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ABSTRACT: This study investigates the effects of crystallographic orientation of titanium substrates on the atomic structure and biological characteristics of hydroxyapatite (HA) coatings. Samples are prepared from extruded rod and rolled sheet of commercially pure titanium having distinct distribution of crystallographic planes. Electrophoresis is used to coat titanium substrates having different microstructures. The biological performance of both HA-coated and non-coated samples is assessed by osteoblast cell attachment, proliferation, differentiation and morphological studies. X-ray diffraction (XRD) analysis of the HA-coated samples indicates the predominant orientation of (002) for HA-coated sheets compared to (211) for the HA-coated rod samples. The numbers of attached and grown cells are higher on the surface of the HA-coated sheet samples. There is also a significant difference in alkaline phosphatase activity on the HA-coated sheet samples. Scanning electron microscopy (SEM) analysis of osteoblast cells grown on HA-coated and non-coated samples demonstrates differences in morphology with respect to spreading and attachment patterns. We believe that the specific atomic structure that is induced in the HA coating by the crystallographic orientation of the sheet substrate causes orientation-dependent coordination with biomolecules and improves cellular interactions. This suggests that crystal orientation of the substrate can be used to both influence the structure of the coating material and improve and control cell-substrate interactions.
Colloids and surfaces. B, Biointerfaces 10/2012; 103C:200-208. · 2.60 Impact Factor
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Wei Song,
Hillel Zukor,
Shih-Hsiung Lin,
Jacob Hascalovici,
Adrienne Liberman,
Ayda Tavitian,
Jeannie Mui, Hojatollah Vali,
Xin-Kang Tong,
Sanjeev K Bhardwaj,
Lalit K Srivastava,
Edith Hamel,
Hyman M Schipper
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ABSTRACT: Delineation of key molecules that act epigenetically to transduce diverse stressors into established patterns of disease would facilitate the advent of preventive and disease-modifying therapeutics for a host of neurological disorders. Herein, we demonstrate that selective overexpression of the stress protein heme oxygenase-1 (HO-1) in astrocytes of novel GFAP.HMOX1 transgenic mice results in subcortical oxidative stress and mitochondrial damage/autophagy; diminished neuronal reelin content (males); induction of Nurr1 and Pitx3 with attendant suppression of their targeting miRNAs, 145 and 133b; increased tyrosine hydroxylase and α-synuclein expression with downregulation of the targeting miR-7b of the latter; augmented dopamine and serotonin levels in basal ganglia; reduced D1 receptor binding in nucleus accumbens; axodendritic pathology and altered hippocampal cytoarchitectonics; impaired neurovascular coupling; attenuated prepulse inhibition (males); and hyperkinetic behavior. The GFAP.HMOX1 neurophenotype bears resemblances to human schizophrenia and other neurodevelopmental conditions and implicates glial HO-1 as a prime transducer of inimical (endogenous and environmental) influences on the development of monoaminergic circuitry. Containment of the glial HO-1 response to noxious stimuli at strategic points of the life cycle may afford novel opportunities for the effective management of human neurodevelopmental and neurodegenerative conditions.
