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[Mechanisms of interaction between DNA and chemical analogues of microbial anabiosis autoinducers].

The alkylhydroxybenzene (AHB) autoregulatory factors d1 (fd1) of microorganisms have been found to directly interact with highly polymeric DNA. This circumstance results in changes, related to alterations in the topology of this macromolecule, in DNA physicochemical properties. The physicochemical properties of DNA in the presence of chemical analogues of microbial AHBs (methylresorcinol; hexylresorcinol; and 2-(4-hydroxyphenyl)ethane-1-ol, also known as tyrosol) were investigated using adsorption spectrophotometry, fluorometry, heat denaturation, viscosimetry, and electrophoresis in agarose gel. A number of concordant effects pointing to DNA-AHB interactions were revealed that manifesed themselves in the hypochromic properties of the resulting complexes, an increase in their melting temperature and viscosity, a decrease in their electrophoretic mobility, and a change in the fluorescent properties of AHBs upon complexation with DNA. Such alterations were particularly significant in the presence of hexylresorcinol, which possessed the maximum alkyl radical length among the fd1 analogues tested. Using atomic force microscopy, we visualized the micelle-like DNA nanostructures forming in the presence of AHBs. The results obtained provided the basis for developing a hypothetical model of the interaction between the biopolymer macromolecule and low-molecular-weight AHBs that takes into account the differences in the hydrophobicity of individual AHB homologues functioning as ligands. In terms of our model, we discuss AHB involvement in the stabilization of DNA and alteration of its topology, i.e., in the process related to intragenomic rearrangements, which account for the intrapopulational variability of bacteria, including dissociation processes.

Animals↗

[Application of the fast chlorophyll fluorescence induction dynamics analysis in photosynthesis study].

The changes in PSII performance of the photosynthetic apparatus caused by environmental stress or senescence have been explored widely by applying the chlorophyll fluorescence technique, mainly by using the modulated fluorometer. In recent years, an alternative approach via analyzing chlorophyll a fluorescence transient using the continuous excitation fluorometer has been developed, which offers more information that cannot be obtained by the modulated fluorometry. The JIP-test based on the theory of energy fluxes in biomembranes has been widely used to analyze the chlorophyll a fluorescence kinetics transient helping researchers to have a deeper insight into the primary photochemical reaction in the photosynthetic apparatus. In this paper, the chlorophyll a fluorescence transient and its significance, the parameters involved and their significance in the JIP-test, and the application of the fast chlorophyll fluorescence induction dynamics analysis in photosynthesis study are introduced combining some results of the author's.

Chlorophyll↗

Estimation of residual DNA in Feulgen reaction. A new correction of DNA determination per nucleus.

For the determination of the residual DNA amount after acid hydrolysis of Feulgen's method, a high salt-fluorochrome assay for DNA (5 microM Hoechst 33258 with 1 M NaCl) was effectively applied. At an optimal time length of acid hydrolysis for Feulgen reaction, the ratio of the residual DNA of non-hydrolysis to total DNA is 10% or more in hepatocyte or lymphocyte nuclei. A lot of residual DNA seems not to be negligible in Feulgen's method. A more accurate determination of DNA can be made by correcting the loss ratio of the residual DNA value to Feulgen DNA value. Thus, the combination assay of Feulgen's method with the present fluorometry is enough to measure separately both the amounts of Feulgen DNA and its residual DNA and successfully determines more accurately the total DNA per nucleus by summing both the amounts. The residual DNA, a resistant portion of the chromatin DNA against acid hydrolysis, is a possible constituent as the physiological component of nuclear structures.

Animals↗

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↗

Time-resolved fluoroimmunoassay for unconjugated estrogen in urine: comparison with a fluorometric assay for total estrogen and application in an in vitro fertilization program.

