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1.
Curr Cancer Drug Targets ; 12(8): 925-39, 2012 Oct.
Article in English | MEDLINE | ID: mdl-22463390

ABSTRACT

Polysialic acid (polySia) is a carbohydrate polymer critical for neuronal cell migration and axon pathfinding in embryonic development. Besides brain regions requiring persistent neuronal plasticity, polySia is essentially absent from the adult body. However, polySia is aberrantly re-expressed on many tumours, where it decorates the surface of NCAM (neuronal cell adhesion molecule) and modulates cell adhesion, migration and invasion. PolySia-NCAM expression is strongly associated with poor clinical prognosis and correlates with aggressive and invasive disease in many cancers, including lung cancer, neuroblastoma and gliomas. The synthesis of polySia is mediated by two polysialyltransferases (polySTs), ST8SiaIV (PST) and particularly ST8SiaII (STX) in cancer cells. The demonstration that polyST knock-down negates events associated with tumour cell dissemination indicates that PST and STX are validated targets. Selective inhibition of polySTs therefore presents a therapeutic opportunity to inhibit tumour invasion and metastasis.


Subject(s)
Neoplasms/metabolism , Neoplasms/pathology , Sialic Acids/metabolism , Sialyltransferases/antagonists & inhibitors , Sialyltransferases/metabolism , Animals , Central Nervous System Neoplasms/metabolism , Central Nervous System Neoplasms/pathology , Drug Design , Enzyme Inhibitors/pharmacology , Glioma/metabolism , Glioma/pathology , Glycocalyx/metabolism , Humans , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Neoplasms/drug therapy , Neural Cell Adhesion Molecules/metabolism , Neuroblastoma/metabolism , Neuroblastoma/pathology , Prognosis , Rhabdomyosarcoma/metabolism , Rhabdomyosarcoma/pathology , Sialic Acids/biosynthesis , Sialyltransferases/genetics
2.
Comput Methods Programs Biomed ; 104(2): 93-103, 2011 Nov.
Article in English | MEDLINE | ID: mdl-20621382

ABSTRACT

A compartmental model for the in vitro uptake kinetics of the anti-cancer agent topotecan (TPT) has been extended from a previously published model. The extended model describes the drug activity and delivery of the pharmacologically active form to the DNA target as well as the catalysis of the aldehyde dehydrogenase (ALDH) enzyme and the elimination of drug from the cytoplasm via the efflux pump. Verification of the proposed model is achieved using scanning-laser microscopy data from live human breast cancer cells. Before estimating the unknown model parameters from the experimental in vitro data it is essential to determine parameter uniqueness (or otherwise) from this imposed output structure. This is formally performed as a structural identifiability analysis, which demonstrates that all of the unknown model parameters are uniquely determined by the output structure corresponding to the experiment.


Subject(s)
ATP-Binding Cassette Transporters/metabolism , Aldehyde Dehydrogenase/metabolism , Drug Resistance, Neoplasm , Models, Theoretical , Neoplasm Proteins/metabolism , ATP Binding Cassette Transporter, Subfamily G, Member 2 , Cell Line, Tumor , Humans , Kinetics , Protein Transport
3.
Comput Methods Programs Biomed ; 89(2): 169-78, 2008 Feb.
Article in English | MEDLINE | ID: mdl-18082908

ABSTRACT

A model describing the response of the growth of single human cells in the absence and presence of the anti-cancer agent topotecan (TPT) is presented. The model includes a novel coupling of both the kinetics of TPT and cell cycle responses to the agent. By linking the models in this way, rather than using separate (disjoint) approaches, it is possible to illustrate how the drug perturbs the cell cycle. The model is compared to experimental in vitro cell cycle response data (comprising single cell descriptors for molecular and behavioural events), showing good qualitative agreement for a range of TPT dose levels.


