Pathways of lanthanum ion transport across the posterior epithelium of the cornea in rabbits

Morphological estimation of the barrier-transport properties of the posterior epithelium in the donor cornea has been performed in the most early time of fanoxic lesions of the cells. Ionized lanthanum, as an effective inhibitor of oxidative phosphorylation and simultaneously--as a marker of transport pathways for particles similar in their size to water molecule, is used in the investigation. The concentration gradient of rare-earth ions is produced by vital injection of lanthanum trichloride into the proper substance (stroma) of the cornea. From the microinjection focus the electron opaque marker is transported through the substrate of the basal substance of the stroma to the posterior epithelium and further along its intercellular and transcellular pathways into the anterior chamber of the eye. The rare-earth ions freely penetrate through the gap and tight junctions. Transcellular transport of lanthanum in the contents of the plasmolemmal microvesicles, vital absorption of the marker on the lateral and luminal parts of the plasmolemma and on the intracellular membranes, lesions of mitochondria and canaliculi of the endoplasmic reticulum of the anoxic character are stated. A suggestion is made on structural preservation of the pathways of convective and dissipative transition of the substance through the posterior epithelium of the cornea during the earliest time of the experimental anoxia.     

Vitamin A dimers trigger the protracted death of retinal pigment epithelium cells

Cellular events responsible for the initiation of major neurodegenerative disorders of the eye leading to blindness, including age-related macular degeneration, Stargardt and Best diseases, are poorly understood. Accumulation of vitamin A dimers, such as N-retinylidene-N-retinylethanolamine (A2E) in the retinal pigment epithelium (RPE), is one of the earliest measurable events preceding retinal degeneration. However, the extent to which these dimers contribute to tissue degeneration is not clear. To determine if A2E could trigger morphological changes associated with the degenerating RPE and subsequent cell death, we evaluated its toxicity to cultured human RPE cells (ARPE-19). We show that A2E triggered the accumulation of debris followed by a protracted death. A2E was up to≈14-fold more toxic than its precursor, retinaldehyde. Measurements reveal that the concentration of A2E in the aged human eye could exceed the concentration of all other retinoids, opening the possibility of A2E-triggered cell death by several reported mechanisms. Findings suggest that accumulation of vitamin A dimers such as A2E in the human eye might be responsible for the formation of ubiquitous RPE debris, an early indication of retinal degeneration, and that preventing or reducing the accumulation of vitamin A dimers is a prudent strategy to prevent blindness.The elucidation of environmental and genetic drivers of RPE senescence has been a persistent goal toward understanding and preventing degenerative disease of the retina. Since their structural elucidation in the 1990s, dimers of dietary vitamin A, in particular N-retinylidene-N-retinylethanolamine (A2E), have been postulated as chemical triggers, driving retinal senescence and associated degeneration. The eye uses vitamin A as a cofactor to sense light. A striking chemical signature of the aging and degenerating retina is the accumulation of vitamin A dimers in the retinal pigment epithelium (RPE)^{1, 2} and underlying Bruch''s membrane.³ In rodent models of macular degeneration,^{4, 5, 6, 7, 8} high levels of vitamin A dimers correlate with poor retinal health and a variety of mechanisms have been proposed by which dimers of vitamin A may induce retinal toxicity ranging from non specific to direct antagonistic/protagonistic mechanisms.^{9, 10, 11, 12, 13, 14, 15, 16} As a cationic ambiphilic pyridinium, A2E has been shown to solubilize lipid membranes, inactivate lysosomes by increasing lysosomal pH, and accumulates in the negatively charged mitochondrial compartment. Once dimerized, the special orientation of the polyene chains make them especially susceptible to oxidative degradation¹⁶ leading to secondary reactive aldehyde and epoxide toxicants.¹⁷ Direct reported mechanisms of A2E toxicity include, acting as an agonist for retinal pigment epithelium-specific 65-kDa protein,¹⁸ retinoic acid receptors,¹⁰ cyclooxygenase-2,¹⁹ and covalent modification of biomolecules,^{20, 21} among others.Despite data demonstrating that dimers of vitamin A can disrupt cellular hemostasis, there is less direct evidence supporting their role as primary drivers of retinal senescence. More recently, based on studies in ARPE-19 cells, it has been proposed that A2E''s chemical precursor, vitamin A aldehyde (retinaldehyde), might play a role in the degenerative process and that A2E might be a benign biomarker of increased levels of retinaldehyde.^{16, 17, 18} To determine if A2E could chemically trigger the degenerative process in the retina, we explored the acute and long-term toxicity of A2E to human retinal pigment epithelium (RPE) cells in vitro. ARPE-19 cells treated with A2E showed degraded mitochondria, accumulated glycogen and lipofuscin debris, and underwent a protracted, dose-dependent death over several days to months. These data suggest that A2E can trigger the accumulation of lipofuscin-like debris in the in vivo RPE and can be detrimental to the retina''s health. Data further reveal that increasing concentrations of A2E in the RPE potentially plays a larger role in retinal senescence than previously thought.     

