxyuridine dye techniques with flow fluorometry.

Fluorometric detection of the biosynthetic incorporation of 5-bromodeoxyuridine (BrdU) into deoxyribonucleic acid has permitted cytologic studies of chromosome structure, replication, and repair. Some of these phenomena, previously detected using BrdU-dye techniques on fixed microscopic preparations, should be particularly amenable to analogous experimentation in fluorescence flow systems. Problems involved in interfacing BrdU-dye methodology with flow fluorometry are discussed. The effects of certain chemical modifications of bisbenzimidazole dyes on their spectroscopic properties and potential use for detecting BrdU incorporation into unfixed cells are described. Data on the use and energy transfer characteristics of a pair of deoxyribonucleic acid-binding dyes (33258 Hoechst and ethidium bromide) capable of simultaneously providing information about BrdU substitution and total deoxyribonucleic acid content are presented.

Bisbenzimidazole↗
PubMed2024

Comparative Studies on Bulky DNA Damage Binding by Nucleotide Excision Repair Proteins Using Surface Plasmon Resonance, Differential Scanning Fluorometry, and DNase I Footprinting.

Nucleotide excision repair is a crucial cellular mechanism that ensures genomic stability, thereby preventing mutations that can lead to cancer. The human XPC and its yeast ortholog Rad4 protein complexes are central to this process and were the focus of the study. We used surface plasmon resonance and differential scanning fluorimetry to study the binding characteristics of XPC and Rad4 when bound to the bulky cluster di-FAAF-containing 55-mer duplex DNA. Our findings revealed that XPC binds 10 times more significant affinity to control and di-FAAF-modified DNA than Rad4 with greater protein-DNA interactions. Differential scanning fluorimetry indicates that Rad4 causes comparatively more significant conformational changes upon complexation with the damaged DNA. We conducted DNase I footprinting of the Rad4/DNA complex for the first time by determining the regions protected from DNase I digestion. The DNA at the lesion is entirely resistant to digestion by DNase I in the absence of Rad4 several nucleotides to the 3'-side of the first FAAF lesion. The lack of DNase I cleavage at the lesions did not change upon adding Rad4. However, in the presence of Rad4, a footprint is observed on the 7-nucleotide region (5'-TGGTGAT-3') of the complementary strand to the 3' side of the lesion.

Surface Plasmon Resonance↗
PubMed1975

Fluorometry of citrate in serum, with use of citrate (pro-3S)-lyase.

We describe a procedure for enzymatic assay of citrate in human serum. The citrate is degraded to acetate and oxaloacetate with citrate oxaloacetate-lyase (pro-3S-CH2-COO- yields acetate) (EC 4.1.3.6). Some oxaloacetate loses CO2 to form pyruvate. Addition of malate and lactate dehydrogenases (EC 1.1.1.37 and 1.1.1.27) permits determination of the oxaloacetate and pyruvate generated, and thus of the citrate concentration. The decrease in NADH concentration is measured fluorometrically. Results obtained for 30 consecutive human sera by this procedure were compared to the procedure in which the citrate is converted to pentabromoacetone. There was no statistically significant difference in values obtained by the two procedures. The range of values (mean plus or minus 2 SD) found for sera from 25 blood donors by this procedure was 12.8-27.2 mg/liter (mean, 19.0 mg/liter). Serum citrate as measured by both procedures during a glucose tolerance test was decreased from initial values under the influence of administered glucose (and endogenous insulin). Insulin concentrations were also measured during these glucose-tolerance tests. Citrate concentrations remain subnormal after the glucose and insulin concentrations return to their initial values. This accords with published reports.

Acetone↗
PubMed1975

Microscope fluorometric investigations on the reticulocytic maturation distribution as diagnostic criterion of disordered erythropoiesis.

