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1.
Graefes Arch Clin Exp Ophthalmol ; 239(9): 698-704, 2001 Sep.
Article in English | MEDLINE | ID: mdl-11688671

ABSTRACT

BACKGROUND: To evaluate the ocular toxicity of trypan blue (TB) injected into the vitreous cavity of rabbit eyes. TB is a dye that could be useful for staining epiretinal membranes during vitrectomy surgery. METHODS: Ten New Zealand White (NZW) rabbits underwent gas-compression vitrectomy. Rabbits were divided into three groups to receive injections of 0.1 ml basic salt solution, 0.1 ml of a 0.06% TB solution or 0.1 ml of a 0.2% TB solution. Ocular toxicity was assessed by slit-lamp biomicroscopy, ophthalmoscopy, electroretinography and histology. RESULTS: Transient posterior capsule opacification was noted in all animals. No significant reductions in a-wave or b-wave amplitudes were found in any of the animals. Light and electron microscopic examination of the inferior retina in the 0.2% TB-treated eyes showed damaged photoreceptors and marked disorganization. Immunohistochemical staining for rhodopsin was strongly reduced in those sections and staining for proliferation with Ki-67 was positive. No histological abnormalities were found in the upper retina of the 0.2% TB-treated eyes or in any part of the retina of the 0.06% TB-treated or control eyes. No histological abnormalities were found in any of the anterior chamber angle specimens. CONCLUSIONS: Although no signs of toxicity were found after the prolonged presence of TB at a concentration of 0.06% in the vitreous cavity of rabbit eyes, marked damage occurred in the lower retina of 0.2% TB-treated eyes. The short-term presence of TB at a concentration of 0.06% in the vitreous cavity is harmless to the rabbit eye but a higher concentration of TB could be unsafe.


Subject(s)
Cataract/chemically induced , Coloring Agents/toxicity , Lens Capsule, Crystalline/drug effects , Retina/drug effects , Retinal Degeneration/chemically induced , Trypan Blue/toxicity , Vitreous Body/drug effects , Animals , Electroretinography , Immunohistochemistry , Ki-67 Antigen/metabolism , Ophthalmic Solutions , Rabbits , Retina/metabolism , Retina/pathology , Retinal Degeneration/metabolism , Retinal Degeneration/pathology , Rhodopsin/metabolism , Vitrectomy
2.
J Biol Chem ; 275(45): 35122-8, 2000 Nov 10.
Article in English | MEDLINE | ID: mdl-10960478

ABSTRACT

We report on the molecular, biochemical, and functional characterization of Cucurbita maxima phloem serpin-1 (CmPS-1), a novel 42-kDa serine proteinase inhibitor that is developmentally regulated and has anti-elastase properties. CmPS-1 was purified to near homogeneity from C. maxima (pumpkin) phloem exudate and, based on microsequence analysis, the cDNA encoding CmPS-1 was cloned. The association rate constant (k(a)) of phloem-purified and recombinant His(6)-tagged CmPS-1 for elastase was 3.5 +/- 1.6 x 10(5) and 2.7 +/- 0.4 x 10(5) m(-)(1) s(-)(1), respectively. The fraction of complex-forming CmPS-1, X(inh), was estimated at 79%. CmPS-1 displayed no detectable inhibitory properties against chymotrypsin, trypsin, or thrombin. The elastase cleavage sites within the reactive center loop of CmPS-1 were determined to be Val(347)-Gly(348) and Val(350)-Ser(351) with a 3:2 molar ratio. In vivo feeding assays conducted with the piercing-sucking aphid, Myzus persicae, established a close correlation between the developmentally regulated increase in CmPS-1 within the phloem sap and the reduced ability of these insects to survive and reproduce on C. maxima. However, in vitro feeding experiments, using purified phloem CmPS-1, failed to demonstrate a direct effect on aphid survival. Likely roles of this novel phloem serpin in defense against insects/pathogens are discussed.


