'IntraCell' plugin for assessment of intracellular localization of nano-delivery systems and their targeting to the individual organelles

Efficient intracellular targeting of drugs and drug delivery systems (DDSs) is a major challenge that should be overcome to enhance the therapeutic efficiency of biopharmaceuticals and other intracellularly-acting drugs. Studies that quantitatively assess the mechanisms, barriers, and efficiency of intracellular drug delivery are required to determine the therapeutic potential of intracellular targeting of nano-delivery systems. In this study we report development and application of a novel ‘IntraCell’ plugin for ImageJ that is useful for quantitative assessment of uptake and intracellular localization of the drug/DDS and estimation of targeting efficiency. The developed plugin is based on threshold-based identification of borders of cell and of the individual organelles on confocal images and pixel-by-pixel analysis of fluorescence intensities.We applied the developed ‘IntraCell’ plugin to investigate uptake and intracellular targeting of novel endoplasmic reticulum (ER)-targeted delivery system based on PLGA nanoparticles decorated with ER-targeting or control peptides and encapsulating antigenic peptide and fluorescent marker. Decoration of the nanoparticles with peptidic residues affected their uptake and intracellular trafficking in HeLa cells, indicating that the targeting peptide was identified as ER-targeting signal by the intracellular trafficking mechanisms in HeLa cells and that these mechanisms can handle nano-DDS of the size comparable to some intracellular vesicles (hundreds of nanometers in diameter).We conclude that decoration of nanoparticles with peptidic residues affects their intracellular localization and trafficking and can be potentially used for intracellularly-targeted drug delivery. ‘IntraCell’ plugin is an useful tool for quantitative assessment of efficiency of uptake and intracellular drug targeting. In combination with other experimental approaches, it will be useful for the development of intracellularly-targeted formulations with enhanced and controlled drug pharmacological activities, such as delivery of antigenic peptides for anticancer vaccination and for other applications.     

Reversible Dihydropyridine Isothiocyanate Binding to Brain Calcium Channels

Voltage-dependent calcium channels from ileal smooth muscle can be affinity-labeled with a [3H]dihydropyridine isothiocyanate radioligand. We examined the binding of this agent to brain membranes, to compare the properties of calcium channel drug binding sites in brain with those previously described in ileum. In brain, the [3H]dihydropyridine isothiocyanate labels sites that correspond in number and pharmacologic characteristics to binding sites for the classic calcium entry blocker, [3H]nitrendipine. However, in contrast to the covalent nature of dihydropyridine isothiocyanate binding in ileum, brain calcium channels are labeled reversibly. This difference in binding properties may reflect structural variations in voltage-dependent calcium channels in different tissues.     

The effects of ruthenium red,lanthanum, fluorescein isothiocyanate and trifluoperazine on vesicle transport,vesicle fusion and tip extension in pollen tubes

The effects of ruthenium red, lanthanum, fluorescein isothiocyanate and trifluoperazine, all antagonists of Ca²⁺ function in cells, have been studied in growing pollen tubes of Tradescantia virginiana. All four drugs inhibit pollen-tube growth but bring about different ultrastructural changes at the growing tips and within the cytoplasm. The results strongly support the hypothesis that Ca²⁺ plays a vital role in the mechanism of pollen-tube tip growth. The effect of ruthenium red provides evidence that sequestration of Ca²⁺ by mitochondria critically adjusts the concentration of these ions at tube tips. Fluorescein isothiocyanate appears to be a potent inhibitor of vesicle fusion at the plasma membrane, with vesicles accumulating in the tip at rates equivalent to those determined previously for their production. Both vesicle fusion and tip extension are regulated by Ca²⁺ but appear to be independently controlled processes.     

Double Coupling Edman Chemistry for High-Sensitivity Automated Protein Sequencing

Capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) is a promising new method for the analysis of protein sequencing products. It gives 10 zmol (1 zmol = 10^–21 mol) limits of detection (3) for fluorescein thiohydantoin (FTH) amino acids. We have developed a separation for the (FTH)-amino acid products generated from 18 of the 20 coded amino acids. The extremely low volume requirement associated with CE-LIF makes it incompatible with commercial sequencers. For this reason, we have also been developing a miniaturized sequencer that can be more easily coupled to our detection system. Both the CE-LIF system and the miniaturized sequencer are described.     

Intraspecific variation in defense against a generalist lepidopteran herbivore in populations of Eruca sativa (Mill.)

