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Intracellular Compartments (intracellular + compartment)
Selected AbstractsMolecular characterization of novel progranulin (GRN) mutations in frontotemporal dementia,HUMAN MUTATION, Issue 4 2008Odity Mukherjee Abstract Frontotemporal dementia (FTD) is a clinical term encompassing dementia characterized by the presence of two major phenotypes: 1) behavioral and personality disorder, and 2) language disorder, which includes primary progressive aphasia and semantic dementia. Recently, the gene for familial frontotemporal lobar degeneration (FTLD) with ubiquitin-positive, tau-negative inclusions (FTLD-U) linked to chromosome 17 was cloned. In the present study, 62 unrelated patients from the Washington University Alzheimer's Disease Research Center and the Midwest Consortium for FTD with clinically diagnosed FTD and/or neuropathologically characterized cases of FTLD-U with or without motor neuron disease (MND) were screened for mutations in the progranulin gene (GRN; also PGRN). We discovered two pathogenic mutations in four families: 1) a single-base substitution within the 3, splice acceptor site of intron 6/exon 7 (g.5913A>G [IVS6,2A>G]) causing skipping of exon 7 and premature termination of the coding sequence (PTC); and 2) a missense mutation in exon 1 (g.4068C>A) introducing a charged amino acid in the hydrophobic core of the signal peptide at residue 9 (p.A9D). Functional analysis in mutation carriers for the splice acceptor site mutation revealed a 50% decrease in GRN mRNA and protein levels, supporting haploinsufficiency. In contrast, there was no significant difference in the total GRN mRNA between cases and controls carrying the p.A9D mutation. Further, subcellular fractionation and confocal microscopy indicate that although the mutant protein is expressed, it is not secreted, and appears to be trapped within an intracellular compartment, possibly resulting in a functional haploinsufficiency. Hum Mutat 29(4), 512,521, 2008. © 2008 Wiley-Liss, Inc. [source] Delivery of Nucleic Acids via Disulfide-Based Carrier SystemsADVANCED MATERIALS, Issue 32-33 2009Sonja Bauhuber Abstract Nucleic acids are not only expected to assume a pivotal position as "drugs" in the treatment of genetic and acquired diseases, but could also act as molecular cues to control the microenvironment during tissue regeneration. Despite this promise, the efficient delivery of nucleic acids to their side of action is still the major hurdle. One among many prerequisites for a successful carrier system for nucleic acids is high stability in the extracellular environment, accompanied by an efficient release of the cargo in the intracellular compartment. A promising strategy to create such an interactive delivery system is to exploit the redox gradient between the extra- and intracellular compartments. In this review, emphasis is placed on the biological rationale for the synthesis of redox sensitive, disulfide-based carrier systems, as well as the extra- and intracellular processing of macromolecules containing disulfide bonds. Moreover, the basic synthetic approaches for introducing disulfide bonds into carrier molecules, together with examples that demonstrate the benefit of disulfides at the individual stages of nucleic acid delivery, will be presented. [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] Adaptation of the brucellae to their intracellular nicheMOLECULAR MICROBIOLOGY, Issue 3 2004R. Martin Roop II Summary Members of the bacterial genus Brucella are facultative intracellular pathogens that reside predominantly within membrane-bound compartments within two host cell types, macrophages and placental trophoblasts. Within macrophages, the brucellae route themselves to an intracellular compartment that is favourable for survival and replication, and they also appear to be well-adapted from a physiological standpoint to withstand the environmental conditions encountered during prolonged residence in this intracellular niche. Much less is known about the interactions of the Brucella with placental trophoblasts, but experimental evidence suggests that these bacteria use an iron acquisition system to support extensive intracellular replication within these host cells that is not required for survival and replication in host macrophages. Thus, it appears that the brucellae rely upon the products of distinct subsets of genes to adapt successfully to the environmental conditions encountered within the two cell types within which they reside in their mammalian hosts. [source] Water diffusion in the different microenvironments of breast cancerNMR IN BIOMEDICINE, Issue 4 2004Yael Paran Abstract The parameters that characterize the intricate water diffusion in tumors may serve to reveal their distinct pathology. Specifically, the application of diffusion magnetic resonance imaging (MRI) can aid in characterizing breast cancer, as well as monitoring response to therapy. We present here a non-invasive, quantitative MRI investigation, at high spatial resolution, of water diffusion in hormonal dependent MCF7 breast tumors implanted orthotopically in immunodeficient mice. Distinctive MRI protocols were designed in this study, utilizing a broad range of diffusion times and diffusion gradient strengths. Application of these protocols allowed water diffusion in the tissue extracellular and intracellular compartments to be distinguished, and the effect of restricted diffusion and water exchange on the water diffusion in these compartments to be evaluated. Pixel-by-pixel analysis yielded parametric maps of the estimated volume fraction and apparent diffusion coefficient of each compartment. The diffusion of the water in the extracellular microenvironment was approximately two fold slower than that of free water, and in the intracellular compartment was about one order of magnitude slower than that of free water