Spectroscopic investigations on the binding of Pyronin Y to human serum albumin.

The interaction of Pyronin Y with human serum albumin (HSA) has been investigated systematically by fluorescence, absorption, fluorescence decay lifetime measurements, FTIR, synchronous fluorescence spectroscopy, and molecular modeling method. The spectroscopic and fluorescence quenching experiments show that Pyronin Y may show a static quenching mechanism with HSA. The specific binding distance of 1.96 nm between HSA and Pyronin Y was obtained via Förster non-radiation energy transfer method. The thermodynamic parameters indicate that the electrostatic interactions play a significant role during the binding process. In addition, synchronous fluorescence and FT-IR spectra indicated that the conformation and microenvironment of HSA were not influenced with the addition of Pyronin Y. The obtained results can be of biological significance in photodynamic therapy.

Holarrhena antidysenterica Extract and Its Steroidal Alkaloid, Conessine, as Resistance-Modifying Agents Against Extensively Drug-Resistant Acinetobacter baumannii.

Emergence and spread of antibiotic-resistant Acinetobacter baumannii have become a major public health concern. This study was designed to investigate the efficacy of Holarrhena antidysenterica extract and its major steroidal alkaloid conessine as resistance-modifying agents (RMAs) on the susceptibility of A. baumannii to novobiocin and rifampicin. A significant synergistic activity of both the extract and conessine in combination with either novobiocin or rifampicin with fractional inhibitory concentration index ≤0.5 was demonstrated. Fluorescent dyes and different efflux pump inhibitors were used to further investigate the synergism. Increase in the uptake of 1-N-phenylnaphthylamine in the bacterial cells treated with the extract and conessine was not observed indicating that both substances did not act as permeabilizers. With regard to efflux pump inhibition, no accumulation in ethidium bromide (EtBr) was noticed suggesting that the AdeABC pump was not involved. In contrast, accumulation in Pyronin Y was significantly increased (p < 0.05) demonstrating that the synergism was due to interference with the AdeIJK pump. Study on frequencies of the spontaneous mutational resistance to the extract in combination with antibiotics demonstrated attenuation in drug-resistant organisms. Thus, H. antidysenterica extract and conessine as RMAs may offer a combinatory therapy to restore antibiotic susceptibility in the extensively drug-resistant A. baumannii.

Non-antibiotic agent ginsenoside 20(S)-Rh2 enhanced the antibacterial effects of ciprofloxacin in vitro and in vivo as a potential NorA inhibitor.

The aim of this study is to explore the potential enhancing effect of ginsenoside 20(S)-Rh2 (Rh2) towards ciprofloxacin (CIP) against Staphylococcus aureus (S. aureus) infection in vitro and in vivo, and analyze the possible mechanisms through NorA inhibition from a target cellular pharmacokinetic view. In combination with non-toxic dosage of Rh2, the susceptibilities of S. aureus strains to CIP were significantly augmented, and the antibacterial kinetics of CIP in the S. aureus strains were markedly promoted. This enhancing effect of Rh2 towards CIP was also observed in S. aureus infected peritonitis mice, with elevated survival rate and reduced bacteria counts in blood. However, Rh2 did not influence the plasma concentrations of CIP. Further analysis indicated that Rh2 significantly promoted the accumulations of CIP in S. aureus, and inhibited the NorA mediated efflux of pyronin Y. The expressions of NorA gene on S. aureus were positively correlated with the enhancing effect of Rh2 with CIP. This is the first report of the enhancing effect of Rh2 with CIP for S. aureus infection in vitro and in vivo, of which it is probably that Rh2 inhibited NorA-mediated efflux and promoted the accumulation of CIP in the bacteria.

Cell cycle measurement of mouse hematopoietic stem/progenitor cells.

Lifelong production of blood cells is sustained by hematopoietic stem cells (HSC). HSC reside in a mitotically quiescent state within specialized areas of the bone marrow (BM) microenvironment known as the hematopoietic niche (HN). HSC enter into active phases of cell cycle in response to intrinsic and extrinsic biological cues thereby undergoing differentiation or self-renewal divisions. Quiescent and mitotically active HSC have different metabolic states and different functional abilities such as engraftment and BM repopulating potential following their transplantation into conditioned recipients. Recent studies reveal that various cancers also utilize the same mechanisms of quiescence as normal stem cells and preserve the root of malignancy thus contributing to relapse and metastasis. Therefore, exploring the stem cell behavior and function in conjunction with their cell cycle status has significant clinical implications in HSC transplantation and in treating cancers. In this chapter, we describe methodologies to isolate or analytically measure the frequencies of quiescent (G0) and active (G1, S, and G2-M) hematopoietic progenitor and stem cells among murine BM cells.

