Studies on corneal endothelial growth and repair. II. Increased transcription as detected by incorporation of 3H-uridine and 3H-actinomycin D

Endothelial cells from injured frog corneas undergo increased ³H-uridine and ³H-actinomycin D (³H-AMD) incorporation as judged by autoradiography. The increase in ³H-AMD binding occurs when living endothelium is labeled in vitro or when fixed preparations are exposed to the drug. The changes in ³H-AMD incorporation detected by the two methods are comparable (55 and 62 % for living and pre-fixed tissue respectively). However, when fixed endothelium is also de-histonized with 2 N HCl, differential binding of ³H-AMD is eliminated. This result suggests that the enhanced incorporation of ³H-AMD into nuclei is at least partly due to a modification in the association of chromosomal proteins with DNA and not entirely to cell permeability changes that may accompany wound repair. This contrasts with observations of cells that are killed outright by the injury. Such cells bind very large amounts of ³H-AMD compared with their living neighbors. Here the difference in incorporation is eliminated by prefixation. Thus, in the dead cells increased binding may be due to a reduction of cell surface permeability barriers which accompanies cell morbidity.     

Studies on corneal endothelial growth and repair. IV. Changes in the surface during cell division as revealed by scanning electron microscopy

Changes in the surface morphology of regenerating rabbit, rat and frog corneal endothelial cells in vivo have been investigated by scanning electron microscopy. In adult tissue these cells do not normally divide unless given a stimulus, such as injury. Surfaces of quiescent rabbit and rat cells are devoid of microvilli but display globular projections and surface pits up to 300 nm in diameter. However, regenerating endothelia are characterized by the appearance of microvilli which attain their greatest length when the cells are rounded. At this stage, cells also possess filopodia and broad processes. In cytokinesis, the microvilli have shortened and blebs and ruffles appear for the first time. In contrast to rabbits and rats, frog endothelial cells of noninjured tissue are covered by microvilli and smaller surface pits of 60-70 nm diameters. During regeneration, these cells have reduced numbers of microvilli and extensive foldings of the membrane. Neither blebs nor filopodia occur during the mitotic cycle and ruffles are not detected until cytokinesis.     

DNA-polymerase activity detected in situ. Relationship between incorporation of 3H-deoxytriphosphates and 3H-actinomycin D binding during the cell cycle in antheridial filaments of Chara vulgaris L. Effect of auxin and cytokinins

DNA-polymerase activity during the cell cycle (S + G2 + M + C type) in antheridial filaments cells of Chara vulgaris was studied using the autoradiographic method. Incorporation of 3H-deoxytriphosphates (3H-dTPs) during the whole of interphase indicates, that the cell cycle is not accompanied by distinct changes in enzyme activity. Incorporation of 3H-dTPs was also observed in spermatids and in early stages of spermatogenesis. Intensity of 3H-dTPs incorporation during interphase and spermatogenesis is similar to the intensity of 3H-actinomycin D (3H-AMD) binding. Auxin (IAA) and kinetin stimulate both 3H-AMD binding and 3H-dTPs incorporation; benzyladenine does not affect any of these processes. The in situ autoradiographic method of detecting DNA-polymerase activity reveals availability of DNA template for the enzyme rather than DNA polymerase activity itself.     

Increased incorporation of aspartate and decreased incorporation of orotate in fibroblasts from Lesch-Nyhan patients as revealed by autoradiography

While it has been known for many years that tissues from Lesch-Nyhan patients are deficient in hypoxanthine-guanine phosphoribosyl transferase, it has been demonstrated more recently that erythrocyte lysates show an increase in the levels of enzymes which participate in “de novo” synthesis of pyrimidines. Also, we were able to demonstrate, using autoradiography, an increased rate of incorporation of aspartate into the nucleus of intact cultured fibroblasts from a Lesch-Nyhan patient. We have now confirmed this finding. In this paper we show increased incorporation of aspartate into the nucleus of cells from three other patients. Interestingly, the incorporation of orotate is decreased in the fibroblasts from the four patients studied. The incorporation of leucine is the same in Lesch-Nyhan fibroblasts as in normal fibroblasts. The significance of these findings is briefly discussed.     

Increased level of serum vascular endothelial growth factor by long-term exposure to hypergravity.

We have previously demonstrated that short-term exposure to hypergravity at 2G for 4 h induces expression of cyclooxygenase-2 (COX-2) in the mouse heart. Moreover, expression of vascular endothelial growth factor (VEGF) is also induced in the heart in a COX-2-dependent manner. Here, we demonstrate that long-term exposure of mice to 2G for 24 h resulted in a significant increase of serum VEGF level, although expression of COX-2 and VEGF in the heart decreased to the 1G-control level. Moreover, increase of serum VEGF was not suppressed by treatment with COX-2 inhibitor, indicating that VEGF was induced in a COX-2-independent manner. These results suggest that gravitational force contributes to maintenance of the serum VEGF level.     