and demonstrated restriction of water diffusion at long diffusion times. Mapping of the water fraction in each compartment was further employed to monitor changes during tumor progression and to assess tumor response to hormonal manipulation with a new antiestrogenic drug, tamoxifen methiodide (TMI). It was found that, in parallel to the growth arrest by this drug, the volume fraction of the slowly diffusing water increased, suggesting a TMI-induced cell swelling. This study can serve as a basis for extending diffusion breast MRI in the clinical setting. Copyright © 2004 John Wiley & Sons, Ltd. [source] Plasma membrane delivery, endocytosis and turnover of transcobalamin receptor in polarized human intestinal epithelial cellsTHE JOURNAL OF PHYSIOLOGY, Issue 2 2007Santanu Bose Cells that are metabolically active and in a high degree of differentiation and proliferation require cobalamin (Cbl: vitamin B12) and they obtain it from the circulation bound to transcobalamin (TC) via the transcobalamin receptor (TC-R). This study has investigated the plasma membrane dynamics of TC-R expression in polarized human intestinal epithelial Caco-2 cells using techniques of pulse-chase labelling, domain-specific biotinylation and cell fractionation. Endogenously synthesized TC-R turned over with a half-life (T1/2) of 8 h following its delivery to the basolateral plasma membrane (BLM). The T1/2 of BLM delivery was 15 min and TC-R delivered to the BLM was endocytosed and subsequently degraded by leupeptin-sensitive proteases. However, about 15% of TC-R endocytosed from the BLM was transcytosed (T1/2, 45 min) to the apical membranes (BBM) where it underwent endocytosis and was degraded. TC-R delivery to both BLM and BBM was inhibited by Brefeldin A and tunicamycin, but not by wortmannin or leupeptin. Colchicine inhibited TC-R delivery to BBM, but not BLM. At steady state, apical TC-R was associated with megalin and both these proteins were enriched in an intracellular compartment which also contained Rab5 and transferrin receptor. These results indicate that following rapid delivery to both plasma membrane domains of Caco-2 cells, TC-R undergoes constitutive endocytosis and degradation by leupeptin-sensitive proteases. TC-R expressed in apical BBM complexes with megalin during its transcytosis from the BLM. [source] Model-Based Analysis of Potassium Removal During HemodialysisARTIFICIAL ORGANS, Issue 10 2009Andrea Ciandrini Abstract Potassium ion (K+) kinetics in intra- and extracellular compartments during dialysis was studied by means of a double-pool computer model, which included potassium-dependent active transport (Na-K-ATPase pump) in 38 patients undergoing chronic hemodialysis. Each patient was treated for 2 weeks with a constant K+ dialysate concentration (K+CONST therapy) and afterward for 2 weeks with a time-varying (profiled) K+ dialysate concentration (K+PROF therapy). The two therapies induced different levels of K+ plasma concentration (K+CONST: 3.71 ± 0.88 mmol/L vs. K+PROF: 3.97 ± 0.64 mmol/L, time-averaged values, P < 0.01). The computer model was tuned to accurately fit plasmatic K+ measured in the course and 1 h after K+CONST and K+PROF therapies and was then used to simulate the kinetics of intra- and extracellular K+. Model-based analysis showed that almost all the K+ removal in the first 90 min of dialysis was derived from the extracellular compartment. The different K+ time course in the dialysate and the consequently different Na-K pump activity resulted in a different sharing of removed potassium mass at the end of dialysis: 56% ± 17% from the extracellular compartment in K+PROF versus 41% ± 14% in K+CONST. At the end of both therapies, the K+ distribution was largely unbalanced, and, in the next 3 h, K+ continued to flow in the extracellular space (about 24 mmol). After rebalancing, about 80% of the K+ mass that was removed derived from the intracellular compartment. In conclusion, the Na-K pump plays a major role in K+ apportionment between extracellular and intracellular compartments, and potassium dialysate concentration strongly influences pump activity. [source] Morphogenesis of hepatitis B virus and its subviral envelope particlesCELLULAR MICROBIOLOGY, Issue 11 2009Romuald Patient Summary After cell hijacking and intracellular amplification, non-lytic enveloped viruses are usually released from the infected cell by budding across internal membranes or through the plasma membrane. The enveloped human hepatitis B virus (HBV) is an example of virus using an intracellular compartment to form new virions. Four decades after its discovery, HBV is still the primary cause of death by cancer due to a viral infection worldwide. Despite numerous studies on HBV genome replication little is known about its morphogenesis process. In addition to viral neogenesis, the HBV envelope proteins have the capability without any other viral component to form empty subviral envelope particles (SVPs), which are secreted into the blood of infected patients. A better knowledge of this process may be critical for future antiviral strategies. Previous studies have speculated that the morphogenesis of HBV and its SVPs occur through the same mechanisms. However, recent data clearly suggest that two different processes, including constitutive Golgi pathway or cellular machinery that generates internal vesicles of multivesicular bodies (MVB), independently form these two viral entities. [source] Neuron-specific expression of atp6v0c2 in zebrafish CNSDEVELOPMENTAL DYNAMICS, Issue 9 2010Ah-Young Chung Abstract Vacuolar ATPase (V-ATPase) is a multi-subunit enzyme that plays an important role in the acidification of a variety of intracellular compartments. ATP6V0C is subunit c of the V0 domain that forms the proteolipid pore of the enzyme. In the present study, we investigated the neuron-specific expression of atp6v0c2, a novel isoform of the V-ATPase