A microscope fluorometric technique is described which permits not only the visual identification of reticulocytes under the fluorescence microscope but also the determination of their relative stage of maturation to normocytes. The technique is based on a specific staining procedure which results in a fluorescent complex between the reticulocytic RNA and acridine orange. Thus, the relative mass of RNA in the individual reticulocytes can be measured by means of mciroscope fluorometry. As the reticulocytic RNA content decreases and finally disappears during the final maturation process of reticulocytes after their release into the peripheral blood stream, the fluorescence signal indicates the relative degree of this maturation. A characteristic frequency distribution of this parameter can be obtained for a given blood sample by microscope fluorometry measuring 200 to 300 reticulocytes. The preliminary use of this technique for following up the course of two cases of hemolytic anemia and one of pernicious megaloblastic anemia during their treatment demonstrates the potential diagnostic value of this technique of identifying the change of the reticulocyte maturation distribution in addition to the reticulocyte count. Satisfactory agreement between the microscope fluorometric results and those obtained by counting separately the four reticulocytic maturation stages according to Heilmeyer and Wesbäuser has been achieved. The possibility of obtaining quantitative and comparable results by use of this method may be considered a general advantage and a promising basis for the development of an automated technique.

Acridines↗
PubMed1978

[Computerized scanning analyses of FITC-labelled microscopic slides (author's transl)].

FITC-labelled virus antigen was analysed in infected cells by means of scanning-fluorometry. According to the predetermined classification the values were computerized and printed out by a mosaic printer or by a coordinate-recorder as a profile graph or a perspective image. The total impression of the 3 dimensional print-out of the quantificated topographic distribution of antigen accumulations within the cells can be optimated by variation of the angle of view.

Animals↗
PubMed1979

Interference of intravenous lipid emulsion with the determination of calcium in serum.

A lipid emulsion in concentrations similar to those attained after therapeutic dosages were found to interfere with the determination of total serum calcium measured by a fluorometric method. Calcium results determined by atomic absorption and by fluorometry at different concentrations of lipid emulsion were compared. When lipid concentrations were less than 100 mg/dl, centrifugation was effective in eliminating the artifactural elevation in total serum calcium. Possible reasons for the observed differences are discussed.

Calcium↗
PubMedDate not supplied

Computer-operated scanning analysis of FITC-labelled measles virus in cell cultures.

FITC-labelled measles virus antigen in infected cells was analysed by computer-operated scanning fluorometry, printed out by a mosaic printer according to a predetermined classification, and by a coordinate-recorder as a perspective image. The three-dimensional print-out of the topographic distribution of antigen accumulations in the cells can be optimized by a variation of the angle of view.

Animals↗
PubMedDate not supplied

Kinetics of drug decomposition. Part 45. Logk--pH profile for rolitetracycline degradation.

UV spectrophotometry and fluorometry were applied for an examination of rolitetracycline (RT) degradation at pH's ranged from ca 0-8 to 13-0. It was demonstrated that these methods enable to follow the rate of degradation of tetracycline formed in the course of RT hydrolysis. However, they do not allow to monitor the hydrolysis of RT to tetracycline. Application of the so-called "subtraction technique" permits to calculate the rate constants for the total, reversible epimerization reaction and for the subsequent degradation of the resultant products. A rate equation derived for RT degradation in an alkaline medium beginning at pH 10-5 contains a term second-order in the hydroxide ion. Such a relationship was not yet observed in the course of an antibiotic degradation.

Catalysis↗
PubMed1976

Chromosomal studies of human prostatic tumors in vitro.

Chromosomal studies were performed on primary cultures from human prostatic adenomas and carcinomas. All cells from both tumor types showed diploid chromosomal counts with a normal human karyotype. Chromosomal analysis was also performed on cells of the permanent epithelial line EB 33 derived from a human prostatic carcinoma in this laboratory. In contrast to the primary cultures, hypotriploid chromosome numbers were found during continuous culture for more than 1 year. The range of chromosome numbers increased with time in culture and was reduced to the original model number of 64 by only one animal passage ("nude" mice). Feulgen fluorometry on different passages of the permanent cell line EB 33 showed tetraploidy. This discrepancy to the hypotriploid chromosome number can be explained by different Feulgen hydrolytic properties of the different cell types.

