Responses of bullfrog tadpoles to hypoxia and predators

Low dissolved oxygen concentrations present numerous challenges for non-air-breathing aquatic organisms. Amphibian larvae and their predators can respond to oxygen levels by altering their behavior and physiology, but the ecological consequences of these responses are generally unknown. We conducted two laboratory experiments to study the effects of dissolved oxygen on respiratory behavior and susceptibility to predation of larval bullfrogs (Rana catesbeiana). In the first, we exposed small, lungless tadpoles to a predatory salamander larva (Ambystoma tigrinum) under high and low oxygen conditions. More tadpoles were consumed in high oxygen tanks than in low ones, presumably because salamanders remained near the surface in the low oxygen tanks while most tadpoles rested on the bottom. Tadpole activity depended on both oxygen and predator presence: swimming decreased after addition of salamanders under high oxygen, but increased under low oxygen. In the second experiment, we examined the effect of predator chemical cues on the air-breathing rate of large tadpoles with well-developed lungs under low oxygen conditions. In the presence of chemical cues produced by dragonfly larvae consuming bullfrog tadpoles, air-breathing and swimming were significantly reduced relative to controls. These experiments demonstrate the potential impact of dissolved oxygen on predator-prey interactions, and suggest that outcomes depend on the respiratory ecology of both predator and prey.     

Magnetic properties of human liver and brain ferritin

Human brain (globus pallidus) and liver tissues were investigated by means of electron microscopy (EM), Mössbauer spectroscopy (MS) and SQUID magnetometry techniques. Based on MS measurements, the iron present was identified to be in the ferritin-like form (61–88%) and in the form of a low-spin iron species (the balance). Its overall concentration was estimated as 1.5(3) mg in the brain and 2.4(5) mg in the liver, per gram of lyophilized tissue. The average core diameter was determined by EM measurements to be equal to 7.5(1.3) nm for the liver and 3.3(5) nm for the brain. Magnetization measurements carried out between 5 and 300 K yielded an estimation of an average blocking temperature, KT _BL, as equal to 6.7 K and 8.5 K for the liver and the brain, respectively. From the dependence of KT _BL on the external magnetic field it was concluded that the ferritin-like cores in the studied samples can be regarded as non-interacting particles. Finally, the uniaxial magnetic anisotropy constant was determined to be 6×10³ J/m³ for the liver and 4×10⁴ J/m³ for the brain.     

Mechanical properties of plant cell walls probed by relaxation spectra

Transformants and mutants with altered cell wall composition are expected to display a biomechanical phenotype due to the structural role of the cell wall. It is often quite difficult, however, to distinguish the mechanical behavior of a mutant's or transformant's cell walls from that of the wild type. This may be due to the plant's ability to compensate for the wall modification or because the biophysical method that is often employed, determination of simple elastic modulus and breakstrength, lacks the resolving power necessary for detecting subtle mechanical phenotypes. Here, we apply a method, determination of relaxation spectra, which probes, and can separate, the viscoelastic properties of different cell wall components (i.e. those properties that depend on the elastic behavior of load-bearing wall polymers combined with viscous interactions between them). A computer program, BayesRelax, that deduces relaxation spectra from appropriate rheological measurements is presented and made accessible through a Web interface. BayesRelax models the cell wall as a continuum of relaxing elements, and the ability of the method to resolve small differences in cell wall mechanical properties is demonstrated using tuber tissue from wild-type and transgenic potatoes (Solanum tuberosum) that differ in rhamnogalacturonan I side chain structure.     

Increased phosphorylation in red cell membranes of subjects affected by essential hypertension

In hemolysates of red cells from hypertensive patients the proteolytic activity of calpain is expressed at a rate approximately three fold higher than in red cells of normotensive subjects. Susceptibility to lysis upon exposure to ionophore A23187 and calcium, conditions that increase intracellular calpain activity, is also significantly enhanced in erythrocytes of hypertensive patients. In inside-out vesicles prepared from erythrocytes of these patients band 3 region undergoes a high extent of phosphorylation which is 1.5 fold higher than that occurring in control red cells from normotensive subjects. This increased phosphorylation can be reproduced in inside-out vesicles from erythrocytes of normal subjects following pretreatment with calpain. Taken together, these results suggest that the presence in erythrocytes of hypertensive subjects of an unregulated calpain dependent proteolytic activity may affect the structure of plasma membranes and determine an increased phosphorylation of intrinsic membrane proteins.     