Subject(s)
Serine Proteinase Inhibitors/chemistry , Serpins/chemistry , Amino Acid Sequence , Animals , Aphids , Binding Sites , Blotting, Western , Chymotrypsin/pharmacology , Cloning, Molecular , Cucurbitaceae/chemistry , DNA, Complementary/metabolism , Electrophoresis, Polyacrylamide Gel , Glycine/chemistry , Kinetics , Molecular Sequence Data , Polymerase Chain Reaction , Protein Binding , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Serine/chemistry , Serpins/genetics , Serpins/metabolism , Thrombin/pharmacology , Time Factors , Trypsin/pharmacology , Valine/chemistry
3.
Graefes Arch Clin Exp Ophthalmol ; 238(6): 515-9, 2000 Jun.
Article in English | MEDLINE | ID: mdl-10943677

ABSTRACT

BACKGROUND: The pathogenesis of central serous chorioretinopathy is poorly understood. It is believed to be due to dysfunction of the retinal pigment epithelium and/or choroid and has been associated with elevated levels of epinephrine and administration of corticosteroids. Epinephrine and corticosteroids have previously been shown to induce apoptosis (programmed cell death) in various types of cells. The objective of this study was to investigate whether these agents can induce apoptosis in cultured retinal pigment epithelium cells. This may help elucidate the pathogenesis of central serous chorioretinopathy. METHODS: Third-passage porcine retinal pigment epithelium cells were grown to confluence and incubated for 1-7 days in culture medium containing epinephrine (10(2)-10(9) pg/ml) or a corticosteroid, dexamethasone (4-4x10(4) ng/ml). The cultures were evaluated for apoptosis by phase-contrast microscopy and in situ terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling. RESULTS: Epinephrine (7x10(7)-10(9) pg/ml) induced apoptosis in a dose- and time-dependent manner. Exposure to lower concentrations of epinephrine (10(2)-6x10(7) pg/ml) and all tested levels of dexamethasone did not result in apoptosis. CONCLUSION: Retinal pigment epithelium cells may undergo apoptosis following exposure to elevated levels of epinephrine. These findings suggest a possible pathophysiologic mechanism for the development of central serous chorioretinopathy.


Subject(s)
Apoptosis/drug effects , Choroid Diseases/etiology , Dexamethasone/pharmacology , Epinephrine/pharmacology , Pigment Epithelium of Eye/pathology , Retinal Diseases/etiology , Animals , Cells, Cultured , Choroid Diseases/pathology , Exudates and Transudates , Glucocorticoids/pharmacology , In Situ Nick-End Labeling , Microscopy, Phase-Contrast , Pigment Epithelium of Eye/drug effects , Retinal Diseases/pathology , Swine , Vasoconstrictor Agents/pharmacology
4.
EMBO J ; 19(12): 2856-68, 2000 Jun 15.
Article in English | MEDLINE | ID: mdl-10856231

ABSTRACT

In plants, cell-to-cell transport of endogenous and viral proteins and ribonucleoprotein complexes (RNPCs) occurs via plasmodesmata. Specificity of this transport pathway appears to involve interaction between such proteins/RNPCs and plasmodesmal chaperones/receptors. Here, KN1 and the cucumber mosaic virus movement protein (CMV-MP) were used, in a modified phage-display screening system, to identify peptides capable of interacting with proteins present in a plasmodesmal-enriched cell wall fraction. Binding/competition assays and microinjection experiments revealed that these phage-displayed peptides and homologous synthetic oligopeptides function as ligand-specific antagonists of macromolecular trafficking through plasmodesmata. A KN1 peptide antagonist had the capacity to interact with a motif involved in the dilation of plasmodesmal microchannels. Although KN1 could still achieve limited movement through plasmodesmata when this SEL motif was blocked, KN1-mediated transport of KN1-sense RNA was fully inhibited. These findings provide direct support for the hypothesis that KN1 requires, minimally, two physically separated signal motifs involved in the dilation of, and protein translocation through, plasmodesmal microchannels, and provide direct proof that plasmodesmal dilation is a prerequisite for the cell-to-cell transport of an RNPC.