Lanthanum-induced alterations in cellular electrolytes and membrane potential in Ehrlich ascites tumor cells

We have investigated the effect of varying La⁺³ concentrations (0.01 mM to 2.0 mM) on membrane potential and electrolyte composition of Ehrlich ascites tumor cells. La⁺³ concentrations less than 0.02 mM had no effect. Above 0.02 mM, La⁺³ induced concentration-dependent loss of electrolytes and water from the cells. At 1.0 mM the effect was maximal and resulted in an 87% reduction in cellular K⁺, 79% in Cl^? and 21% in Na⁺ within 4.8 minutes. The Na⁺ loss occurred even in the face of an electrochemical potential gradient favoring Na⁺ entry. La⁺³ increased the recorded values of membrane potential; the magnitude of the effect was related to the external La⁺³ concentration, and was maximal at 1.0 mM. Studies using ¹⁴⁰La showed that La⁺³ binds rapidly to the cell surface and does not enter the cells. The amount of La⁺³ bound to the cells was related to the external La⁺³ concentration by a sigmoidal curve and was maximal at about 1.0 mM. The bound La⁺³ could not be displaced by either added La⁺³ or Ca⁺². Agents known to effect the integrity of the cell membrane, such as phospholipase C, neuraminidase, pronase and Hg⁺² were tested for their ability to displace bound La⁺³. Only pronase displaced bound La⁺³, indicating that La⁺³ associates with cell protein. It is hypothesized that La⁺³ rapidly interacts with membrane protein causing alterations in membrane permeability and capacity to actively transport ions.     

Olfactory epithelium consisting of supporting cells and horizontal basal cells in the posterior nasal cavity of mice

The olfactory epithelium of mice generally consists of olfactory cells, progenitors of olfactory cells (globose basal cells), supporting cells, and horizontal basal cells. However, in the dorsal fossa (the roof) of the posterior nasal cavity of mice, we found seven epithelial patches consisting of only non-neuronal cell types, i.e., supporting cells and horizontal basal cells, among the normal olfactory epithelium. The supporting cells occupied three or four layers in the apical to middle regions; in the basal region, horizontal basal cells were localized in a single row adjacent to the basement membrane. Bowman's gland ducts were also present in the epithelium. Neuronal cells (olfactory cells and globose basal cells) were totally absent. The ultrastructure of the supporting cells, horizontal basal cells, and Bowman's glands was essentially similar to that in the normal olfactory epithelium. In the early postnatal period (P1-P7), cell types in the epithelium were the same as those in the normal olfactory epithelium. From P10 to P21, olfactory cells and globose basal cells had disappeared from the olfactory epithelium. At this period, the number of TUNEL-positive cells was significantly higher than that in the surrounding olfactory epithelium; ultrastructurally, many apoptotic figures were observed. This suggests that the epithelium consisting of supporting cells and horizontal basal cells is generated by the apoptotic death of olfactory cells and globose basal cells during postnatal development.     

Ezrin promotes morphogenesis of apical microvilli and basal infoldings in retinal pigment epithelium

Ezrin, a member of the ezrin/radixin/moesin (ERM) family, localizes to microvilli of epithelia in vivo, where it bridges actin filaments and plasma membrane proteins. Here, we demonstrate two specific morphogenetic roles of ezrin in the retinal pigment epithelium (RPE), i.e., the formation of very long apical microvilli and of elaborate basal infoldings typical of these cells, and characterize the role of ezrin in these processes using antisense and transfection approaches. In the adult rat RPE, only ezrin (no moesin or radixin) was detected at high levels by immunofluorescence and immunoelectron microscopy at microvilli and basal infoldings. At the time when these morphological differentiations develop, in the first two weeks after birth, ezrin levels increased fourfold to adult levels. Addition of ezrin antisense oligonucleotides to primary cultures of rat RPE drastically decreased both apical microvilli and basal infoldings. Transfection of ezrin cDNA into the RPE-J cell line, which has only trace amounts of ezrin and moesin, sparse and stubby apical microvilli, and no basal infoldings, induced maturation of microvilli and the formation of basal infoldings without changing moesin expression levels. Taken together, the results indicate that ezrin is a major determinant in the maturation of surface differentiations of RPE independently of other ERM family members.     