Journal of Neuroscience 08/2012; 32(32):10841-53. · 7.11 Impact Factor
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Wei Song,
Hillel Zukor,
Shih-Hsiung Lin,
Adrienne Liberman,
Ayda Tavitian,
Jeannie Mui, Hojatollah Vali,
Carine Fillebeen,
Kostas Pantopoulos,
Ting-Di Wu,
Jean-Luc Guerquin-Kern,
Hyman M Schipper
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ABSTRACT: The mechanisms responsible for pathological iron deposition in the aging and degenerating mammalian CNS remain poorly understood. The stress protein, HO-1 mediates the degradation of cellular heme to biliverdin/bilirubin, free iron, and CO and is up-regulated in the brains of persons with Alzheimer's disease and Parkinson's disease. HO-1 induction in primary astroglial cultures promotes deposition of non-transferrin iron, mitochondrial damage and macroautophagy, and predisposes cocultured neuronal elements to oxidative injury. To gain a better appreciation of the role of glial HO-1 in vivo, we probed for aberrant brain iron deposition using Perls' method and dynamic secondary ion mass spectrometry in novel, conditional GFAP.HMOX1 transgenic mice that selectively over-express human HO-1 in the astrocytic compartment. At 48 weeks, the GFAP.HMOX1 mice exhibited increased deposits of glial iron in hippocampus and other subcortical regions without overt changes in iron-regulatory and iron-binding proteins relative to age-matched wild-type animals. Dynamic secondary ion mass spectrometry revealed abundant FeO(-) signals in the transgenic, but not wild-type, mouse brain that colocalized to degenerate mitochondria and osmiophilic cytoplasmic inclusions (macroautophagy) documented by TEM. Sustained up-regulation of HO-1 in astrocytes promotes pathological brain iron deposition and oxidative mitochondrial damage characteristic of Alzheimer's disease-affected neural tissues. Curtailment of glial HO-1 hyperactivity may limit iron-mediated cytotoxicity in aging and degenerating neural tissues.
Journal of Neurochemistry 08/2012; 123(2):325-36. · 4.06 Impact Factor
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ABSTRACT: The potential role of clay minerals in the abiotic origin of life has been the subject of ongoing debate for the past several decades. At issue are the clay minerals found in a class of meteorites known as carbonaceous chondrites. These clay minerals are the product of aqueous alteration of anhydrous mineral phases, such as olivine and orthopyroxene, that are often present in the chondrules. Moreover, there is a strong correlation in the occurrence of clay minerals and the presence of polar organic molecules. It has been shown in laboratory experiments at low temperature and ambient pressure that polar organic molecules, such as the oxalate found in meteorites, can catalyze the crystallization of clay minerals. In this study, we show that oxalate is a robust catalyst in the crystallization of saponite, an Al- and Mg-rich, trioctahedral 2:1 layer silicate, from a silicate gel at 60°C and ambient pressure. High-resolution transmission electron microscopy analysis of the saponite treated with octadecylammonium (n(C)=18) cations revealed the presence of 2:1 layer structures that have variable interlayer charge. The crystallization of these differently charged 2:1 layer silicates most likely occurred independently. The fact that 2:1 layer silicates with variable charge formed in the same gel has implications for our understanding of the origin of life, as these 2:1 clay minerals most likely replicate by a mechanism of template-catalyzed polymerization and transmit the charge distribution from layer to layer. If polar organic molecules like oxalate can catalyze the formation of clay-mineral crystals, which in turn promote clay microenvironments and provide abundant adsorption sites for other organic molecules present in solution, the interaction among these adsorbed molecules could lead to the polymerization of more complex organic molecules like RNA from nucleotides on early Earth.
Astrobiology 06/2012; 12(6):549-61. · 2.15 Impact Factor
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ABSTRACT: Microbes in natural settings typically live attached to surfaces in complex communities called biofilms. Despite the many advantages of biofilm formation, communal living forces microbes to compete with one another for resources. Here we combine mathematical models with stable isotope techniques to test a reaction-diffusion model of competition in a photosynthetic biofilm. In this model, a nutrient is transported through the mat by diffusion and is consumed at a rate proportional to its local concentration. When the nutrient is supplied from the surface of the biofilm, the balance between diffusion and consumption gives rise to gradients of nutrient availability, resulting in gradients of nutrient uptake. To test this model, a biofilm was incubated for a fixed amount of time with an isotopically labeled nutrient that was incorporated into cellular biomass. Thus, the concentration of labeled nutrient in a cell is a measure of the mean rate of nutrient incorporation over the course of the experiment. Comparison of this measurement to the solution of the reaction-diffusion model in the biofilm confirms the presence of gradients in nutrient uptake with the predicted shape. The excellent agreement between theory and experiment lends strong support to this one-parameter model of reaction and diffusion of nutrients in a biofilm. Having validated this model empirically, we discuss how these dynamics may arise from diffusion through a reactive heterogeneous medium. More generally, this result identifies stable isotope techniques as a powerful tool to test quantitative models of chemical transport through biofilms.