Subject(s)
Antineoplastic Agents/pharmacokinetics , Cell Cycle/drug effects , Dose-Response Relationship, Drug , Models, Biological , Topotecan/pharmacokinetics , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/therapeutic use , Cell Cycle/genetics , Humans , Topotecan/administration & dosage , Topotecan/therapeutic use , United Kingdom
5.
J Biomed Biotechnol ; 2007(7): 54169, 2007.
Article in English | MEDLINE | ID: mdl-18273410

ABSTRACT

Colloidal quantum dots (QDs) are now commercially available in a biofunctionalized form, and Förster resonance energy transfer (FRET) between bioconjugated dots and fluorophores within the visible range has been observed. We are particularly interested in the far-red region, as from a biological perspective there are benefits in pushing to approximately 700 nm to minimize optical absorption (ABS) within tissue and to avoid cell autofluorescence. We report on FRET between streptavidin- (STV-) conjugated CdTe quantum dots, Qdot705-STV, with biotinylated DY731-Bio fluorophores in a donor-acceptor assay. We also highlight the changes in DY731-Bio absorptivity during the streptavidin-biotin binding process which can be attributed to the structural reorientation. For fluorescence beyond 700 nm, different alloy compositions are required for the QD core and these changes directly affect the fluorescence decay dynamics producing a marked biexponential decay with a long-lifetime component in excess of 100 nanoseconds. We compare the influence of the two QD relaxation routes upon FRET dynamics in the presence of DY731-Bio.

6.
Appl Opt ; 45(9): 2115-23, 2006 Mar 20.
Article in English | MEDLINE | ID: mdl-16579582

ABSTRACT

A study of the practicality a simple technique for obtaining time-domain information that uses continuous wave detection of fluorescence is presented. We show that this technique has potential for use in assays for which a change in the lifetime of an indicator occurs in reaction to an analyte, in fluorescence resonance energy transfer, for example, and could be particularly important when one is carrying out such measurements in the scaled-down environment of a lab on a chip (biochip). A rate-equation model is presented that allows an objective analysis to be made of the relative importance of the key measurement parameters: optical saturation of the fluorophore and period of the excitation pulse. An experimental demonstration of the technique that uses a cuvette-based analysis of a carbocyanine dye and for which the excitation source is a 650 nm wavelength, self-pulsing AlGaInP laser diode is compared with the model.


Subject(s)
Algorithms , Fluorescence Resonance Energy Transfer/methods , Microscopy, Fluorescence/methods , Spectrometry, Fluorescence/methods , Stroboscopy/methods , Fluorescent Dyes , Half-Life , Kinetics
7.
Adv Drug Deliv Rev ; 57(1): 153-67, 2005 Jan 02.
Article in English | MEDLINE | ID: mdl-15518927

ABSTRACT

Many anticancer drugs require interaction with DNA or chromatin components of tumor cells to achieve therapeutic activity. Quantification and exploration of drug targeting dynamics can be highly informative in the rational development of new therapies and in the drug discovery pipeline. The problems faced include the potential infrequency and transient nature of critical events, the influence of micropharmacokinetics on the drug-target equilibria, the dependence on preserving cell function to demonstrate dynamic processes in situ, the need to map events in functional cells and the confounding effects of cell-to-cell heterogeneity. We demonstrate technological solutions in which we have integrated two-photon laser scanning microscopy (TPLSM) to track drug delivery in subcellular compartments, with the mapping of sites of critical molecular interactions. We address key design concepts for the development of modular tools used to uncover the complexity of drug targeting in single cells. First, we describe the combination of two-photon excitation with fluorescence lifetime imaging microscopy (FLIM) to map the nuclear docking of the anticancer drug topotecan (TPT) at a subset of DNA sites in nuclear structures of live breast tumor cells. Secondly, we demonstrate how we incorporate the smart design of a two-photon 'dark' DNA binding probe, such as DRAQ5, as a well-defined quenching probe to uncover sites of drug interaction. Finally, we discuss the future perspectives on introducing these modular kinetic assays in the high-content screening arena and the interlinking of the consequences of drug-target interactions with cellular stress responses.