A time-resolved fluoroimmunoassay (TR-FIA) for unconjugated estrogens in human urine is described. 6-Keto-17 beta-estradiol-6-(O-carboxymethyl)oxime:bovine serum albumin is immobilized onto microtiter strip wells and the coated wells are incubated with 17 beta-estradiol standard preparations or unknowns with a polyclonal antiserum to 17 beta-estradiol-16,17-monosuccinyl:albumin. The antiserum-bound estrogen is detected by incubation with a europium-labeled anti-rabbit IgG that serves as both second antibody and tracer. After the immunoreactions, the bound portion of the labeled antiserum is quantified by dissociating the Eu3+ in a fluorescence-enhancement solution and measuring its fluorescence with a time-resolved fluorometer. The detection limit of the TR-FIA is 24 pmol of 17 beta-estradiol per liter; the analytical range extends to 1.8 nmol/L. This assay is a convenient alternative to radioimmunoassay and to the automated Kober-Ittrich fluorometry of total estrogen. Its advantages include short counting times; use of nonradioactive, stable reagents, all of which are commercially available; and more nearly complete automation. We conclude that this TR-FIA, compared with the Kober-Ittrich fluorometric assay (J Endocrinol 1957; 16:49-56), provides the clinician with equivalent information during follicular development therapy as part of an in vitro fertilization program.

Autoanalysis↗

Effects of intraocular pressure change on movement of FITC-dextran across vitreous-aqueous interface.

In an attempt to study the effect of change in the intraocular pressure (IOP) on the status of the vitreous-aqueous interface, the effect of topical bunazosin was studied in the rabbit eye using the new Johnson-Maurice method. This entails an intravitreal injection of FITC-dextran and measurements of its concentration changes in the anterior chamber many days after the injection. Repeated instillations of bunazosin solution reduced the IOP by about 5 mmHg, without changing the aqueous flow rate as determined by the method of fluorometry. On the other hand, the FITC-dextran concentration in the anterior chamber increased, indicating that the movement of FITC-dextran from the vitreous into the aqueous was greater when the IOP was lowered. This increase in the FITC-dextran movement into the anterior chamber was thought to be mainly attributable to an increase in the anteriorly directed fluid movement across the vitreous-aqueous interface. The increase in the fluid movement was estimated to be about 1.3 microliter/hour. This study suggested that a reduction in the IOP generated a small fluid flow from the vitreous into the aqueous, and that the new Johnson-Maurice method can be a useful means for noninvasive investigations of the physiology of the vitreous-aqueous interface.

Adrenergic alpha-Antagonists↗

Assay of pyridoxal-5'-phosphate, pyridoxal and pyridoxic acid in biological material.

The two vitamin B6-vitamers having an aldehyde function are oxidised to the corresponding acids and subjected to an HPLC separation on an RP 18 phase with a solvent consisting of 5% methanol in phosphate buffer at pH 3.5. The detection is carried out by fluorometry with excitation at 318 nm and emission at 418 nm. The peaks obtained correspond to pyridoxic acid 5'-phosphate and pyridoxic acid. Pyridoxal-5'-phosphate is determined as pyridoxic acid 5'-phosphate. Pyridoxal is determined as pyridoxic acid by subtracting the amount of pyridoxic acid already existing before oxidation.

Amniotic Fluid↗

Pyridine nucleotides and phosphorylation potential of rabbit corneal epithelium and endothelium.

In order to validate in situ corneal redox fluorometry, the redox state and phosphorylation potential of freeze trapped rabbit corneal epithelium and endothelium were studied using quantitative histochemical methods. The results were compared with noninvasive measurements using an optically sectioning fluorometer microscope. Enucleated rabbit eyes were either frozen in Freon-12, cooled by liquid nitrogen or exposed for 1 hr in 1 mM NaCN to block oxidation and then freeze trapped. Corneas were sectioned, freeze-dried, samples of individual layers dissected, weighed, and analyzed for: NADH, NAD+, NADPH, NADP+, ATP, ADP, and Pi. The aerobic epithelium showed a ratio for NAD+/NADH of 1.85 +/- 0.08 (9). In anoxia this ratio decreased to 1.06 +/- 0.07 (8). The NAD+/NADH ratio of aerobic endothelium was 3.25 +/- 0.28 (6); in anoxia this ratio was 0.68 +/- 0.14 (5). The values of phosphorylation potential ATP/(ADP X Pi)M-1 were: 447.9 +/- 40.2 (9) in aerobic epithelium, 378.2 +/- 24.7 (5) in anoxic epithelium; 308.4 +/- 25.2 (7) in aerobic endothelium and 225.4 +/- 19.1 (5) in anoxic endothelium. Aerobic-anoxic transitions alter the concentrations of NADH and NAD+ but did not affect the concentration of NADPH and NADP+. The microhistochemical data indicate that the redox state of rabbit epithelium is less sensitive to hypoxia than the endothelium. This difference between the two limiting layers is reflected in alterations of phosphorylation potential induced by hypoxia. The similarly high efficiencies of both layers in maintaining relatively high ATP levels during histotoxic hypoxia is most likely a result of compensatory ATP generation by enhanced glycolysis.

