A LIGHT AND ELECTRON MICROSCOPE STUDY OF THE MORPHOLOGICAL CHANGES INDUCED IN RAT LIVER CELLS BY THE AZO DYE 2-ME-DAB

The cytological changes induced in rat liver cells by the aminoazo dye 2-Me-DAB have been examined by light and electron microscopy. It is observed that this non-carcinogenic compound duplicates most of the morphological alterations produced by other hepatotoxins, some of which, such as the closely related aminoazo dye 3'-Me-DAB, are potent carcinogens. These non-specific effects involve both the granular and agranular forms of the endoplasmic reticulum as well as the glycogen content of hepatic cells. The arrays of cisternal profiles of the granular reticulum in normal hepatic cells become disorganized and the dispersed cisternae often appear fragmented and irregular. Large cytoplasmic inclusions, consisting of loosely organized tubules and vesicles, are also observed which result from a hypertrophy of the agranular reticulum. The glycogen in the cells progressively decreases in amount. The most specific effect of 2-Me-DAB is to induce an increase in the number of mitochondria per cell. Many of these organelles are characterized by the presence of a median double membrane continuous with the inner limiting membrane of the mitochondrial envelope. Evidence is presented in favor of the view that this partition is directly related to the phenomenon of mitochondrial division. It was noted also in the course of the experiment that an increasing number of cells appear which stain quite intensely with methylene blue and appear denser than normal under electron microscopy. The significance of these cells is not known.     

Composition of nuclear dense bodies and nucleolus-associated bodies in interphase nuclei of the unicellular green alga Chlamydomonas reinhardtii

Summary— The interphase nucleus of the green alga, Chlamydomonas reinhardtii, displayed two types of bodies some of them, the dense bodies, lying apparently free in the nucleoplasm while the others were attached to the nucleolus and were, therefore, referred to as nucleolus-associated bodies (NABs). The presence of DNA, RNA and histones in dense bodies was investigated by means of post-embedding immunocytochemistry and cytochemistry using a monoclonal antibody to single and double stranded DNA, a polyclonal antibody to rye H3 histones and RNase A-gold complexes. The dense bodies were shown to contain significant amounts of RNA but neither DNA nor histones were detected; their composition was thus similar to that of the dense bodies described in higher plant cells. We propose that dense bodies might be implicated in the assembly of the 25 to 45 nm granules observed throughout the nucleoplasm of Chalamydomonas interphase nuclei. The composition of NABs was found to be distinct from that of the dense bodies since they were labeled by the antibody to DNA, specially in cryofixed and cryosubstituted specimens. The presence of DNA in NABs together with their intimate association to the nucleolus suggest that they may correspond to specific segments of chromosomes.     

IL-1 production by human thymic dendritic cells: studies on the interrelation with DC accessory function

Thymic dendritic cells (DC) have been proposed to play a critical role in the generation of immunocompetent T lymphocytes. Since IL-1 is widely considered to be an important second signal in T cell stimulation, we have studied the ability of isolated human thymic DC to produce IL-1. Using the EL4/CTLL conversion assay standardized with recombinant IL-1 beta (rIL-1 beta), we demonstrate that upon LPS-stimulation thymic DC produce small amounts of IL-1 as compared to peripheral blood monocytes (PBM). In contrast with PBM, DC IL-1 production is not influenced by indomethacin. IL-1 activity was detected in the supernatants of DC cultures from all thymuses tested, although quantitative variability was noted among individual thymic donors. The specificity of the active factor was confirmed by neutralization assays with anti-IL-1 beta mAb. On the other hand, we demonstrate that rIL-1 beta cannot substitute for nor amplify the accessory function of thymic DC and that anti-IL-1 beta mAb fails to block the DC accessory function. Thus we conclude that IL-1 beta might not be a major factor for the efficient DC accessory function toward mature thymocytes recently demonstrated in our laboratory. Of interest, IL-1 beta was also detected in the supernatants of DC-thymocyte cocultures in the absence of mitogenic factor, suggesting that thymocyte contacts can constitute a sufficient signal to induce DC to produce IL-1. These observations indicate that human thymic DC represent an intrathymic source of IL-1 whose role in thymocyte proliferation or maturation remains to be understood.     

Ultrastructural localization ofl -fucose residues in nuclei of root primordia of the green peaPisum sativum

Summary Sugars have been demonstrated in animal cell nuclei, but only a few studies have mentioned their presence in plant cell nuclei. In this studyl-fucose residues were localized at the ultrastructural level, usingUlex europeaus agglutinin I lectin, during the early stages of germination ofPisum sativum and in mature root tip cells. This sugar was present after 1 h of germination, and its concentration was found to vary during 3 to 6 h imbition; after 72 h of imbition its concentration had more than doubled. Furthermore, labelling was particularly abundant in the nucleolus, nucleolus-associated bodies and dense nuclear bodies. The possibility that some of thel-fucose residues are associated with proteins is discussed.     