Journal of Theoretical Biology 08/2011; 289:90-5. · 2.21 Impact Factor
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ABSTRACT: Magnetotactic bacteria contain nanometre-sized, membrane-bound organelles, called magnetosomes, which are tasked with the biomineralization of small crystals of the iron oxide magnetite allowing the organism to use geomagnetic field lines for navigation. A key player in this process is the HtrA/DegP family protease MamE. In its absence, Magnetospirillum magneticum str AMB-1 is able to form magnetosome membranes but not magnetite crystals, a defect previously linked to the mislocalization of magnetosome proteins. In this work we use a directed genetic approach to find that MamE, and another predicted magnetosome-associated protease, MamO, likely function as proteases in vivo. However, as opposed to the complete loss of mamE where no biomineralization is observed, the protease-deficient variant of this protein still supports the initiation and formation of small, 20 nm-sized crystals of magnetite, too small to hold a permanent magnetic dipole moment. This analysis also reveals that MamE is a bifunctional protein with a protease-independent role in magnetosome protein localization and a protease-dependent role in maturation of small magnetite crystals. Together, these results imply the existence of a previously unrecognized 'checkpoint' in biomineralization where MamE moderates the completion of magnetite formation and thus committal to magneto-aerotaxis as the organism's dominant mode of navigating the environment.
Molecular Microbiology 03/2011; 80(4):1075-87. · 5.01 Impact Factor
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ABSTRACT: Intracellular magnetite crystal formation by magnetotactic bacteria has emerged as a powerful model for investigating the cellular and molecular mechanisms of biomineralization, a process common to all branches of life. Although magnetotactic bacteria are phylogenetically diverse and their crystals morphologically diverse, studies to date have focused on a few, closely related species with similar crystal habits. Here, we investigate the process of magnetite biomineralization in Desulfovibrio magneticus sp. RS-1, the only reported species of cultured magnetotactic bacteria that is outside of the alpha-Proteobacteria and that forms bullet-shaped crystals. Using a variety of high-resolution imaging and analytical tools, we show that RS-1 cells form amorphous, noncrystalline granules containing iron and phosphorus before forming magnetite crystals. Using NanoSIMS (dynamic secondary ion mass spectroscopy), we show that the iron-phosphorus granules and the magnetite crystals are likely formed through separate cellular processes. Analysis of the cellular ultrastructure of RS-1 using cryo-ultramicrotomy, cryo-electron tomography, and tomography of ultrathin sections reveals that the magnetite crystals are not surrounded by membranes but that the iron-phosphorus granules are surrounded by membranous compartments. The varied cellular paths for the formation of these two minerals lead us to suggest that the iron-phosphorus granules constitute a distinct bacterial organelle.
Proceedings of the National Academy of Sciences 07/2010; 107(27):12263-8. · 9.68 Impact Factor
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ABSTRACT: Although membrane-bounded compartments are commonly considered a unique eukaryotic characteristic, many species of bacteria have organelles. Compartmentalization is well studied in eukaryotes; however, the molecular factors and processes leading to organelle formation in bacteria are poorly understood. We use the magnetosome compartments of magnetotactic bacteria as a model system to investigate organelle biogenesis in a prokaryotic system. The magnetosome is an invagination of the cell membrane that contains a specific set of proteins able to direct the synthesis of a nanometer-sized magnetite crystal. A well-conserved region called the magnetosome island (MAI) is known to be essential for magnetosome formation and contains most of the genes previously implicated in magnetosome formation. Here, we present a comprehensive functional analysis of the MAI genes in a magnetotactic bacterium, Magnetospirillum magneticum AMB-1. By characterizing MAI deletion mutants, we show that parts of its conserved core are not essential for magnetosome biogenesis and that nonconserved genes are important for crystal formation. Most importantly, we show that the mamAB gene cluster encodes for factors important for magnetosome membrane biogenesis, for targeting of proteins to this compartment and for several steps during magnetite production. Altogether, this genetic analysis defines the function of more than a dozen factors participating in magnetosome formation and shows that magnetosomes are assembled in a step-wise manner in which membrane biogenesis, magnetosome protein localization, and biomineralization are placed under discrete genetic control.