Animals↗
PubMed2026

DNA Extraction Optimisation for Minute Land Snails of Vertigo Müller, 1773 (Gastropoda: Vertiginidae): A Comparative Evaluation of Six Methods, Including a Non-Destructive Shell-Preserving Protocol.

No systematic comparison of DNA extraction strategies exists for minute Vertiginidae (shell height <&#x2009;3&#x2009;mm), a group posing a dual analytical challenge: extremely low tissue input and co-purified PCR-inhibitory mucus. For legally protected species, an additional requirement to preserve the shell voucher further constrains available protocols. Using Vertigo antivertigo as the model species, we compared six approaches applied to specimens preserved in 96% ethanol (n&#x2009;=&#x2009;10 per method): two HotSHOT alkaline-lysis protocols (destructive and non-destructive shell-preserving variants), a modified CTAB protocol supplemented with PVP-40 and DTT, and three commercial silica-column kits (GeneJET Genomic, DNeasy Blood & Tissue, QIAamp DNA Micro). DNA yields were quantified by QuantiFluor fluorometry, and PCR performance was subsequently assessed across four loci (COI barcode, COI mini-barcode, ITS1, ITS2). DNeasy Blood & Tissue produced the highest fluorometric concentrations; QIAamp DNA Micro and CTAB&#x2009;+&#x2009;PVP-40 gave intermediate values. The shell-preserving HotSHOT variant yielded lower concentrations but improved A260/230 ratios. BSA and trehalose supplementation increased PCR success in inhibition-prone HotSHOT extracts from 70% to 100%. ITS1 Sanger sequencing of three Vertigo species listed in Annex II of the EU Habitats Directive, all extracted with the shell-preserving protocol, confirmed species-level identification (99.8%-100% BLASTn identity; mean Phred Q&#x2009;>&#x2009;51). The shell-preserving non-destructive HotSHOT protocol yields sequenceable DNA from protected Vertiginidae while retaining the morphological voucher, making it the preferred option for conservation-genetic monitoring. The practical decision framework documented here-integrating voucher preservation, amplification robustness and per-sample cost-has broad applicability to other minute terrestrial gastropods processed in large-scale biodiversity surveys.

Habitats Directive↗
PubMed1975

Molecular basis of chromosome banding. II. The effect of silver and mercury ions on the fluorescence intensity of acranil-DNA complexes.

Silver and mercury ions are known to react with the bases of nucleic acids in solution. At low cation/base ratios Ag+ has an affinity for GC pairs in DNA, whereas Hg++ is preferentially bound to AT-rich nucleic acids. We have used fluorometry to measure the effect of these cations on the fluorescence intensity of preformed complexes of acranil and DNA in solution. The results are: 1) Ag+ enhances the fluorescence intensity presumably by affecting the dye intercalated in the vicinity of GC-pairs. 2) The addition of Hg++ leads to a quenching of the fluorescence intensity of the complex at low ion/base ratios, suggesting an effect on the dye molecules bound to AT pairs. At high GC-content of the nucleic acid, slight enhancement of the fluorescence intensity occurs with Hg++. 3) With both metals there is a correlation between base content of DNA and effect on the intensity of fluorescence indicating base specificity of the dye-polymer interaction.

Acridines↗
PubMed1978

Regenerative proliferation of mouse epidermal cells following application of a carcinogenic skin irritant (MCA). Micro-flow fluorometric DNA measurements and 3H-TdR incorporation studies of isolated basal cells.