Subject(s)
Intercellular Junctions/drug effects , Nicotiana/metabolism , Nicotiana/ultrastructure , Oligopeptides/pharmacology , Plants, Toxic , Amino Acid Sequence , Biological Transport/drug effects , Desmosomes , Homeodomain Proteins/metabolism , Microinjections , Molecular Chaperones/metabolism , Molecular Sequence Data , Peptide Library , Plant Proteins/metabolism , Plant Viral Movement Proteins , Protein Binding , Receptors, Cell Surface/metabolism , Viral Proteins
5.
Toxicol In Vitro ; 14(3): 265-74, 2000 Jun.
Article in English | MEDLINE | ID: mdl-10806377

ABSTRACT

We have established a new test concept for in vitro pharmacological trials. Our model employs tissue explants to test compounds for toxicity which arises with the metabolic interactions among different cell types. Microsurgical preparation of tissue explants avoids the destruction of the organ-specific tissue architecture. Explants were mounted in tissue carriers to improve nutrition and handling of the sample. To allow for the omission of serum supplementation of the culture medium, explants were cultured under continuous medium flow. Test substances are applied considering the polar architecture of most tissues in vivo, for example, covering the apical aspect of epithelia. In principal, all tissues obtained from any species, including man, can be used in this system. A trial application was performed with vitreous body substitutes, substances used in ophthalmology. One compound had passed cell culture tests, but caused massive blood vessel deterioration in vivo. Using our test system based on the developing renal vessel system, we could confirm, within 24 hours, severe vessel damage which resembles the injury suffered by the rabbit retina. We demonstrate that an improved tissue culture assay is a suitable tool for the detection of toxicity that remained unidentified in cell culture tests.


Subject(s)
Toxicology/methods , Animals , Culture Media , Kidney/drug effects , Perfusion , Rabbits , Tissue Preservation , Vitreous Body/drug effects
6.
Science ; 283(5398): 94-8, 1999 Jan 01.
Article in English | MEDLINE | ID: mdl-9872750

ABSTRACT

CmPP16 from Cucurbita maxima was cloned and the protein was shown to possess properties similar to those of viral movement proteins. CmPP16 messenger RNA (mRNA) is present in phloem tissue, whereas protein appears confined to sieve elements (SE). Microinjection and grafting studies revealed that CmPP16 moves from cell to cell, mediates the transport of sense and antisense RNA, and moves together with its mRNA into the SE of scion tissue. CmPP16 possesses the characteristics that are likely required to mediate RNA delivery into the long-distance translocation stream. Thus, RNA may move within the phloem as a component of a plant information superhighway.


Subject(s)
Cucurbitaceae/metabolism , Plant Proteins/metabolism , RNA, Messenger/metabolism , RNA, Plant/metabolism , Amino Acid Sequence , Biological Transport , Cloning, Molecular , Cucumis sativus , Cucurbitaceae/genetics , Microinjections , Molecular Sequence Data , Plant Leaves/metabolism , Plant Proteins/chemistry , Plant Proteins/genetics , Plant Roots/metabolism , Plant Stems/metabolism , Plant Viral Movement Proteins , RNA, Antisense/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction , Viral Proteins/chemistry , Viral Proteins/metabolism
8.
Cell Tissue Res ; 287(1): 193-201, 1997 Jan.
Article in English | MEDLINE | ID: mdl-9011395

ABSTRACT

Kidney function depends on a well-developed vascular system. Any impairment of the blood supply disturbs the integrity and function of the organ. The differentiation of renal vessels has been investigation for many years, but little is known about the relationship between nephrogenesis and vessel development. In the present work the spatial organization of the differentiating vessels was analyzed in precisely oriented tissue sections and in optical sections acquired by laser scan microscopy. Developing vessels as well as small capillaries were visualized with two endothelium-detecting antibodies. Small vessels running in parallel towards the organ capsule were detected in numerous cortico-medullary-oriented tissue sections. Cross-sections of the nephrogenic zone showed a regularly arranged network, which was composed of cells detected by both monoclonal antibodies. Parts of this network were localized in regions of the nephrogenic zone which have been assumed to be free of vessels or vessel-like structures for a long time. These results were confirmed by the laser-scan-microscopic analysis of complete cortex explants. The extraordinarily regular arrangement of the endothelial network in the nephrogenic zone allowed us to reconstruct the developing vascular system. The results presented here underline the close relationship between nephrogenesis and vessel development.