Purification and properties of the manganese superoxide dismutase from the liver of bullfrog, Rana catesbeiana

A manganese superoxide dismutase (Mn-SOD) from the liver of bullfrog, Rana catesbeiana, was purified to electrophoretic homogeneity. The enzyme has a molecular weight of about 84,000 and is composed of four identical subunits, each containing one manganese atom. The amino acid composition of the enzyme is similar to that of Mn-SODs isolated from human and chicken livers, but differs considerably from that of the Escherichia coli enzyme (D. Barra et al. (1984) J. Biol. Chem. 259, 12595-12601; R. A. Weisiger and I. Fridovich (1973) J. Biol. Chem. 248, 3582-3592; H. M. Steinman (1978) J. Biol. Chem. 253, 8708-8720). The N-terminal amino acid is lysine. The sequence of 23 amino acid residues in the N-terminal region was determined. It shows excellent homologies with those of the human and chicken enzymes (H. M. Steinmam and R. L. Hill (1973) Proc. Natl. Acad. Sci. USA 70, 3725-3729; C. Ditlow et al. (1982) Carlsberg Res. Commun. 47, 81-91). The frog liver enzyme is also located exclusively in the mitochondrial matrix. Immunologically the same enzyme is also found in the tadpole liver, in an amount of about one-half of that in the adult bullfrog.     

In vitro activation of rat liver phenylalanine hydroxylase by phosphorylation.

Essentially pure phenylalanine hydroxylase from rat liver can be activated between 2.5- and 3.0-fold by treatment with Mg2+, ATP, protein kinase, and cyclic AMP. The activation is seen when the hydroxylase is assayed in the presence of tetrahydrobiopterin, but not in the presence of 2-amino-4-hydroxy-6,7-dimethyltetrahydropteridine. In the presence of [gamma-32P]ATP, activation is accompanied by incorporation of 32P into the protein to the extent of 0.7 mol/mol of hydroxylase subunit (Mr = 50,000). Cehmical analysis of the untreated enzyme shows that it already contains about 0.3 mol of Pi/mol of hydroxylase. These results suggest that the activity of the hydroxylase may be regulated by phosphorylation.     

Thermal acclimation and hardening in tadpoles of the bullfrog, Rana catesbeiana

1. 1.|Critical thermal maxima (CTMax) and minima (CTMin) were measured to evalute thermal hardening in Rana catesbeiana.

2. 2.|Tadpoles show heat hardening and CTMax acclimation, and both responses are influenced by developmental stage.

3. 3.|The first evidence of cold hardening in vertebrates is reported here.

4. 4.|Heat hardening significantly reduces cold tolerance, but there is otherwise no evidence of a cross-hardening effect.

Association of ferritin with liver cell membrane fractions.

Following intraperitoneal injection of rats with a large dose of ferric ammonium citrate containing ⁵⁹Fe, some 35 to 50% of the dose was deposited in the liver within the first 1 to 4 h. Almost all of the deposited iron could be precipitated with a ferritin antiserum from a homogenate of liver heated to 75 °C, but only half of this was precipitable when the unheated homogenate was treated with antiserum. The remainder of the ferritin iron was made available to the antiserum by treatment with deoxycholate, and was therefore presumed to be associated with membranous components of the cell.Subcellular fractionation of the liver following administration of ⁵⁹Fe-labeled ferric ammonium citrate showed that most of the radioactivity deposited within the first 4 h was equally divided between the cell sap and a light microsome (membrane-rich) fraction. Ferritin in this latter fraction was made available to antibody following deoxycholate treatment. The liver microsome fraction of the young rat contains little unavailable ferritin, but with aging there is an accumulation of ferritin in the microsomal fraction which is unavailable to antibody until the membrane is removed.It is suggested that at least part of the injected iron salt is taken up by pinocytotic vesicles and transferred to ferritin within this fraction, possibly followed by release of some of this ferritin into the cell sap.     

Protein chain initiation in vitro by liver cell components from DMNA-treated rats.