Entropy and Mg2+ control ligand affinity and specificity in the malachite green binding RNA aptamer.

The binding of small molecule targets by RNA aptamers provides an excellent model to study the versatility of RNA function. The malachite green aptamer binds and recognizes its ligand via stacking and electrostatic interactions. The binding of the aptamer to its original selection target and three related molecules was determined by isothermal titration calorimetry, equilibrium dialysis, and fluorescence titration. The results reveal that the entropy of complex formation plays a large role in determining binding affinity and ligand specificity. These data combined with previous structural studies show that metal ions are required to stabilize the complexes with non-native ligands whereas the complex with the original selection target is stable at low salt and in the absence of divalent metal ions.

SmvA, and not AcrB, is the major efflux pump for acriflavine and related compounds in Salmonella enterica serovar Typhimurium.

OBJECTIVES: The aim was to study the role played by SmvA pump in the efflux of quaternary ammonium compounds (QACs) in Salmonella enterica serovar Typhimurium (Salmonella Typhimurium). METHODS: Mutants in the smvA, acrB and tolC genes were constructed by the red swap method. P22 was used to transduce tolC to acrB and smvA mutant strains. The susceptibility of these strains to acriflavine and a variety of QACs was determined by MIC assays. RESULTS: In comparison with the Salmonella Typhimurium wild-type strain, the smvA mutant was more susceptible to QACs than the acrB mutant strain. A tolC single mutant was more susceptible than an acrB mutant to QACs, acriflavine, ethidium bromide, malachite green and pyronin B. The tolC-acrB double mutant was as susceptible as the single tolC mutant to QACs. Additionally, the smvA mutant strain was more susceptible to acriflavine than the acrB mutant (MICs = 31.3 versus 125 mg/L, i.e. 4-fold). Finally, the tolC-smvA double mutant (3.9 mg/L) was approximately 10 times more susceptible to acriflavine than either smvA (31.3 mg/L) or tolC (31.3 mg/L) single mutants. CONCLUSIONS: It is the SmvA efflux pump, and not AcrB, that plays the major role in the efflux of acriflavine and other QACs from Salmonella Typhimurium. This apparently conflicting report is due to the fact that in Escherichia coli the smvA gene does not exist. Our results suggest that tolC and smvA genes encode components of two different efflux systems with overlapping specificities that work in parallel to export acriflavine and other QACs.

Neural cell death is induced by neutralizing antibody to nerve growth factor: an in vivo study.

The central nervous system (CNS) of vertebrates originates from neuroepithelial cells located within the embryonic neural tube. Coincidental with the processes of proliferation, migration and differentiation in the developing CNS, cell death is also a major phenomenon during normal development. The investigation of neural cell death in development has focused on the role of target-derived survival factors such as nerve growth factor (NGF). In this study, the effects of anti-NGF antibody on neural cell death in the cerebral cortex have been investigated. Injection of anti-NGF antibody into the cisterna magnum of mouse pups increased the number of neural cell deaths and resulted in thinning of the cerebral cortex compared with a control group. It is concluded that endogenous NGF is essential for cortical cell survival in the cerebral cortex of the newborn mouse. Moreover, this method may be applied to the other factors and different CNS regions, allowing identification of molecules and signals involved in neural cell survival.

A simple and high sensitive spectrophotometric method for ultra trace determination of ruthenium with its catalytic effect on the oxidation of pyronin B by periodate.

A new simple, selective, high sensitive and rapid method has been developed for spectrophotometric determination of ultra trace amounts of ruthenium based on its catalytic effect on the oxidation of pyronin B by periodate at lambdamax=555 nm. The described method is able to quantify ruthenium in the range of 0.1-100 ng ml-1 (r=0.9973), with a detection limit (S/N=3) of 0.036 ng ml-1. Under optimum conditions, this procedure has been successfully applied to determine the trace levels of ruthenium in the environmental and biological samples. The precision, expressed as relative standard deviation of three measurements, is better than 2.44%.

