14 results for Jacobs, MD

  • 3D finite element modeling of avascular circulation in the ocular lens

    Vaghefi Rezaei, Seyed; Hunter, Peter; Jacobs, MD (2008)

    Journal article
    The University of Auckland Library

    The ability to see is dependent on the actions of several structures in and around the eyeball. By looking at an object, light rays are reflected from the object to the cornea. Light rays are refracted and focused by the cornea, lens, and ???vitreous???. The lens function is to ensure that the light rays come to a sharp focus point on the retina. A comprehensive, quantitative model of lens functionality would be of great utility for the development of any anticataract medicine. Yet most of the research to date has focused on isolated and specific features of the crystalline lens. Thus a full integrated 3D model of the lens is needed in order to facilitate development of therapies and prevention procedures for cataracts. Here we developed an initial 3D model of the lens circulation using a finite element modeling approach based on the CMISS simulation environment created at the Auckland Bioengineering Institute. This work represents the first multiscale integrative numerical model of the avascular circulation system. The model we have developed correctly simulates and predicts the available empirical data on lens physiology.

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  • Application of two-photon flash photolysis to measure microscopic diffusion and calcium fluxes

    Cannell, Mark; Jacobs, MD; Donaldson, Paul; Soeller, Christian (2005)

    Journal article
    The University of Auckland Library

    Two-photon excitation (TPE) via a microscope objective lens produces a spatially confined excitation volume where UV-excited caged molecules may be broken (uncaged) to release active products. We describe an optical system that creates a stationary parfocal TPE uncaging spot on the stage of a conventional confocal microscope. With this system, we have examined the ability of two dyes to track microscopic calcium changes produced by TPE photolysis of DM-nitrophen. We find that, even when EGTA is used with a low affinity indicator, the dye signals are complicated by diffusion of both indicator-Ca complex and CaEGTA to produce a signal that does not simply report the spatial dimensions of the calcium release site. In addition, the time course of calcium release is poorly reported. This suggests that considerable caution must be applied to the interpretation of spatially resolved calcium signals inside cells. We have also used TPE of CMND-caged fluorescein to measure the rate of fluorescein production in test solution (2500 s-1) as well as the diffusion of fluorescein in drops of solution and within and between between eye lens fiber cells. While diffusion of uncaged fluorescein was about an order of magnitude slower inside fiber cells than in aequeous solution, slower diffusion between cells could also be detected and could be explained by the gap junctions joining the cells behaving as a barrier to diffusion. By using a computer model, parameter fits to experimental data gave estimates for both intracellular and intercellular diffusion coefficients. From this analysis, the gap junctions in eye lens fiber cells permit exchange of low molecular weight compounds between cells at about 0.4% of the rate of free diffusion.

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  • Cytological characterization of heterochromatin and rDNA in Pinus radiata and P. taeda

    Jacobs, MD; Gardner, Richard; Murray, Brian (2000)

    Journal article
    The University of Auckland Library

    Fluorochrome C-banding of Pinus radiata and P. taeda metaphase chromosomes showed many pericentromeric DAPI bands and interstitial CMA bands in P. radiata, and centromeric and interstitial CMA bands in P. taeda. Giemsa C-band patterns differed between the species with centromeric bands in P. radiata but no consistent bands in P, taeda. A karyotype of P. radiata was developed based on banding patterns that distinguished all but two of the 12 pairs of chromosomes. Insitu hybridization (ISH) using probes for high-copy ribosomal DNA (rDNA) showed 10 pairs of 18S-25S sites and two pairs of 5S sites in both species. Most of the sites were interstitial or centromeric.

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  • Insertion of MP20 into lens fibre cell plasma membranes correlates with the formation of an extracellular diffusion barrier

    Grey, Angus; Jacobs, MD; Tamir, G; Kistler, Joerg; Donaldson, Paul (2003)

    Journal article
    The University of Auckland Library

    It is known that during lens differentiation a number of fibre cell specific membrane proteins change their expression profiles. In this study we have investigated how the profiles of the two most abundant fibre cell membrane proteins AQP0 (formerly

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  • Quantifying changes in gap junction structure as a function of lens fiber cell differentiation

    Jacobs, MD; Soeller, Christian; Cannell, Mark; Donaldson, Paul (2001)

    Journal article
    The University of Auckland Library

    The ocular lens is an ideal model system for studying gap junction structure-function relationships. Here we apply novel methods to quantitatively compare connexin expression over macroscopic distances while simultaneously resolving the intracellular

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  • Molecular identification of p-glycoprotein: a role in lens circulation?

