Interaction of extrinic fluoresence probes with E. coli glutamine synthetase

Summary Binding of 2-p-toluidinylnaphthalene-6-sulfonate (TNS) to adenylylated glutamine synthetase is cooperative and time-dependent, with 3 dye sites per subunit. In fluorescence polarization experiments TNS and pyrene butyrate give normalized Perrin plots that indicate a symmetrical arrangement of dye excited state dipoles, relative to the rotational axis of the oblate ellipsoid of the dodecameric native enzyme.Acknowledgment. Drs G. Weber and L. Brand are thanked for helpful comments on this work. — Supported in part by grants from the National Science Foundation (BMS 72-02165 and GU-3182). Portions of this work were presented in preliminary form at the 9th International Congress of Biochemistry, Stockholm, Sweden, July 1973.     

Kinetik der hepatischen Farbstoffaufnahme von Indocyaningrün. Einfluss von Bilirubin und Natriumglykocholat

Summary Kinetics of hepatic uptake of indocyanine green, a dye which is used for evaluation of liver function, were studied in the rat. The results indicate that the relationship between ICG-dose and initial hepatic dye uptake obeys Michaelis-Menten kinetics suggesting an interaction of the dye with a carrier or fixed site in the liver cell. Thus it was possible to calculate maximum ICG-uptake (v _max ) and the Michaelis constant (K _m ) of this transport system from several submaximal values.v _max was 7.65 (6-06-9.65)²² mg per 100 g liver/min and K _m 0.56 (0.31–0.81)²². Under the influence of substances which inhibit the elimination of dyes by the liver the parametersv _max and K _m showed changes which allowed characterization of the type of inhibition. While sodium glycocholate had no influence on maximum hepatic ICG-uptake and the Michaelis constant bilirubin caused a significant increase of K _m to 1.29 (0.68–1.90)²² without significantly changingv _max . These data suggest that bilirubin interferes with hepatic uptake of indocyanine green by competitive inhibition and that uptake of bile acids is dependent on a different mechanism.     

Colored filters for microphotography: a costless 'do-it-yourself' solution.

Black and white microphotography makes use of several colored filters for an increased contrast. These filters are sometimes expensive and do not correspond exactly to the stain to be masked on the negative. Our specific colored filters contain a sufficient and adjusted amount of the dye used in the staining technic in an Agar medium.     

Physiological dissection of various effects of ruthenium red dye on Paramecium cells.

Polycationic dye ruthenium red, but not alcian blue, if externally applied to Paramecium cells quickly inhibits their phagocytosis. Ruthenium red combined with the cell surface diminishes frequency and duration of ciliary reversals and gradually inactivates the Ca++ gating mechanism. This effect persists for 1-3 after ruthenium red removal from the culture medium.     

The pre-history of hemocyanin. The discovery of copper in the blood of molluscs

The presence of copper in molluscs was discovered in 1833 by Bartolomeo Bizio, a Venetian chemist, who found copper in marine gastropods of the family Muricidae during his studies on the purple dye he had isolated from these animals. Bizio was so surprised by this finding that he extended his analysis to several other invertebrate species in which he identified the metal.     

Effect of insulin on lateral diffusion of pyrene in rat liver plasma membrane.

The yield of excimer formation by pyrene molecules inserted in rat liver plasma membranes in sensibly decreased in the presence of 1 nM insulin. This effect can be interpreted as indicating a decrease of the value of the translational diffusion coefficient of the dye within the membrane.     

Simultaneous monitoring by optical techniques of respiratory chain and intracellular pH in toad ventricle strip.

Intracellular pH and oxidative metabolism can be measured in toad ventricle strips simultaneously by the use of the pH indicator dye, neutral red, and a rapid scanning spectrophotometer. The effects of hypoxia and acidification on mechanical function are approximately additive. The decrease in tension due to slight acidification is probably through an effect on the portion of the twitch tension supported by anaerobic metabolism.     

Intercellular dye-coupling in intestinal smooth muscle. Are gap junctions required for intercellular coupling?