Subject(s)
Antineoplastic Agents/administration & dosage , Drug Delivery Systems/methods , Microscopy, Fluorescence/methods , Models, Molecular , Technology, Pharmaceutical , Topotecan/administration & dosage , Antineoplastic Agents/metabolism , Antineoplastic Agents/pharmacokinetics , Humans , Topotecan/metabolism , Topotecan/pharmacokinetics
8.
Br J Cancer ; 88(10): 1649-58, 2003 May 19.
Article in English | MEDLINE | ID: mdl-12771935

ABSTRACT

The links between low-dose range taxol-induced mitotic arrest and the subsequent engagement of apoptosis are important for identifying the routes to therapeutic action. Here we have investigated the timing of cell-cycle perturbation and cell death responses following continuous exposure to clinically relevant drug concentrations (1-20 nM). Following 8 h of exposure to taxol, the cell line DoHH2 (p53 wild type) exhibited mitotic arrest and engagement of apoptosis, whereas the cell line SU-DHL-4 (p53 mutant) breached cell-cycle arrest with progression to an abnormal cycle and a 24 h delay in the engagement of apoptosis. Imaging showed equivalent dysfunction of mitotic spindles in both cell lines. The results of kinetic analyses indicated that although cell death may occur at different stages of progression through mitosis and subsequent cell cycles, the overall kinetics of cell death relate to the rate of arrival at a critical event window in the cell cycle. We propose a simple model of low-dose taxol-induced cell death for cycling populations in which mitotic stress acts as a primary trigger for apoptosis with equivalent but potentially delayed outcomes. This view provides a rationale for the clinical effectiveness of this agent, independent of the initial capacity of the tumour cell to engage apoptosis due, for example, to mutant p53 expression. The results provide a perspective for the design of combination regimens that include low-dose taxol and a component that may disturb mitotic delivery.


Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Apoptosis/drug effects , Lymphoma, B-Cell/pathology , Lymphoma, Follicular/pathology , Mitosis/drug effects , Paclitaxel/pharmacology , Gene Expression Regulation, Neoplastic/drug effects , Humans , Immunohistochemistry , Time Factors , Tumor Cells, Cultured , Tumor Suppressor Protein p53/biosynthesis
9.
Br J Cancer ; 88(8): 1310-7, 2003 Apr 22.
Article in English | MEDLINE | ID: mdl-12698201

ABSTRACT

The anticancer agent topotecan is considered to be S-phase specific. This implies that cancer cells that are not actively replicating DNA could resist the effects of the drug. The cycle specificity of topotecan action was investigated in MCF-7 cells, using time-lapse microscopy to link the initial cell cycle position during acute exposures to topotecan with the antiproliferative consequences for individual cells. The bioactive dose range (0.5-10 microM) for 1-h topotecan exposures was defined by rapid drug delivery and topoisomerase I trapping. Topotecan caused pan-cycle induction and activation of p53. Lineage analysis of the time-lapse sequences identified cells initially in S-phase and G2, and defined the time to mitosis for cells originating from G2, S-phase and G1. Topotecan prevented all mitoses from S-phase cells and G1 cells (half-maximal effects at 0.14 microM and 0.96 microM, respectively). No dose of topotecan completely prevented mitosis among G2 cells, and at saturating doses of topotecan about half the cells of G2 origin continued dividing (the half-maximal effects was at 0.31 microM). Overall, topotecan differentially targeted G1-, S- and G2-phase cells, but many G2 cells were resistant to topotecan, presenting a clear route for cell cycle-mediated drug resistance.


