Home About us Contact
|
Home About us Contact | ||
|
|
||
Intracellular Distribution (intracellular + distribution)
Selected AbstractsIntracellular Distribution of Macrophage Targeting Ferritin,Iron Oxide NanocompositeADVANCED MATERIALS, Issue 4 2009Masaki Uchida Intracellular distribution of iron oxide nanoparticles incorporated within a ferritin mutant that displays genetically introduced cell-targeted peptides (RGD-4C) on its exterior surface are investigated using scanning transmission electron microscopy with a high-angle annular dark-field detector. The particles (indicated by arrows) internalized into macrophages much more effectively than those with noncell-targeted ferritin. [source] Intracellular distribution of peroxynitrite during doxorubicin cardiomyopathy: evidence for selective impairment of myofibrillar creatine kinaseBRITISH JOURNAL OF PHARMACOLOGY, Issue 3 2002Michael J Mihm Cardiac peroxynitrite and protein nitration are increased during doxorubicin cardiotoxicity, but the intracellular targets and functional consequences have not been defined. We investigated the intracellular distribution of protein nitration during doxorubicin cardiotoxicity in mice. Following in vivo cardiac function assessments by echocardiography, cardiac tissues were prepared for immunohistochemistry and electron microscopy 5 days after doxorubicin (20 mg kg,1) or vehicle control. Increased cardiac 3-nitrotyrosine was observed using light microscopy in doxorubicin treated animals. Immunogold electron microscopy (55,000×) revealed increased myofibrillar and mitochondrial 3-nitrotyrosine levels following doxorubicin, but cellular 3-nitrotyrosine density was 2 fold higher in myofibrils. We therefore investigated the actions of peroxynitrite on intact cardiac contractile apparatus. Skinned ventricular trabeculae were exposed to physiologically relevant peroxynitrite concentrations (50 or 300 nM) for 1 h, then Ca2+ induced contractile responses were measured in the presence of ATP (4 mM) or phosphocreatine (12 mM) as high energy phosphate supplier. ATP maximal force generation was unaltered after 50 nM peroxynitrite, but phosphocreatine/ATP response was reduced (0.99±0.63 vs 1.59±0.11), suggesting selective inactivation of myofibrillar creatine kinase (MM-CK). Reduction of ATP maximal force was observed at 300 nM peroxynitrite and phosphocreatine/ATP response was further reduced (0.64±0.30). Western blotting showed concentration dependent nitration of MM-CK in treated trabeculae. Similarly, cardiac tissues from doxorubicin treated mice demonstrated increased nitration and inactivation of MM-CK compared to controls. These results demonstrate that peroxynitrite-related protein nitration are mechanistic events in doxorubicin cardiomyopathy and that the cardiac myofibril is an important oxidative target in this setting. Furthermore, MM-CK may be a uniquely vulnerable target to peroxynitrite in vivo. British Journal of Pharmacology (2002) 135, 581,588; doi:10.1038/sj.bjp.0704495 [source] Human and Drosophila UDP-galactose transporters transport UDP- N -acetylgalactosamine in addition to UDP-galactoseFEBS JOURNAL, Issue 1 2002Hiroaki Segawa A putative Drosophila nucleotide sugar transporter was characterized and shown to be the Drosophila homologue of the human UDP-Gal transporter (hUGT). When the Drosophila melanogaster UDP-Gal transporter (DmUGT) was expressed in mammalian cells, the transporter protein was localized in the Golgi membranes and complemented the UDP-Gal transport deficiency of Lec8 cells but not the CMP-Sia transport deficiency of Lec2 cells. DmUGT and hUGT were expressed in Saccharomyces cerevisiae cells in functionally active forms. Using microsomal vesicles isolated from Saccharomyces cerevisiae expressing these transporters, we unexpectedly found that both hUGT and DmUGT could transport UDP-GalNAc as well as UDP-Gal. When amino-acid residues that are conserved among human, murine, fission yeast and Drosophila UGTs, but are distinct from corresponding ones conserved among CMP-Sia transporters (CSTs), were substituted by those found in CST, the mutant transporters were still active in transporting UDP-Gal. One of these mutants in which Asn47 was substituted by Ala showed aberrant intracellular distribution with concomitant destabilization of the protein product. However, this mutation was suppressed by an Ile51 to Thr second-site mutation. Both residues were localized within the first transmembrane helix, suggesting that the structure of the helix contributes to the stabilization and substrate recognition of the UGT molecule. [source] The centrosomal protein Lats2 is a phosphorylation target of Aurora-A kinaseGENES TO CELLS, Issue 5 2004Shingo Toji Human Lats2, a novel serine/threonine kinase, is a member of the Lats