Hypoxia as pathogenic factor affecting the eye tissues: The selective apoptotic damage of the conjunctiva and anterior epithelium of the cornea

The effect of acute hypoxia on the occurrence of apoptosis in eye cells in rats placed in a pressure chamber was studied. Selective primary lesion of cells of the conjunctiva and the anterior corneal epithelium was found. A possible role of the simulated hypoxic conditions in the dry eye syndrome pathogenesis, which is accompanied by primary lesion of cells in the anterior eye surface tissues is discussed.     

Observations on the morphology of the developing primate cornea: epithelium, its innervation and anterior stroma.

A survey is made of some ultrastructural features of the developing cornea of Macaca mulatta. The observations are confined to the anterior central area, starting with the lens vesicle stage and progressing through midgestation, when the morphologic characteristics of the cornea are fully established. Subepithelial filaments and some partially aggregated collagen fibrils are present in the earliest embryo and are of a size and appearance similar to those in the future vitreous cavity. Epithelial secretory activity points to, but does not prove direct contribution to the deposition of the acellular matrix components beneath it. No trace of a structured, orthogonal collagenous stroma can be visualized. The primitive endothelium forms prior to the fibroblast invasion of the distended filamentous matrix. Bowman's layer has undoubted epithelial contributions. Its aggregated collagen fibrils have approximately the same diameter as those of the anterior stroma. Intraepithelial appearance of single nerve fibers and fascicles takes place during the first trimester of gestation, as soon as the two continuous epithelial layers are formed. Terminal areas approach closely to the basal cell's nucleus, without touching it. The plasmalemma of the invaginating nerve fiber is surrounded by that of the epithelial cell in a mesaxon-like manner, with occasional gap junctions uniting adjoining neural and epithelial cell membranes. The fetal neurites contain microtubules, some clear vesicles and dense vacuoles resembling those of mature monamine and non-monamine neurons. Mitochondria are small and compact, their presence indicating a high rate of metabolic activity in the immature terminal area.     

Basolateral membrane K permselectivity and regulation in bullfrog cornea epithelium

Summary Ion channels permeable to barium and calcium were reconstituted from theAplysia nervous system into phospholipid bilayers formed on the tips of patch electrodes. With asymmetrical concentrations of barium or calcium on the two sides of the bilayer, the single-channel currents reversed at the calculated barium or calcium reversal potentials, indicating that the channels were cation selective. Channels with conductances of 10, 25 and 36 pS were routinely observed. Calcium and barium were equally effective as charge carriers for the 36-pS channel, whereas magnesium was at least fifteenfold less effective. The gating of all three channels was independent of the voltage across the bilayer, but was affected by the dihydropyridine calcium channel agonist Bay K 8644 (Bay K). In the presence of Bay K but not in its absence, long discrete gating events were routinely observed, suggesting that the dihydropyridine increased the probability of long open states as it does for calcium channels in other systems.Bilayers invariably contained more than a single channel (or conductance state). This was observed even when theAplysia nervous system membranes were prepared in the presence of cytoskeleton disrupting agents, or when the membrane proteins were diluted extensively with exogenous phospholipid. Furthermore, transitions between conductance levels were observed with high frequency. These findings, together with the fact that all of the conductance states share certain properties including voltage-independence and sensitivity to Bay K, suggest that the apparent multiple channel types may in fact represent subconductance states of a single ion channel.     

Structural reactions to polarized light of microvilli in photoreceptor cells of the moth Spodoptera

Summary Intact armyworm moths (Spodoptera exempta, Farn. Noctuidae) were illuminated by polarized monochromatic light to induce structural changes in the rhabdomeres of the compound eyes. The degree of distortion of their microvilli depends on the light energy absorbed per time unit. Under polarized light, the number of quanta absorbed varies with the position of the plane of polarization relative to the axis of the microvilli (intrinsic dichroism). Therefore, in Spodoptera, different degrees of deformations could be demonstrated in differently oriented rhabdomeres of both types of ommatidia. Moreover, in rhabdoms of the lobed type with fan-like arranged microvilli, different reactions were regularly seen in differently oriented microvilli of one rhabdomere. This indicates that microvilli may react to light individually.Supported by Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 114 (Bionach)     

Proliferation and apoptosis in the epithelium of the developing human cornea and conjunctiva.

