CHANGES IN PIGMENT EPITHELIUM CELLS AND IRIS PIGMENT CELLS OF RANA PIPIENS INDUCED BY CHANGES IN ENVIRONMENTAL CONDITIONS

1. Retina pigment epithelium cells and iris pigment cells are placed in an identical medium. 2. The differentiation into two different parts, as well as the pigment strains of the migrating pigment epithelium cells, is lost. The normally highly specialized cuticular structures disappear. The cells begin to move and assume a spindle shape, thus losing their original hexagonal shape. 3. The migrating iris pigment cells begin to show a distribution of the pigment, similar to that exhibited by the migrating pigment epithelium cells. The cuticular structures of this cell type also disappear. These cells also assume the ability of free movement and become spindle-shaped. 4. By means of these changes both cell types not only become more similar to the ordinary culture cell type, but also in certain respects, more like each other, whereas before explantation they were very unlike. In order to accomplish this the pigment epithelium cells must change much more thoroughly than the iris pigment cells. 5. The changes, particularly those of the pigment epithelium cells, show a definite relation to the changes of external factors, as they are the expression of the transmission from dissimilar conditions existing on different sides of the cells into conditions which are uniform on all sides

RELATION BETWEEN THYROID GLAND, METAMORPHOSIS, AND GROWTH

1. Two substances are involved in amphibian metamorphosis as studied in Ambystoma opacum: first, iodine, which is taken up by the food, and second, an excretor substance, which is evolved during the processes of growth and serves to induce the excretory function of the thyroid gland. 2. This explains why in larvæ, whose metamorphosis is inhibited by lack of iodine, growth is checked at the time when metamorphosis should occur; for at this time the excretor substance commences to act and this results, if iodine is absent, in the excretion by the thyroid of toxic substances which cause the breakdown of proteins and consequently a decrease in size of the larvæ. 3. Larvæ whose metamorphosis is inhibited by extirpation of the thyroid or by the hereditary lack of a thyroid (as is the case in Typhlomolge) can grow normally, since in them the action of the excretor substance cannot result in the excretion by the thyroid of a toxic growth-inhibiting substance. 4. At low temperature less excretor substance is produced than at high temperature during an equal rate of growth; therefore larvæ kept at low temperature reach a larger size than larvæ kept at high temperature, before they metamorphose

NATURE OF THE RETARDING INFLUENCE OF THE THYMUS UPON AMPHIBIAN METAMORPHOSIS

1. Though thymus-fed salamander larvæ often metamorphose normally, thymus feeding sometimes retards and in rare cases inhibits metamorphosis completely. 2. The addition of normal food to a thymus diet abolishes the inhibitory effect of the thymus. 3. Addition of a small quantity of iodothyrin leads rapidly to precocious metamorphosis of thymus-fed larvæ. 4. The inhibitory effect of the thymus is not due to a specific inhibiting substance in the thymus, but to the absence from the thymus of a substance required to develop the thyroid to the secretory state

THE FORM OF THE EPITHELIAL CELLS IN CULTURES OF FROG SKIN, AND ITS RELATION TO THE CONSISTENCY OF THE MEDIUM

1. Fragments of skin from the leopard frog (Rana pipiens) were cultivated in media of varying consistency. A mixture of frog plasma, frog muscle extract, chicken plasma, and chicken embryo extract usually produced a very firm medium; a mixture of frog plasma, frog muscle extract, and chicken plasma, one less firm (semi-firm); and a mixture of frog plasma and frog muscle extract a medium of a consistency varying from soft to liquid. 2. (a) In a firm medium the cells which migrate into the medium are polyhedral (polygonal when seen from above) in form, which shape they retain permanently. They remain united in a compact membrane, the central parts of which consist of several (three to four) layers of cells. Migration of isolated cells into the medium does not take place. (b) In the semi-firm media the cells situated at the edge of the membrane become fusiform in shape, gradually detach themselves from the membrane, and stray out individually into the medium. This causes the membrane to become loose in character, and to contain holes, while its edges at the same time become very irregular and send out pointed projections. (c) In a soft medium the cells are fusiform or thread-like in shape. The migration of isolated cells is much more pronounced than in the semi-firm media, as a result of which the membrane undergoes constant and rapid loosening up. By this means whole portions of the membrane become detached and their separate parts are at first united by the thread-like columnar cells, which become drawn out in the form of long threads upon the separation of the individual sections of the membrane. The loosening up of the membrane is further assisted by liquefaction and the consequent formation of vacuoles; the latter process likewise results in the formation of thread-like columnar cells. (d) Liquid media contain only round cells. 3. This serves to explain numerous internal processes of the organism, especially certain changes of form observed by Leo Loeb in transplantations of wound scabs and of skin; the conditions artificially produced by Leo Loeb must have effected a change in the consistency of the medium. 4. It has been shown that it is unnecessary, for a satisfactory explanation of the above findings, to have recourse to the theories of "functional stimulus" or "inhibiting influences," by means of which Champy wished to account for the variations in the morphological character of the cells

