Frog Tissues Research Articles

Studies on isozymes of fructose-1,6-diphosphate aldolase during amphibian development. II. Characterization of isozymes in the egg of Rana pipiens

1. 1. The types of fructose-1,6-diphosphate aldolases (ketose-1-phosphate aldehyde-lyase, EC 4.1.2.7; fructose-1,6-diphosphate d-glyceraldehyde-3-phosphate-lyase, EC 4.1.2.13) found in the unfertilized frog egg, as well as in early stages of development, have been investigated to gain insight into the biological significance of aldolases of the A, B, and C types in early embryogenesis. 2. 2. The relative electrophoretic mobilities of the isozymes from egg and adult frog tissues indicated that five-membered hybrid sets of the AC types are present in the unfertilized egg. 3. 3. The five isozymes in egg and in brain have been separated by chromatographic procedures and their properties compared. The substrate specificities, electrophoretic mobilities, and immunological properties of the individual isozymes from both sources were comparable and differed significantly from those of frog liver aldolase (type B). Dissociation and reassociation of a given hybrid isozyme from egg led to the formation of the other four isozymes of aldolase. 4. 4. These studies show that A and C type aldolases, together with their hybrids, are present in the unfertilized frog egg. 5. 5. No marked changes in the relative proportions of the AC isozymes were observed in embryos up to hatching at which time muscle aldolase (type A) increased.

Oxygen consumption of frog tissues under high hydrostatic pressure

The rate of oxygen consumption of some frog tissues was measured under various hydrostatic pressures up to 500 atm. The tissues either intact, minced, or homogenized were immersed in oxygenated Ringers solution in 10 ml syringes without any gas bubbles. The syringes were immersed in water in a pressure vessel with a control in a similar vessel at atmospheric pressure. The partial pressure of the oxygen in the solution was measured with an oxygen electrode before and after a period of a few hours under pressure. In skin, liver, kidney and lung, pressure caused a decrease in oxygen consumption, but in skeletal muscle there was a 25% increase because the muscles went into a contracture. This increased the demand for oxygen in spite of a presumed inhibition of the oxidative process itself. To account for the inhibition there is presumably some point in the oxidative process which involves an increase in volume. The burning of glucose to CO 2and water does result in a slight increase in volume.

Influence of temperature on the composition of fatty acids and on lipogenesis in frog tissues

1. 1. Frogs ( Rana esculenta) were adapted to various temperatures and the fatty acid composition of tissues and lipogenesis from radioactive precursors were studied. The activities of NADPH-generating enzyme systems were also compared. 2. 2. Liver and adipose tissue from cold-adapted frogs contained more unsaturated fatty acids and less saturated, than the corresponding tissues from frogs kept at higher temperatures. However, differences were found mostly in the proportions of polyenoic acids and not in those of the monoenoic acids. The changes in fatty acid composition of phospholipids and of neutral lipids were similar. 3. 3. As a result of in vitro lipogenesis from 1-C 14-acetate mostly saturated and monoenoic fatty acids were formed and there was no indication of de novo formation of linoleic acid in tissues from either cold-adapted frogs or those kept at 20°C. 4. 4. The temperature of adaptation did not influence either the extent of hepatic lipogenesis from C 14-acetate or the distribution of radioactivity in lipid fractions. On the other hand, in adipose tissue of frogs kept at 20°C both lipogenesis and esterification of newly formed fatty acids were strongly inhibited as compared with the tissue from cold-adapted animals. 5. 5. No correlation between the rate of lipogenesis and the activities of NADPH-generating enzymes could be observed and under the conditions of depressed lipogenesis formation of NADPH did not seem to be rate limiting.

Studies on avian and mammalian heart tissues

1. 1. In a study of in vitro amino acid utilization avian heart tissues were found to utilize glutamate and alanine, and release glutamine, whereas mammalian tissues did the reverse. 2. 2. These differences were attributable to the differences in the activities of l-glutamate: ammonia ligase, glutamine: glutamyl glutamyltransferase and alanine: 2-oxoglutarate aminotransferase in the above species. 3. 3. Studies of heart tissue of cat, rabbit, rat, bat, pigeon, chick, sparrow, calotees, frog and fish showed a significant correlation between respiratory rate, heart beats/min, AMP aminohydrolase activity, l-glutamate: ammonia ligase and glutamate: NAD oxidoreductase activities together, and heart weight in relation to body weight. 4. 4. The results support the view that l-glutamate: ammonia ligase and glutamate: NAD oxidoreductase serve as an ammonia removal mechanism in heart tissue.