Subject(s)
Breast Neoplasms/pathology , Cell Cycle/drug effects , Topotecan/toxicity , Antineoplastic Agents/toxicity , Biological Transport , Cell Line, Tumor , Female , Humans , Kinetics , Topotecan/pharmacokinetics , Tumor Suppressor Protein p53/metabolism
10.
J Cell Sci ; 114(Pt 21): 3845-55, 2001 Nov.
Article in English | MEDLINE | ID: mdl-11719551

ABSTRACT

The assembly of gap junctions was investigated in mammalian cells expressing connexin (Cx) 26, 32 and 43 fused to green, yellow or cyan fluorescent proteins (GFP, YFP, CFP). Targeting of Cx32-CFP and 43-GFP to gap junctions and gap junctional communication was inhibited in cells treated with Brefeldin A, a drug that disassembles the Golgi. However gap junctions constructed of Cx26-GFP were only minimally affected by Brefeldin A. Nocodazole, a microtubule disruptor, had little effect on the assembly of Cx43-GFP gap junctions, but perturbed assembly of Cx26-GFP gap junctions. Co-expression of Cx26-YFP and Cx32-CFP in cells treated with Brefeldin A resulted in assembly of gap junctions constructed of Cx26-YFP. Two amino acids that distinguish Cx26 from Cx32 in transmembrane domains were mutated in Cx32 to investigate underlying mechanisms determining trafficking routes to gap junctions. One mutation, Cx32I28L, conferred on it partial Cx26-like trafficking properties as well the post-translational membrane insertion characteristics of Cx26, suggesting that a key determinant regulating trafficking was present in the first transmembrane domain. The results provide a protein trafficking basis for specifying and regulating connexin composition of gap junctions and thus selectivity of intercellular signaling, with Cx32 and 43 trafficking through the secretory pathway and Cx26 also following an alternative pathway.


Subject(s)
Connexin 43/metabolism , Connexins/metabolism , Endoplasmic Reticulum/metabolism , Gap Junctions/metabolism , Intracellular Membranes/metabolism , Signal Transduction , Animals , Biological Transport , Brefeldin A/pharmacology , COS Cells , Chlorocebus aethiops , Connexin 26 , Connexin 43/genetics , Connexins/genetics , Cytoskeleton/drug effects , Cytoskeleton/metabolism , HeLa Cells , Humans , Nocodazole/pharmacology , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Gap Junction beta-1 Protein
11.
Biophys J ; 81(1): 57-65, 2001 Jul.
Article in English | MEDLINE | ID: mdl-11423394

ABSTRACT

An important aspect of Ca(2+) signaling is the ability of cells to generate intracellular Ca(2+) waves. In this study we have analyzed the cellular and subcellular kinetics of Ca(2+) waves in a neuroendocrine transducer cell, the melanotrope of Xenopus laevis, using the ratiometric Ca(2+) probe indo-1 and video-rate UV confocal laser-scanning microscopy. The purpose of the present study was to investigate how local Ca(2+) changes contribute to a global Ca(2+) signal; subsequently we quantified how a Ca(2+) wave is kinetically reshaped as it is propagated through the cell. The combined kinetics of all subcellular Ca(2+) signals determined the shape of the total cellular Ca(2+) signal, but each subcellular contribution to the cellular signal was not constant in time. Near the plasma membrane, [Ca(2+)](i) increased and decreased rapidly, processes that can be described by a linear and exponential function, respectively. In more central parts of the cell slower kinetics were observed that were best described by a Hill equation. This reshaping of the Ca(2+) wave was modeled with an equation derived from a low-pass RC filter. We propose that the differences in spatial kinetics of the Ca(2+) signal serves as a mechanism by which the same cellular Ca(2+) signal carries different regulatory information to different subcellular regions of the cell, thus evoking differential cellular responses.