Subject(s)
Blood Vessels/ultrastructure , Kidney/blood supply , Animals , Blood Vessels/growth & development , Blood Vessels/metabolism , Kidney/metabolism , Kidney/ultrastructure , Mice , Mice, Inbred BALB C , Rabbits
9.
Eur J Cell Biol ; 71(2): 216-20, 1996 Oct.
Article in English | MEDLINE | ID: mdl-8905300

ABSTRACT

In search for the cellular mechanisms of graviperception in basidiomycete fruit bodies, nuclear and vacuole motility was investigated in the fungus, Flammulina velutipes. In this organism, hyphal nuclei are closely linked with the actin cytoskeleton, which is involved in gravity perception. Active motion of the nuclei appears aligned with the axis of gravity, and is maintained after spatial reorientation of the cell. The vacuoles showed low motility not aligned with the gravity axis at all. The nuclear density was determined with 1.22 g/cm3. Calculation of the forces exerted by the nuclei shows that nuclear displacement in the submicrometer range already fulfills the physical minimum condition for a statolith. Based on these findings, a function of nuclei as statoliths in basidiomycete hyphae is proposed.


Subject(s)
Basidiomycota/physiology , Gravity Sensing/physiology , Cell Nucleus/physiology , Microscopy, Video , Movement/physiology , Vacuoles/physiology
10.
Mycol Res ; 100 Pt 3: 257-75, 1996 Mar.
Article in English | MEDLINE | ID: mdl-11541308

ABSTRACT

The shape changes which occur in agaric fruit bodies in response to change in the direction of gravity, usually referred to as gravitropism are morphogenetic changes. Our interest in what we prefer to call gravimorphogenesis is to use it to examine morphogenesis experimentally. We are examining two agarics, Coprinus cinereus and Flammulina velutipes, and applying the best available technologies, including video analysis, all forms of electron microscopy, computer-aided image analysis and experiments in orbit in Spacelab. Responses to gravity of the two organisms differ in ways which can be related to their ecological and structural adaptations. C. cinereus reacts extremely rapidly; its fruit body can regain the vertical within 3 h of being placed horizontal, whereas F. velutipes requires 12 h to bend through 90 degrees. The fungi also differ in the bulk of tissue involved in the response. In Coprinus, a zone extending several cm down from the apex is normally involved in bending. In Flammulina, gravisensing is limited to a region just a few mm immediately below the cap, although curvature is performed in a zone of up to 2 cm below. Flammulina cultures were flown on the Spacelab D-2 mission in 1993, and fruit body disorientation in orbit provides the first definitive proof that 'gravitropism' really is a response to the unidirectional gravity vector. Experiments with different clinostat rotation rates in Flammulina indicate that the perception threshold is about 10(-4) x g. Analysis of different times of exposure to an altered gravity vector prior to clinorotation in Coprinus reveals that the perception time is 7 minutes and that continued response requires continued exposure. Cell size determinations in Coprinus demonstrate that cells of the stem increase in length, not diameter, to produce the growth differential. In Flammulina a unique population of highly electron-transparent microvacuoles changes in distribution; decreasing in upper cells and increasing in the lower cells in a horizontal fruit body within a few minutes of disorientation. These are thought to contribute to vacuolar expansion which accompanies/drives cell elongation. Application of a variety of metabolic inhibitors indicates that the secondary messenger calcium is also involved in regulating the growth differentials of gravimorphogenesis but that gravity perception is unaffected by inhibitors of calcium signalling. In both Flammulina and Coprinus, gravity perception seems to be dependent on the actin cytoskeleton since cytochalasin treatment suppresses gravitropic curvature in Flammulina and, in Coprinus, significantly delays curvature without affecting stem extension. This, together with altered nuclear motility observed in living hyphae during reorientation suggests that gravity perception involves statoliths (possibly nuclei) acting on the actin cytoskeleton and triggering specific vesicle/microvacuole release from the endomembrane system.