    Merriman-Smith, Barbara; Young, MA; Jacobs, MD; Kistler, Joerg; Donaldson, Paul (2002)

    Journal article
    The University of Auckland Library

    PURPOSE. To determine whether P-glycoprotein is expressed in the rat lens and to assess what type of damage occurs when P-glycoprotein inhibitors are applied to organ-cultured lenses. METHODS. An initial screening for the P-glycoprotein isoforms multidrug resistance (mdr)1a, mdr1b, and mdr2 was performed by RT-PCR on RNA extracted from rat lens fiber cells. Northern blot analysis was used to determine whether transcript levels detected by RT-PCR were significant. The presence of P-glycoprotein in the lens was confirmed by Western blot analysis and immunocytochemistry. Organ-cultured lenses, maintained in isotonic artificial aqueous humor, were exposed to various concentrations of the P-glycoprotein inhibitor tamoxifen. Lens opacification was assessed by dark-field microscopy, and the underlying cellular changes were visualized by confocal microscopy of lens sections, using a fluorescent membrane marker. Initial cellular damage was assessed after a 6-hour exposure to 100 ??M tamoxifen. Other P-glycoprotein inhibitors, verapamil, and 1,9-dideoxyforskolin (DDFK) were assessed, and the damage phenotypes were compared with those seen for tamoxifen. RESULTS. Transcript for all three P-glycoprotein isoforms was detected with RT-PCR, but only mdr1a and mdr2 could be detected by Northern blot analysis. P-glycoprotein was localized in the plasma membrane of lens epithelial and fiber cells. Treatment of organ-cultured lenses with increasing doses of the P-glycoprotein inhibitor tamoxifen for 18 hours showed that two distinct damage phenotypes were evident. At a dose of 20 ??M tamoxifen, tissue damage was found in a discrete zone that initially started approximately 100 ??m from the capsule, whereas at higher doses (60???100 ??M tamoxifen), extensive vesiculation of fiber cell membranes occurred throughout the entire lens cortex. Decreasing tamoxifen (100 ??M) exposure to 6 hours showed that the inner zone of damage was caused by the dilation of extracellular space between fiber cells. The extracellular space dilution and fiber cell vesiculation could be reproduced by varying the concentrations of other P-glycoprotein inhibitors, verapamil and DDKF. CONCLUSIONS. The P-glycoproteins mdr1a and mdr2 are expressed in the lens and appear to be functional. The initial cellular damage phenotype of extracellular space dilations caused by the P-glycoprotein inhibitors was identical with that caused by chloride channel inhibitors, indicating that P-glycoprotein may play a role in regulating cell volume in the lens. Whether the secondary damage phenotype of fiber cell vesiculation, induced by high doses of P-glycoprotein inhibitors, was due to the inhibition of additional regulatory activities of P-glycoprotein or to nonspecific effects of the drugs remains to be determined. However, regardless of the precise mode of action, these results indicate that P-glycoprotein should be considered in the regulatory mechanisms associated with the control of lens volume and in the initiation of osmotic cataract.

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  • MP20, the second most abundant lens membrane protein and member of the tetraspanin superfamily, joins the list of ligands of galectin-3

    Gonen, T; Grey, Angus; Jacobs, MD; Donaldson, Paul; Kistler, Joerg (2001)

    Journal article
    The University of Auckland Library

    Background: Although MP20 is the second most highly expressed membrane protein in the lens its function remains an enigma. Putative functions for MP20 have recently been inferred from its assignment to the tetraspanin superfamily of integral membrane proteins. Members of this family have been shown to be involved in cellular proliferation, differentiation, migration, and adhesion. In this study, we show that MP20 associates with galectin-3, a known adhesion modulator.Results: MP20 and galectin-3 co-localized in selected areas of the lens fiber cell plasma membrane. Individually, these proteins purified with apparent molecular masses of 60 kDa and 22 kDa, respectively. A 104 kDa complex was formed in vitro upon mixing the purified proteins. A 102 kDa complex of MP20 and galectin-3 could also be isolated from detergent-solublized native fiber cell membranes. Binding between MP20 and galectin-3 was disrupted by lactose suggesting the lectin site was involved in the interaction.Conclusions: MP20 adds to a growing list of ligands of galectin-3 and appears to be the first representative of the tetraspanin superfamily identified to possess this specificity.