Adenine Nucleotides↗

Europium and samarium as labels in time-resolved immunofluorometric assay of follitropin.

This time-resolved immunofluorometric assay for human follitropin involves use of europium- or samarium-labeled monoclonal antibodies, with an average incorporation ratio of 3 mol of Eu3+ or Sm3+ per mole of antibody. These lanthanide ions are bound to the antibody molecules by means of the anhydride of diethylenetriaminepentaacetic acid. The solid-phase antibody is immobilized inside polystyrene tubes in which plasma samples were assayed in a one-step procedure. After incubation, the fluorescence intensity of Eu3+ or Sm3+ label is measured by time-resolved fluorometry, with a nitrogen laser as the pulsed excitation source. The sensitivity of the assay is largely better with Eu3+ than with Sm3+ because of the difference in their intrinsic luminescence properties. Results obtained with the proposed methods correlated well with those by an immunoradiometric method.

Adolescent↗

Noninvasive monitoring of tissue viability.

Noninvasive monitoring techniques are procedures of little or no morbidity that may be repeated frequently to assess tissue viability. The ideal noninvasive technique would be safe, sensitive, reliable, reproducible, simple to use, and inexpensive. Although no such ideal technique yet exists, several currently available methods have clinical usefulness. Visual fluorescein, fluorescein photography, and surface fluorometry all work by the same principle. We prefer the surface fluorometer because of its greater sensitivity, the more objective nature of the data it provides, and the greater frequency with which it can be repeated. Ultrasound Doppler techniques are useful preoperatively in locating vessels and assessing their patency, and we have found the bidirectional Doppler quite helpful in the postoperative monitoring of replanted or revascularized digits. The laser Doppler, photoplethysmograph, and surface thermometer have not been as helpful in our experience. Transcutaneous oxygen measurement shows great promise for the postoperative monitoring of replants and free tissue transfers.

Animals↗

[Changes in the venular epithelium of the mesentery in rats after application of hydrogen peroxide].

The change of the venular permeability and endothelial structure was studied with the aid of fluorometry and electron microscopy after application of H2O2. Transmural transfer rate of FITC-albumin++ and permeable fraction of the vessel walls were increased. There were formed the "leaks" and transendothelial canals in the venular endothelium. Numerous local membrane injuries of the endotheliocytes and pericytes, degranulation of the mast cells and destruction of thrombocytes into the vessel lumen were detected.

Animals↗

Experimental definition of latissimus dorsi, gracilis, and rectus abdominus musculocutaneous flaps in the dog.

Dissection and injection studies in canine cadavers and in anesthetized dogs were conducted to determine the feasibility of using the latissimus dorsi, gracilis, and rectus abdominus muscles as musculocutaneous free flaps. Lengths of vascular pedicles for the latissimus dorsi (2 +/- 0.8 cm), gracilis (1.8 +/- 0.8 cm), and rectus abdominus (1.9 +/- 0.9-cm cranial deep epigastric, 1.7 +/- 0.5-cm caudal deep epigastric), as well as arterial diameters (1.28 +/- 0.31-mm thoracodorsal for the latissimus dorsi, 1.10 +/- 0.33-mm muscular branch for the gracilis, 1.25 +/- 0.25-mm cranial deep epigastric and 1.26 +/- 0.32-mm caudal deep epigastric for the rectus abdominus) were considered satisfactory for microvascular transfer. Fluorometry demonstrated overlying cutaneous perfusion in all flaps based on their muscle vascular pedicles, with the exception of the rectus abdominus flap based on the caudal deep epigastric artery. In this instance, up to 20% of the cutaneous element had questionable or no perfusion.