c-subunit, during the development of the zebrafish CNS. Zebrafish atp6v0c2 was isolated from a genome-wide analysis of the zebrafish mibta52b mutant designed to identify genes differentially regulated by Notch signaling. Whole-mount in situ hybridization revealed that atp6v0c2 is expressed in a subset of CNS neurons beginning several hours after the emergence of post-mitotic neurons. The ATP6V0C2 protein is co-localized with the presynaptic vesicle marker, SV2, suggesting that it is involved in neurotransmitter storage and/or secretion in neurons. In addition, the loss-of-function experiment suggests that ATP6V0C2 is involved in the control of neuronal excitability. Developmental Dynamics 239:2501,2508, 2010. © 2010 Wiley-Liss, Inc. [source] Diverse regulatory roles for lysosomal proteases in the immune responseEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 11 2009Jeff D. Colbert Abstract The innate and adaptive immune system utilise endocytic protease activity to promote functional immune responses. Cysteine and aspartic proteases (cathepsins) constitute a subset of endocytic proteases, the immune function of which has been described extensively. Although historically these studies have focused on their role in processes such as antigen presentation and zymogen processing within the endocytic compartment, recent discoveries have demonstrated a critical role for these proteases in other intracellular compartments, and within the extracellular milieu. It has also become clear that their pattern of expression and substrate specificities are more diverse than was first envisaged. Here, we discuss recent advances addressing the role of lysosomal proteases in various aspects of the immune response. We pay attention to reports demonstrating cathepsin activity outside of its canonical endosome/lysosome microenvironment. [source] Alanine screening of the intracellular loops of the human bradykinin B2 receptor , effects on receptor maintenance, G protein activation and internalizationFEBS JOURNAL, Issue 13 2009Alexander Faussner The bradykinin B2 receptor is coupled to G protein Gq/11 and becomes sequestered into intracellular compartments after activation. To more closely define the receptor sequences involved in these processes and their functions, we systematically mutated all three intracellular loops (ICLs), either as point mutations or in groups of three to five amino acids to Ala, obtaining a total of 14 mutants. All constructs were stably expressed in HEK 293 cells and, with the exception of triple mutant DRY , AAA, retained the ability to specifically bind [3H]bradykinin. The binding affinities at 4 or 37 °C of several mutants differed considerably from those determined for the wild-type receptor, indicating an allosteric connection between the conformation of the binding site and that of the ICLs. Mutations in ICL-1 strongly reduced surface expression without affecting G protein signaling or [3H]bradykinin internalization. Two cluster mutants in the middle of ICL-2 containing basic residues displayed considerably reduced potencies, whereas two mutations in ICL-3 resulted in receptor conformations that were considered to be semi-active, based on the observation that they responded with phosphoinositide hydrolysis to compounds normally considered to be antagonists. This, and the fact that a cluster mutant at the C-terminal end of ICL-3 was signaling incompetent, hint at the involvement of ICL-2 and ICL-3 in Gq/11 activation, albeit with different functions. None of the mutants displayed reduced ligand-induced receptor internalization, indicating that the loops are not essential for this process. No conclusion could be drawn, however, with regard to the role of the DRY sequence, as the corresponding triplet mutation lacked binding capability. [source] Regulated expression by PPAR, and unique localization of 17,-hydroxysteroid dehydrogenase type 11 protein in mouse intestine and liverFEBS JOURNAL, Issue 18 2007Yasuhide Yokoi 17,-Hydroxysteroid dehydrogenase type 11 (17,-HSD11) is a member of the short-chain dehydrogenase/reductase family involved in the activation and inactivation of sex steroid hormones. We recently identified 17,-HSD11 as a gene that is efficiently regulated by peroxisome proliferator-activated receptor-, PPAR, in the intestine and the liver [Motojima K (2004) Eur J Biochem271, 4141,4146]. In this study, we characterized 17,-HSD11 at the protein level to obtain information about its physiologic role in the intestine and liver. For this purpose, specific antibodies against 17,-HSD11 were obtained. Western blotting analysis showed that administration of a peroxisome proliferator-activated receptor-, agonist induced 17,-HSD11 protein in the jejunum but not in the colon, and to a much higher extent than in the liver of mice. A subcellular localization study using Chinese hamster ovary cells and green fluorescent protein-tagged 17,-HSD11 showed that it was mostly localized in the endoplasmic reticulum under normal conditions, whereas it was concentrated on lipid droplets when they were induced. A pulse-chase experiment suggested that 17,-HSD11 was redistributed to the lipid droplets via the endoplasmic reticulum. Immunohistochemical analysis using tissue sections showed that 17,-HSD11 was induced mostly in intestinal epithelia and hepatocytes, with heterogeneous localization both in the cytoplasm and in vesicular structures. A subcellular fractionation study of liver homogenates confirmed that 17,-HSD11 was localized mostly in the endoplasmic reticulum when mice were fed a normal diet, but was distributed in both the endoplasmic reticulum and the lipid droplets of which formation was induced by feeding a diet containing a proliferator-activated receptor-, agonist. Taken together, these data indicate that 17,-HSD11 localizes both in the endoplasmic reticulum and in lipid droplets, depending on physiologic