0.025 ml of a 1% solution of the complete skin carcinogen 20-methylcholanthrene (MCA) dissolved in benzene was applied to the back skin of hairless mice. At different time intervals up to 3 days after the carcinogen application groups of animals were injected i.p. with 30 muCi 3H-TdR 30 min before they were killed. Single cell suspensions of epidermal basal cells were prepared by a combined enzymatic and mechanical separation method, and the DNA frequency distribution pattern from each cell suspension was measured by means of micro-flow fluorometry. Smears for autoradiography were made from each cell suspension and the labeling index and mean grain count assessed. After a short initial delay, MCA induced an increase in the labeling index similar to that observed after non-specific cell injury and cell loss. Thereafter, the cells were considerably delayed in their progression through the S phase, with a low exit from S resulting in a transient emptying of the G2 compartment, without indications of any significant delay of the passage through G2 phase. The cells that had been injured by the MCA application in or just before S phase proceeded into the G2 phase and mitosis more than 24 h after the initiation of DNA synthesis. The cell kinetic reaction of epidermis to a single application of MCA is thus very different from that caused by a nonspecific cell damage, e.g. application of the vesicant agent cantharidin or removal of surface cells by cellophane tape stripping.

Animals↗
PubMed1976

Studies on the binding of FMN by apoflavodoxin from Peptostreptococcus elsdenii, pH and NaCl concentration dependence.

1. The pH and ionic strength dependence of the interaction of FMN with apoflavodoxin has been studied by fluorometry in the pH region 2-5, at 22 degrees C. 2. The rate constant of dissociation and the dissociation constant were experimentally determined; the rate constants of association were claculated at a given pH value. These constants depend on the ionic strength. The plots of these constants against the square root of the ionic strength are straight. 3. Our data have been interpreted in terms of the Brönsted theory, which relates chemical reaction rates to ionic strength. The data indicate that the apoenzyme reaches its maximum net positive charge at pH 2.0-2.6. The calculated net charge in this pH region is between 11 and 12 and is in agreement with the theoretical value of 12 as deduced from the primary structure of the protein. The isoelectric point of the holoenzyme is about 4. 4. The rate constant of association extrapolated to zero ionic strength is 3.2-10(5)M-1-s-1 and is pH-independent. 5. The rate constant of dissociation and the dissociation constant extrapolated to zero ionic strength depend on the pH. The results are explained by assuming that there are two protein ionizations with a pK value of 3.4; these ionizing groups are possibly close to the FMN binding site.

Apoproteins↗
PubMed1975

Inorganic saltsin the fluorometric detection of presticides.

A method is described for the detection and quantification of pesticides on silica gel ghromatograms by in situ fluorometry. Simple inorganic salts are used to induce fluorescence in a number of pesticidal compounds. Intensified fluorescence is shown for one naturally fluorescent pesticide. Spectral data and instrumental limits of detection are given.

Chromatography, Thin Layer↗
PubMed1978

Beta-blockade and blood-levels after low-dose oral propranolol: The hepatic "first-pass" threshold revisited.

Heart-rate, arterial pressure, and plasmarenin activity were determined in six normal subjects at rest and after an injection of 8 microgram isoprenaline with and without prior propranolol administered orally in a dose of 5 mg 8-hourly for a total of five doses. After propranolol, resting heart-rate, systolic pressure, and plasma-renin activity all fell significantly (P less than 0.05 to less than 0.001). When the isoprenaline-induced changes of heart-rate, diastolic pressure, and plasma renin activity without propranolol were compared to those with propranolol, these responses were greatly diminished (P less than 0.01 to less than 0.001). The percent blockade by propranolol of the isoprenaline-induced changes ranged from 65% for diastolic pressure to 77% for heart rate and 78% for plasma-renin activity. Propranolol levels determined by conventional fluorometry were below accurate detection limits, whereas those determined by gas-liquid chromatography ranged from 2.3 to 8.5 ng/ml. These findings, which demonstrate beta-blockade with low-dose propranolol, are not consistent with the existence of a postulated threshold for the hepatic "first-pass effect" in man, which is said to require saturation by single doses of 30 mg or more before propranolol enters the systemic circulation.

Administration, Oral↗