kinase family that includes the Drosophila tumour suppressor lats/warts. Lats1, a counterpart of Lats2, is phosphorylated in mitosis and localized to the mitotic apparatus. However, the regulation, function and intracellular distribution of Lats2 remain unclear. Here, we show that Lats2 is a novel phosphorylation target of Aurora-A kinase. We first showed that the phosphorylated residue of Lats2 is S83 in vitro. Antibody that recognizes this phosphorylated S83 indicated that the phosphorylation also occurs in vivo. We found that Lats2 transiently interacts with Aurora-A, and that Lats2 and Aurora-A co-localize at the centrosomes during the cell cycle. Furthermore, we showed that the inhibition of Aurora-A-induced phosphorylation of S83 on Lats2 partially perturbed its centrosomal localization. On the basis of these observations, we conclude that S83 of Lats2 is a phosphorylation target of Aurora-A and this phosphorylation plays a role of the centrosomal localization of Lats2. [source] Effect of immune serum and role of individual Fc, receptors on the intracellular distribution and survival of Salmonella enterica serovar Typhimurium in murine macrophagesIMMUNOLOGY, Issue 2 2006Hazel Uppington Summary Immune serum has a protective role against Salmonella infections in mice, domestic animals and humans. In this study, the effect of antibody on the interaction between murine macrophages and S. enterica serovar Typhimurium was examined. Detailed analysis at the single-cell level demonstrated that opsonization of the bacteria with immune serum enhanced bacterial uptake and altered bacterial distribution within individual phagocytic cells. Using gene-targeted mice deficient in individual Fc gamma receptors it was shown that immune serum enhanced bacterial internalization by macrophages via the high-affinity immunoglobulin G (IgG) receptor, Fc gamma receptor I. Exposure of murine macrophages to S. enterica serovar Typhimurium opsonized with immune serum resulted in increased production of superoxide, leading to enhanced antibacterial functions of the infected cells. However, opsonization of bacteria with immune serum did not increase either nitric oxide production in response to S. enterica serovar Typhimurium or fusion of phagosomes with lysosomes. [source] Subcellular distribution of key enzymes of lipid metabolism during the euthermia-hibernation-arousal cycleJOURNAL OF ANATOMY, Issue 6 2009Anna Suozzi Abstract Mammalian hibernation is a natural, fully reversible hypometabolic state characterized by a drastic reduction of body temperature and metabolic activity, which ensures survival to many species under adverse environmental conditions. During hibernation, many hibernators rely for energy supply almost exclusively on lipid reserves; the shift from carbohydrate to lipid metabolism implies profound rearrangement of the anabolic and catabolic pathways of energetic substrates. However, the structural counterpart of such adaptation is not known. In this study we investigated, by using immunoelectron microscopy, the fine intracellular distribution of two key enzymes involved in lipid metabolism, namely, the fatty acid synthase (FAS) and the long-chain fatty acyl-CoA synthetase (ACSL), in hepatocytes of euthermic, hibernating and arousing hazel dormice. Our results show that the two enzymes are differentially distributed in cellular compartments (cytoplasm, mitochondria and cell nuclei) of hepatocytes during euthermia. Quantitative redistribution of both enzymes among cellular compartments takes place during hibernation and arousal, in accordance with the physiological changes. Interestingly, this redistribution follows different seasonal patterns in cytoplasm, mitochondria and nuclei. In conclusion, our data represent the first quantitative morphological evidence of lipid enzyme distribution in a true hibernator throughout the year cycle, thus providing a structural framework to biochemical changes associated with the hypometabolism of hibernation. [source] Involvement of protein kinase C-, in DNA damage-induced apoptosisJOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 4 2003Alakananda Basu Abstract Apoptosis is a highly orchestrated cell suicidal program required to maintain a balance between cell proliferation and cell death. A defect in apoptotic machinery can cause cancer. Many anticancer drugs are known to kill tumor cells by inducing apoptosis, and a defect in apoptosis can lead to anticancer drug resistance. Apoptosis is regulated by a complex cellular signaling network. Several members of the protein kinase C (PKC) family serve as substrates for caspases and PKC, isozyme has been intimately associated with DNA damage-induced apoptosis. It can act both upstream and downstream of caspases. In response to apoptotic