conditions, and that lipid droplet 17,-HSD11 is not merely an endoplasmic reticulum contaminant or a nonphysiologically associated protein in the cultured cells, but a bona fide protein component of the membranes of both intracellular compartments. [source] Formation of cholesterol-enriched structures by aberrant intracellular accumulation of ATP-binding cassette transporter A1GENES TO CELLS, Issue 8 2008Arowu R. Tanaka ATP-binding cassette transporter A1 (ABCA1) is a key transporter associated with excess cellular lipid efflux. Here, we report that in HEK293 cells ABCA1 functions in intracellular compartments along the endocytic pathway. Inhibition of ABCA1-GFP degradation with proteasome inhibitors induced the internalization of ABCA1 and the formation of intracellular round-shaped structures, designated "A1 bodies". Importantly, cholesterol was selectively accumulated in A1 bodies, and this depended on the cholesterol efflux activity of ABCA1. Treatment with either lactacystin or acetylated LDL, which reduces proteasome activity, resulted in internalization of ABCA1 in mouse peritoneal macrophages. By performing array analysis on macrophages treated with these reagents, we identified Rab4 as a key protein involved in the internalization and aberrant accumulation of ABCA1 in HEK cells. Treatment of the cells with proteasome inhibitors inhibited the degradation of Rab4, and Rab4 over-expression induced the formation of small A1 bodies. Furthermore, A1 bodies formation was substantially inhibited by silencing of the endogenous Rab4 gene. Taken together, our findings suggest that the endocytic ABCA1 possesses cholesterol efflux activity, and thus the cellular control of post-endocytic sorting, retention or recycling of functional ABCA1 in the endocytic vesicles, which is in part regulated by Rab4, is important for cholesterol metabolism in living cells. [source] Delivery of Nucleic Acids via Disulfide-Based Carrier SystemsADVANCED MATERIALS, Issue 32-33 2009Sonja Bauhuber Abstract Nucleic acids are not only expected to assume a pivotal position as "drugs" in the treatment of genetic and acquired diseases, but could also act as molecular cues to control the microenvironment during tissue regeneration. Despite this promise, the efficient delivery of nucleic acids to their side of action is still the major hurdle. One among many prerequisites for a successful carrier system for nucleic acids is high stability in the extracellular environment, accompanied by an efficient release of the cargo in the intracellular compartment. A promising strategy to create such an interactive delivery system is to exploit the redox gradient between the extra- and intracellular compartments. In this review, emphasis is placed on the biological rationale for the synthesis of redox sensitive, disulfide-based carrier systems, as well as the extra- and intracellular processing of macromolecules containing disulfide bonds. Moreover, the basic synthetic approaches for introducing disulfide bonds into carrier molecules, together with examples that demonstrate the benefit of disulfides at the individual stages of nucleic acid delivery, will be presented. [source] Tissue edema does not change gadolinium-diethylenetriamine pentaacetic acid (Gd-DTPA)-enhanced T1 relaxation times of viable myocardium,,JOURNAL OF MAGNETIC RESONANCE IMAGING, Issue 6 2005Gang Li MD Abstract Purpose To determine whether tissue edema changes gadolinium-diethylenetriamine pentaacetic acid (Gd-DTPA)-enhanced T1 relaxation times of the viable myocardium. Materials and Methods A total of 16 isolated pig hearts were divided into four groups (N = 4/group) and perfused in a Langendorff apparatus. Gd-DTPA was injected into the aortic perfusion line. Tissue edema was then induced by two hours of simultaneous arterial/venous perfusion (SAVP). Myocardial water content and T1 relaxation times were monitored throughout SAVP. The volumes of the extracellular and intracellular compartments were assessed using 31P MRS-detectable markers, phenylphosphonic acid (PPA) and dimethyl methylphosphonate (DMMP). Results Tissue water content in both viable and infarcted myocardium increased significantly during two-hour SAVP. However, Gd-DTPA-enhanced T1 relaxation times of the viable myocardium remained relatively unchanged. Infarcted myocardium, on the other hand, exhibited significant T1 shortening during SAVP. Furthermore, SAVP resulted in significant expansions of both extracellular and intracellular compartments, but the ratio of the volumes of the two compartments remained relatively constant. Conclusion Tissue edema in the viable myocardium does not increase the relative distribution volume of the contrast agent. As a result, edema does not change Gd-DTPA-enhanced T1 relaxation times of the viable myocardium. J. Magn. Reson. Imaging 2005;21:744,751. Published 2005 Wiley-Liss, Inc. [source] Localization of the membrane-anchored MMP-regulator RECK at the neuromuscular junctionsJOURNAL OF NEUROCHEMISTRY, Issue 2 2008Satoshi Kawashima Abstract Nerve apposition on nicotinic acetylcholine receptor clusters and invagination of the post-synaptic membrane (i.e. secondary fold formation) occur by embryonic day 18.5 at the neuromuscular junctions (NMJs) in mouse skeletal muscles. Finding the molecules expressed at the NMJ at this stage of development may help elucidating how the strong linkage between a nerve terminal and a muscle fiber is established. Immunohistochemical analyses indicated that the membrane-anchored matrix metalloproteinase regulator RECK was enriched at the NMJ in adult skeletal muscles. Confocal and electron microscopy revealed the localization of RECK immunoreactivity in secondary folds and subsynaptic intracellular compartments in muscles. Time course studies indicated that RECK immunoreactivity becomes associated with the NMJ in the diaphragm at around embryonic day 18.5 and thereafter. These