H. Rothstein

Radiation Cataract and Mitosis

X-irradiation of frog eyes with natural and experimentally induced differences in lenticular mitotic activity reveals cataractogenesis to be dependent on the proliferative activity of the germinative

Synthesis of macromolecules in epithelial cells of the cultured amphibian lens: I. DNA and RNA

Abstract 1. 1. When bullfrog lenses are introduced into a culture system, the epithelial cells undergo bursts of DNA synthesis and mitosis which reach peaks at 52 and 87 hr post-isolation, respectively. 2. 2. These events are preceded by an augmentation of RNA synthesis which begins within 10 hr of isolation and attains peaks at 36 and 72 hr. 3. 3. If the increment in RNA synthesis is blocked by treatment with 10 −1 μg/ml actinomycin D the waves of DNA synthesis and mitosis are suppressed even though uridine incorporation in “blocked” lenses is still significantly higher than it is in freshly isolated material. 4. 4. It was shown by autoradiographic means that when the actinomycin D was supplied in tritiated form it was incorporated almost exclusively into the nucleus. 5. 5. The foregoing results were discussed with reference to the mechanisms which control proliferation.

Response to injury in the lens epithelium of the bullfrog (R. Catesbeiana): II. Spatio-temporal patterns of DNA synthesis and mitosis☆

Abstract The experiments described herein show that mitosis is a normal adjunct of the injury response in bullfrog lens epithelium. In lenses maintained in vivo mitotic activity starts at 64 hr after injury. A greater number of division figures is evident at 96 hr. When lenses are completely isolated from adjoining tissue at 93 1 2 hr and are then incubated for 2 1 2 hr, mitotic activity is virtually absent. This can be obviated if the posterior 1 4 – 1 3 of the globe is removed and the remainder is incubated with the lens in place. Hence, the dearth of mitotic activity which we previously reported can safely be imputed to the isolation procedure employed. We have also tried to determine whether or not the stimulus to DNA synthesis and mitosis in injured frog lenses is propagated as a wave. Tritiated thymidine has been injected into the anterior chamber of the eye according to three different experimental regimens. In no case could it be shown that cells nearer to the site of injury were caused to enter the synthetic stage prior to those situated further out. Taken together with evidence from an earlier study, we construe our data to mean that in the frog lens, the stimulus to DNA synthesis does not move outward from the injury in the manner of a wave. Implications of these findings are discussed.

Stimulation of Mitosis in the Vertebrate Lens in the Presence of Insulin

Rabbit and frog lens epithelial cells can be induced to undergo DNA synthesis and mitosis in a completely defined insulin containing medium. The hormone was added to Medium 199 in these experiments. In general, the insulin aroused division throughout the central region of the epithelium. Insulin added to Medium 199 from a different source with a different formulation, was unable to evoke the response. The non-stimulating 199, could support the activation if it was supplemented with insulin plus rabbit serum dialysate or the low molecular weight serum components obtained by gel filtration. In contrast to the response elicited by whole rabbit serum, marked cellular disorganization did not precede the hyperplasia that occured after culture in Medium 199 plus insulin. The temporal aspects of DNA synthesis and mitosis observed in the presence of insulin, are similar to those obtained employing whole rabbit serum. These experiments have led to the fabrication of a system (for mammalian and amphibian material) in which an organized tissue can be prompted to increase, strikingly, its level of mitosis, while being cultured in a medium whose constituents are known.

Induction of mitosis in ocular tissue by chemotoxic agents

Abstract Ocular inflammation has been induced in a number of species by a variety of chemical agents, including methylene blue. Initial cell destruction and subsequent repopulation by mitogenesis in lens epithelium and corneal endothelium were observed using techniques for living and fixed tissue. During the course of the chemical inflammation response, the lens displayed a prominent, anterior pole subcapsular opacity underlying the area of maximal epithelial destruction. Near-normal transparency returned with recovery of the lens tissue cytoarchitecture. Elevated protein concentrations in aqueous humor were found throughout the course of the experiments. Similar alterations in aqueous humor were observed in the course of immunogenic uveitis although lenticular or corneal endothelial destruction and subsequent proliferation were not detected.