Subject(s)
Calcium Signaling , Calcium/metabolism , Erythrocytes/metabolism , Intracellular Fluid/metabolism , Animals , Calibration , Electrochemistry , Erythrocytes/cytology , Imaging, Three-Dimensional , Kinetics , Models, Biological , Xenopus laevis
12.
Cell Commun Adhes ; 8(4-6): 243-8, 2001.
Article in English | MEDLINE | ID: mdl-12064596

ABSTRACT

The assembly of gap junction channels was studied using mammalian cells expressing connexin (Cx) 26, 32 and 43 in which the carboxyl terminus was fused to green, yellow or cyan fluorescent proteins (GFP, YFP, CFP). Intracellular targeting of Cx32-CFP and 43-GFP to gap junctions was disrupted by brefeldin A treatment and resulted in a severe loss of gap junctional intercellular communication reflected by low intercellular dye transfer. Cells expressing Cx43-GFP exposed to nocodazole showed normal targeting to gap junctions and dye transfer. Cx32 and 43 thus appear to be transported and assembled into gap junctions via the classical secretory pathway. In contrast, we found that assembly of Cx26-GFP into functional gap junctions was relatively unaffected by treatment of cells with brefeldin A, but was extremely sensitive to nocodazole treatment. Coexpression of Cx26-YFP and Cx32-CFP indicated a different intracellular distribution that was accentuated in the presence of brefeldin A, with the gap junctions in these cells constructed predominantly of Cx26-YFP. A site specific mutation in the first transmembrane domain that distinguished Cx32 from Cx26 (Cx32128L) resulted in the adoption of the trafficking properties of Cx26 as well as its unusual post-translational membrane integration characteristics. The results indicate that multiple intracellular connexin trafficking routes exist and provide a further mechanism for regulating the connexin composition of gap junctions and thus specificity in intercellular signalling.


Subject(s)
Connexins/metabolism , Gap Junctions/metabolism , Luminescent Proteins/metabolism , Protein Transport/physiology , Animals , Brefeldin A/pharmacology , COS Cells , Connexin 26 , Connexins/chemistry , Connexins/genetics , Fluorescent Dyes/metabolism , Gap Junctions/chemistry , HeLa Cells , Humans , Luminescent Proteins/genetics , Mutation , Nocodazole/pharmacology , Protein Synthesis Inhibitors/pharmacology , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism
13.
Cytometry ; 40(4): 280-91, 2000 Aug 01.
Article in English | MEDLINE | ID: mdl-10918279

ABSTRACT

BACKGROUND: The multiparameter fluorometric analysis of intact and fixed cells often requires the use of a nuclear DNA discrimination signal with spectral separation from visible range fluorochromes. We have developed a novel deep red fluorescing bisalkylaminoanthraquinone, DRAQ5 (Ex(lambdamax) 646 nm; Em(lambdamax) 681 nm; Em(lambdarange) 665->800 nm), with high affinity for DNA and a high capacity to enter living cells. We describe here the spectral characteristics and applications of this synthetic compound, particularly in relation to cytometric analysis of the cell cycle. METHODS: Cultured human tumor cells were examined for the ability to nuclear locate DRAQ5 using single and multiphoton laser scanning microscopy (LSM) and multiparameter flow cytometry. RESULTS: Multiparameter flow cytometry shows that the dye can rapidly report the cellular DNA content of live and fixed cells at a resolution level adequate for cell cycle analysis and the cycle-specific expression of cellular proteins (e.g., cyclin B1). The preferential excitation of DRAQ5 by laser red lines (633/647 nm) was found to offer a means of fluorescence signal discrimination by selective excitation, with greatly reduced emission overlap with UV-excitable and visible range fluophors as compared with propidium iodide. LSM reveals nuclear architecture and clearly defines chromosomal elements in live cells. DRAQ5 was found to permit multiphoton imaging of nuclei using a 1,047-nm emitting mode-locked YLF laser. The unusual spectral properties of DRAQ5 also permit live cell DNA analysis using conventional 488 nm excitation and the single-photon imaging of nuclear fluorescence using laser excitation between 488 nm and low infrared (IR; 780 nm) wavelengths. Single and multiphoton microscopy studies revealed the ability of DRAQ5 to report three-dimensional nuclear structure and location in live cells expressing endoplasmic reticulum targeted-GFP, MitoTracker-stained mitochondria, or a vital cell probe for free zinc (Zinquin). CONCLUSION: The fluorescence excitation and emission characteristics of DRAQ5 in living and fixed cells permit the incorporation of the measurement of cellular DNA content into a variety of multiparameter cytometric analyses.