findings, together with known properties of RECK, support the hypothesis that RECK participates in NMJ formation and/or maintenance, possibly by protecting extracellular components, such as synaptic basal laminae, from proteolytic degradation. [source] Multifunctioning pH-responsive nanoparticles from hierarchical self-assembly of polymer brush for cancer drug deliveryAICHE JOURNAL, Issue 11 2008Youqing Shen Abstract Polymer nanoparticles are extensively explored as drug carriers but they generally have issues of premature burst drug release, slow cellular uptake, and retention in acidic intracellular compartments. Herein, we report multifunctioning three-layered nanoparticles (3LNPs) that can overcome these problems. The 3LNPs have a poly(,-caprolactone) (PCL) core, a pH-responsive poly[2-(N,N-diethylamino)ethyl methacrylate](PDEA) middle layer and a polyethylene glycol (PEG) outer layer. The pH-responsive PDEA layer is insoluble at pH above 7 but becomes positively charged and soluble via protonation at pH lower than 6.5. Thus, this layer has three functions: it covers on the PCL core inhibiting the premature burst drug release at the physiological pH, becomes positively charged and thus promotes endocytosis for fast cellular internalization in the acidic interstitium of solid tumors, and is highly positively charged in lysosomes to disrupt the lysosomal membrane and release the nanoparticle into the cytosol. The multifunctioning nanoparticles are an efficient carrier for cancer cytosolic drug delivery. © 2008 American Institute of Chemical Engineers AIChE J, 2008 [source] The protein secretory pathway of Candida albicansMYCOSES, Issue 4 2009William A. Fonzi Summary Virulence of the opportunistic pathogen, Candida albicans, relies on an assemblage of attributes. These include the secretion of hydrolytic enzymes, cell surface adhesins, morphological transition between yeast and hyphae, phenotypic switching and biofilm formation. These diverse features are united by their dependence on the protein secretory apparatus for expression. Although the secretory apparatus of C. albicans has been studied limitedly, it appears to conform to the well-conserved eukaryotic system of vesicle-mediated transport between intracellular compartments and the cell surface. Genome comparison with Saccharomyces cerevisiae, however, shows multiple differences whose functional significance is yet unstudied. A unique aspect of the secretory pathway of C. albicans is its structural, and perhaps functional, rearrangement in hyphal vs. yeast cells. This, and evidence of non-conserved secretion mechanism(s), suggest that there is much fundamental knowledge to be derived from the analysis of secretion in C. albicans, which will be relevant to its ability to cause disease. [source] Water diffusion in the different microenvironments of breast cancerNMR IN BIOMEDICINE, Issue 4 2004Yael Paran Abstract The parameters that characterize the intricate water diffusion in tumors may serve to reveal their distinct pathology. Specifically, the application of diffusion magnetic resonance imaging (MRI) can aid in characterizing breast cancer, as well as monitoring response to therapy. We present here a non-invasive, quantitative MRI investigation, at high spatial resolution, of water diffusion in hormonal dependent MCF7 breast tumors implanted orthotopically in immunodeficient mice. Distinctive MRI protocols were designed in this study, utilizing a broad range of diffusion times and diffusion gradient strengths. Application of these protocols allowed water diffusion in the tissue extracellular and intracellular compartments to be distinguished, and the effect of restricted diffusion and water exchange on the water diffusion in these compartments to be evaluated. Pixel-by-pixel analysis yielded parametric maps of the estimated volume fraction and apparent diffusion coefficient of each compartment. The diffusion of the water in the extracellular microenvironment was approximately two fold slower than that of free water, and in the intracellular compartment was about one order of magnitude slower than that of free water and demonstrated restriction of water diffusion at long diffusion times. Mapping of the water fraction in each compartment was further employed to monitor changes during tumor progression and to assess tumor response to hormonal manipulation with a new antiestrogenic drug, tamoxifen methiodide (TMI). It was found that, in parallel to the growth arrest by this drug, the volume fraction of the slowly diffusing water increased, suggesting a TMI-induced cell swelling. This study can serve as a basis for extending diffusion breast MRI in the clinical setting. Copyright © 2004 John Wiley & Sons, Ltd. [source] ,Smart' delivery systems for biomolecular therapeuticsORTHODONTICS & CRANIOFACIAL RESEARCH, Issue 3 2005PS Stayton Structured Abstract Authors ,, Stayton PS, El-Sayed MEH, Murthy N, Bulmus V, Lackey C, Cheung C, Hoffman AS Objective ,, There is a strong need for drug delivery systems that can deliver biological signals from biomaterials and tissue engineering scaffolds, and a particular need for new delivery systems that can efficiently deliver biomolecules to intracellular targets. Viruses and pathogens have evolved potent molecular machinery that sense the lowered pH gradient of the endosomal compartment and become activated to destabilize the endosomal membrane, thereby enhancing protein or DNA transport to the cytoplasmic compartment. A key feature of many of these biological delivery systems is that they are reversible, so that the delivery systems are not directly toxic. These delivery systems have the ability to change their structural and functional properties and thus display remarkable ,smart' material properties. The objective of this presentation is to review the initial development of smart polymeric carriers that mimic these biological delivery