A novel subpopulation lacking Oct4 expression in the testicular side population.

We characterized murine spermatogonial stem cells (SSCs) using a multi-parameter selection strategy, combining Oct4 expression determined by monitoring green fluorescent protein (GFP) expression, and the testicular side population (SP) showing weak fluorescence on Hoechst 33342 dye staining, as markers of stem cell purification. Testicular cells were collected from Oct4/GFP transgenic mice and analyzed using a fluorescence-activated cell sorter (FACS). SP was detected in testicular cell suspensions at an average rate of 0.10%. Multicolor analysis indicated that 96% of SP cells were negative for Oct4. The cells did not express SSC marker genes, but expressed Bcrp1. While the main population was 93% positive for pyronin Y staining, this was limited to 51% in SP. We found a novel subpopulation with reduced RNA content lacking Oct4 expression in testicular SP. These results suggest that the cells isolated by FACS represent a novel population of SSCs in the G0 quiescent state.

Kinetic spectrofluorimetric determination of trace ascorbic acid based on its inhibition on the oxidation of pyronine Y by nitrite.

A highly sensitive spectrofluorimetric method is proposed for the determination of trace amount of ascorbic acid using a new indication. The method is based on the inhibition of ascorbic acid on the oxidation of pyronine Y (PRY) by nitrite. The detection limit for ascorbic acid is 0.012 microg ml(-1), the linear range of the determination is 0.02-0.36 microg ml(-1). Analytical parameters, such as reagent concentration, pH, reaction temperature and time, were optimized. The relative standard deviations of eleven replication determinations of 0.12 and 0.24 microg ml(-1) ascorbic acid were 1.4 and 0.72%, respectively. This method has been used to determine ascorbic acid in pharmaceuticals, vegetables, fruits and soft drink with satisfactory results.

Photo-induced inactivation of viruses: adsorption of methylene blue, thionine, and thiopyronine on Qbeta bacteriophage.

The adsorption of cationic organic dyes (methylene blue, thionine, and thiopyronine) on Qbeta bacteriophage was studied by UV-visible and fluorescence spectroscopy. The dyes have shown a strong affinity to the virus and some have been used as sensitizers for photo-induced inactivation of virus. In the methylene blue concentration range of 0.1-5 microM and at high ratios of dye to virus (greater than 1000 dye molecules per virion), the dyes bind as aggregates on the virus. Aggregation lowers the efficiency of photoinactivation because of self-quenching of the dye. At lower ratios of dye to virus (lower than 500 dye molecules per virion), the dye binds to the virus as a monomer. Fluorescence polarization and time-resolved studies of the fluorescence support the conclusions based on fluorescence quenching. Increasing the ionic strength (adding NaCl) dissociates bound dye aggregates on the virus and releases monomeric dye into the bulk solution.

Cold-induced conformational changes of ribonuclease A as investigated by subzero transverse temperature gradient gel electrophoresis.

Subzero temperature gradient gel electrophoresis is a new approach which allows to measure the transition temperature of low temperature-induced subtle conformational changes of proteins and to detect the different conformational states, including unfolded states. Using this technique under destabilizing conditions, i.e., in the presence of 4 M urea, bovine pancreas ribonuclease A exhibited two transitions: (i) a continuous transition with a midpoint temperature of -14 degrees C corresponding to a rapid equilibrium between the initial enzyme state and a conformational state more compact than the initial one; (ii) a discontinuous transition at -22.5 degrees C from intermediate to a non migrating species. Under reducing conditions this second transition was shifted toward high temperatures (-18.5 degrees C). We attempted to detect these two transitions by differential scanning calorimetry, UV spectrophotometry and circular dichroism measurements. These transitions have been ascribed to subtle cold-induced conformational changes.

[Experimental and theoretical studies of the formation of DNA complexes with biologically active ligands containing chromophore groups, depending on the ionic strength of the solution]. / Eksperimental'nye i teoreticheskie issledovaniia obrazovaniia kompleksov DNK s biologicheski aktivnymi ligandami, soderzhashchimi khromofornye gruppy, v zavisimosti ot ionnoi sily rastvora.

