Lens Organ Culture Research Articles

Effects of lipid peroxidation products on the rat lens in organ culture: A possible mechanism of cataract initiation in retinal degenerative disease

Rat lenses in organ culture which are exposed to bovine rod outer segments (ROS) or to the major fatty acid of ROS, docosahexaenoic acid, are impaired in their ability to accumulate radiolabeled compounds which lenses normally accumulate by active processes. The extent of lens damage correlates well with the extent of lipid peroxidation in the culture medium as assessed by the thiobarbituric acid assay. Addition of vitamin E to the medium inhibits the effect on the lens while addition of Fe-ADP complexes potentiates the effect. Thus, the lens damage appears to be attributable to toxic species generated by peroxidation of the polyunsaturated lipid added to the culture medium. Toxic aldehyde products appear to be major mediators of the lens damage, since semi-carbazide, which avidly reacts with aldehydes, can protect lenses in this system. These findings may have relevance to the cataracts clinically associated with retinal degenerative diseases such as retinitis pigmentosa. The highly membranous photoreceptor cells are extremely rich in polyunsaturated lipid. Degeneration of these cells, which is the primary pathology in such diseases, would likely lead to peroxidation with generation of toxic products within the eye. Such products could potentially produce secondary damage to other ocular structures including the lens.

H2O2-induced uncoupling of bovine lens Na+,K+-ATPase.

A 1-hr exposure of bovine lenses in organ culture to H2O2 concentrations in the range found in the aqueous fluid of patients with cataracts inhibits 86Rb+ influx. At 1 mM H2O2, complete inhibition was observed and further investigated. Membrane permeability is slightly decreased. Although lactate concentrations increase 2-fold, lens ATP concentrations decrease approximately equal to 10%, suggesting that glycolysis may be stimulated but ATP production is not able to keep up with the demand for energy. Examination of epithelial cell Mg2+-stimulated Na+,K+-ATPase isolated from the cultured lenses indicates H2O2-induced modification. At 5 mM MgATP, ATP hydrolysis is accelerated 30%; at 3 mM MgATP, hydrolysis is normal; and at 0.75 mM MgATP, it is inhibited 75%. p-Nitrophenyl phosphate hydrolysis and eosin maleimide binding indicate that K+ control of the enzyme is modified. Thus, a very early effect of H2O2 upon the lens, well before the formation of opacity, appears to be the uncoupling of Na+ and K+ transport from ATP hydrolysis.

Photodynamic cross-linking of polypeptides in intact rat lens

During aging and particularly during cataractogenesis, crystallin polypeptides in the human lens become cross-linked by non-disulfide covalent bonds. Recently it has been hypothesized that singlet oxygen generated photodynamically within the lens oxidized certain amino acid residues in lens crystallins leading to cross-link formation. That singlet oxygen can produce cross-links in lens protein solutions has been established. In the present work we demonstrate by electrophoresis in sodium dodecyl sulfate that photodynamic cross-linking of crystallins can occur within the intact rat lens in organ culture. The cross-linking was most pronounced in the urea insoluble fraction of the peripheral lens cortex. Urea insoluble protein was increased in the irradiated lenses relative to non-irradiated controls. The process can be supported by either the photosensitizing dye rose bengal or kynurenine, an oxidation product of tryptophan which is endogenous to human lens. Based on inhibition by scavengers we conclude that the cross-linking is mediated by singlet oxygen. Cross-linking within the intact lens appeared to have greater specificity with respect to the polypeptides involved than did cross-linking in protein solutions.

Serum proteins neutralize the toxic effect of lysophosphatidyl choline.

Lysophosphatidyl choline (LPC), which has been shown to be toxic to the lens in organ culture, is markedly elevated in uveitic eyes. These findings have led to the suggestion that LPC is an initiator of cataractogenesis in uveitis. We report here that a factor not considered by previous investigators, i.e. the presence of serum proteins, can modulate LPC toxicity in vitro. First, the toxicity of LPC to lens epithelial cells or to whole lens in organ culture was effectively neutralized by addition of serum or serum albumin to the test cultures, thus suggesting that the potential toxicity of the increased LPC in the uveitic eye might be neutralized by the concomitant increase in serum protein. Secondly, the susceptibility to LPC toxicity of lens epithelial cells was no greater than that of lymphocytes which are normally exposed to LPC levels higher than those in the uveitic eye.

Incorporation of 14C acetate into the sterols of rabbit lens in organ culture.