To determine the distribution of proliferating and apoptotic cells in the human cornea during prenatal and early postnatal development, we examined sections of the bulbar conjunctiva, the limbus as well as the central and peripheral cornea between 11 weeks of gestation and 6 months after birth. The objective was to localize dividing cells by proliferating cell nuclear antigen-like immunoreactivity (PCNA-LI) and apoptotic cells by terminal transferase-mediated nick-end labeling (TUNEL). Before the 17th gestational week, PCNA-LI was absent in all 4 regions examined, indicating negligible cell proliferation during early development. After 20 weeks, strong PCNA-labeling was observed in all regions examined suggestive of high proliferative activity not only in the limbus and the bulbar conjunctiva, but also in the central and peripheral cornea. This rise in proliferative activity was followed by a steady decline: after 28 weeks, anti-PCNA staining gradually disappeared in the central and peripheral cornea, so that, at 6 months after birth, it was confined to the limbus and the bulbar conjunctiva, resembling the picture described for the adult cornea. TUNEL-positive cells were virtually absent in all 4 regions examined before the 38th gestational week. Apoptotic cells only started to appear at 38 weeks; at this stage, they were confined to the bulbar conjunctival epithelium. At 6 months after birth, TUNEL-positive cells were observed in the bulbar conjunctival epithelium and the entire cornea; the limbus, however remained devoid of apoptotic cells throughout the entire prenatal and early postnatal period. The present study for the first time localizes proliferating and apoptotic cells in the epithelium of the developing human cornea. Three stages of development can be distinguished: Minimal proliferation (until 17th week), vigorous proliferation over the entire cornea including the limbus and the bulbar conjunctiva (until 28th week) and gradual decrease in proliferative activity (after 28th week) accompanied by the appearance of apoptotic cells.     

Biophysical mechanism of radiation damage to mammalian cells by X- and gamma-rays

Published information on the reproductive death in mammalian cells irradiated by a wide range of X- and gamma-ray energies has been re-analysed to extract intrinsic efficiencies of damage for the secondary electrons in transient equilibrium. On a log-log plot, a linear dependence on the track average l.e.t. and the average specific primary ionization is found, indicating that either serves as a good quality parameter. The soft X-ray data are consistent with this conclusion. Upon comparison with data for fast heavy ion irradiations, the average specific primary ionization is shown to be applicable independently of radiation type whereas track average l.e.t. is not. Furthermore it is revealed that electrons are most damaging near the end of their range but their efficiency is only about 10-20 per cent of that of fast ions at the same quality, possibly due to the influence of multiple scatter on the electron penetration depth. It is deduced that, for the dose rates involved, the damage by electrons is predominantly by intra-track action and not inter-track action. The results are consistent with the suggestion that optimum damage occurs when the mean free path between ionizations is equivalent to the strand separation in the double-stranded DNA.     

Desmoplakin controls microvilli length but not cell adhesion or keratin organization in the intestinal epithelium

Maintaining proper cell-cell adhesion in the intestine is essential for tissue homeostasis and barrier function. This adhesion is thought to be mediated by cell adhesion structures, including tight junctions, adherens junctions, and desmosomes, which concentrate in the apical junctional region. While clear roles for adherens and tight junctions have been established in simple epithelia, the function of desmosomes has not been addressed. In stratified epithelia, desmosomes impart mechanical strength to tissues by organizing and anchoring the keratin filament network. In this paper, we report that the desmosomal protein desmoplakin (DP) is not essential for cell adhesion in the intestinal epithelium. Surprisingly, when DP is lacking, keratin filament localization is also unperturbed, although keratin filaments no longer anchor at desmosomes. Unexpectedly, DP is important for proper microvillus structure. Our study highlights the tissue-specific functions of desmosomes and reveals that the canonical functions for these structures are not conserved in simple epithelium.     

Identification of cytoskeletal markers for the different microvilli and cell types of the rat vomeronasal sensory epithelium