Summary The dye Lucifer Yellow was injected into single smooth muscle cells in the guinea pig small intestine in order to study intercellular coupling. Dye-coupling was observed in both the circular and longitudinal muscle layers and was markedly reduced when the intercellular pH was lowered. These results suggest the presence of gap junctions among intestinal muscle cells, but are inconsistent with previous ultrastructural studies that failed to demonstrate such junctions in the longitudinal muscle.     

Intercellular dye-coupling in intestinal smooth muscle. Are gap junctions required for intercellular coupling?

The dye Lucifer Yellow was injected into single smooth muscle cells in the guinea pig small intestine in order to study intercellular coupling. Dye-coupling was observed in both the circular and longitudinal muscle layers and was markedly reduced when the intercellular pH was lowered. These results suggest the presence of gap junctions among intestinal muscle cells, but are inconsistent with previous ultrastructural studies that failed to demonstrate such junctions in the longitudinal muscle.     

Gastrointestinal transit and digestibility of maltitol, sucrose and sorbitol in rats: a multicompartmental model and recovery study.

Using data obtained with a dye marker and the gavage technique, the kinetics of gastrointestinal transit of different loads of sugar substitutes (maltitol, sorbitol) and sugar (sucrose) in the rat were analysed using a linear multicompartmental model over a range from the realistic to the non-physiologic high, of carbohydrate intake levels and using only a few experimental time points. The model gave detailed insight into intestinal propulsion and gastrocecal transit time. Rate constants of transport between the compartments investigated were determined; they showed characteristics which could be related to the substance and the dosage administered. Analyses of the gastrointestinal content and calculations of the intestinal net water movement showed that the digestibility and absorption of the disaccharide sugar alcohol, maltitol, in the small gut depended inversely on the dose ingested. For all substances tested, caloric availability in the small intestine was calculated. At a physiological low level of maltitol intake, the results also indicated an insignificant calorie-saving effect in comparison to sucrose, an effect based mainly on the slow absorption rate of the maltitol cleavage product sorbitol.     

Visualization of the mitochondria of Toxoplasma gondii-infected mouse fibroblasts by the cationic permeant fluorescent dye rhodamine 123

The mitochondria of living mouse fibroblasts infected with Toxoplasma gondii were monitored with the cationic permeant fluorescent dye rhodamine 123. Fluorescence microscopy revealed that host cell mitochondria accumulated at the cytoplasmic surface of parasitophorous vacuoles and increased the dye uptake in the periparasitophorous vacuole as T. gondii multiplied.     

Visualization of the mitochondria ofToxoplasma gondii-infected mouse fibroblasts by the cationic permeant fluorescent dye rhodamine 123

Summary The mitochondria of living mouse fibroblasts infected withToxoplasma gondii were monitored with the cationic permeant fluorescent dye rhodamine 123. Fluorescence microscopy revealed that host cell mitochondria accumulated at the cytoplasmic surface of parasitophorous vacuoles and increased the dye uptake in the periparasitophorous vacuole asT. gondii multiplied.The author wishes gratefully to acknowledge the work of Miss Mayumi Kato and Mr. Isao Kimata in preparing the photographs. The publication cost was supported by a grant of the Ohyama Health Foundation.     

Biliary excretion of sulfobromophthalein in isolated perfused livers from normal and spironolactone-treated rats

Summary Bile flow and biliary excretion of sulfobromophthalein (BSP) was examined in isolated perfused livers from normal and spironolactone(SP)-treated rats. BSP biliary excretion contributed to the bile production in both groups. Moreover SP increased BSP biliary excretion but transfer of dye from plasma into liver was not affected.Acknowledgments. This work was supported by a grant from Consejo Nacional de Investigaciones Científicas y Técnicas República Argentina (EFIHER-CONICET). Isolated rat liver preparations were performed by Miss Graciela Patricia Rodriguez. The assistance of Carlos Guillermo Grieve is also gratefully acknowledged.     