Subject(s)
DNA Probes , DNA, Neoplasm/analysis , Fluorescent Dyes , Melanoma/genetics , Nitrogen Oxides , Anthraquinones , CDC2 Protein Kinase/metabolism , Cell Cycle , Cyclin B/metabolism , Cyclin B1 , Diagnostic Imaging/methods , Flow Cytometry/methods , Fluorescent Antibody Technique , Gene Expression , Green Fluorescent Proteins , Humans , Infrared Rays , Luminescent Proteins/metabolism , Melanoma/metabolism , Melanoma/pathology , Microscopy, Confocal/methods , Microscopy, Fluorescence/methods , Molecular Structure , Quinolones/metabolism , Spectrophotometry/methods , Tosyl Compounds/metabolism , Tumor Cells, Cultured
15.
J Anat ; 192 ( Pt 3): 369-78, 1998 Apr.
Article in English | MEDLINE | ID: mdl-9688503

ABSTRACT

We examined cells from the nucleus pulposus and annulus fibrosus of adult bovine intervertebral discs, using confocal laser scanning microscopy on living unfixed tissue. These cells were visualised using chloromethyl fluorescein diacetate, a membrane-impermeant fluorescent dye. The organisation of cells from the outer annulus was also determined using confocal microscopy after fixation and staining the actin-filaments with FITC-phalloidin. We found that cellular processes were a dominant feature of cells from all regions of the disc including the cells of the nucleus pulposus and inner annulus. These processes were also visible in histological sections of disc examined both at the light and electron microscope level, even though cells from the nucleus and inner annulus appeared chondrocyte-like, being rounded and enclosed in a capsule. The function of these processes is at present unknown. We suggest that they may serve to sense mechanical strain.


Subject(s)
Image Processing, Computer-Assisted , Intervertebral Disc/cytology , Actins/analysis , Animals , Cattle , Cytoplasm/ultrastructure , Intervertebral Disc/chemistry , Intervertebral Disc/ultrastructure , Microscopy, Confocal , Microscopy, Electron , Staining and Labeling
16.
J Am Soc Nephrol ; 8(10): 1493-501, 1997 Oct.
Article in English | MEDLINE | ID: mdl-9335376

ABSTRACT

Water transport across the mammalian collecting tubule is regulated by vasopressin-dependent aquaporin-2 insertion into and retrieval from the apical cell membrane. To establish a cell line that properly expresses aquaporin-2 and its hormone-dependent shuttling, Madin-Darby canine kidney cells were stably transfected with an aquaporin-2 expression construct. Cells of a representative clone (wild-type 10 [WT-10]) were grown on semipermeable supports, and transcellular osmotic water permeability (Pf; in microm/s +/- SEM) was measured. The basal Pf of WT-10 cells, which was lowered with indomethacin, increased from 10.6 +/- 0.8 to 35.7 +/- 1.2 upon incubation with 1-desamino-8-D-arginine vasopressin (dDAVP). This increase coincided with the translocation of aquaporin-2 from an intracellular compartment to the apical membrane. The Pf of untransfected cells (6.5 +/- 0.8) was unchanged by dDAVP. Kinetic studies with WT-10 cells revealed that maximal Pf was obtained within 30 min after dDAVP addition, which remained elevated for at least 90 min. Intracellular cAMP levels peaked within 5 min after dDAVP admission and decreased to basal levels within 45 min. After preincubation with dDAVP, the Pf decreased within 15 min after dDAVP washout and returned to basal levels within 75 min. In conclusion, the WT-10 cells mimic the vasopressin-regulated transcellular water transport and aquaporin-2 translocation as found in collecting duct cells to a great extent, and therefore constitute an in vitro cell model that can be used to study the regulation of transcellular water transport in detail and provide a simplified test system for screening putative aquaporin-2 blockers.