Proceedings of the National Academy of Sciences 03/2010; 107(12):5593-8. · 9.68 Impact Factor
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ABSTRACT: The influence of an electrochemical surface passivation technique (cyclic potentiodynamic polarization, CPP) on the physico-chemical surface properties of SS316LS and its subsequent response to fibronectin (Fn) and pre-osteoblasts were investigated. Contact angle and zeta-potential measurements showed that the CPP-modified surface is more hydrophilic and more positively charged than the unmodified surface. Polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS) was used to investigate the interaction of Fn with both surfaces. The saturated surface concentration of adsorbed Fn was higher on the CPP-modified surface. As well, significant changes were identified in the secondary structure of Fn adsorbed on both surfaces, in comparison to its native state. This data also indicated a higher degree of Fn unfolding on the CPP-modified surface. Cell studies indicated that the attachment, proliferation and morphology of pre-osteoblasts were significantly improved on the CPP-modified surface, which was attributed to the more open conformation of Fn on the CPP-modified surface. Thus, the CPP surface passivation method was demonstrated to yield a SS316LS surface of enhanced biocompatibility.
Physical Chemistry Chemical Physics 09/2009; 11(29):6218-24. · 3.57 Impact Factor
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ABSTRACT: Calcium oxalate dihydrate (COD) mineral and the urinary protein osteopontin/uropontin (OPN) are commonly found in kidney stones. To investigate the effects of OPN on COD growth, COD crystals were grown with phosphorylated OPN or a polyaspartic acid-rich peptide of OPN (DDLDDDDD, poly-Asp(86-93)). Crystals grown with OPN showed increased dimensions of the {110} prismatic faces attributable to selective inhibition at this crystallographic face. At high concentrations of OPN, elongated crystals with dominant {110} faces were produced, often with intergrown, interpenetrating twin crystals. Poly-Asp(86-93) dose-dependently elongated crystal morphology along the {110} faces in a manner similar to OPN. In crystal growth studies using fluorescently tagged poly-Asp(86-93) followed by imaging of crystal interiors using confocal microscopy, sectoral (compositional) zoning in COD was observed resulting from selective binding and incorporation (occlusion) of peptide exclusively into {110} crystal sectors. Computational modeling of poly-Asp(86-93) adsorption to COD {110} and {101} surfaces also suggests increased stabilization of the COD {110} surface and negligible change to the natively stable {101} surface. Ultrastructural, colloidal-gold immunolocalization of OPN by transmission electron microscopy in human stones confirmed an intracrystalline distribution of OPN. In summary, OPN and its poly-Asp(86-93) sequence similarly affect COD mineral growth; the {110} crystallographic faces become enhanced and dominant attributable to {110} face inhibition by the protein/peptide, and peptides can incorporate into the mineral phase. We, thus, conclude that the poly-Asp(86-93) domain is central to the OPN ability to interact with the {110} faces of COD, where it binds to inhibit crystal growth with subsequent intracrystalline incorporation (occlusion).
Journal of Biological Chemistry 08/2009; 284(35):23491-501. · 4.77 Impact Factor
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ABSTRACT: Modification of a biomedical-grade stainless steel 316LS surface by electrochemical cyclic potentiodynamic passivation (CPP) and the response of fibrinogen (Fg), platelets, endothelial cells (ECs) and smooth muscles cells (SMCs) to this surface was investigated. Polarization modulation infrared reflection absorption spectroscopy revealed a significant difference between the secondary structure of Fg adsorbed on the unmodified and CPP surface, the latter being closer to that of native Fg. This was postulated as the origin of the significantly lower surface density of attached platelets on the CPP surface. The competitive interaction of ECs and SMCs with the surface showed that the ECs/SMCs surface density ratio is significantly higher on the CPP surface over the first 2h of attachment, suggesting faster initial attachment kinetics of ECs on the CPP surface. The presented results thus clearly demonstrate an increase in biocompatibility of the CPP 316LS surface.
