Biomedical subjects

Marc D Jacobs

Publications and source records attributed to Marc D Jacobs.

6 recordsLinked to original sources

PubMed2005

Pim-1 ligand-bound structures reveal the mechanism of serine/threonine kinase inhibition by LY294002.

Pim-1 is an oncogene-encoded serine/threonine kinase primarily expressed in hematopoietic and germ cell lines. Pim-1 kinase was originally identified in Maloney murine leukemia virus-induced T-cell lymphomas and is associated with multiple cellular functions such as proliferation, survival, differentiation, apoptosis, and tumorigenesis (Wang, Z., Bhattacharya, N., Weaver, M., Petersen, K., Meyer, M., Gapter, L., and Magnuson, N. S. (2001) J. Vet. Sci. 2, 167-179). The crystal structures of Pim-1 complexed with staurosporine and adenosine were determined. Although a typical two-domain serine/threonine protein kinase fold is observed, the inter-domain hinge region is unusual in both sequence and conformation; a two-residue insertion causes the hinge to bulge away from the ATP-binding pocket, and a proline residue in the hinge removes a conserved main chain hydrogen bond donor. Without this hydrogen bond, van der Waals interactions with the hinge serve to position the ligand. The hinge region of Pim-1 resembles that of phosphatidylinositol 3-kinase more closely than it does other protein kinases. Although the phosphatidylinositol 3-kinase inhibitor LY294002 also inhibits Pim-1, the structure of the LY294002.Pim-1 complex reveals a new binding mode that may be general for Ser/Thr kinases.

Adenosine↗

PubMed2004

Functional imaging: new views on lens structure and function.

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 lens, we have localized precisely histological sites of connexin 46 cleavage, quantitatively mapped changes in gap junction distribution and fibre cell morphology and correlated these changes to differences in intercellular dye transfer. 3. Profiling of glucose transporter isoform expression showed that lens epithelial cells express GLUT1, whereas deeper cortical fibre cells express the higher-affinity GLUT3 isoform. Near the lens periphery, GLUT3 was located in the cytoplasm of fibre cells, but it underwent a differentiation-dependent membrane insertion. 4. Similarly, the putative adhesion protein membrane protein 20 is inserted into fibre cell membranes at the stage when the cells lose their nuclei. This redistribution is strikingly rapid in terms of fibre cell differentiation and correlates with a barrier to extracellular diffusion. 5. Our imaging-orientated approach has facilitated new insights into the relationships between fibre cell differentiation and lens function. Taken together, our results indicate that a number of strategies are used by the lens during the course of normal differentiation to change the subcellular distribution, gross spatial location and functional properties of key membrane transport proteins.

Animals↗

PubMed2004

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

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 membrane marker wheat germ agglutinin, and the nuclear stain propidium iodide. Two-photon microscopy and digital image analysis were used to quantify label and cell morphology as a function of radial distance (r/a) across the lens. Loop- and tail-specific Cx46 antibodies were used to identify regions of posttranslational modification. Local fiber cell coupling was imaged in situ using two-photon flash photolysis of caged fluorescein. RESULTS: Antibody labeling showed that the cytoplasmic tail of Cx46 was removed in two zones (r/a approximately 0.9 and r/a approximately 0.7). In addition, with increasing depth, the large radially aligned plaques of peripheral fiber cells became fragmented and dispersed around the cell membrane, and cells became more circular in cross section. Fluorescein transfer between peripheral fiber cells was highly anisotropic and occurred predominantly within a column of fiber cells, resulting in radially directed transport. In regions beyond the zone of nuclear loss, transport was more isotropic and occurred across columns of fiber cells. CONCLUSIONS: The cleavage of Cx46 is associated with a spatial redistribution of gap junction plaques. The distribution of gap junction plaques around the cell membrane can explain the observed directionality of intercellular solute transfer. The findings suggest that the processing and redistribution of gap junction proteins is central to controlling radial and circumferential solute gradients in different regions within the lens.

Animals↗

PubMed2003

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

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, we show that by combining two-photon microscopy with novel image montage methods (fast beam blanking coupled with mathematical alignment tools) we have extended the limited field of view of laser scanning microscopes. To illustrate the utility of our approach, the distribution of connexin-46 was visualized across equatorial sections of the rat mammalian lens. By optimizing fixation protocols, good morphological preservation could be achieved over the thickness of the lens (approximately 4 mm) while preserving antigenicity of lens proteins. Using the same image data, changes in lens fiber cell morphology were mapped quantitatively by automatic image analysis routines. The methods presented should be generally applicable to any tissue system where changes in antibody labeling and tissue structure occur over large and small distances.

Animals↗

PubMed2003

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

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 known as Major Intrinsic Protein, MIP) and MP20 change as a function of fibre cell differentiation. While AQP0 was always found associated with fibre cell membranes, MP20 was initially found in the cytoplasm of peripheral fibre cells before becoming inserted into the membranes of deeper fibre cells. To determine at what stage in fibre cell differentiation MP20 becomes inserted into the membrane, sections were double-labelled with an antibody against MP20, and propidium iodide, a marker of cell nuclei. This showed that membrane insertion of MP20 occurs in a discrete transition zone that coincided with the degradation of cell nuclei. To test the significance of the membrane insertion of MP20 to overall lens function, whole lenses were incubated for varying times in a solution containing either Texas Red-dextran or Lucifer yellow as markers of extracellular space. Lenses were fixed and then processed for immunocytochemistry. Analysis of these sections showed that both tracer dyes were excluded from the extracellular space in an area that coincided with insertion of MP20 into the plasma membrane. Our results suggest that the insertion of MP20 into fibre cell membranes coincides with the creation of a barrier that restricts the diffusion of molecules into the lens core via the extracellular space.

Animals↗

PubMed2002

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

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 micro 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 micro M tamoxifen, tissue damage was found in a discrete zone that initially started approximately 100 micro m from the capsule, whereas at higher doses (60-100 micro M tamoxifen), extensive vesiculation of fiber cell membranes occurred throughout the entire lens cortex. Decreasing tamoxifen (100 micro 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.

ATP Binding Cassette Transporter, Subfamily B↗