Subject(s)
Aquaporins , Deamino Arginine Vasopressin/metabolism , Ion Channels/genetics , Ion Channels/metabolism , Kidney Tubules, Collecting/metabolism , Animals , Aquaporin 2 , Aquaporin 6 , Biological Transport, Active , Cell Line , Cyclic AMP/metabolism , Dogs , Immunohistochemistry , Kidney Tubules, Collecting/cytology , Kinetics , Models, Biological , Osmosis , Permeability , RNA, Messenger/genetics , Transfection , Water/metabolism
17.
Cell Biol Int ; 21(4): 201-12, 1997 Apr.
Article in English | MEDLINE | ID: mdl-9204532

ABSTRACT

We have used isoform-specific antibodies against the Na+, K(+)-ATPase alpha (alpha 1, alpha 2 and alpha 3) and beta (beta 1 and beta 2) subunit isoforms in order to establish their specific localization in isolated bovine articular chondrocytes. Immunoblotting confirmed the presence of the alpha 1 and alpha 3 isoforms, although alpha 1 expression was significantly greater than alpha 3 as assessed by immunofluorescence confocal laser scanning microscopy and PCR. A similar approach revealed the presence of the beta 1 and beta 2 isoforms in chondrocytes, although beta 2 immunostaining on the plasma membrane was more punctate than beta 1 which in contrast predominated in a subcellular compartment. The plasma membrane abundance of the Na+, K(+)-ATPase was found to be sensitive to the extracellular ionic concentration and long-term elevation of extracellular Na+ concentration significantly upregulated Na+, K(+)-ATPase density as measured by specific 3H-ouabain binding. Our observations suggest that the expression of alpha 3 and beta 2 is not restricted to excitable tissues as previously reported. The physiological relevance of alpha 3 expression in chondrocytes may be related to its low affinity for intracellular Na+ in an extracellular environment where Na+ concentration is unusually high (260-350 mM) compared to other cell types (140 mM). Glycoproteins and their branched carbohydrates have been implicated in cell recognition events, thus the beta 2 subunit glycoprotein may allow the chondrocyte to detect changes in its extracellular environment by physically interacting with components of the cellular cytoskeleton and matrix macromolecules.


Subject(s)
Cartilage, Articular/enzymology , Isoenzymes/metabolism , Sodium-Potassium-Exchanging ATPase/metabolism , Animals , Antibodies , Blotting, Western , Cattle , Isoenzymes/chemistry , Isoenzymes/isolation & purification , Polymerase Chain Reaction , Sodium-Potassium-Exchanging ATPase/chemistry , Sodium-Potassium-Exchanging ATPase/isolation & purification
18.
Am J Physiol ; 272(3 Pt 1): C1040-51, 1997 Mar.
Article in English | MEDLINE | ID: mdl-9124506

ABSTRACT

Regulation of cell volume is a fundamental cellular homeostatic mechanism in the face of osmotic stress. In normal articular cartilage, chondrocytes are exposed to a changing osmotic environment. We present a comprehensive protocol for studying the volume regulatory behavior of chondrocytes within intact cartilage tissue using confocal laser-scanning microscopy. Our data acquisition regime optimizes both signal-to-noise and cell viability during time-lapsed three-dimensional (3-D) (x, y, z, t) imaging. The porcine cartilage is treated as an integrated component of the imaging system, and we demonstrate methods for the direct assessment of tissue-induced axial attenuation and image distortion. Parameterized functions describing these two components of image degradation are used to correct experimental data. The current study also highlights the problems associated with the analysis and visualization of four-dimensional (4-D) images. We have devised two new types of data reconstruction. The first compresses each 3-D time point into a single quantitative view, termed a coordinate view. From these reconstructions we are able to simultaneously view and extract cell measurements. A second type, a 4-D reconstruction, uses color to represent relative changes in cell volume, again while maintaining the morphological and spatial information. Both these approaches of image analysis and visualization have been implemented to study the morphology, spatial distribution, and dynamic volume behavior of chondrocytes after osmotic perturbation. We have mapped chondrocyte shape, arrangement, and absolute volume in situ, which vary significantly from the tissue surface through to the underlying bone. Despite the rigid nature of the extracellular matrix, cartilage cells are osmotically sensitive and respond to stimulation of volume regulatory mechanisms. The combined techniques of confocal laser-scanning microscopy and vital cell labeling have enabled us to study, for the first time, the response of chondrocytes in situ to changes in interstitial osmotic pressure.