stimuli, the full-length and the catalytic fragment of PKC, may translocate to distinct cellular compartments, including mitochondria and the nucleus, to reach their targets. Both activation and intracellular distribution of PKC, may have significant impact on apoptosis. This review intends to assimilate recent views regarding the involvement of PKC, in DNA damage-induced apoptosis. [source] Role of phospholipases A2 in growth-dependent changes in prostaglandin release from 3T6 fibroblastsJOURNAL OF CELLULAR PHYSIOLOGY, Issue 2 2001Teresa Sánchez Previously, we reported a growth-dependent change in prostaglandin production as a consequence of a marked growth-dependent alteration in arachidonic acid (AA) mobilization from phospholipids. Our present results show that fetal calf serum (FCS) and 4,-phorbol-12-myristate acetate (PMA) caused an enhancement of phospholipase A2 (PLA2) activity in the membrane fraction of non-confluent cells allowing PLA2 access to its substrate and the release of AA. Western blot analysis has shown that FCS and PMA increased secreted PLA2 (sPLA2) expression in non-confluent 3T6 fibroblast cultures. Moreover, FCS and PMA induced dithiothreitol-sensitive and bromoenol lactone-sensitive PLA2 activities in cytosol and membrane fraction. However, these stimuli did not modify significantly the PLA2 activity in both fractions when 3T6 fibroblasts reached a high cell density. This could be associated with the impairment of AA mobilization in these cell culture conditions. On the other hand, we observed that FCS and PMA induced the same prostaglandin H synthase-2 induction in non-confluent and confluent culture conditions. Moreover, the prostaglandin E2 levels reached in cell culture supernatants were independent of the degree of confluence when AA was added exogenously. These results suggest that the changes of intracellular distribution of PLA2 activity of sPLA2 and iPLA2 stimulated by exogenous stimuli may be controlled by cell density conditions which constitute an important mechanism in the regulation of prostaglandin release.© 2001 Wiley-Liss, Inc. [source] Anoxia leads to a rapid translocation of human trypsinogen 4 to the plasma membrane of cultured astrocytesJOURNAL OF NEUROCHEMISTRY, Issue 2 2010Krisztián Tárnok J. Neurochem. (2010) 115, 314,324. Abstract Trypsinogen 4 is specifically expressed in the human brain, mainly by astroglial cells. Although its exact role in the nervous tissue is yet unclear, trypsin 4-mediated pathological processes were suggested in Alzheimer's disease, multiple sclerosis and ischemic injury. In the present study, we analyzed the intracellular distribution of fluorescently tagged human trypsinogen 4 isoforms during normal and anoxic conditions in transfected mouse primary astrocytes. Our results show that initiation of anoxic milieu by the combined action of KCN treatment and glucose deprivation rapidly leads to the association of leader peptide containing trypsinogen 4 constructs to the plasma membrane. Using rhodamine 110 bis-(CBZ-L-isoleucyl-L-prolyl-L-arginine amide), a synthetic chromogen peptide substrate of trypsin, we show that anoxia can promote extracellular activation of trypsinogen 4 indicating that extracellular activation of human trypsinogen 4 can be an important component in neuropathological changes of the injured human brain. [source] Expression of gp130 and leukaemia inhibitory factor receptor subunits in adult rat sensory neurones: regulation by nerve injuryJOURNAL OF NEUROCHEMISTRY, Issue 1 2002Natalie J. Gardiner Abstract Members of the interleukin-6 (IL-6) family of cytokines have been implicated as major mediators of the response of the adult nervous system to injury. The basis for an interaction of IL-6 cytokines with adult sensory neurones has been established by analysing the levels and distribution of the two signal-transducing receptor subunits, glycoprotein 130 (gp130) and leukaemia inhibitory factor receptor (LIFR), in the dorsal root ganglion (DRG) of male adult rats before and following nerve injury. All sensory neurones express gp130-immunoreactivity (IR) in the cytoplasm and on the plasma membrane. Levels of gp130 and its intracellular distribution remained unchanged up to 14 days following sciatic nerve axotomy. LIFR-IR was largely absent from the cytoplasm and plasma membrane of sensory neurones, but confined almost exclusively to the nuclear compartment. However, following axotomy, punctate cytoplasmic LIFR-IR was detected which persisted up to 28 days following axotomy. The expression of cytoplasmic LIFR 2 days post-axotomy was proportionally greater in a subset of small diameter sensory neurones which expressed either the sensory neuropeptide CGRP or the cell surface marker isolectin B4. The coexpression of gp130 and LIFR in