The binding of two ligands--dyes pyronin G (PG) and methylene blue (MB)--to DNA depending on the Na+ concentration was studied spectrophotometrically in the visible and UV ranges. The theoretical model taking into account the binding of a dye to DNA by means of aggregation and intercalation on two independent lattices was used for computation of the complexation parameters. The results of optimization of spectrophotometric concentration dependences show that the ligand occupies two phosphates in the process of aggregation on a negatively charged matrix. In is shown that the position of isobestic points can serve as an additional criterion for choosing the best values of the cooperativity parameters, absorption spectra of ligands in the aggregated state, and other parameters of the complex formation. The obtained intercalation constants show a linear dependence of 1gK(int) vs 1g C0Na for both PG and MB. These equations were used in a modified version of the computer program which allows optimization of the absorption spectra for DNA-ligand mixtures in a wide range of Na+ concentrations simultaneously, and estimation of the constant for Na+ binding to DNA phosphate (K1 < 10).

Interactions of nucleic acids with fluorescent dyes: spectral properties of condensed complexes.

Interaction of cations with nucleic acids (NA) often results in condensation of the product. The driving force of aromatic cation-induced condensation is the cooperative interaction between ligand and single-stranded (ss) NA. This type of reaction is highly specific with regard to the primary and secondary structure of NA, and results in destabilization of the latter. The spectral properties of fluorescent intercalating and non-intercalating ligands [acridine orange, pyronin Y(G), DAPI, Hoechst 33258, and Hoechst 33342]-NA complexes were studied in both the relaxed and condensed form. The changes in absorption, excitation, and fluorescence emission spectra and fluorescence yield that followed the condensation were examined. Although some of these effects can be explained by changes in solvation of the fluorophore and its interaction with NA bases and the solvent, the overall effect of condensation on spectral properties of the complex is unpredictable. In particular, no correlation was found between these effects and the ds DNA binding mode of these ligands. Nevertheless, the spectral data associated with polymer condensation can yield information about the composition and structure of NA and can explain some nonspecific interactions of these probes.

Staining with pyronin Y detects changes in conformation of RNA during mitosis and hyperthermia of CHO cells.

Cellular RNA in Chinese hamster ovary (CHO) cells synchronized in mitosis (M) or G2 phase, as well as in interphase cells subjected to hyperthermia (42 degrees C, 10 min), was stained with acridine orange (AO), ethidium bromide (EB), or pyronin Y (PY) and the resultant fluorescence was measured by flow cytometry. Total RNA content detected after staining with AO increased in M as compared to G2-phase cells, consistent with continued RNA synthesis during G2 phase. The content of double-stranded RNA, stained with EB (after DNase treatment), was also somewhat higher in M cells. In contrast, the stainability of RNA with PY decreased by 27% in M- compared to G2-phase cells. Furthermore, a decrease in stainability of RNA with PY was observed in G2 cells compared to cells in G1 phase. In separate experiments, RNA stainability with AO or EB was generally unaffected when interphase CHO cells were exposed to 42 degrees C for 10 min, though this same treatment resulted in a 26% decrease in RNA stainability with PY. The decreased PY stainability of cellular RNA in M or heat-treated cells was observed at a relatively narrow range of dye concentration (1.0-2.0 micrograms/ml). The observed hypochromicity of RNA coincides with dissociation of polyribosomes into single ribosomes known to occur during mitosis and following exposure to hyperthermia. It is presumed that the phenomenon involves selective denaturation and condensation of ribosomal (r) RNA by PY in single ribosomes which does not occur in polyribosomes. While the molecular mechanisms responsible for stabilization of rRNA in polyribosomes preventing its denaturation and condensation by PY are unknown, PY appears to be a sensitive probe that can be used to detect and study these changes in rRNA confirmation in situ.

Characterization of granulosa cell subpopulations from avian preovulatory follicles by multiparameter flow cytometry.