Changes in the pathways for serine biosynthesis in the developing amphibian

1. 1. The pathways of serine biosynthesis utilizing either phosphorylated or nonphosphorylated intermediates, as well as the levels of activity of phosphoglycerate and d-glycerate dehydrogenases, have been investigated in tissues of frog at various stages of development. 2. 2. Serine biosynthesis occurs via the phosphorylated pathway in the unfertilized egg but by the nonphosphorylated route in liver of tadpoles undergoing metamorphosis and in adult liver. 3. 3. Phosphoglycerate dehydrogenase is present in unfertilized eggs and its activity remains relatively constant during the first the first 30 days following fertilization. Appreciable d-glycerate dehydrogenase activity is not detected until several days following hatching.

Multiple adenosine deaminases in the frog ( Rana catesbeiana)

1. 1. Three classes of adenosine deaminases have been found in frog tissues. Types A, B and C have molecular weights in the range of 180,000, 100,000 and 35,000 respectively. Studies of tissues from a wide variety of vertebrates have shown that all of these enzyme types are widespread among vertebrates (Ma & Fisher, unpublished results). 2. 2. Tissue specific differences in the frog result from different proportions of the three types of enzymes in the liver and duodenum. In the liver a type B enzyme accounts for most activity while in the duodenum a type C enzyme predominates.

Effect of ACTH and hypophysectomy on the interrenal tissue in the common frog, Rana temporaria.

Histological and histochemical changes have been studied in the interrenal tissue of the male common frog, Rana temporaria, following ACTH administration and ablation of the pars distalis of the pituitary. A combination of the following parameters appeared to be useful for the determination of interrenal activity in histological material: 1. the cellular area; 2. the nuclear area, shape and stainability of the nuclei, and the presence and size of the nucleoli; 3. the amount of lipoidal material in the cytoplasm, the cholesterol content of the lipids, and in connection with these the size of the lipid droplets; 4. the mitotic activity.

The effect of metopirone and aldactone on the interrenal tissue of intact and pars distalis-ectomized common frogs, Rana temporaria.

The effects of metopirone (SU-4885) and aldactone (SC-9420) on the histology and histochemistry of the interrenal tissue of male common frogs, Rana temporaria, have been studied. From the changes in nuclear and cellular size, mitotic rate, and lipid and cholesterol content it has been concluded that in intact animals the administration of metopirone as well as of aldactone resulted in an increase of the interrenal secretory activity. The effects were very similar to that of ACTH. Ablation of the pars distalis prevented the interrenal activation by metopirone. The effect of aldactone was markedly reduced in operated frogs, but some stimulation remained. It is suggested that both substances act by way of enhancing pituitary ACTH ouput. The dependence of the secretion of corticosteroids on pituitary ACTH is discussed.

Parathyroid glands of amphibians. II. Structural and biochemical changes in amphibian tissues elicited by parathyroid hormone under varying conditions of season and temperature.

The parathyroid glands have received relatively little attention with regard to their impact on the internal milieu in amphibians. Present information, however, clearly indicates that distribution of mineral in frog tissues and body fluids is under hormonal regulation; further modifications are imposed by season and temperature. Under conditions of cold acclimation or during winter normal amphibian bone shows minimal rates of carbohydrate utilization: simultaneously, phosphate is conserved by reduced excretion and increased incorporation in tissue. Exogenous parathyroid hormone, injected under these conditions when endogenous titers may be low due to glandular degeneration, reverses these effects, and phosphate is lost in a hyperphosphaturic urine. During summer or periods of warm acclimation, hormone promotes incorporation of phosphate in tissues, and increases rates of carbohydrate metabolism in bone; citric acid, which may act in solubilizing bone mineral, is elevated in the bone. Concurrently, pre-existing sulfated mucopolysaccharides of “old” bone may be degraded, while epiphyseal zones enlarge and may exhibit accelerated or abnormal elaboration of sulfated components of cartilage and/or osteoid matrix.