Acta biomaterialia 08/2009; 6(2):695-701. · 3.98 Impact Factor
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ABSTRACT: Oxidative stress, deposition of non-transferrin iron, and mitochondrial insufficiency occur in the brains of patients with Alzheimer disease (AD) and Parkinson disease (PD). We previously demonstrated that heme oxygenase-1 (HO-1) is up-regulated in AD and PD brain and promotes the accumulation of non-transferrin iron in astroglial mitochondria. Herein, dynamic secondary ion mass spectrometry (SIMS) and other techniques were employed to ascertain (i) the impact of HO-1 over-expression on astroglial mitochondrial morphology in vitro, (ii) the topography of aberrant iron sequestration in astrocytes over-expressing HO-1, and (iii) the role of iron regulatory proteins (IRP) in HO-1-mediated iron deposition. Astroglial hHO-1 over-expression induced cytoplasmic vacuolation, mitochondrial membrane damage, and macroautophagy. HO-1 promoted trapping of redox-active iron and sulfur within many cytopathological profiles without impacting ferroportin, transferrin receptor, ferritin, and IRP2 protein levels or IRP1 activity. Thus, HO-1 activity promotes mitochondrial macroautophagy and sequestration of redox-active iron in astroglia independently of classical iron mobilization pathways. Glial HO-1 may be a rational therapeutic target in AD, PD, and other human CNS conditions characterized by the unregulated deposition of brain iron.
Journal of Neurochemistry 03/2009; 109(3):776-91. · 4.06 Impact Factor
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ABSTRACT: To date the cellular and molecular mechanisms by which liver pathological calcifications occur and are regulated are poorly investigated. To study the mechanisms linked to their appearance, we performed a proteomics analysis of calcified liver samples. To this end, human liver biopsies collected in noncalcified (N), precalcified (P), and calcified (C) areas of the liver were subjected to weak ion exchange chromatography, SDS-PAGE, and LC-ESI MS/MS analyses. As we previously demonstrated that alpha-smooth muscle actin (α-SMA) expressing myofibroblasts were involved in liver pathological calcification, we performed a targeted analysis of actin cytoskeleton remodeling-related proteins. This revealed dramatic changes in protein expression patterns in the periphery of the calcified areas. More particularly, we found that IQGAP1 and IQGAP2 proteins were subjected to major expression changes. We show that IQGAP1 expression within P and C areas of the liver correlates with the high abundance of myofibroblasts and that IQGAP1 is specifically expressed in these cells. In addition, we find that IQGAP1 is part of a protein complex including β-catenin and Rac1 mainly in P and C regions of the liver. These results suggest that IQGAP1 may play a critical role in the regulation of cytoskeleton remodeling in liver myofibroblasts in response to liver injury and consequently impact on their function.
PROTEOMICS - CLINICAL APPLICATIONS 02/2009; 3(3):307 - 321. · 1.81 Impact Factor
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ABSTRACT: Cell adhesion is a fundamental process that controls cell proliferation, migration, and differentiation and is crucial for biomaterial-tissue integration. Osteoblast attachment on the surfaces of implant materials is, therefore, essential for the proper function of any implant in which osseointegration is required. Although many reports are available on osteoblast attachment using different surface modification, there is no specific report, so far, that investigates the effect of atomic order of specific crystallographic orientation of substrates on cell behavior. A novel coculture system is proposed to show the differential response of preosteoblast and fibroblast cell lines to the titanium single-crystal substrates. Our investigation has shown that surface recognition by the cell is influenced by the atomic structure of the surface leading to cell-type-specific adhesion. The degree of preosteoblast attachment is significantly higher on the Ti-(1120), whereas the fibroblast adhesion is increased on the Ti-(1010). This demonstrates that the three distinct faces of titanium substrates differ greatly in their capacity to serve as cell adhesive substrates. It also provides clear evidence for the role of crystal structure in regulating and improving cell-substrate interactions relevant for the optimal function of bone implant materials.