systems and combine similar pH-sensitive, membrane-destabilizing activity for the delivery of therapeutic biomolecules. Design ,, We have developed new ,smart' polymeric carriers to more effectively deliver and broaden the available types of biomolecular therapeutics. The polymers are hydrophilic and stealth-like at physiological pH, but become membrane-destabilizing after uptake into the endosomal compartment where they enhance the release of therapeutic cargo into the cytoplasm. They can be designed to provide a range of pH profiles and membrane-destabilizing activities, allowing their molecular properties to be matched to specific drugs and loading ranges. A versatile set of linker chemistries is available to provide degradable conjugation sites for proteins, nucleic acids, and/or targeting moieties. Results ,, The physical properties of several pH-responsive polymers were examined. The activity and pH profile can be manipulated by controlling the length of hydrophobic alkyl segments. The delivery of poly(propyl acrylic acid) (PPAA)-containing lipoplexes significantly enhanced wound healing through the interconnected effects of altered extracellular matrix organization and greater vascularization. PPAA has also been shown to enhance cytoplasmic delivery of a model protein therapeutic. Polymeric carriers displaying pH-sensitive, membrane-destabilizing activity were also examined. The pH profile is controlled by the choice of the alkylacrylic acid monomer and by the ratio of the carboxylate-containing alkylacrylic acid monomer to alkylacrylate monomer. The membrane destabilizing activity is controlled by the lengths of the alkyl segment on the alkylacrylic acid monomer and the alkylacrylate monomer, as well as by their ratio in the final polymer chains. Conclusion ,, The molecular mechanisms that proteins use to sense and destabilize provide interesting paradigms for the development of new polymeric delivery systems that mimic biological strategies for promoting the intracellular delivery of biomolecular drugs. The key feature of these polymers is their ability to directly enhance the intracellular delivery of proteins and DNA, by destabilizing biological membranes in response to vesicular compartment pH changes. The ability to deliver a wide variety of protein and nucleic acid drugs to intracellular compartments from tissue engineering and regenerative scaffolds could greatly enhance control of important processes such as inflammation, angiogenesis, and biomineralization. [source] Insights into the membrane proteome of rat liver peroxisomes: Microsomal glutathione-S-transferase is shared by both subcellular compartmentsPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 3 2006Markus Islinger Dr. Abstract Peroxisomes are ubiquitous "multipurpose" organelles of eukaryotic cells. Their matrix enzymes catalyze mainly catabolic and anabolic reactions of lipid metabolism, thus contributing to the regulation of lipid homeostasis. Since most metabolites must be actively transported across the peroxisomal membrane and since individual proteins and protein complexes play functional roles in such transport processes, we analyzed the peroxisomal membrane proteome. Benzyldimethyl- n -hexadecylammoniumchloride (16-BAC)/SDS-2-D-PAGE and mass spectrometry were used to characterize the proteomes of highly purified "light" and "heavy" peroxisomes of rat liver obtained by density gradient centrifugation. In both populations, the major integral membrane proteins could be detected in high concentrations, verifying 16-BAC/SDS-2-D-PAGE as a suitable tool for the preparation of membrane proteomes destined for mass spectrometric analysis. Both reliable and reproducible detection of a distinct set of microsomal (ER) membrane proteins, including microsomal glutathione-S-transferase (mGST), in light and heavy peroxisomal fractions was also possible. Compared with the abundance of most microsomal membrane proteins, we found mGST to be specifically enriched in peroxisomal membrane fractions. Furthermore, C terminus epitope-tagged mGST versions were localized at least in part to peroxisomes in different mammalian cell lines. Taken together, these data suggest that the peroxisomal GST is not a mere ER-contaminant, but a bona fide protein comprising the membrane proteome of both intracellular compartments. In addition, we could detect several mitochondrial proteins in light peroxisome fractions. This finding may likely indicate a physical association of light peroxisomes with mitochondria, since the organelles could be partly separated by mechanical stress. Whether this association is of functional importance awaits further investigation. [source] Secreted proteome of the murine multipotent hematopoietic progenitor cell line DKmixRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 5 2010Nina Luecke Administration of the multipotent hematopoietic progenitor cell (HPC) line DKmix improved cardiac function after myocardial infarction and accelerated dermal wound healing due to paracrine mechanisms. The aim of this study was to analyse the secreted proteins of DKmix cells in order to identify the responsible paracrine factors and assess their relevance to the wide spectrum of therapeutic effects. A mass spectrometry (MS)-based approach was used to identify secreted proteins of DKmix cells. Serum free culture supernatants of DKmix-conditioned medium were collected and the proteins present were separated, digested by trypsin and the resulting peptides were then analyzed by matrix-assisted laser desorption/ionization tandem time-of-flight (MALDI-TOF/TOF) MS. Overall 95 different proteins were identified. Among them, secretory proteins galectin-3 and gelsolin were identified. These proteins are known to stimulate cell migration and influence wound healing and cardiac remodelling. The remaining proteins originate from intracellular compartments like cytoplasm (69%), nucleus (12%), mitochondria (4%), and cytoplasmic membrane (3%) indicating permeable or leaky DKmix cells in the conditioned medium. Additionally, a sandwich immunoassay was used to detect and quantify cytokines and chemokines. Interleukin-6 (IL-6), interleukin-13 (IL-13), monocyte-chemoattractant protein-1 (MCP-1), monocyte-chemoattractant protein-3 (MCP-3), monocyte-chemoattractant protein-1, (MIP-1,) and monocyte-chemoattractant protein-1, (MIP-1,) were detected in low concentrations. This study identified a subset of proteins present in the DKmix-conditioned medium that act as paracrine modulators of tissue repair. Moreover, it suggests that DKmix-derived conditioned medium might have therapeutic potency by promoting tissue regeneration. Copyright © 2010 John Wiley & Sons, Ltd. [source] Model-Based Analysis of Potassium Removal During HemodialysisARTIFICIAL ORGANS, Issue 10 2009Andrea Ciandrini Abstract Potassium ion (K+) kinetics in intra- and extracellular compartments during dialysis was studied by means of a double-pool computer model, which included potassium-dependent active transport (Na-K-ATPase pump) in 38 patients undergoing chronic hemodialysis. Each patient was treated for 2 weeks with a constant K+ dialysate concentration (K+CONST therapy) and afterward for 2 weeks with a time-varying (profiled) K+ dialysate concentration (K+PROF therapy). The two therapies induced different levels of K+ plasma concentration (K+CONST: 3.71 ± 0.88 mmol/L vs. K+PROF: 3.97 ± 0.64 mmol/L, time-averaged values, P < 0.01). The computer model was tuned to accurately fit plasmatic K+ measured in the course and 1 h after K+CONST and K+PROF therapies and was then used to simulate the kinetics of intra- and extracellular K+. Model-based analysis showed that almost all the K+ removal in the first 90 min of dialysis was derived from the extracellular compartment. The different K+ time course in the dialysate and the consequently different Na-K pump activity resulted in a different sharing of removed potassium mass at the end of dialysis: 56% ± 17% from the extracellular compartment in K+PROF versus 41% ± 14% in K+CONST. At the end of both therapies, the K+ distribution was largely unbalanced, and, in the next 3 h, K+ continued to flow in the extracellular space (about 24 mmol). After rebalancing, about 80% of the K+ mass that was removed derived from the intracellular compartment. In conclusion, the Na-K pump plays a major role in K+ apportionment between extracellular and intracellular compartments, and potassium dialysate concentration strongly influences pump activity. [source] Key Factors in Alzheimer's Disease: ,-amyloid Precursor Protein Processing, Metabolism and Intraneuronal TransportBRAIN PATHOLOGY, Issue 1 2001Thomas A. Bayer During the last years it has become evident that the ,-amyloid (A,) component of senile plaques may be the key molecule in the pathology of Alzheimer's disease (AD). The source and place of the neurotoxic action of A,, however, is still a matter of controversy. The precursor of the ,-amyloid peptide is the predominantly neuronal ,-amyloid precursor protein. We, and others, hypothesize that intraneuronal misregulation of APP leads to an accumulation of A, peptides in intracellular compartments. This accumulation impairs APP trafficking, which starts a cascade of pathological changes and causes the pyramidal neurons to degenerate. Enhanced A, secretion as a function of stressed neurons and remnants of degenerated neurons provide seeds for extracellular A, aggregates, which induce secondary degenerative events involving neighboring cells such as neurons, astroglia and macrophages/microglia. [source] Effects of chlorpromazine on excitation,contraction coupling events in fast-twitch skeletal muscle fibres of the ratBRITISH JOURNAL OF PHARMACOLOGY, Issue 4 2004R Wagner Single mechanically skinned fibres from the rat extensor digitorum longus muscle, which allow access to intracellular compartments, were used to examine the effects of 0.5,100 ,M chlorpromazine hydrochloride (CPZ) on the major steps of the excitation,contraction (E,C) coupling to elucidate the involvement of skeletal muscle in the neuroleptic malignant syndrome (NMS). At 1 ,M, CPZ caused a 20,30% increase in the force response induced by t-system depolarisation and a marked increase in the rate of caffeine-induced SR Ca2+ release. At [CPZ]2.5 ,M, there was an initial increase followed by a marked decrease of the t-system depolarisation-induced force responses, while the potentiating effect on the caffeine-induced SR Ca2+ release remained. These effects were reversible. CPZ had no effect on the maximum Ca2+ -activated force, but caused reversible, concentration-dependent increases in the Ca2+ sensitivity of the contractile apparatus at [CPZ] 10 ,M, with a 50% predicted shift of 0.11 pCa (,log [Ca2+]) units at 82.3 ,M CPZ. CPZ did not alter the rate of SR-Ca2+ loading at 1 and 10 ,M, but reversibly reduced it by ,40% at 100 ,M by reducing the SR Ca2+ pump. Nevertheless, the SR Ca2+ content was greater when fibres became unresponsive to t-system-induced depolarisation in the presence than in the absence of 100 ,M CPZ. The results show that CPZ has concentration-dependent stimulatory and inhibitory effects on various steps of the E,C coupling, which can explain the involvement of skeletal muscle in NMS and reconcile previous divergent data on CPZ effects on muscle. British Journal of Pharmacology (2004) 141, 624,633. doi:10.1038/sj.bjp.0705655 [source] Mechanisms and consequences of persistence of intracellular pathogens: leishmaniasis as an exampleCELLULAR MICROBIOLOGY, Issue 6 2008Christian Bogdan Summary Lifelong persistence after clinical cure of the primary infection is a characteristic feature of many intracellular pathogens, including viruses, bacteria and protozoa. The underlying mechanisms are complex and range from the passive protection