A Dynamic Cue of the Magnitude of Tactile Sensation

Publications by Nafe, and Nafe and Kenshalo have proposed the movement of cutaneous nerve-endings as the stimulus for all of the cutaneous qualities." Support for this view is found in the search by Nafe and Wagoner who were unable to relate any static stimulus component to verbal reports to stimulus failure (adaptation) and in Nafe and Kenshalo who described afferent neuron excitation by constant velocity contact stimulation of tissues of rat and frog.3

Experimental leptospirosis in frogs.

LEPTOSPIRAL agglutinins or agglutinating factors have been reported in poikilothermic vertebrates collected from the natural environment1–5. Pathogenic leptospires have been isolated from6,7 and experimentally propagated1,8 in lower vertebrates. During follow-up of a human leptospirosis outbreak associated with swimming9,10, leptospires (unidentified) were isolated from the kidney tissue of frogs collected at the place of swimming11. Frogs were inoculated with the leptospiral isolate in an attempt to: (a) produce infection and disease; (b) measure an antibody response; (c) propagate the organism; and (d) develop carriers and shedders.

Isozymes of Aldolase

A new method of starch-gel electrophoresis using a technique of staining dependent on enzyme activity has been employed to demonstrate the isozymes of aldolase from a variety of human, rat, and frog tissues. Five of these isozymes were detected in man, seven in the rat, and at least four in the frog. The abilities of these isozymes to cleave fructose-1 ,6-diphosphate and fructose-1-phosphate were compared.

Viruses and renal carcinoma of Rana pipiens: II. Ultrastructural studies and sequential development of virus isolated from normal and tumor tissue

The sequential development of a virus (FV 1) isolated from cultured frog kidney cells has been studied by electron microscopy in fathead minnow (FHM) and baby hamster kidney (BHK 21/13) cells. Regions of virus synthesis were first seen in the cell cytoplasm at 7 and 10 hours after infection of FHM and BHK 21/13 cells, respectively. These sites contained virus particles in all stages of development from partial capsids to complete capsids with nucleoids. The virus was hexagonal and measured approximately 120 by 130 mμ. At 12 hours after infection, virus was seen budding from the cytoplasmic membrane, thus acquiring an envelope. This process continued throughout the course of the infection. Late in the infection (24–48 hours) one or more virus crystals were regularly seen in the cytoplasm of FHM cells and occasionally in BHK 21/13 cells. FV 1 replication in adult and embryonic frog cell monolayers, cultured Lucké tumor cells, and chick embryo monolayers was identical to that seen in FHM and BHK 21/13 cells. Eleven additional isolates from both normal and tumor (Lucké) frog tissue were examined in FHM cells. The morphology and site of synthesis of these isolates were identical to FV 1 and contrasted with the nuclear site of synthesis of virus seen in the Lucké tumor.

Ectodermal or presumptive skin disintegration in the frog embryo as a response to specific tissue factor(s)

A few hours of exposure of frog embryos at the neurula stage (14–15) to head, ventral, or isolated skin homogenates prepared from donor embryos (stages 13–25) produced an extensive ectodermal disintegration of the recipient embryos, while their endoderm remained intact. No disintegration was observed in neurulae treated with homogenates of embryonic endoderm, of a variety of feeding tadpole or adult frog tissues, or of embryonic newt heads. Frog embryos at stages older than the neurula were also found to be susceptible to the homogenates. In these embryos it was again shown that only the external tissues (ectoderm and underlying mesoderm) were disintegrated; other tissues (somites, peeled belly region) were unaffected. Other test objects tried (feeding tadpoles or adult frog tissues) underwent no disintegration when treated with the embryonic homogenates. Toad embryos were the only other test objects, besides the frog embryos, that were disintegrated by the action of the homogenates. A possible role of the hatching enzyme in the ectodermal disintegration of the frog embryo was excluded by appropriate control experiments.

Localization of a basic protein in the myelin of various species with the aid of fluorescence and electron microscopy.