the same intracellular compartment following axotomy conveys potential responsiveness of injured sensory neurones to members of the IL-6 family of cytokines. [source] Melanophores: A model system for neuronal transport and exocytosis?JOURNAL OF NEUROSCIENCE RESEARCH, Issue 12 2007Sara Aspengren Abstract Black pigment cells, melanophores, from lower vertebrates are specialized in bidirectional and coordinated translocation of pigment granules, melanosomes, in the cytoplasm. Melanophores develop from the neuronal crest and are most abundant in the dermal and epidermal layers of the skin, where the intracellular distribution of the pigment significantly influences the color of the animal. The transport of pigment is dependent on an intact cytoskeleton and motor proteins associated with cytoskeletal components. The easily cultured melanophores have proved to be excellent models for organelle transport because the intracellular movements of pigment can be visualized via light microscopy, and the granules move in response to defined chemical signals. The ease of achieving a combination of morphological and functional transport studies is the advantage of the melanophore system, and studies on pigment cells have revealed new components of the transport machinery, including molecular motors, their adapters, and transfer of vesicles to other cells. Many cellular components are transported with a combination of the actin- and microtubule-based transport systems, and, since all eukaryotic organisms rely on functional intracellular transport and an intact cytoskeleton, studies on melanophores are important for many aspects of cell biology, including axonal transport. In this review, we present an overview of the research on the pigment transport system and the potential use of pigment cells as a model system. © 2006 Wiley-Liss, Inc. [source] Altered expression of collagen XVII in ameloblastomas and basal cell carcinomasJOURNAL OF ORAL PATHOLOGY & MEDICINE, Issue 10 2001Mataleena Parikka Abstract: Background: Collagen XVII (BP180) is an epithelial transmembrane protein, which presumably plays a role in cell migration and differentiation under both physiological and pathological conditions. Ameloblastoma, the most common odontogenic neoplasm, and basal cell carcinoma (BCC) of the skin exhibit similar growth patterns and share histological features. Methods: Here, we examined the distribution and expression of collagen XVII in ameloblastomas and BCCs using immunohistochemistry and non-radioactive in situ hybridization. In both tumors, the distribution of collagen XVII varied in different parts of the lesions. Results: In ameloblastomas, immunostaining for collagen XVII was usually localized in the basal and suprabasal cells of the tumor nests, although in some tumors, a diffuse intracellular staining was detected in the central cells of the neoplastic islands. In BCCs, collagen XVII was mostly seen as diffuse cytoplasmic staining in some central and peripheral cells of the tumor islands and also at the cell membranes in the basal keratinocytes of the epidermis overlying the tumor nests. Double immunostaining with antibody against ,2 chain of laminin-5 showed that these two components of the keratinocyte adhesion complex are usually co-localized in ameloblastomas and BCCs. In both tumors, collagen XVII mRNA was found in the basal epithelial cells and in some central and peripheral cells of the tumor islands, while the stromal cells were negative. Conclusions: These findings indicate that the expression of collagen XVII may be differentially regulated in various parts of the tumor. Diffuse intracellular distribution of collagen XVII and a consequent loss of critical cellular attachments may contribute to the infiltrative and progressive growing potential of tumors. [source] Lysosomal sequestration of amine-containing drugs: Analysis and therapeutic implicationsJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 4 2007Allyn M. Kaufmann Abstract Amine-containing drugs represent a very important class of therapeutic agents, with the majority of all drugs containing at least one basic nitrogen. For many decades, it has been known that weakly basic compounds can be sequestered into acidic organelles such as lysosomes. Some amines can achieve very high concentrations and induce a dramatic expansion (vacuolization) of the compartment. In the early 70s, Nobel laureate and discoverer of lysosomes, Christian de Duve et al. wrote an elegant commentary describing the theoretical basis for lysosomal sequestration of amines, referring to the process as pH-partitioning and the substrates as lysosomotropics. Recently, a resurgence of interest in the intracellular distribution of drugs has occurred considering its therapeutic importance. Specifically, lysosomal