THE INFLUENCE OF FEEDING THE ANTERIOR LOBE OF THE HYPOPHYSIS ON THE SIZE OF AMBYSTOMA TIGRINUM

1. Animals of the species Ambystoma tigrinum when fed anterior lobe can reach a size far in excess of that of animals fed earthworms and presumably also of that of liver-fed animals. 2. Liver produces a rate of growth as high as that resulting from anterior lobe-feeding, but maintains growth only, until the animals reach a definite size far below that of anterior lobe-fed animals

EXPERIMENTAL PRODUCTION OF GIGANTISM BY FEEDING THE ANTERIOR LOBE OF THE HYPOPHYSIS

1. Metamorphosed salamanders of the species Ambystoma opacum and Ambystoma tigrinum were fed on a pure diet of the anterior lobe of the hypophysis of cattle; the controls were fed on an abundant diet of earthworms. 2. The rate of growth of the animals fed on the anterior lobe of the hypophysis was greatly increased over the rate of growth of normal animals. 3. Growth of the animals fed on anterior lobe did not cease after they had reached the normal "maximum" size of the species, and experimental giants were produced. 4. The largest animal of the species Ambystoma opacum fed on anterior lobe of the hypophysis was 19 mm. larger than the largest normal animal of this species known to the writer; the largest animal of the species Ambystoma tigrinum fed on anterior lobe is at present about 28 mm. larger than the largest normal animal of the eastern race of this species known to the writer

THE ANTAGONISM BETWEEN THYROID AND PARATHYROID GLANDS

From the facts stated in this paper it is evident that the thymus gland of mammals contains a substance which is capable of producing tetany when fed to the larvæ of certain species of salamanders (Ambystoma opacum and Ambystoma maculatum). As long as the larvæ have not developed their own thymus glands, they are able, by means of some mechanism, to counterbalance the tetanic action of the thymus substance introduced in their food. When, however, the secretion from their own thymus glands is added to the thymus material introduced with the food, this mechanism of preventing tetany becomes inadequate and tetany ensues. In the larva of a third species of salamander, Ambystoma tigrinum, this mechanism will prevent tetany even when the larvæ are fed on thymus. In mammals the parathyroids are known to prevent tetany and are supposed either to absorb the tetany-producing substance and thus prevent its action or to change it into another non-toxic substance. It is at least probable that in the amphibians the parathyroids play the same rôle. Larvæ of anuran amphibians, which develop their parathyroids soon after hatching, never show tetanic convulsions if they are fed on thymus, but in certain species of salamanders, whose parathyroids develop only during metamorphosis, the larvæ invariably have tetanic convulsions upon thymus feeding, while the metamorphosed animals never show tetany. But in addition to the parathyroids the salamanders must possess still another mechanism which during the larval period inhibits the production of tetany by the animal's own thymus glands. In the larvæ of Ambystoma opacum and Ambystoma maculatum this mechanism is sufficient only to prevent tetany from the animal's own thymus, while in the larvæ of Ambystoma tigrinum it is capable of preventing tetany even when the larvæ are fed with thymus. If the thymus is the organ by whose action tetany is produced, we can understand why tetany in human beings occurs far more frequently in children than in adults, since in the latter the thymus gland is replaced, at least to a great extent, by connective tissue. The relation of thymus to tetany may also possibly explain the occurrence of tetany during pregnancy; while the parathyroids of the mother may be sufficient to prevent tetany from her largely atrophied thymus, they may not be sufficient to prevent tetany from the excess of thymus substance furnished by the fetus to the blood of the mother