Animals↗

Noninvasive measurements of pyridine nucleotide and flavoprotein in the lens.

Abnormalities in glucose metabolism are thought to be among the main causes of cataract formation. The authors have made noninvasive biochemical measurements of the lens that provide information concerning glucose metabolism in the lens epithelium. The autofluorescence of reduced pyridine nucleotides (PN) and oxidized flavoproteins (Fp) within the rabbit lens were noninvasively measured as a function of depth using redox fluorometry. The peak of the autofluorescence at 440 nm (excited at 360 nm) and 540 nm (excited at 460 nm) were determined at the lens epithelium. When 8 mM sodium pentobarbital, a known inhibitor of mitochondrial respiration, was applied to the lens, the autofluorescence peak at 440 nm increased and that at 540 nm decreased. The 440 nm autofluorescence is thought to be from reduced pyridine nucleotides, whereas the 540 nm autofluorescence is from the oxidized flavoprotein. Blocking lens respiration with pentobarbital caused an increase in the PN/Fp ratio by a factor of 3 within 3.5 hr after pentobarbital application.

Animals↗

Diffusion coefficients of quenchers in proteins from transient effects in the intensity decays.

We used 2-GHz frequency-domain fluorometry to examine the intensity decays of N-acetyl-L-tryptophamide (NATA) and the protein staphylococcal nuclease in the presence and absence of quenching by oxygen or acrylamide. When analyzed with a multiexponential model, the decays of NATA and nuclease both become more heterogeneous in the presence of quenching. We attribute the increased complexity to transient effects in quenching or equivalently a time-dependent rate constant for quenching. The frequency-domain data were analyzed using the Smoluchowski model (exp(-t/tau-2b square root t)) and the radiation model, which is known to correct some flaws in the more approximate Smoluchowski model. The radiation model provides improved fits to the data, as evidenced by average 10-fold decreases in chi R2. The radiation model also provides an estimate of the sum of the diffusion coefficients and the specific rate constant for quenching. The apparent diffusion coefficients for acrylamide and oxygen in nuclease, as seen by its single tryptophan (residue 140) are 15- and 11-fold lower than in water, respectively. The apparent values of the oxygen diffusion coefficient in water, as seen by NATA, are 2- to 3-fold larger than expected from earlier steady-state measurements. The ability to recover the detailed form of the intensity decays by the frequency-domain method should allow comparison of experimental results with calculated trajectories of quenchers in proteins.

Acrylamide↗

Human corneal endothelial permeability to fluorescein and fluorescein glucuronide.

The corneal endothelial permeability coefficient (Pac) for fluorescein and fluorescein glucuronide was determined in ten normal young volunteers. After oral administration of fluorescein, the apparent concentrations of both dyes in the corneal stroma and the anterior chamber were measured by differential fluorometry. The apparent dye levels calculated directly from the in vivo fluorometric measurements were converted to the true ones, based on the result of a normalization experiment performed in rabbit eyes. The value of Pac averaged 5.44 +/- 1.77 X 10(-4) cm/min for fluorescein and 3.77 +/- 1.10 X 10(-4) cm/min for fluorescein glucuronide (mean +/- SD, N = 20); the former was significantly greater than the latter (paired t-test, P less than 0.001). The aqueous-cornea distribution ratio was 0.50 +/- 0.14 for fluorescein and 0.66 +/- 0.16 for fluorescein glucuronide; the latter was significantly greater than the former (paired t-test, P less than 0.001). It was suggested that the previously reported values of Pac for fluorescein in the human eye were underestimates.

Administration, Oral↗

Evaluation of a direct fluorometric method for determination of serum retinol.