sequestration of amines has received considerable attention for reasons including its involvement in drug resistance, inducement of phospholipidosis, and its influence on whole body distribution/pharmacokinetics. Moreover, the sequestration phenomenon has been recently exploited in the development of a novel drug targeting strategy. This review will focus on these occurrences/developments and conclude with a commentary on the expected impact that knowledge regarding the intracellular distribution of drugs will likely have on future drug development processes. © 2006 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 96: 729,746, 2007 [source] Platinum-based anticancer agents: Innovative design strategies and biological perspectivesMEDICINAL RESEARCH REVIEWS, Issue 5 2003Yee-Ping Ho Abstract The impact of cisplatin on cancer chemotherapy cannot be denied. Over the past 20 years, much effort has been dedicated to discover new platinum-based anticancer agents that are superior to cisplatin or its analogue, carboplatin. Most structural modifications are based on changing one or both of the ligand types coordinated to platinum. Altering the leaving group can influence tissue and intracellular distribution of the drug, whereas the carrier ligand usually determines the structure of adducts formed with DNA. DNA,Pt adducts produced by cisplatin and many of its classical analogues are almost identical, and would explain their similar patterns of tumor sensitivity and susceptibility to resistance. Recently some highly innovative design strategies have emerged, aimed at overcoming platinum resistance and/or to introduce novel mechanisms of antitumor action. Platinum compounds bearing the 1,2-diaminocyclohexane carrier ligand; and those of multinuclear Pt complexes giving rise to radically different DNA,Pt adducts, have resulted in novel anticancer agents capable of circumventing cisplatin resistance. Other strategies have focused on integrating biologically active ligands with platinum moieties intended to selectively localizing the anticancer properties. With the rapid advance in molecular biology, combined with innovation, it is possible new Pt-based anticancer agents will materialize in the near future. © 2003 Wiley Periodicals, Inc. Med Res Rev, 23, No. 5, 633,655, 2003 [source] Immunocytochemical analysis of the circadian clock protein in mouse hepatocytesMICROSCOPY RESEARCH AND TECHNIQUE, Issue 5 2003Manuela Malatesta Abstract Many biochemical, physiological, and behavioral processes in organisms ranging from prokaryotes to humans exhibit circadian rhythms, defined as cyclic oscillations of about 24 hours. The mechanism of the cellular circadian clock relies on interlocking positive and negative transcriptional/translational feedback loops based on the regulated expression of several genes. Clock is one of these genes and its transcript, CLOCK protein, is a transcription factor belonging to the bHLH-PAS family. In mammals the clock gene is expressed in several tissues, including the liver. In the present study, we analyzed by means of quali-quantitative immunoelectron microscopy the fine intracellular distribution of the CLOCK protein in mouse hepatocytes during the daily cycle. We demonstrated that CLOCK protein is mostly located in the cell nucleus, where it accumulates on perichromatin fibrils, representing the in situ form of nascent pre-mRNA, while condensed chromatin and nucleoli contain lower amounts of protein. Moreover, we found that CLOCK protein shows circadian oscillations in these nuclear compartments, peaking in late afternoon. At this time the hepatic transcriptional rate reaches the maximal level, thus suggesting an important role of CLOCK protein in the regulation of liver gene expression. Microsc. Res. Tech. 61:414,418, 2003. © 2003 Wiley-Liss, Inc. [source] Branching sites and morphological abnormalities behave as ectopic poles in shape-defective Escherichia coliMOLECULAR MICROBIOLOGY, Issue 4 2004Trine Nilsen Summary Certain mutants in Escherichia coli lacking multiple penicillin-binding proteins (PBPs) produce misshapen cells containing kinks, bends and branches. These deformed regions exhibit two structural characteristics of normal cell poles: the peptidoglycan is inert to dilution by new synthesis or turnover, and a similarly stable patch of outer membrane caps the sites. To test the premise that these aberrant sites represent biochemically functional but misplaced cell poles, we assessed the intracellular distribution of proteins that localize specifically to bacterial poles. Green fluorescent protein (GFP) hybrids containing polar localization sequences from the Shigella flexneri IcsA protein or from the Vibrio cholerae EpsM protein formed foci at the poles of wild-type E. coli and at