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  • Probing microscopic diffusion by 2-photon flash photolysis: Measurement of isotropic and anisotropic diffusion in lens fiber cells

    Cannell, MB; Jacobs, MD; Donaldson, Paul; Soeller, C (2004)

    Journal article
    The University of Auckland Library

    Two-photon excited flash photolysis (TPEFP) was used to photorelease caged fluorescein in test solutions and inside fiber cells of the eye lens. Accurate alignment between the focus of the IR beam and the probe beam from the confocal microscope was a

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  • Resolving morphology and antibody labeling over large distances in tissue sections

    Jacobs, MD; Donaldson, Paul; Cannell, MB; Soeller, C (2003)

    Journal article
    The University of Auckland Library

    Protein expression patterns are a primary determinant of tissue function and in this study we developed methods to study protein expression over macroscopic distances at subcellular levels of detail. Using the mammalian lens as a model tissue system,

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  • Application of two-photon flash photolysis to reveal intercellular communication and intracellular Ca2+ movements

    Soeller, Christian; Jacobs, MD; Donaldson, Paul; Cannell, Mark (2003)

    Journal article
    The University of Auckland Library

    One print or electronic copy may be made for personal use only. Systematic reproduction and distribution, duplication of any material in this paper for a fee or for commercial purposes, or modification of the content of the paper are prohibited. Two-photon excitation makes it possible to excite molecules in volumes of much less than 1 fl. In two-photon flash photolysis (TPFP) this property is used to release effector molecules from caged precursors with high three-dimensional resolution. We

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  • Uptake and distribution of gadolinium in the ocular lens

    Vaghefi Rezaei, Seyed; Jacobs, MD (2008)

    Journal article
    The University of Auckland Library

    The lens of the eye has no blood vessels. Although necessary for transparency, this feature of the ocular lens implies that any circulation in the lens tissue must be avascular. A range of previous studies attests to the metabolic activity of the fiber cells that make up the body of the lens. It is also established that the continuing transparency of the lens depends upon this metabolic activity. When metabolism is disturbed, cataracts (lens opacities) result. It has been proposed that metabolism occurs throughout the lens, enabled by an intercellular micro-circulation system driven by ion pumps and cell volume-regulation mechanisms. The present study attempted directly to trace micro-circulation in the ocular lens on a spatially coarse scale. High field strength magnetic resonance imaging was used to record the movement of gadolinium into the lens and the global distribution patterns that result. Our data lend new support to previous attempts at documenting, by other techniques, differential micro-circulation mechanisms in the ocular lens.

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  • Functional imaging: New views on lens structure and function

    Donaldson, Paul; Grey, Angus; Merriman-Smith, Barbara; Sisley, Aran; Soeller, Christian; Cannell, Mark; Jacobs, MD (2004)

    Journal article
    The University of Auckland Library

    1. We have developed an experimental imaging approach that allows the distribution of lens membrane proteins to be mapped with subcellular resolution over large distances as a function of fibre cell differentiation. 2. Using this approach in the rat

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  • Gap junction processing and redistribution revealed by quantitative optical measurements of connexin46 epitopes in the lens

    Jacobs, MD; Soeller, Christian; Sisley, Aran; Cannell, Mark; Donaldson, Paul (2004)

    Journal article
    The University of Auckland Library

    PURPOSE. To map changes in the structure and function of fiber cell gap junctions that occur with lens differentiation. METHODS. Equatorial lens sections were fluorescently labeled with antibodies to the gap junction protein connexin (Cx)46, the memb

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  • Spatially transformed fluorescence image data for ERK-MAPK and selected proteins within human epidermis

    Cursons, J; Angel, Catherine; Hurley, DG; Print, Cristin; Dunbar, Peter; Jacobs, MD; Crampin, EJ (2015-12-14)

    Journal article
    The University of Auckland Library

    Background: Phosphoprotein signalling pathways have been intensively studied in vitro, yet their role in regulating tissue homeostasis is not fully understood. In the skin, interfollicular keratinocytes differentiate over approximately 2 weeks as they traverse the epidermis. The extracellular signal-regulated kinase (ERK) branch of the mitogen-activated protein kinase (MAPK) pathway has been implicated in this process. Therefore, we examined ERK-MAPK activity within human epidermal keratinocytes in situ. Findings: We used confocal microscopy and immunofluorescence labelling to measure the relative abundances of Raf-1, MEK1/2 and ERK1/2, and their phosphorylated (active) forms within three human skin samples. Additionally, we measured the abundance of selected proteins thought to modulate ERK-MAPK activity, including calmodulin, ??1 integrin and stratifin (14-3-3??); and of transcription factors known to act as effectors of ERK1/2, including the AP-1 components Jun-B, Fra2 and c-Fos. Imaging was performed with sufficient resolution to identify the plasma membrane, cytoplasm and nucleus as distinct domains within cells across the epidermis. The image field of view was also sufficiently large to capture the entire epidermis in cross-section, and thus the full range of keratinocyte differentiation in a single observation. Image processing methods were developed to quantify image data for mathematical and statistical analysis. Here, we provide raw image data and processed outputs. Conclusions: These data indicate coordinated changes in ERK-MAPK signalling activity throughout the depth of the epidermis, with changes in relative phosphorylation-mediated signalling activity occurring along the gradient of cellular differentiation. We believe these data provide unique information about intracellular signalling as they are obtained from a homeostatic human tissue, and they might be useful for investigating intercellular heterogeneity.

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