Subject(s)
Basidiomycota/growth & development , Coprinus/growth & development , Gravitropism/physiology , Gravity Sensing/physiology , Space Flight , Weightlessness , Basidiomycota/cytology , Basidiomycota/physiology , Coprinus/cytology , Coprinus/physiology , Gravitation , Plant Stems/cytology , Plant Stems/growth & development , Signal Transduction/physiology
11.
Eur J Cell Biol ; 66(2): 151-6, 1995 Feb.
Article in English | MEDLINE | ID: mdl-7774601

ABSTRACT

Inhibitor studies demonstrate a major role of the actin cytoskeleton in cellular graviperception of the basidiomycete Flammulina velutipes. Treatment of explanted fruiting body stipes with 10(-4) M of the actin filament-disrupting agent cytochalasin D causes specific suppression of gravitropic curvature to 21% of the control value. Elongation growth is depressed to 48%. In contrast, curvature and elongation remain almost unaffected by 10(-4) M of the microtubule inhibitor oryzalin. Immunohistochemical labeling of actin filaments in stipe hyphae of Flammulina reveals a close colocalization with the nuclei. The label pattern is destroyed upon 10(-4) M cytochalasin treatment. The role of the actin cytoskeleton in graviperception in Flammulina parallels aspects of gravisensing in Chara rhizoids and thus points at basic similarities between fungal and plant mechanisms of cellular gravity perception.


Subject(s)
Actins/ultrastructure , Basidiomycota/physiology , Gravity Sensing/physiology , Basidiomycota/ultrastructure , Cytoskeleton/ultrastructure
12.
Exp Mycol ; 18(2): 150-8, 1994 Jun.
Article in English | MEDLINE | ID: mdl-11541305

ABSTRACT

Differential elongation of stipe hyphae drives the gravitropic reorientation of Flammulina velutipes (Agaricales) fruiting bodies. The gravitropic curvature is strictly dependent on the presence of the transition zone between pileus and stipe. Elongation growth, providing the driving force for curvature, is also promoted by the pileus. Gravitropic curvature is successfully suppressed by clinostatic rotation, but the elongation rate is not affected. Explantation of fruiting body stipes lowers curvature and elongation rates corresponding to explant size reduction. In Flammulina, 25 mm length of transition zone explants is an efficient size for reproducible curvature and elongation during 48- to 72-h curvature tests. Submersion of specimens in aqueous medium causes cessation of the gravitropic curvature, but does not affect elongation. Thus the involvement of a diffusible factor in transmission of the curvature signal is probable. Splitting the fruiting body stipe in segments of 1/8 diameter does not suppress the gravitropic response, and the segments are individually reoriented to the vertical. It is concluded that the graviresponse of the Flammulina fruiting body is based on cellular perception of the gravistimulus and that a differential growth signal is transmitted in the stipe by a soluble factor that regulates hyphal elongation.


Subject(s)
Basidiomycota/growth & development , Gravitropism/physiology , Gravity Sensing/physiology , Plant Stems/growth & development , Basidiomycota/physiology , Gravitation , Gravitropism/drug effects , Immersion , Plant Stems/physiology , Rotation , Water/pharmacology
13.
Exp Mycol ; 18(1): 7-19, 1994 Mar.
Article in English | MEDLINE | ID: mdl-11541306

ABSTRACT

The fruiting body of the basidiomycete Flammulina velutipes shows a distinct negative gravitropic response. Maturing fruiting bodies in the rapid elongation phase become graviresponsive with basidiospore differentiation. Lateral gravistimulation by horizontal arrangement of the fruiting body results in unilateral growth regulation. Elongation in the upper Stipe side decreases to 40% during gravitropic reorientation of the fruiting body. Overshooting of the gravitropic response during reorientation is precisely regulated. The graviresponsiveness is concentrated to the apical area of the stipe, the transition zone between pileus and stipe, which features a prominent elongation capability. The small size and low vacuolization of the transition zone hyphae compared with differentiated basal stipe hyphae correspond with this physiological function on the light and electron microscopical levels. Curvature experiments using intact and explanted fruiting bodies demonstrated the graviperceptive role of the transition zone. The excision of various amounts of pilear tissue, even the disruption of the whole pileus, had no severe effect on gravitropic curvature, until the transition zone was damaged. Removal of the transition zone resulted in a dramatic loss of graviresponse, whereas the decrease of elongation was less drastic.


Subject(s)
Basidiomycota/physiology , Gravitropism/physiology , Gravity Sensing/physiology , Basidiomycota/growth & development , Basidiomycota/ultrastructure , Gravitation , Microscopy, Electron , Plant Stems/growth & development , Plant Stems/physiology , Plant Stems/ultrastructure
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