the poles and morphological abnormalities in PBP mutants. In addition, secreted wild-type IcsA localized to the outer membrane overlying these aberrant domains. We conclude that the morphologically deformed sites in these mutants represent fully functional poles or pole fragments. The results suggest that prokaryotic morphology is driven, at least in part, by the controlled placement of polar material, and that one or more of the low-molecular-weight PBPs participate in this process. Such mutants may help to unravel how particular proteins are targeted to bacterial poles, thereby creating important biochemical and functional asymmetries. [source] Differential cellular compartmentalization of the nuclear receptor SpSHR2 splicing variants in early sea urchin embryosMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 2 2001Aikaterini Kontrogianni-Konstantopoulos Abstract SpSHR2 is a member of the nuclear receptor superfamily, expressed in embryos, larvae, and adult tissues of sea urchin. During embryonic development, two receptor isoforms are produced via alternative splicing. One exhibits the typical structure of nuclear receptors (SpSHR2-full length), whereas the other is missing the entire LBD (SpSHR2-splice variant). DNA-constructs encoding these isoforms and two additional in vitro generated deletion mutants were engineered in an expression vector carrying the myc-tag. Expression of the tagged isoforms in S. purpuratus embryos showed that the exogenous SpSHR2 full-length protein displays a similar subcellular localization as the endogenous receptor. In early cleavage stages (4-cells), the full-length isoform is predominantly localized in the nucleus, whereas two cell divisions later (16-cells) protein accumulations are detected in both the nucleus and cytoplasm. To the contrary, the SpSHR2-splice variant is confined in the embryonic nuclei both at 4- and 16-cell stage embryos. Analysis of the intracellular distribution of two receptor mutants, one having a deletion within the DBD (,P) and the other a truncation of the C-terminal F-domain (,F), revealed that ,P is localized similarly to full-length receptor, whereas ,F is maintained in the nucleus, similar to the SpSHR2 splice variant. Investigation of the DNA binding and dimerization properties of the two SpSHR2 isoforms demonstrated that they recognize and bind to a DR1-element as monomers, whereas ,P does not bind DNA and ,F binds to DR1 poorly. These results suggest that the receptor's putative LBD is responsible for the differential subcellular localization of the two natural SpSHR2-isoforms in early development. Mol. Reprod. Dev. 60: 147,157, 2001. © 2001 Wiley-Liss, Inc. [source] Spatial Distribution of Protein Damage by Singlet Oxygen in KeratinocytesPHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 1 2008Yu-Ying He Singlet oxygen may be generated in cells by either endogenous or exogenous photosensitizers as a result of exposure to UV or visible irradiation. We have used immuno-spin trapping (Free Radic. Biol. Med. 36: 1214, 2004) to identify the subcellular targets of singlet oxygen generated by rose bengal (RB). Confocal fluorescence microscopy of HaCaT keratinocytes incubated with RB clearly showed that the dye entered the cells and was located mainly in the perinuclear region, probably associated with the Golgi apparatus and endoplasmic reticulum. Previous studies by Wright et al. (Free Radic. Biol. Med.34: 637, 2003) have shown that long-lived protein hydroperoxides (POOH) are present in cells exposed to singlet oxygen-generating dyes. The addition of reducing metal ions such as Cu+ to POOH results in the generation of protein-derived radicals, POO, and PO,, which react with the spin trap 5,5-dimethyl-1-pyrroline N -oxide (DMPO) to give relatively stable spin adducts. In order to determine the subcellular localization of the protein-DMPO adducts, we exposed keratinocytes to RB/light exposure and then incubated the cells with Cu+ and DMPO. After staining with antibody against DMPO followed by a secondary Alexa Fluor 488 goat anti-rabbit IgG, the intracellular distribution of protein-DMPO adducts was determined by confocal microscopy. The subcellular localization of the protein DMPO adducts was coincident with that of RB. This approach may provide information on the spatial distribution of singlet oxygen generated in cells. [source] Photosensitizing and Radiosensitizing Effects of Hypericin on Human Renal Carcinoma Cells in VitroPHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 1 2008Johannes Theodor Wessels The renal cell carcinoma (RCC) is extremely resistant to chemotherapy and radiotherapy. The prognosis of patients with metastatic RCC still remains poor, the median survival is less than 12 months. Therefore, new therapeutic options are