We evaluated the usefulness of a fluorometric method for determining serum retinol (Futterman et al., Invest Ophthalmol Vis Sci 1975;14:125-30) in which fluorescence (excitation 335 nm; emission 460 nm) of retinol is directly measured in unextracted, diluted serum. Using serum from 466 individual donors, we compared values so obtained with those by a "high-performance" liquid-chromatographic method. The correlation coefficient (r) was 0.74. When we compared fluorometric retinol values with retinol-binding protein values for the 466 samples, r was 0.71. About 1% of the 466 samples had markedly higher values by fluorometry than by chromatography, the result of positive interferences. For two serum pools, we obtained CVs of 1.58% (n = 57) and 1.79% (n = 57) in long-term precision studies lasting 60 days. Although the fluorometric method of Futterman et al. has not been widely adopted, we find that it is simple to perform and that results compare favorably with the chromatographic method in precision and accuracy. It is unique among commonly used serum retinol methods in that the serum need not be extracted with organic solvents.

Chromatography, High Pressure Liquid↗

Quantitative tumor cytochemistry--G.H.A. Clowes Memorial Lecture.

Quantitative optical cytochemistry permits the determination of many different parameters in whole cells or parts of cells. Total amounts of DNA, RNA, and protein and/or the amounts of these substances in nucleus or nucleolus are examples. Techniques in this field have contributed considerably to the development of our present conceptions of normal cell growth and regulation. Normal cell study has, however, predominated so that tumor work has not yet been very extensive, even though quite eqrly on, in the 1940's, a conspicuous cytochemical peculiarity was found in cancers, namely, a pronounced variability between cells with regard to several measurable parameters. The result has been different kinds of technical difficulties, which are especially great in work on tumor material. However, as years passed, a fair amount of information was accumulated from studies of different parameters in tumors. Only a few years ago this experience clearly indicated that, provided very considerable improvements were made in technique, several cytochemical methods could be used as efficient supplements to morphological cytodiagnostic work, especially in such important clinical fields as preneoplasia identification, malignancy grading, and judgment of the state of growth of for instance, hormone-stimulated populations and also the distinction, so difficult in morphology, between "atypical hyperplasia" and "truly malignant" states. In an effort to eliminate the foremost of these technical difficulties, during the last few years we have developed a comprehensive system for large-scale uni- and multiparameter measurements aimed specifically at the types of specimens encountered in cytopathological work. The system is based on cytophotometry in ultraviolet and visible light, interferometry, and fluorometry and also encompasses special arrangements to simplify the identification and recovery of individual cells for different types of study. Instruments for facilitation of the comparison of cytochemical results with morphological findings are also included.

Animals↗

Lipid mobility in the assembly and expression of the activity of the prothrombinase complex.

A phospholipid or membrane surface is a required component of the prothrombinase complex, yet little is known about the influence of the lipid on the assembly and expression of this complex. Vesicles composed of synthetic phospholipids were used to investigate the effects of membrane "fluidity" on the prothrombinase complex. All vesicle types studied were capable of supporting the prothrombinase reaction which in each case was characterized by a similar apparent Km. The binding constants for the interaction of Factor Va and prothrombin with synthetic phospholipid vesicles were not significantly affected by temperature. The rate of thrombin production, however, increased with increasing temperature. The fluidity of the vesicles was assessed by measuring the fluorescence lifetimes, steady state anisotropies, and differential phase fluorometry of diphenylhexatriene embedded in the vesicles. No correlation was observed between the fluidity of the vesicles and the steady-state rate of thrombin production, even when the enzymatic activity was monitored below and above the phase transition temperature of the lipid vesicles. A distinct correlation, however, was found between the fluidity of the vesicle and the time required to reach the maximum rate of thrombin production (pre-steady-state interval). We believe that this "lag" time corresponds to the time required for the assembly of the prothrombinase complex. Thus, although lipid fluidity does affect the assembly of the prothrombinase complex, after the complex is assembled, this property has little effect on the catalytic process itself.

Animals↗