Subject(s)
Cartilage/cytology , Microscopy, Confocal/methods , Water-Electrolyte Balance , Animals , Cell Size , Image Processing, Computer-Assisted , In Vitro Techniques , Swine
19.
J Cell Sci ; 107 ( Pt 7): 1753-60, 1994 Jul.
Article in English | MEDLINE | ID: mdl-7983145

ABSTRACT

The repair of damage induced in DNA by ultraviolet light involves excision of the damaged sequence and synthesis of new DNA to repair the gap. Sites of such repair synthesis were visualized by incubating permeabilized HeLa or MRC-5 cells with the DNA precursor, biotin-dUTP, in a physiological buffer; then incorporated biotin was immunolabeled with fluorescent antibodies. Repair did not take place at sites that reflected the DNA distribution; rather, sites were focally concentrated in a complex pattern. This pattern changed with time; initially intense repair took place at transcriptionally active sites but when transcription became inhibited it continued at sites with little transcription. Repair synthesis in vitro also occurred in the absence of transcription. Repair sites generally contained a high concentration of proliferating cell nuclear antigen but not the tumour-suppressor protein, p53.


Subject(s)
Cell Nucleus/radiation effects , DNA Damage , DNA Repair , DNA/biosynthesis , Proliferating Cell Nuclear Antigen/metabolism , Transcription, Genetic , Tumor Suppressor Protein p53/metabolism , Ultraviolet Rays , Amanitins/pharmacology , Cell Line , Cell Membrane Permeability , Cell Nucleus/metabolism , Cell Nucleus/ultrastructure , DNA/radiation effects , HeLa Cells , Humans , Kinetics , Phosphorus Radioisotopes , Thymine Nucleotides/metabolism , Transcription, Genetic/radiation effects
20.
J Cell Sci ; 107 ( Pt 2): 425-34, 1994 Feb.
Article in English | MEDLINE | ID: mdl-7515893

ABSTRACT

HeLa cells synchronized at different stages of the cell cycle were permeabilized and incubated with analogues of nucleotide triphosphates; then sites of incorporation were immunolabeled with the appropriate fluorescent probes. Confocal microscopy showed that sites of replication and transcription were not diffusely spread throughout nuclei, reflecting the distribution of euchromatin; rather, they were concentrated in 'foci' where many polymerases act together. Transcription foci aggregated as cells progressed towards the G1/S boundary; later they dispersed and became more diffuse. Replication was initiated only at transcription sites; later, when heterochromatin was replicated in enlarged foci, these remained sites of transcription. This illustrates the dynamic nature of nuclear architecture and suggests that transcription may be required for the initiation of DNA synthesis.


Subject(s)
DNA Replication , Ribonucleoproteins, Small Nuclear , Transcription, Genetic , Autoantigens/metabolism , Biotin/analogs & derivatives , Biotin/pharmacology , Cell Cycle , Cell Nucleus/immunology , Cell Nucleus/metabolism , Cell Nucleus/ultrastructure , DNA/biosynthesis , DNA Replication/drug effects , Deoxyuracil Nucleotides/pharmacology , HeLa Cells , Humans , Image Processing, Computer-Assisted , RNA/biosynthesis , Transcription, Genetic/drug effects , snRNP Core Proteins
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