The objective of the present study was to characterize the granulosa cell populations from individual hen (Gallus domesticus) preovulatory follicles at defined stages of follicular maturation using multiparameter flow cytometry. Granulosa cells were fixed and stained with three fluorochromes that selectively bind to DNA (Hoechst 33342, blue), RNA (pyronin Y, red), or protein (fluorescein isothiocyanate, green). A flow cytometer equipped with a three-laser excitation system was used to analyze three colors of fluorescence from stained cells. Forward angle light scatter and axial light loss measurements were made on each cell to determine relative cell size. In addition, the ratios of RNA to protein and DNA to protein were measured. The major findings obtained from correlated measurements of cell cycle (DNA), protein, RNA, cell size, and ratios were: 1) the percentage of proliferating cells decreased while cell size increased during follicular maturation; 2) two subpopulations of granulosa cells were identified within each follicle based on relatively high and low protein contents; the fraction of cells in the high protein subpopulation increased, and the fraction of cells in the low protein subpopulation decreased during follicular maturation; 3) the high and low protein subpopulations also differed in cell cycle distribution, RNA content, and cell size; and 4) the distribution of cells into the two subpopulations and the degree of proliferation were influenced by stage of the ovulatory cycle, primarily in the most mature follicles. The results demonstrate the dynamic heterogeneity of the granulosa cell populations from individual ovarian follicles and show the influences of follicular maturation and stage of the ovulatory cycle on cell growth and metabolism.

A rapid method for measuring drug enrichment in epidermis.

A rapid and simple method is described for measuring the enrichment of small molecules in epidermal tissue. To measure such an enrichment, a small tissue sample (2-10 mg) is allowed to equilibrate with a buffered solution of a labelled substance for periods of 12-36 h. The concentration of the radioactive molecule in the tissue is measured as a decrease of radioactivity in the solution. Concentration measurements in the tissue itself can be performed, but are not required to detect enrichment in the tissue or to assess its magnitude. The specific density of appendage free human epidermis has been determined and was found to be 1.20 g/cm3. Using this value, tissue weight can be translated into volume and concentraton changes in the solution can be recalculated to yield the concentration of the substance in the tissue itself. Close agreement was found between the calculated tissue concentration and the values actually measured, following digestion of the epidermis with NaOH and measuring the activity in the tissue digest. The enrichment of five substances in human epidermis was measured: alpha-estradiol, thiopyronincce, 8-methoxypsoralen (8-MOP), 5-methoxypsoralen (5-MOP), and theophylline. Of these substances, the first four are concentrated by human epidermis and the concentrations reached within the tissue are 10-500 times higher than the concentration of the same substance in the surrounding buffer. The enrichment data has been analysed in an attempt to distinguish between reversible affinity binding to specific tissue sites and partitioning of the substances between buffer and tissue components (lipids, membranes, etc.). In the case of thiopyronin and 8-MOP, reversible binding is indicated with dissociation constants of 10(-7) M and 10(-5) M, respectively, while partitioning distribution could account for the behavior of 5-MOP and alpha-estradiol. The method can be used either as a rapid screening method or as a quantitative analysis for the characterization of tissue enrichment with specific drugs.

[Stimulation of post-traumatic regeneration of rat spleen under conditions of gravitational stress]. / O stimuliatsii posttravmaticheskoi regeneratsii selezenki krys v usloviiakh gravitationnoi peregruzki

Post-traumatic regeneration of the rat spleen was studied after resection of half the organ, under gravitation overloading (11 units) using spleen tissue extract prepared by Filatov's method. Gravitation overloading caused a decrease in the size of the spleen nodules, smooths their contours, increases the red pulp infiltration by the lymphocytes, reduces the number of labeled cells and the intensity of the label in the reactive centers of the spleen nodules, decelerates the capsule formation in the resection area. Application of the stimulant normalized the structure of white pulp, increased the number of labeled cells, and accelerated the capsule formation. The tissue extract used in gravitation overloading brought the restoration process nearer to the usual course of the posttraumatic spleen regeneration (by the character and periods of tissue differentiation development).

Decay of methyl green-pyroninophilia in Burkitt's lymphoma.

The spontaneous decay of methyl green-pyroninophilia in Burkitt's lymphoma was studied at four temperatures (0 degrees C to 56 degrees C) in four biopsies using Kurnick's method. The decay is temperature and time related and is presumably due to intrinsic enzymatic action, probably ribonuclease. Imprints from tumor tissue preserved at 0 degrees C lose pyroninophilia by six hours; from tumor preserved at room temperature pyroninophilia, in most instances, would have been lost by three hours, from tumor tissue maintained at 37 degrees C, by two hours in most instances and by 15 minutes at 56 degrees C in most instances. The absence of pyroninophilia after these intervals at the appropriate temperatures should not detract from the diagnosis of Burkitt's Lymphoma.