Journal of Biomedical Materials Research Part A 12/2008; 91(3):656-62. · 2.63 Impact Factor
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Dirk Schumann,
Timothy D Raub,
Robert E Kopp,
Jean-Luc Guerquin-Kern,
Ting-Di Wu,
Isabelle Rouiller,
Aleksey V Smirnov,
S Kelly Sears,
Uwe Lücken,
Sonia M Tikoo,
Reinhard Hesse,
Joseph L Kirschvink, Hojatollah Vali
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ABSTRACT: We report the discovery of exceptionally large biogenic magnetite crystals in clay-rich sediments spanning the Paleocene-Eocene Thermal Maximum (PETM) in a borehole at Ancora, NJ. Aside from previously described abundant bacterial magnetofossils, electron microscopy reveals novel spearhead-like and spindle-like magnetite up to 4 microm long and hexaoctahedral prisms up to 1.4 microm long. Similar to magnetite produced by magnetotactic bacteria, these single-crystal particles exhibit chemical composition, lattice perfection, and oxygen isotopes consistent with an aquatic origin. Electron holography indicates single-domain magnetization despite their large crystal size. We suggest that the development of a thick suboxic zone with high iron bioavailability--a product of dramatic changes in weathering and sedimentation patterns driven by severe global warming--drove diversification of magnetite-forming organisms, likely including eukaryotes.
Proceedings of the National Academy of Sciences 11/2008; 105(46):17648-53. · 9.68 Impact Factor
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ABSTRACT: In contrast to physiologic biomineralization occurring in bones, teeth and otoconia in vertebrates, calcification of soft tissues occurs in many pathologic conditions. Although similarities have been noted between the two processes, and despite the important clinical consequences of ectopic calcification, the molecular mechanisms regulating ectopic calcification are poorly understood. Although calcification is mainly extracellular, intracellular calcification has been reported and might indeed contribute to pathologic calcification of soft tissues. To better understand the process of intracellular calcification as a potential origin for pathologic calcification, and to examine the role of proteoglycans in this process, we investigated a pattern of intracellular nucleation and growth of hydroxyapatite in Madin-Darby Canine Kidney (MDCK) epithelial cells using electron microscopy, secondary ion mass spectroscopy (NanoSIMS), cytochemical staining, immunolabeling and biochemical analysis. We report here that under mineralizing cell culture conditions where beta-glycerophosphate (betaGP) was added as an exogenous organic source of phosphate, betaGP-cleaving alkaline phosphatase activity increased and hydroxyapatite crystals subsequently nucleated within intracellular, membrane-bounded compartments. The small, leucine-rich proteoglycan decorin was also upregulated and associated with mineral in these cultures. Such information provides insight into the mechanisms leading to pathologic calcification and describes a process whereby hydroxyapatite deposition in cells might lead to ectopic calcification.
Journal of Structural Biology 07/2008; 162(3):468-79. · 3.41 Impact Factor
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ABSTRACT: Ultra-fine surface features are commonly used to modulate cellular activity on a variety of materials. The continuing challenge for materials in contact with bone is the development of a material with both favorable surface and bulk properties to modulate not only the cell-substrate interactions, but also to ensure the long-term stability of the implant. In a combined approach involving material sciences and cell and molecular biology, the nature and mechanism of cell-substrate interaction, in particular, the molecular machinery controlling cell response to the surface of the nanostructured titanium based material produced by the high pressure torsion (HPT) process is assessed. The degree of pre-osteoblast attachment and rate of growth, which are regulated through the activity and interaction of proteins present in the extracellular matrix and associated with cytoskeleton and focal adhesion, are notably increased on the HPT-processed titanium substrates. The improved cell activity is attributed to the nanostructured feature of these substrates consisting of ultra-fine crystals (<50 nm) and a distinct surface oxide layer which provide higher degree of surface wettability. These findings demonstrate the advantages of HPT-processed titanium over the conventional and coated titanium implants, as both mechanical properties and cellular response are improved.