Imaging oxygen in neural cell and tissue models by means of anionic cell-permeable phosphorescent nanoparticles

Cell-permeable phosphorescent probes enable the study of cell and tissue oxygenation, bioenergetics, metabolism, and pathological states such as stroke and hypoxia. A number of such probes have been described in recent years, the majority consisting of cationic small molecule and nanoparticle structures. While these probes continue to advance, adequate staining for the study of certain cell types using live imaging techniques remains elusive; this is particularly true for neural cells. Here we introduce novel probes for the analysis of neural cells and tissues: negatively charged poly(methyl methacrylate-co-methacrylic acid)-based nanoparticles impregnated with a phosphorescent Pt(II)-tetrakis(pentafluorophenyl)porphyrin (PtPFPP) dye (this form is referred to as PA1), and with an additional reference/antennae dye poly(9,9-diheptylfluorene-alt-9,9-di-p-tolyl-9H-fluorene) (this form is referred to as PA2). PA1 and PA2 are internalised by endocytosis, result in efficient staining in primary neurons, astrocytes, and PC12 cells and multi-cellular aggregates, and allow for the monitoring of local O₂ levels on a time-resolved fluorescence plate reader and PLIM microscope. PA2 also efficiently stains rat brain slices and permits detailed O₂ imaging experiments using both one and two-photon intensity-based modes and PLIM modes. Multiplexed analysis of embryonic rat brain slices reveals age-dependent staining patterns for PA2 and a highly heterogeneous distribution of O₂ in tissues, which we relate to the localisation of specific progenitor cell populations. Overall, these anionic probes are useful for sensing O₂ levels in various cells and tissues, particularly in neural cells, and facilitate high-resolution imaging of O₂ in 3D tissue models.     

A preliminary report on gamma-irradiated protein-dye complexes

In spectral studies of gamma-irradiated protein-dye complexes, influences of concentrations of the components and changes in dye character are mainly noted.     

Lysine estimation with the modified Udy-dye binding method in hexaploid wheat

Summary Lysine content in bread wheat (Triticum aestivum L. m. Thell) was determined by a modified Udy-dye binding method and was compared with that obtained from the amino acid analyzer. The values obtained from the 2 methods were correlated and the co-efficient was found to be +0.91 at 0.450 mg/ml dye concentration. The modified method is quick, less expensive and quite helpful for screening lysine at earlier generation in wheat breeding for improved grain quality content.The author is grateful to Dr V.A. Johnson, University of Nebraska, Lincoln, USA, for supplying wheat cultivars with known protein and lysine values.     

Zur Bestimmung der Lymphozytotoxizität

Summary The technique for testing lymphocytotoxicity by staining with Trypan-blue has been reevaluated. It was found that both cell suspension and aqueous stock solution (0.5%) of Trypan-blue must be adjusted to a pH of 7.3 prior to mixing in order to avoid a toxic effect of the dye itself, accounting for the lysis up to 35% of the cell population. This modification permitted a reproducibility of the results with a variation of less than 5%. This Trypan-blue test gives identical results as are obtained with the⁵¹Cr releasing method. The results may also be important for testing the viability of cells with Trypan-blue.     

Skeletal muscle capillary densities during reactive hyperemia

Reactive hyperemia was induced in hindlimbs of rats by occlusion of the femoral artery. Using fluorescein dye as a peripheral vascular marker, we observed that there was an increase in the number of flowing capillaries supplying the muscle fibres following release of the occlusion. The results indicate that the number of flowing capillaries is not dependent on the duration of occlusion (2-10 min).     

Inhibition of thiamine transport in baker's yeast by methylene blue

Summary Methylene blue was found to inhibit thiamine transport competitively (K_i=0.63 M) in baker's yeast. The dye was also effective in abolishing the growth inhibition ofSaccharomyces cerevisiae by pyrithiamine which is known to be taken up by a common transport system for thiamine in yeast cells. A possible mechanism for the inhibition by methylene blue of the thiamine transport system in baker's yeast is discussed.