Rabbit lenses were incubated in organ culture with 14C acetate for 3, 6, 12 and 20 hours and the nonsaponifiable fraction, composed primarily of sterols, was isolated. The incorporation of acetate into the nonsaponifiable fraction was linear for periods of 3 to 20 hours incubation. The nonsaponifiable fraction was subjected to thin-layer chromatography and 6 bands were observed. On the basis of Rf values, one band was tentatively identified as squalene, another as lanosterol and a third as a cholesterol-desmosterol mixture. The percentage of the radioactivity incorporated into the cholesterol-desmosterol band increased with incubation time. Conversely the percentage of the radioactivity incorporated into the squalene and lanosterol bands decreased with incubation time, indicating a precursor-product relationship.

Incorporation of 14C stearic acid in lens organ culture.

14C stearic acid was incubated in organ culture of rabbit lens for 6, 12 and 20 hours. Stearic acid did penetrate the lens and was incorporated into triacyl-glycerides and phospholipids. It also served as a carbon source for cholesterol synthesis. Individual fatty acids were isolated and radioactivity measurements were made. Incorporation of label was found in C12:0, C14:0 and C16:0 fatty acids, indicating de novo synthesis in the lens; incorporation was found in C16:1 and C18:1, indicating delta 9 desaturase activity, and incorporation was found in the fatty acids of 20 carbons or longer, indicating the presence of a fatty acid elongation system in the lens.

Synthesis of cytoskeletal and membrane polypeptides by the organ cultured chicken lens.

The synthesis of cytoskeletal and membrane polypeptides of the chick lens epithelium, superficial cortical fiber cells and deep cortical fiber cells was studied in the organ-cultured lens. The synthetic activity of the cytoskeletal polypeptides was restricted to the epithelium and superficial cortical fiber cells while membrane polypeptides were synthesized by deep cortical fiber cells as well. None of the polypeptides was observed to be synthesized by the lens nuclear fiber cells.

Suckling mouse cataract agent is a helical wall-free prokaryote (spiroplasma) pathogenic for vertebrates.

THE suckling mouse cataract agent (SMCA) is a filterable organism originally isolated from a pooled extract of rabbit ticks (Haemaphysalis leporis-palustris) collected near Atlanta, Georgia in April 1961 (ref. 1). The agent grows to high titre in the eyes and brains when inoculated intracerebrally into newborn mice in which it induces cataract, uveitis and chronic brain infection1–3. SMCA has also been grown to high titre in embryonated hen's eggs, in which it produces a lethal infection in 4–9 d (ref. 1). The agent has not been grown in tissue culture or in artificial media, but growth in a rabbit lens organ culture has been reported4. Stained SMCA-infected tissues failed to show the presence of rickettsiae and, as a result of such negative inferences and other characteristics of the agent3–5, it was considered to be a candidate slow virus6. Within the past few years however, ultrastructural and biological studies showed that a wall-free prokaryote was consistently associated with the acute and chronic disease in mice and with lethality in chick embryos7,8. Although the agent resembled mycoplasmas morphologically, it differed from them in its apparent non-cultivability on conventional mycoplasma medium. We now report that the organism associated with the suckling mouse cataract syndrome is a prokaryote similar to the spiroplasmas—a group of mycoplasmas known previously only from plants and insects.

Differential formation of desmosomes and hemidesmosomes in epidermal cell cultures treated with retinoic acid.

IN various epithelial tissues, desmosomes (D) as well as hemidesmosomes (HD) are common specialisations of the plasma membrane1. Both membrane differentiations are thought to play an important role either in cell–cell adhesion (D) or in the attachment to biological substrata (HD)2. In vitro formation of D and HD has been observed in embryonic lens cells3 or organ culture outgrowth of skin4. We have now found that in cultured post-embryonic epidermal cells (PEC) both membrane specialisations axe formed independently of each other. We show that in cells stimulated to grow, an increased number of attachment devices contacting the substratum is formed. At the dorsal surface of the same cell, however, morphological expression of tissue-specific differentiation is present.