The mechanism of inhibition of protein synthesis in rat liver after dimethylnitrosamine (DMNA) administration was studied at the level of peptide-chain initiation by use of initiation-dependent amino acid incorporating systems. Ribosomal monomers, poly(A)-concontaining loss of acticity due to the DMNA treatment. The poly(A) RNA from monosomes and polysomes, and crude initiation factors from microsomes were prepared 2 h after a single dose of DMNA (75 mg/kg), and their activities in the production of new protein chains determined under conditions of nearly linear response. Monosomes and crude initiation factors from DMNA-treated rats were at least as active as those from controls. Preparations of poly(A)-containing RNA had a consistently higher template activity when prepared from polysomes instead of monosomes. However, in neither case was there any ltaining RNA was methylated by DMNA to about the same extent as the 18S and 28S rRNA. The methylation was consistently somewhat higher in the RNA preparations from monosomes than in those from polysomes.     

Studies on the in vitro phosphorylation of 6-phosphofructo-1-kinase from rat liver

Rat hepatic 6-phosphofructo-1-kinase (ATP:d-fructose-6-phosphate 1-phosphotransferase) was purified to homogeneity and its phosphorylation by the catalytic subunit of the cyclic AMP-dependent protein kinase examined. Up to 4 mol of phosphate could be incorporated per mole of tetrameric enzyme, and the phosphate was incorporated into seryl residues. Phosphorylation did not alter the affinity of the enzyme for fructose 6-phosphate or fructose 2,6-bisphosphate. The rate of phosphorylation was enhanced by allosteric activators of 6-phosphofructo-1-kinase such as AMP and fructose 2,6-bisphosphate, and it was decreased by the allosteric inhibitors ATP and H⁺. The phosphopeptide region of the enzyme subunit was susceptible to limited proteolysis by trypsin. Removal of the phosphopeptide did not affect the subunit molecular weight nor the maximum activity of the enzyme, but it enhanced the apparent affinity of the enzyme for both fructose 6-phosphate and fructose 2,6-bisphosphate. It is concluded that the phosphopeptide region of the enzyme subunit is an important determinant of the affinity of the enzyme for its substrate as well as for the allosteric activator fructose 2,6-bisphosphate.     

Separation and properties of a red cell sensitizing substance from streptococci

Moskowitz, Merwin (Purdue University, Lafayette, Ind.). Separation and properties of a red cell sensitizing substance from streptococci. J. Bacteriol. 91:2200-2204. 1966.-An antigen that binds onto red cells and causes them to be agglutinated by antiserum was separated from streptococci. Various procedures to extract the antigen from streptococci were investigated, and the greatest amount of antigen was obtained by extraction of cells with a phenol-water mixture. The reaction of the antigen with red cells was shown to be reversible by use of the Ashby mixed agglutination technique. The antigen also combines with a number of different tissues, and it was demonstrated that the antigen could be transferred from red cells to tissues and vice versa. An hypothesis is presented on the basis of these findings which suggests a possible role for this antigen in the etiology of rheumatic fever.     

Adrenergic receptors and the regulation of vascular resistance in bullfrog tadpoles (Rana catesbeiana)

Summary Vascular adrenergic sensitivity to exogenous catecholamines was examined in tadpoles of the American bullfrog (Rana catesbeiana), ranging from stage III to XIV. Central arterial blood pressure was measured in decerebrate bullfrog tadpoles to determine a reasonable initial infusion pressure. Solutions of epinephrine and phenylephrine were infused into the vasculature of pithed tadpoles, and the resulting changes in vascular resistance (R _v) were used to construct log dose-response relationships. Epinephrine infusion produced a dose-dependent increase in R _v (EC₅₀=5.3·10^-7 M), which could be reversed by sodium nitroprusside (a smooth muscle relaxant) and blocked by phenoxybenzamine (an -adrenergic antagonist). Larval R _v also increased with infusion of the -agonist phenylephrine (EC₅₀=7.4·10⁸ M). Infusion of 10^-6 M isoproterenol (a -agonist) largely reversed the phenylephrine-induced increase in R _v. These results indicate that the capacity exists for both -mediated vasoconstriction and -mediated vasodilation early in bullfrog ontogeny. Neither initial R _v nor the responses to infused epinephrine or phenylephrine were significantly correlated to development over the range of larval stages used in this study.Abbreviations ECG electrocardiogram - EPI epinephrine - ISO isoproterenol - PHE phenylephrine - POB phenoxybenzamine - R _v vascular resistance - SNP sodium nitroprusside