Cytosolic Compartment Of Cells Research Articles

Unesterified Cholesterol Accumulation in Late Endosomes/Lysosomes Causes Neurodegeneration and Is Prevented by Driving Cholesterol Export from This Compartment

While unesterified cholesterol (C) is essential for remodeling neuronal plasma membranes, its role in certain neurodegenerative disorders remains poorly defined. Uptake of sterol from pericellular fluid requires processing that involves two lysosomal proteins, lysosomal acid lipase, which hydrolyzes C esters, and NPC1 (Niemann-Pick type C1). In systemic tissues, inactivation of either protein led to sterol accumulation and cell death, but in the brain, inactivation of only NPC1 caused C sequestration and neurodegeneration. When injected into the CNS of the npc1(-/-) mouse, 2-hydroxypropyl-β-cyclodextrin (HP-β-CD), a compound known to prevent this C accumulation, diffused throughout the brain and was excreted with a t(½) of 6.5 h. This agent caused suppression of C synthesis, elevation of C esters, suppression of sterol regulatory-binding protein 2 (SREBP2) target genes, and activation of liver X receptor-controlled genes. These findings indicated that HP-β-CD promoted movement of the sequestered C from lysosomes to the metabolically active pool of C in the cytosolic compartment of cells in the CNS. The ED(50) for this agent in the brain was ∼0.5 mg/kg, and the therapeutic effect lasted >7 d. Continuous infusion of HP-β-CD into the ventricular system of npc1(-/-) animals between 3 and 7 weeks of age normalized the biochemical abnormalities and completely prevented the expected neurodegeneration. These studies support the concept that neurons continuously acquire C from interstitial fluid to permit plasma membrane turnover and remodeling. Inactivation of NPC1 leads to lysosomal C sequestration and neurodegeneration, but this is prevented by the continuous, direct administration of HP-β-CD into the CNS.

Mesoporous magnetic hollow nanoparticles—protein carriers for lysosome escaping andcytosolic delivery

It is important for a controlled release system to determine whether nanoparticles canpenetrate cell membranes and deliver protein into the nuclear or cytosolic compartments ofcells, and thus function as carriers. Here, we prepared different functionalizedmesoporous magnetic hollow nanoparticles (MMHs) and chose bovine serum albumin(BSA) as a model protein to detect the intracellular trafficking of MMHs. Theresults showed that MMHs modified with amino groups (AMMHs) were efficient inprotein loading and that the loading was dependent on the pH, temperature andionic strength. Furthermore, we found that the AMMHs not only transportedBSA into the cells but also released the BSA carried into the nuclear or cytosoliccompartments of the cells. In addition, the nanoparticles were biocompatible,and the encapsulation of BSA in AMMHs did not affect their bioactivity. Takentogether, AMMHs are excellent carriers for releasing protein into the cytosol andnucleus, and they have the potential to be used in a controlled release system.

Characterization and temperature-dependent quantification of heat shock protein 60 of the immunogenic fungusAlternaria alternata

Heat shock proteins or chaperones are found in mitochondrial and cytosolic compartments of cells. They are responsible for the correct folding of proteins and are up-regulated in reaction to various stressors. In addition, when released or presented on the surface of cells, they may play an important role in inflammatory and immunomodulating processes. To identify and characterize hsp60 in the common environmental mold Alternaria alternata, the fungus was cultivated and incubated at different temperatures to induce a possible heat shock response. Fully automated RNA extraction was followed by quantitative real-time RT-PCR targeting A. alternata specific Hsp60 mRNA and subsequent sequencing of the amplicon. While Hsp60 mRNA was constitutively expressed in all samples tested, a temperature-dependent expression of Hsp60 mRNA was observed. Sequencing revealed an identity of more than 85% to other fungal hsp60, indicating the existence of this protein in A. alternata.

Identification of small non-coding RNAs from mitochondria and chloroplasts

Small non-protein-coding RNAs (ncRNAs) have been identified in a wide spectrum of organisms ranging from bacteria to humans. In eukarya, systematic searches for ncRNAs have so far been restricted to the nuclear or cytosolic compartments of cells. Whether or not small stable non-coding RNA species also exist in cell organelles, in addition to tRNAs or ribosomal RNAs, is unknown. We have thus generated cDNA libraries from size-selected mammalian mitochondrial RNA and plant chloroplast RNA and searched for small ncRNA species in these two types of DNA-containing cell organelles. In total, we have identified 18 novel candidates for organellar ncRNAs in these two cellular compartments and confirmed expression of six of them by northern blot analysis or RNase A protection assays. Most candidate ncRNA genes map to intergenic regions of the organellar genomes. As found previously in bacteria, the presumptive ancestors of present-day chloroplasts and mitochondria, we also observed examples of antisense ncRNAs that potentially could target organelle-encoded mRNAs. The structural features of the identified ncRNAs as well as their possible cellular functions are discussed. The absence from our libraries of abundant small RNA species that are not encoded by the organellar genomes suggests that the import of RNAs into cell organelles is of very limited significance or does not occur at all.

The tum− antigens P91A and P198 derive from proteins located in the cytosolic compartment of cells

To characterize the proteins P91Ap and P198p, of which mutants generate the tum- antigens P91A and P198, respectively, rabbit antisera were raised with ovalbumin-coupled synthetic peptides that correspond to their respective C terminus. In immunoadsorption tests using immobilized protein A the antisera recognized the translation products synthesized by rabbit reticulocyte lysates programmed with the SP6 polymerase transcripts of the P91A and P198 cDNA. The presence of the two proteins was demonstrated by SDS-PAGE and immunoblotting in all the mouse cells and organs examined. P91Ap is a constituent of the cytosol; despite a remarkable homology to the Drosophila diphenol oxidase DOX-A2, it separates from murine catechol oxidase activity in rate zonal sedimentation analysis. P198p is a ribosomal constituent, or a factor firmly linked to both the free and membrane-bound ribosomes. These subcellular localizations strengthen other evidence that the antigens presented to T lymphocytes by class I products of the major histocompatibility complex derive from proteins of the cytosol, or in direct contact with it.

Measurement of electrically-induced Ca 2+ transients in quin2-loaded cardiac myocytes

Quin2 is a fluorescent Ca2+-sensitive indicator which in its acetoxymethyl ester form can be loaded into the cytosolic compartment of cells without any disruption of the plasma membrane. Quin2 has been used with many cell types to measure changes of [Ca2+]i in response to hormones and other stimuli. In this report we demonstrate that Quin2 can be used to follow Ca2+ transients induced by electrical depolarization of excitable cells such as myocytes. Unlike microelectrodes and the microinjection of Ca2+ indicators, the use of Quin2 allows the collection of signals from large numbers of cells simultaneously. Ca2+ transients measured using Quin2 show a rapid rise from a basal [Ca2+]i of 200 nM to peak values of up to 500 nM within 40 ms in the presence of the beta-adrenergic agonist isoproterenol. As the intracellular Quin2 load is increased, Quin2 acts to buffer the free Ca2+ change, causing a decrease of the peak Ca2+ concentration and an increase of the time required for relaxation of the transient. Thus, Quin2 can serve both to monitor and to perturb Ca2+ transients in heart cells.