Intramolecular folding in human ILPR fragment with three C-rich repeats

Enrichment of four tandem repeats of guanine (G) rich and cytosine (C) rich sequences in functionally important regions of human genome forebodes the biological implications of four-stranded DNA structures, such as G-quadruplex and i-motif, that can form in these sequences. However, there have been few reports on the intramolecular formation of non-B DNA structures in less than four tandem repeats of G or C rich sequences. Here, using mechanical unfolding at the single-molecule level, electrophoretic mobility shift assay (EMSA), circular dichroism (CD), and ultraviolet (UV) spectroscopy, we report an intramolecularly folded non-B DNA structure in three tandem cytosine rich repeats, 5'-TGTC4ACAC4TGTC4ACA (ILPR-I3), in the human insulin linked polymorphic region (ILPR). The thermal denaturation analyses of the sequences with systematic C to T mutations have suggested that the structure is linchpinned by a stack of hemiprotonated cytosine pairs between two terminal C4 tracts. Mechanical unfolding and Br(2) footprinting experiments on a mixture of the ILPR-I3 and a 5'-C4TGT fragment have further indicated that the structure serves as a building block for intermolecular i-motif formation. The existence of such a conformation under acidic or neutral pH complies with the strand-by-strand folding pathway of ILPR i-motif structures.     

A CORRELATED LIGHT AND ELECTRON MICROSCOPE STUDY OF THE NUCLEOLAR MATERIAL DURING MITOSIS IN VICIA FABA

Root meristematic cells of Vicia faba were examined, with both light and electron microscopes, in order to study the behaviour of the nucleolar material during the mitotic process. Under light microscopy, the preprophase nucleolus is seen to consist of a densely stained material in which are embedded several unstained vacuole-like structures of varying size. The electron microscope reveals that the dense nucleolar material is formed of two structurally distinct components, each segregated into irregularly shaped zones blending with one another. One of these components is represented by 150 A granules which, in places, are arranged into thread-like structures approximately 0.1 µ in diameter; the other component apparently consists of fibrils 60 to 100 A in diameter. The large and medium sized intranucleolar vacuoles contain loosely scattered granules and fibrils similar to those just described. The granular and fibrillar components of the denser portion of the nucleolus persist as such during prophase and disperse throughout the nuclear cavity at the time of nucleolar disintegration. After nuclear membrane breakdown, these granules and fibrils, as well as those of the nucleoplasm, mix freely with similar elements already present within the forming spindle. No evidence has been obtained that, during or after nucleolar disintegration, the structural components of the nucleolus become associated as such with the chromosomes to form an external or internal matrix. Our observations suggest the existence, of a matrix substance within late prophase, metaphase, and anaphase chromosomes, the fine structure of which bears strong resemblance to that of their constituent coiled chromonemata. Data are presented, moreover, that indicate that part of this matrix substance, presumably formed at some time during prophase, is released from the chromosomes during their anaphasic movement. A number of observations indicate that the main bulk of the next nucleolus is derived from a prenucleolar fibrillogranular material, arranged into thread-like structures some 0.1 µ in diameter, which collect in the interchromosomal spaces during early and midtelophase. Finally, our data would seem to favour the view that most of this prenucleolar material results from a resumption of the synthetic activity of the early and midtelophase chromosomes rather than from a mere shedding of a preexisting matrix substance.     

Optimization-driven identification of genetic perturbations accelerates the convergence of model parameters in ensemble modeling of metabolic networks

The ensemble modeling (EM) approach has shown promise in capturing kinetic and regulatory effects in the modeling of metabolic networks. Efficacy of the EM procedure relies on the identification of model parameterizations that adequately describe all observed metabolic phenotypes upon perturbation. In this study, we propose an optimization-based algorithm for the systematic identification of genetic/enzyme perturbations to maximally reduce the number of models retained in the ensemble after each round of model screening. The key premise here is to design perturbations that will maximally scatter the predicted steady-state fluxes over the ensemble parameterizations. We demonstrate the applicability of this procedure for an Escherichia coli metabolic model of central metabolism by successively identifying single, double, and triple enzyme perturbations that cause the maximum degree of flux separation between models in the ensemble. Results revealed that optimal perturbations are not always located close to reaction(s) whose fluxes are measured, especially when multiple perturbations are considered. In addition, there appears to be a maximum number of simultaneous perturbations beyond which no appreciable increase in the divergence of flux predictions is achieved. Overall, this study provides a systematic way of optimally designing genetic perturbations for populating the ensemble of models with relevant model parameterizations.