Studies on Scenedesmus acutus growth. The incorporation of 14C-glucose by Scenedesmus acutus under light and dark periods

Qualitative and quantitative estimations of alcohol-extractable compounds from (14)C-glucose-incorporated Scendesmus acutus cells were performed at various intervals of time. After two hours of incubation with (14)C-glucose in light, amino acids were synthesized at 65% and sucrose at 26% of the total input label. In the dark incorporation, 30% of the total radioactivity was found in amino acids and 46% in sucrose. Leucine and valine were detected only during the oxidation of glucose in light. The concentration of serine increased more in the presence of light, as compared to dark. These results on the oxidation of glucose are discussed in this article.     

Hyperoxia elevates Cu,Zn-superoxide dismutase of endothelial cells as detected by a sensitive ELISA.

An enzyme-linked immunosorbent assay (ELISA) was developed for the measurement of bovine Cu,Zn-SOD. Accuracy of the ELISA and specificity of the antibody for cell-free extracts was established by: (1) measurement of antigen levels of bovine endothelial cell extracts reconstituted with pure antigen, and (2) immunoblotting with affinity purified antibody. The ELISA was highly sensitive and 0.05-0.10 ng of pure antigen could be accurately detected, which allowed the measurement of Cu,Zn-SOD in as few as 250 endothelial cells. With utilization of the ELISA for detection, DEAE-cellulose chromatography patterns of endothelial cell Cu,Zn-SOD overlapped those of pure bovine erythrocyte Cu,Zn-SOD. Exposure of cells in culture to 80% O2 for 48 h increased the relative abundance of the Cu,Zn-SOD as measured by the ELISA by 1.8-fold. Thus, endothelial cells in culture respond to hyperoxia by enhanced production of Cu,Zn-SOD protein. The ELISA developed in this study may be useful for assessing other factors that regulate cellular production of Cu,Zn-SOD.     

Vitamin B6 deficiency and the lymphoid system: I. Effects on cellular immunity and in vitro incorporation of 3H-uridine by small lymphocytes

Thoracic duct lymphocytes from vitamin B₆-deficient rats were found to have a reduced capacity to respond to foreign lymphoid cells in the mixed lymphocyte reaction (MLR), to produce normal lymphocyte transfer reactions, and to incorporate ³H-uridine in vitro. These findings indicate that specific nutritional deficiencies may impair cellular immunity and that this impairment can be monitored by the MLR. It is suggested that the reduction in MLR activity and in ³H-uridine uptake by TDL cells reflected either a shift in the proportions of T and B cells in the TDL and/or an impairment in the capacity of such cells to function in the MLR and in the in vitro test for ³H-uridine incorporation.     

Ultrastructural immunocytochemical localization of fibronectin deposition during corneal endothelial wound repair. Evidence for cytoskeletal involvement

The distribution of the extracellular matrix (ECM) protein, fibronectin (FN), has been examined ultrastructurally in noninjured and injured rat corneal endothelium in vivo and in vitro by immunoperoxidase cytochemistry. In noninjured endothelia, FN was observed within the rough endoplasmic reticulum (RER) cisternae but not along the cell-Descemet's membrane (DM) interface. Twenty-four and 48 h after a circular freeze injury, immunoperoxidase reaction product was detected at the cell-DM interface as well as within cytoplasmic vesicles and intercellular spaces. By 1 and 2 wk post-injury, a line of reaction product could still be demonstrated at the cell-DM interface and evidence for newly deposited basement membrane material was observed in this region. In order to understand whether fibronectin deposition during wound repair was dependent on cytoskeletal influences, organ culture experiments were performed in which the media was supplemented with either 10(-8) M colchicine or 2.5 X 10(-3) M cytochalasin B. Without inhibitors, injured corneas cultured for 24 h had FN deposition at the cell-DM interface similar to the in vivo results. Corneas cultured in the presence of cytochalasin B also showed FN deposition at the cell-DM interface. However, when injured endothelia were cultured in the presence of colchicine, no reaction product was observed at the cell-DM interface, although it could be detected intracellularly within RER. Incubating the tissues in the presence of puromycin abolished all extracellular and intracellular staining. These results indicate that during wound repair, corneal endothelial cells produce fibronectin and deposit it upon Descemet's membrane by a mechanism that may be mediated by microtubules.     

Some observations on the use of cultured corneal endothelial cells as a model for intact corneal endothelium

Major differences have been identified between corneal endothelial cells in situ and those grown in culture. Cells in intact porcine corneal endothelium were studied and compared with primary cultures of the same cells either in suspension or in monolayers which had been grown on plastic (Nunc, Permonax). Differences were identified in the organization of the cytoskeleton (filamentous actin) between the cells in situ and in monolayer culture. The ability to withstand exposure to cryoprotective concentrations of Me(2)SO also varied substantially depending on whether the cells were in situ or in culture. These results underline the need for caution in the use of cells in culture as a model for studying the nature of injury to cells during the freezing of whole tissues.