The presence of polyriboadenylic acid sequences in calf lens messenger RNA

The presence of poly(rA) sequences in lens RNA has been demonstrated by the isolation of RNase A and T 1-resistant fragments of approximately 50 nucleotide residues. These poly(rA)-rich sequences, obtained from lenses incubated for six hours in organ culture with [ 3H]adenosine, are located at the 3′ termini of mRNA as determined by 3′ exoribonuclease digestion. Limited digestion of the [ 3H]adenosine-labeled mRNA with the enzyme led to the abolition of binding to poly(rU)-filters and a concomitant loss of template activity with avian myeloblastosis virus RNA-dependent DNA polymerase. Furthermore, after incubation of lenses in organ culture with 3′-deoxyadenosine, the isolated polysomal RNA was unable to function as a template in an avian myeloblastosis virus RNA-dependent DNA polymerase-catalyzed reaction system.

Biochemistry of Bovine Lens Proteins: IV. SYNTHESIS AND AGGREGATION OF α-CRYSTALLIN SUBUNITS IN DIFFERENTIATING LENS CELLS

Abstract The vertebrate lens is composed of two distinct cell types, the epithelial cells and the fiber cells. The absolute rate of synthesis of total protein and of purified α-crystallin, a structural polymeric protein found in both cell types, has been calculated using the kinetics of [3H]leucine incorporation into organ-cultured lenses. The data show that in adult epithelial cells about 2% of α-crystallin is renewed per day, as opposed to 0.3% in cortex fiber cells. The rate of α-crystallin synthesis is about 7 times higher in epithelial cells than in cortex fiber cells; for non-α-crystallin proteins, the corresponding ratio is approximately 24. Both epithelial cells and cortex fiber cells are shown to be highly specialized for the synthesis of α-crystallin; 60% of newly synthesized protein in adult epithelial cells is α-crystallin, whereas in cortex fiber cells the proportion is 75%. The polymeric α-crystallin has been dissociated into its component subunits and the resulting mixture of polypeptide chains has been analyzed by acrylamide gel electrophoresis. Four discrete bands are resolved by this technique, corresponding to two acidic subunits (αA1 and αA2) and two basic subunits (αB1 and αB2). The kinetics of aggregation of these subunits to form the polymeric α-crystallin has been investigated in adult epithelial cells. Two major points emerge from this study. (a) About 60% of the leucine incorporated into α-crystallin is accounted for by subunit αA2 and about 30% by subunit αB2. Since the relative concentrations of αA2 and αB2 within the aggregate are 60% and 30%, respectively, the incorporation data indicate that in adult epithelial cells the composition of newly formed α-crystallin is identical to that of the previously aggregated molecule. (b) No significant uptake of radioactive leucine in the polymeric molecule via subunit αA1 is detectable. This is consistent with our previous report that this subunit is not a product of direct genetic translation.

Experimental Modification of the Temporal Aspects of the Serum-Induced Cell Cycle in the Cultured Adult Rabbit Lens

The adult rabbit lens in organ culture has been used as a model system for studies on the control of cell division in an organized tissue. Freshly isolated lenses, placed immediately in culture media containing serum (M-199-S) undergo a series of characteristic changes in macromolecular synthesis (RNA, protein, DNA) and ultrastructure which herald the onset of mitosis. In the present experiments lenses were preincubated 24 h in a non-stimulating medium which maintains the central epithelial cells in their normal non DNA synthesis proliferating state. They were then transferred to M-199-S. Preincubation for 24 h in the non-stimulating medium decreases the time required between the addition of serum and the initiation of and mitosis, and increases the degree of synchrony with which the cells enter DNA synthesis. It also shortens the time required for the serum-induced increase in RNA production and ribosome formation. Serum-induced ribosome formation in preincubated lenses is relatively intense (as compared with freshly-isolated lenses). The acceleration of serum effects on macromolecular synthesis and mitosis is discussed in terms of the effects of preincubation on the G-0-1 → G-1 transition

Suckling mouse cataract agent (SMCA) in tissue culture.

SummarySuckling mouse cataract agent, a new addition to the “slow” virus group, has shown appreciable growth in explant tissue cultures of SMCA-infected chick embryos. SMCA readily propagated for the first time in significant titers in organ cultures of rabbit lens. In addition, several serial passages of SMCA in rabbit lens organ cultures were made with no significant loss in infectivity titers. The data presented should be of considerable value in further studies on the nature of this agent and its pathogenesis in chronically infected brain.

Lens Organ Culture: I. Methodology and Metabolic Evaluation

Lens Organ Culture: I. Methodology and Metabolic Evaluation

Lens Organ Culture: II. Pharmacologic Evaluation

Lens Organ Culture: II. Pharmacologic Evaluation