In this study, alanine was shown to be the N-terminal amino acid of a basic protein of low molecular weight that was isolated from either human or guinea pig brain. Antibodies prepared against the guinea pig protein were labeled with either fluorescein or ferritin. Studies with the labeled antibodies showed that an immunohistochemically similar protein is found in the myelin sheaths of central and peripheral nervous tissues of chicken and frog and a variety of mammalian species. Loss of integrity of the myelin during processing was shown to enhance markedly the antigen-antibody reaction.

Endothelial vesicles and protein transport.

THE physiological mechanism involved in the transfer of water, solutes and macromolecules across endothelium remains obscure1,2 though it has been subjected to considerable investigation. When permeability is increased, endothelial gaps, believed to be intercellular, are found in the endothelium3–5. In electron microscopic examinations, tracer particles, injected into the blood stream, aggregate in the gaps, which are the sites of plasma leakage and the stigmata and stomata of light microscopy6,7. However, in physiological states, with the exception of lymphatics8,9 and such special vascular beds as the sinusoidal vessels of the liver1,10–13, spleen14, bone marrow13,15 and frog heart16, gaps do not occur in vascular endothelium. This report concerns the finding of a new type of vesicle in endothelium, and it is considered that these specialized micropinocytotic vesicles could be related to protein transport in the lymphatic and vascular endothelium of frog tissues.

Seasonal changes in the functional activity of the interstitial and spermatogenetic tissues of the green frog, Rana esculenta

The testes of Rana esculenta display a well-marked seasonal variation not only in their spermatogenetic activity, but also in the cyclical appearance and disappearance of cholesterol-positive lipids from the interstitial and Sertoli cells. There is a close correlation between the lipid appearance of the Leydig cells and the development of the accessory sex organs. A postnuptial accumulation of dense interstitial lipid, giving a strong cholesterol-positive Schultz reaction, occurs in this species as it does in Rana temporaria and is atriibuted to a cessation of male hormone production by these cells. The evidence for this is discussed. A cycle is also displayed by the Sertoli cells and involves the appearance of cholesterol-positive lipid droplets in the cell cytoplasm at spermiation followed by the detachment of the cell from the tubule wall. Thus, as spermiation progresses there is a gradual accumulation of densely lipoidal discharged Sertoli cell in the tubule lumen. This intratubular lipid material disappears with the resurgence of spermatogenetic activity. The pituitary-gonadal mechanism by which environmental temperature regulates the spermatogenetic activity is discussed.

A Laboratory Exercise: Permanent Microscopic Preparation of the Frog's Tissues

A Laboratory Exercise: Permanent Microscopic Preparation of the Frog's Tissues

Composition and labeling patterns of “free” and protein amino acids in Rana catesbeiana tadpoles and frogs

The composition of free and protein amino acids, patterns of glucose-U-C 14 labeling, and amino acid pools of various tissues of Rana catesbeiana tadpoles and frogs were compared. It was demonstrated that there were no significant variations in the amino acid composition of whole organ proteins within one animal and of corresponding tissues of tadpole and frog. However, marked differences in several free amino acids in muscle were observed, with histidine being unusually abundant. Glucose-U-C 14 was incorporated into the protein amino acids alanine, glutamic acid, and aspartic acid in all tadpole tissues; in addition to these compounds, serine, glycine, and proline were also labeled in tadpole liver. Radioactivity levels of the free amino acids indicated that the synthesis of serine and glycine from glucose occurred more readily in frog liver than in tadpole liver. The time course of glucose-U-C 14 utilization for the formation of free and protein amino acids in tadpole liver was followed. It was observed that the maximum radioactivity in the free amino acids was reached before the peak of radioactivity was seen in the protein amino acids.

Cholinesterase inhibition in tissues and the action of reversible inhibitors

The method is based on determination of the cholinesterase activity in a piece of tissue without preliminary homogenization thereof. This helps to avoid the inhibitor dilution with a marked reduction of its concentration, which invariably occurs in preparing tissue homogenates. With this method cholinesterase inhibition was detected in the muscular and nervous tissue of frogs anesthetized with ethyl alcohol. The usual method of preparation of tissue homogenates failed to demonstrate any inhibition under the same conditions.