against toxic effector molecules of the host and the remodelling of intracellular compartments as safe niches to the active modulation of the immune response at multiple levels. Parasites of the genus Leishmania have been particular helpful in unravelling some of the basic processes and form therefore the centre of the discussion. [source] Lounging in a lysosome: the intracellular lifestyle of Coxiella burnetiiCELLULAR MICROBIOLOGY, Issue 4 2007Daniel E. Voth Summary Most intracellular parasites employ sophisticated mechanisms to direct biogenesis of a vacuolar replicative niche that circumvents default maturation through the endolysosomal cascade. However, this is not the case of the Q fever bacterium, Coxiella burnetii. This hardy, obligate intracellular pathogen has evolved to not only survive, but to thrive, in the harshest of intracellular compartments: the phagolysosome. Following internalization, the nascent Coxiella phagosome ultimately develops into a large and spacious parasitophorous vacuole (PV) that acquires lysosomal characteristics such as acidic pH, acid hydrolases and cationic peptides, defences designed to rid the host of intruders. However, transit of Coxiella to this environment is initially stalled, a process that is apparently modulated by interactions with the autophagic pathway. Coxiella actively participates in biogenesis of its PV by synthesizing proteins that mediate phagosome stalling, autophagic interactions, and development and maintenance of the mature vacuole. Among the potential mechanisms mediating these processes is deployment of a type IV secretion system to deliver effector proteins to the host cytosol. Here we summarize our current understanding of the cellular events that occur during parasitism of host cells by Coxiella. [source] The Vibrio cholerae haemolysin anion channel is required for cell vacuolation and deathCELLULAR MICROBIOLOGY, Issue 7 2002Monica Moschioni Summary Several strains of Vibrio cholerae secrete a haemolytic toxin of 63 kDa, termed V. cholerae cytolysin (VCC). This toxin causes extensive vacuolation and death of cells in culture and forms an anion-selective channel in planar lipid bilayers and in cells. Here, we identify inhibitors of the VCC anion channel and show that the formation of the anion channel is necessary for the development of the vacuoles and for the cell death induced by this toxin. Using markers of cell organelles, we show that vacuoles derive from different intracellular compartments and we identify the contribution of late endosomes and of the trans -Golgi network in vacuole biogenesis. [source] Internalization of Bordetella pertussis adenylate cyclase,haemolysin into endocytic vesicles contributes to macrophage cytotoxicityCELLULAR MICROBIOLOGY, Issue 11 2001Nadia Khelef Bordetella pertussis adenylate cyclase,haemolysin is a critical virulence factor in the murine model of intranasal infection, where it is required for several pathological effects, including macrophage apoptosis. Based on biochemical and immunological properties, it was proposed that the toxin was delivered directly to the cytoplasm of eukaryotic cells without trafficking through the endocytic pathway. In the present study, we analysed the cellular distribution of the adenylate cyclase,haemolysin during intoxication of macrophages. We showed that, shortly after its initial binding to the plasma membrane of macrophages, the toxin gains access to intracellular compartments that become progressively positive for the endosomal marker transferrin, but not for the lysosomal membrane protein CD107a/Lamp1. Importantly, the vesicular trafficking of the adenylate cyclase,haemolysin appears to be required for its ability to induce macrophage death. Inhibitors of actin polymerization and of macropinocytosis, as well as depletion of plasma membrane cholesterol and disruption of the Golgi network, reduce the toxin's ability to kill macrophages. Altogether, these results suggest that internalization of the adenylate cyclase,haemolysin into endocytic vesicles, at least partly through macropinocytosis, contributes to cytotoxicity. [source] Templated assembly of the pH-sensitive membrane-lytic peptide GALACHEMICAL BIOLOGY & DRUG DESIGN, Issue 6 2004D.H. Haas Abstract:, Delivery of protein or nucleic acid therapeutics into intracellular compartments may require facilitation to allow these macromolecules to cross otherwise impermeant cellular membranes. Peptides capable of forming membrane-spanning channels hold promise as just such facilitators, although the requirement for peptide oligomerization to form these channels may limit their effectiveness. Synthetic molecules containing multiple copies of membrane-active peptides attached to a template molecule in a pre-oligomerized form have attracted interest for drug-delivery applications. Using three template designs, we synthesized multimeric versions of the pH-sensitive lytic peptide GALA and compared their performance to monomeric GALA. Template assembly stabilized helix formation: templated GALA retained , -helical structure even at neutral pH, unlike monomeric GALA. In membrane leakage assays, templated GALA retained the pH sensitivity of the monomer, with improved leakage for dimeric GALA. Surprisingly, trimeric GALA was less effective, particularly when synthesized with a larger and more flexible spacer. Surface plasmon resonance analysis indicated that reversible binding of templated GALA to lipid surfaces at acidic conditions was greatly reduced compared with monomeric GALA, but that the amount of irreversibly bound material was similar. We interpreted these results to indicate that templated peptides may cyclize into ,self-satisfied' oligomeric structures, incapable of further aggregation and subsequent pore formation. Future design of templated peptides must be carefully performed to avoid this unwanted consequence. [source] | ||||||||||||||||