Biomaterials 09/2007; 28(27):3887-95. · 7.40 Impact Factor
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ABSTRACT: A microbial process that exploits the ability of iron-reducing microorganisms to produce copious amounts of extra-cellular metal (M)-substituted magnetite nanoparticles using akaganeite and dopants of dissolved form has previously been reported. The objectives of this study were to develop methods for producing M-substituted magnetite nanoparticles with a high rate of metal substitution by biological processes and to identify factors affecting the production of nano-crystals. The thermophilic and psychrotolerant iron-reducing bacteria had the ability to form M-substituted magnetite nano-crystals (M(y)Fe(3-y)O(4)) from a doped precursor, mixed-M iron oxyhydroxide, (M(x)Fe(1-x)OOH, x< or =0.5, M is Mn, Zn, Ni, Co and Cr). Within the range of 0.01< or =x< or =0.3, using the mixed precursor material enabled the microbial synthesis of more heavily substituted magnetite compared to the previous method, in which the precursor was pure akaganeite and the dopants were present as soluble metal salts. The mixed precursor method was especially advantageous in the case of toxic metals such as Cr and Ni. Also this new method increased the production rate and magnetic properties of the product, while improving crystallinity, size control and scalability.
Journal of Microbiological Methods 07/2007; 70(1):150-8. · 2.09 Impact Factor
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ABSTRACT: There is increasing interest in the usefulness of block copolymer micelles as drug delivery vehicles. However, their subcellular distribution has not been explored extensively, mostly because of the lack of adequately labeled block copolymers. In a previous study, we showed that fluorescently labeled block copolymer micelles entered living cells and co-localized with cytoplasmic organelles selectively labeled with fluorescent dyes. The details of the observed co-localizations were, however, limited by the resolution of the fluorescence approach, which is ca. 500 nm. Using transmission electron microscopy (TEM), we established time- and concentration-dependent subcellular distributions of gold-labeled micelles within human embryonic kidney (HEK 293) cells and human lung carcinoma (A549) cells. Gold particles were incorporated into poly(4-vinylpyridine)-block-poly(ethylene oxide) (P4VP21-b-PEO45) micelles. Data from dynamic light scattering (DLS) and TEM analyses revealed that the sizes of the gold particles ranged from 4 to 8 nm. The cells survived up to 24 h in the presence of low gold-labeled micelle concentrations (0.73 microg/mL), but cell death occurred at higher concentrations (i.e., kidney cells are more susceptible than lung cells). Over 24 h periods of equivalent exposure, lung cells internalized significantly more gold-incorporated micelles than kidney cells. Although micelles were added to the cell culture media as dispersed colloidal particles, the presence of serum in these media caused aggregation. These aggregates occurred mainly close to the cell plasma membrane at early times (5-10 min); however, at later times (24 h) aggregated particles were seen inside endosomes and lysozomes. Thus, gold-incorporated (labeled) micelles can serve as a valuable extension of the fluorescence approach to visualizing the localization of micelles in subcellular compartments, improving the resolution by at least 20-fold.
Langmuir 04/2007; 23(9):4830-6. · 4.19 Impact Factor
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ABSTRACT: Recent studies suggest a possible link between calcification and ischemia-reperfusion injury following liver transplantation. Histological staining, immunolabeling, and biochemical and electron microscopy analyses were applied to assess the possible mechanism(s) of calcification in liver tissue. Although light microscopy studies did not reveal the presence of large necrotic or apoptotic areas, electron microscopy showed the presence of membrane-bound vacuolar structures in hepatocytes, indicative of cell damage. Myofibroblasts were abundant in regions surrounding and within calcification. In these precalcified and calcified areas, myofibroblasts expressed bone-specific matrix proteins, such as osteopontin, type 1 collagen and bone sialoprotein. In addition, transforming growth factor beta (TGFbeta)-1 and BMP2, two growth factors implicated in osteoblast differentiation, and Runx2 and Msx2, two transcription factors targets of TGFbeta-1 and BMP2, were also expressed in these myofibroblasts. These data suggest that liver calcification following transplantation may be a consequence of precipitation of hydroxylapatite emanating from necrotic or apoptotic hepatocytes associated with proliferation of myofibroblasts expressing bone-specific matrix proteins.
Modern Pathology 04/2007; 20(3):357-66. · 4.79 Impact Factor