REGENERATION AND NEOTENY

It is apparently quite certain that removal of parts of the body (limbs, tail) followed by regeneration of these parts (1) does not produce neoteny in the larvae of salamanders, and (2) has no influence upon metamorphosis

PARATHYROIDS AND CALCIUM METABOLISM

The experiments reported in this article are in full agreement with the facts known about the action of Ca and Mg salts in tetanic animals. In the concentrations used here both Ca lactate and Mg lactate suppressed the muscular convulsions in the tetanic salamander larvæ. The Mg lactate, however, appears to be more effective than the Ca lactate. At any rate the suppression of the tetanic convulsions does not seem to be a specific action of the calcium. The most important result seems to be the fact that the salts used, though they prevented the muscular convulsions, did not prevent the other symptoms of tetany which in the salamander larvæ are very definite and constant. The permanent spasmodic contractions and the paralysis of the muscles developed in spite of the presence of the Ca and Mg, Furthermore, the muscular contractions and the paralysis developed even in such thymus-fed animals in which the convulsions had been suppressed completely; this was the case in one of the animals of the Mg series. From the experiments of Biedl and others it is likely that the tetanic convulsions are due to lesions of the central nervous system, since convulsions of a leg can be prevented by isolating it from the central nervous system by cutting the nerves which connect the muscles with the central nervous system. Evidently these lesions of the central nervous system are the chief factor in tetany, while the convulsions of the muscles are only an effect. In the larvæ of salamanders these lesions find a definite expression in the permanent paralysis of almost the entire muscular system. In the writer's opinion, MacCallum's hypothesis that the tetany toxin has a special affinity for Ca, thereby diminishing the Ca content of the organism, cannot be disproved at present. But the present experiments seem to prove, first, that the tetany-producing substance causes permanent lesions of the nervous system, which lead to permanent spasmodic contractions and paralysis of the muscle even in the absence of tetanic convulsions, and second, that these cannot be prevented by either Ca or Mg. For the most part they result in an early death of the animals no matter whether or not Ca or Mg has been applied. In connection with this fact we wish to mention Biedl's claim that no one has yet succeeded in prolonging the life of parathyroidectomized animals by the application of Ca. From MacCallum's paper, on account of the lack of controls, it cannot be seen whether his parathyroidectomized dogs lived longer with Ca treatment than without. That in spontaneous tetany Ca treatment may effect a cure, as is evident from the report by Howland and Marriott, does not prove that in this case Ca has inhibited tetany as a disease. In spontaneous tetany the period of the action of the tetany-producing substance may be a very short one and the mere prevention of the tetanic convulsions may keep the patient alive until normal function of the glands involved has been restored. The pathological changes which the central nervous system undergoes in this short period may not be severe enough to endanger the life of the patient after the cessation of the action of the tetany toxin. In the light of the facts presented our experiments lead to the following conclusions: 1. The thymus gland excretes a tetany-producing substance which in the normal animal is antagonized in an unknown way by the parathyroids. 2. In animals devoid of parathyroids (salamander larvæ, parathyroidectomized mammals) this substance may, according to MacCallum, reduce the Ca content of the organism; but by far the most dangerous and important quality of this substance is its highly injurious effect upon the central nervous system, which causes permanent spasmodic contractions of the muscles and paralysis of almost the entire muscular system. 3. It is possible to prevent the muscular contractions by introducing Ca salts into the body, though this can be done more effectively by means of Mg salts. 4. No substance, however, has been found so far to antagonize the tetany toxin and to prevent the development of the lesions of the central nervous system caused by the tetany toxin. 5. This explains why in spite of the application of Ca or Mg and in spite of the suppression by these substances of the tetanic convulsions the other symptoms of tetany develop and frequently lead to the death of the animal. 6. Accordingly the most important function of the parathyroids is to prevent the tetany toxin, by antagonizing it, from coming into contact with the central nervous system