Effect of Lincocin Treatment on the Greening Process in Bean (Phaseolus vulgaris) Leaves

The effect of lincocin (a plastid protein synthesis inhibitor) treatment on the greening process of bean (Phaseolus vulgaris L.) leaves have been studied. In comparison with control leaves treated ones had a decreased rate of chloroplast development. They had a marked chlorophyll deficiency and a decreased chlorophyll a/b ratio. Some long and short wavelength forms of chlorophyll a were lacking as evidenced from the absorption spectra at 25°C and the fluorescence spectra at 77°K. The –¹⁴CO₂ fixation was inhibited by 80–90% in treated leaves. The fluorescence induced by the measuring light was greater in the treated leaves than in the control ones, and the kinetics of the decline of the relative fluorescence intensity were also different. Electron microscopic studies showed macrogranum-like structures and incomplete membrane vesicles in the treated plastids. After longer treatment a destruction of membranes was observed. The results indicate some structural and functional membrane deficiencies and instability of the membranes.     

Lecithotrophic development and metamorphosis in the Indo-West Pacific brittle star Ophiomastix venosa (Echinodermata: Ophiuroidea)

Summary

The larval development of the ophiocomid ophiuroid Ophiomastix venosais described using SEM. The gastrula transforms into a uniformly ciliated early larva which progressively changes into a lecithotrophic late premetamorphic larva with a continuous bilateral ciliated band. This stage is short-lived and equivalent to a highly reduced ophiopluteus. Comparisons between O. venosa and other ophiuroid species whose development has been investigated suggest that, whatever the developmental mode (lecithotrophic or planktotrophic), a pluteus stage always occurs in ophiuroids with planktonic development. Two metamorphic stages were identified, the late metamorphic larva differing from the early one by the closure of the larval mouth. The appearance of the permanent mouth marks the end of the metamorphosis. The postlarva still possesses remnants of larval features. The transformation of the reduced ophiopluteus into a barrel-shaped metamorphic larva with transverse ciliated bands, a vitellaria larva, is followed. The possible occurrence of a unique type of metamorphic larva in non-brooding ophiuroids is discussed. Verification of this, however, needs further SEM investigations on metamorphic larva from species having “regular” planktotrophic development.     

FRETcalc plugin for calculation of FRET in non-continuous intracellular compartments

FRET has emerged as an important tool for studying intracellular processes and interactions between biomolecules. Intracellular donor and acceptor molecules are distributed in individual organelles that usually have complex non-continuous shape. Consequently, background pixels arising from fluorophore-free regions of the cell are proximal to FRET-positive pixels, leading to systemic errors in the estimated FRET values. This study introduces a new FRET(TH) algorithm for FRET estimation by acceptor photobleaching that separates the FRET-positive pixels from the background by applying user-defined thresholds for pixel selection. The FRET(TH) algorithm was validated by analysis of interactions between fluorescently tagged proteins in the endoplasmic reticulum using acquired and simulated images. The novel algorithm showed superior performance to the regular FRET calculation algorithm in acquired images and in most simulations. The developed algorithm was incorporated into the FRETcalc plugin for ImageJ program that enables user-defined choices of thresholds for calculation of FRET by acceptor photobleaching.     

'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.     

Aggregate formation by ERp57-deficient MHC class I peptide-loading complexes

The endoplasmic reticulum (ER)-resident proteins TAP, tapasin and ERp57 are the core components of the major histocompatibility complex (MHC) class I peptide-loading complex and play an important role in peptide loading by MHC class I-beta(2)microglobulin dimers. ERp57 and tapasin form a stable disulfide-linked heterodimer within the peptide-loading complex. We demonstrate that ERp57-deficient loading complexes, obtained by expression in a tapasin-negative cell line of a tapasin mutant (C95A) that is not able to form a disulfide bond with ERp57, are prone to aggregation. We studied the assembly, stability and aggregation of the core loading complex using cell lines stably expressing fluorescently tagged tapasin (wild type or C95A mutant) and TAP1. Part of the loading complexes containing the tagged C95A tapasin and TAP1 were sequestered in the ER, without change of their ER transmembrane topology, and were surrounded by a mesh of filaments at the cytosolic side, resulting in formation of protein aggregates with characteristic morphology. Protein aggregates were associated with changes in ER protein turnover but did not affect the cell viability and did not induce the unfolded protein response. Fluorescence resonance energy transfer analysis of the aggregate-free ER fraction revealed that lack of ERp57 did not affect the stoichiometry or stability of tapasin-TAP1 interactions in the assembled 'soluble' core loading complexes. We conclude that the presence of ERp57 is important for the stability of core loading complexes, and that in its absence, the core loading complexes may form stable aggregates within the ER.