desirable. The aim of this study was to investigate the photosensitizing and radiosensitizing effects of hypericin on human RCC cells in vitro. First the RCC-derived cell lines A498 and ACHN were incubated with different concentrations of hypericin. In vitro uptake and intracellular distribution of hypericin were confirmed by fluorescence microscopy. Subsequently cells were illuminated and irradiated with a dose of 2,8 Gy, respectively. Finally, metabolic activity, apoptosis and clonogenic survival were investigated. Uptake of hypericin was observed for almost all cells. Hypericin treatment combined with illumination led to a 94,97% decrease in metabolic activity and caused apoptosis in nearly 100% of RCC cells. Hypericin enhanced the radiosensitivity of A498 cells in vitro. The clonogenic survival after irradiation was significantly reduced by hypericin treatment. Taken together, the photosensitizing and radiosensitizing effects of hypericin on human RCC cells we found in this investigation could be of clinical relevance, e.g. for radiotherapy and intraoperative photodynamic therapy, respectively. [source] Influence of cell cycle on ecdysteroid receptor in CHO-K1 cellsARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY (ELECTRONIC), Issue 3 2009Katarzyna Betanska Abstract CHO-K1 cells are routinely used for characterization of ecdysone receptor (EcR) function, because these vertebrate cells are devoid of endogenous ecdysone receptor protein. Moreover, the endogenous expression of RXR, the vertebrate orthologue of Ultraspiracle (Usp), the most important heterodimerization partner, is neglectable. In contrast to insect cells, there is also no influence of moulting hormone on CHO-K1 cells on cell proliferation either in the absence or presence of transiently expressed EcR. In contrast to Usp, which is exclusively found in nuclei, EcR is heterogeneously distributed between cytoplasm and nuclei in non-synchronized cells. Synchronization of CHO-K1 cells by nocodazole revealed that the cell cycle influences receptor concentration with lowest amounts in late S-phase and G2/M phase and intracellular distribution of the receptor protein showing a minimum of receptors present in nuclei during S-phase. EcR, but not Usp reduces cyclin D1 expression and cyclin D1 concentration is impaired by cyclin D1. Coimmunoprecipitation studies reveal physical interaction of EcR and cyclin D1. © 2009 Wiley Periodicals, Inc. [source] Intracellular distribution of peroxynitrite during doxorubicin cardiomyopathy: evidence for selective impairment of myofibrillar creatine kinaseBRITISH JOURNAL OF PHARMACOLOGY, Issue 3 2002Michael J Mihm Cardiac peroxynitrite and protein nitration are increased during doxorubicin cardiotoxicity, but the intracellular targets and functional consequences have not been defined. We investigated the intracellular distribution of protein nitration during doxorubicin cardiotoxicity in mice. Following in vivo cardiac function assessments by echocardiography, cardiac tissues were prepared for immunohistochemistry and electron microscopy 5 days after doxorubicin (20 mg kg,1) or vehicle control. Increased cardiac 3-nitrotyrosine was observed using light microscopy in doxorubicin treated animals. Immunogold electron microscopy (55,000×) revealed increased myofibrillar and mitochondrial 3-nitrotyrosine levels following doxorubicin, but cellular 3-nitrotyrosine density was 2 fold higher in myofibrils. We therefore investigated the actions of peroxynitrite on intact cardiac contractile apparatus. Skinned ventricular trabeculae were exposed to physiologically relevant peroxynitrite concentrations (50 or 300 nM) for 1 h, then Ca2+ induced contractile responses were measured in the presence of ATP (4 mM) or phosphocreatine (12 mM) as high energy phosphate supplier. ATP maximal force generation was unaltered after 50 nM peroxynitrite, but phosphocreatine/ATP response was reduced (0.99±0.63 vs 1.59±0.11), suggesting selective inactivation of myofibrillar creatine kinase (MM-CK). Reduction of ATP maximal force was observed at 300 nM peroxynitrite and phosphocreatine/ATP response was further reduced (0.64±0.30). Western blotting showed concentration dependent nitration of MM-CK in treated trabeculae. Similarly, cardiac tissues from doxorubicin treated mice demonstrated increased nitration and inactivation of MM-CK compared to controls. These results demonstrate that peroxynitrite-related protein nitration are mechanistic events in doxorubicin cardiomyopathy and that the cardiac myofibril is an important oxidative target in this setting. Furthermore, MM-CK may be a uniquely vulnerable target to peroxynitrite in vivo. British Journal of Pharmacology (2002) 135, 581,588; doi:10.1038/sj.bjp.0704495 [source] | ||||||||||||||||||||||||||||