Mitotic Variations in the Lens Epithelium of the Frog

Proteins corresponding to growth hormone and prolactin have been electrophoretically separated from the pituitary gland of the frog (Rana pipiens berlandieri and Rana catesbeiana). These substances can independently stimulate mitosis in the lenses of animals where it had previously been inhibited by means of hypophysectomy. DNA synthesis is greatly augmented six days after the start of hormone administration to intact animals. Nine days are required before this occurs in hypophysectomized ones. No other anterior pituitary hormone has been found to achieve the effect described. Initial attempts at amino acid analysis, though incomplete, indicate that the two frog hormones are different from each other as well as from their bovine counterparts. Pituitary factors do not affect mitotic activity in either corneal epi or endothelium of the frog.

Chapter 3 Experimental Techniques for Investigation of the Amphibian Lens Epithelium

Publisher Summary This chapter presents experimental techniques for the study of the amphibian lens epithelium. Such techniques can be applied, with slight modifications, to the investigations of lens epithelium in almost any vertebrate organism. Many techniques can be applied to investigate the lens epithelium of amphibians (and other animals as well), and newer approaches will be applicable with the passage of time. The question of whether such techniques can pave the way for a more acute understanding of cellular activities is important. It is a homogeneous group of cells existing in as simple an array as has been found in any multicellular organism. Thus, when proliferation is stimulated—for example, by mechanical injury, the trouble related to the possibility of other types of cell complicating the picture dissappear. Routine tissue culture should render possible the investigation of almost any question relating to cellular function—for example, work on membrane transport. The lens, therefore, emerges as an extraordinarily versatile kind of experimental system, a system upon which it is possible to execute biochemical, histochemical, and microscopic investigations at almost all technical levels in use.

Inhibition of DNA synthesis and cell division by actinomycin D

Nach in-vitro-Kultur oder Verwundung in vivo der Froschlinsen wird zuerst meistens, im Epithel, eine intensive DNS-Synthese gefunden auf die später zahlreiche Kernteilungsfiguren folgen. Nach Behandlung mit Actinomycin D werden diese Reaktionen stark gehemmt. Autoradiographische Untersuchungen mit dem DNS-Vorläufer H-3-Thymidin und dem RNS-Vorläufer H-3 Uridin erlauben den Schluss, dass die DNS-Synthese (und damit Zellteilung) von einer früheren RNS-Synthese abhängt.

Mitotic variations in the lens epithelium of the frog ☆: III. Somatotropin

Abstract Hypophysectomy reduces mitotic activity of the frog ( Rana pipiens and Rana catesbeiana ) lens to zero within 3–4 wk after surgery. The effects are reversed by pituitary powder or bovine growth hormone. Although TSH and T 3 are mitogenic in intact organisms, they are unable to reverse the effect of hypophysectomy. The data suggest they may not be natural regulators of mitotic activity in frogs. Growth hormone is a more probable candidate.

Cell cycle variations in chromosomal proteins of the lens epithelium

Abstract A method has been developed for the isolation and electrophoretic separation of chromosomal proteins from rabbit lens epithelium. We have analysed these proteins at a number of times after explantation of the lens into serum containing culture media. Under these conditions, the G1-arrested lens cells are induced to undergo DNA synthesis and mitosis. Qualitatively, the electrophoretic banding pattern for all the chromosomal proteins was remarkably constant throughout the times studied. The chromosomal protein labeling patterns obtained from lenses exposed to a 1 h pulse of tritiated amino acids showed increased incorporation into non-histone chromosomal proteins (NHCPs) during the latter part of the prereplicative and S phase. Histone labeling was confined to the S phase. Absorbance scanning of the stained gels reveals characteristic ratio changes between the total stainable NHCPs and histones during the cell cycle. A 62% inhibition of DNA synthesis does not seem to decrease the incorporation of [ 3 H]leucine into histones or NHCPs.