The vomeronasal organ (VNO) of the mammal nose is specialized to detect pheromones. The presumed site of the chemosensory signal transduction of pheromones is the vomeronasal brush border of the VNO sensory epithelium, which has been shown to contain two different sets of microvilli: (i) the tall microvilli of supporting cells and (ii) the short microvilli of the chemoreceptive VNO neurons that branch and intermingle with the basal portions of the longer supporting cell microvilli. A key problem when studying the subcellular distribution of possible VNO signal transduction molecules at the light microscope level is the clear discrimination of immunosignals derived from dendritic microvilli of the VNO neurons and surrounding supporting cell structures. In the present study we therefore looked for cytoskeletal marker proteins, that might help to distinguish at the light microscope level between the two sets of microvilli. By immunostaining we found that the VNO dendritic microvilli can be selectively labelled with antibodies to the calcium-sensitive actin filament-bundling protein villin, whereas supporting cell microvilli contain the actin filament cross-linking protein fimbrin, but not villin. Useful cytoplasmic marker molecules for cellular discrimination were cytokeratin 18 for supporting cells and β-tubulin for dendrites of VNO neurons. A further finding was that the non-sensory epithelium of the rat VNO contains brush cells, a cell type that appears to be involved in certain aspects of chemoreception in the gut. Brush cells or other structures of the vomeronasal brush border did not contain α-gustducin.     

Accelerated shedding of prions following damage to the olfactory epithelium

In this study, we investigated the role of damage to the nasal mucosa in the shedding of prions into nasal samples as a pathway for prion transmission. Here, we demonstrate that prions can replicate to high levels in the olfactory sensory epithelium (OSE) in hamsters and that induction of apoptosis in olfactory receptor neurons (ORNs) in the OSE resulted in sloughing off of the OSE from nasal turbinates into the lumen of the nasal airway. In the absence of nasotoxic treatment, olfactory marker protein (OMP), which is specific for ORNs, was not detected in nasal lavage samples. However, after nasotoxic treatment that leads to apoptosis of ORNs, both OMP and prion proteins were present in nasal lavage samples. The cellular debris that was released from the OSE into the lumen of the nasal airway was positive for both OMP and the disease-specific isoform of the prion protein, PrP(Sc). By using the real-time quaking-induced conversion assay to quantify prions, a 100- to 1,000-fold increase in prion seeding activity was observed in nasal lavage samples following nasotoxic treatment. Since neurons replicate prions to higher levels than other cell types and ORNs are the most environmentally exposed neurons, we propose that an increase in ORN apoptosis or damage to the nasal mucosa in a host with a preexisting prion infection of the OSE could lead to a substantial increase in the release of prion infectivity into nasal samples. This mechanism of prion shedding from the olfactory mucosa could contribute to prion transmission.     

Circardian rhythm of mitoses in the epithelium of the cornea after splenectomy]

In corneal epithelium of CBA mice the index of colchicine mitoses diminished after splenectomy in the day period characterized by rising mitotic activity in control animals. The duration of active phase of cell division rhythm shortened while the maximum of mitotic activity delayed in comparison with control animals. The total amount of cells entering mitosis during 24 hours diminished by 27.7% and the rate of physiological regeneration of corneal epithelium decreased.     

The protective effect and mechanism of soybean oil and its extracts on DNA damage in human ECV304 cells exposed to UV-C

The degree of DNA damage in the human endothelial cell line ECV304 exposed to UV-C, with or without the presence of soybean oil (SBO), was assessed by the Comet assay. After 5-min exposure to UV-C, the %Tail DNA in the ECV304 cells ranged from 0% to 20% for SBO treatment groups and from 50% to 70% for the control group. The result indicated a strong protective effect of SBO against UV-C-induced DNA damage. To clarity the mechanism of this protective effect of SBO, the methanol extract of SBO (MESO) was analyzed and its capacity against UV-C-induced DNA damage was evaluated. Gas chromatography mass spectrometry (GC-MS) analysis confirmed that MESO contained many antioxidants including n-3-polyunsaturated fatty acid (n-3-PUFA), tocopherols and phytosterols. Comet assay revealed that the MESO was also active in reducing the DNA damage dose-dependently (P<0.0001) vs. control in the ECV304 cells. Therefore, we concluded that these potential antioxidants may be responsible for the scavenge of oxidative radicals induced by UV-C irradiation. This study suggested that dietary SBO, which is abundant of antioxidants, may reduce the content or impact of reactive oxygen species (ROS) and lower the risk of diseases caused by ROS.     

Principles of the organization of intestinal epithelium and its recovery from damage revealed by modeling

Using the developed method of modelling the curves of the fraction of labelled indexes (FLI) in the small intestine epithelium, a relationship was found between the FLI shape and duration of the S phase and the whole generation cycle for various generations of proliferating crypt cells. On the basis of the comparison between the theoretical and experimental FLI the normal values of the generation cycles along the crypt were specified for various intestinal parts. FLI were shaped along the crypt at various time intervals after irradiation. As a result, the data were obtained on the correlation between the duration of generation cycles and their phases during the postirradiation period and for various cell generations.