Populations of Eruca sativa (Brassicaceae) from desert and Mediterranean (Med) habitats in Israel differ in their defense against larvae of the generalist Spodoptera littoralis but not the specialist Pieris brassicae. Larvae of the generalist insect feeding on plants of the Med population gained significantly less weight than those feeding on the desert plants, and exogenous application of methyl jasmonate (MJ) on leaves of the Med plants significantly reduced the level of damage created by the generalist larvae. However, MJ treatment significantly induced resistance in plants of the desert population, whereas the generalist larvae caused similar damage to MJ‐induced and noninduced plants. Analyses of glucosinolates and expression of genes in their synthesis pathway indicated that defense in plants of the Med population against the generalist insect is governed by the accumulation of glucosinolates. In plants of the desert population, trypsin proteinase inhibitor activity was highly induced in response to herbivory by S. littoralis. Analysis of genes in the defense‐regulating signaling pathways suggested that in response to herbivory, differences between populations in the induced levels of jasmonic acid, ethylene, and salicylic acid mediate the differential defenses against the insect. In addition, expression analysis of myrosinase‐associated protein NSP2 suggested that in plants of the desert population, glucosinolates breakdown products were primarily directed to nitrile production. We suggest that proteinase inhibitors provide an effective defense in the desert plants, in which glucosinolate production is directed to the less toxic nitriles. The ecological role of nitrile production in preventing infestation by specialists is discussed.     

Membrane mobility agent alters the consequences of lectin-cell interaction in a malignant cell membrane

The membrane mobility agent 2-(2-methoxyethoxy)-ethyl 8-(cis-2-n-octylcyclopropyl)-octanoate promotes cap formation from wheat germ agglutinin-receptor combinations at the expense of agglutination in membranes of malignant mastocytoma cells.     

Estimating viability of plant protoplasts using double and single staining

Summary The utility of numerous dyes for determining the viability of barley (Hordeum vulgare L. cv. Himalaya) aleurone protoplasts was studied. Protoplasts isolated from the barley aleurone layer synthesize and secrete -amylase isozymes in response to treatment with gibberellic acid (GA) and Ca²⁺. These cells also undergo dramatic morphological changes which eventually result in cell death. To monitor the viability of protoplasts during incubation in GA and Ca²⁺, several types of fluorescent and nonfluorescent dyes were tested. Evans blue and methylene blue were selected as nonfluorescent dyes. Living cells exclude Evans blue, but dead cells and cell debris stain blue. Both living and dead cells take up methylene blue, but living cells reduce the dye to its colorless form whereas dead cells and cell debris stain blue. The relatively low extinction coefficient of these dyes sometimes makes it difficult to distinguish blue-stained cells against a background of blue dye. Several types of fluorescent dyes were tested for their ability to differentially stain dead or living cells. Tinopal CBS-X, for example, stains only dead cells, and its high extinction coefficient allows its ultraviolet fluorescence to be recorded even when preparations are simultaneously illuminated with visible light. To double-stain protoplasts, the most effective stain was a combination of fluorescein diacetate (FDA) and propidium iodide (PI). By employing a double-exposure method to record the fluorescence from cells stained with both FDA and PI, dead and living cells could be distinguished on the basis of fluorochromasia.     

Some observations onStrongwellsea castrans [Zygomycetes: Entomophthorales], a parasite of root flies [Delia spp.], in the south of Scotland

Root flies,Delia radicum (L.) andD. floralis (Fallén) (Diptera: Anthomyiidae), trapped in swede crops in the south of Scotland, were infected withStrongwellsea castrans Batko and Weiser (Zygomycetes: Entomophthorales). The pathogen was associated mainly with females of the 1st and 2nd generations ofD. radicum and of the single generation ofD. floralis. Disease incidence showed time-lagged density dependence. Greatest sustained infection levels were recorded in the coolest wettest season. Higher proportions ofD. radicum were infected in the headland than in the crop. Unbaited yellow water traps caught greater proportions of infected flies than traps baited with mustard oil (allyl isothiocyanate). The reasons for high levels of infection in Scotland are discussed.

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Résumé Les mouches des racines,Delia radicum (L.) etD. floralis (Fallén) [Diptera: Anthomyiidae] attrapées dans la culture du rutabaga dans le sud de l'écosse, étaient contaminées parStrongwellsea castrans Batko & Weiser [Zygomycetes: entomophthorales]. Le pathogène était surtout associé aux fe nelles de la lère et de la 2ème générations deD. radicum et de la seule génération deD. floralis. Les cas de maladie montraient avec retard leur dépendance avec la densité des mouches. Les niveaux d'infection les plus grands étaient enregistrés pendant la saison la plus froide et la plus humide. Les bordures en friche des parcelles hébergeaient de plus grandes proportions deD. radicum infectés que les cultures. Des pièges jaunes sans appat ont attrapé de plus grandes proportions de mouches que lorsqu'ils étaient appatés avec de l'huile de moutarde (allyl-isothiocyanate). Les raisons qui expliquent pourquoi les niveaux d'infection en écosse sont plus élevés qu'ailleurs sont discutées.