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Severe IgE-mediated anaphylaxis following consumption of fried frog legs: definition of alpha-parvalbumin as the allergen in cause.

BACKGROUND: IgE-mediated allergic reactions to bullfrog and edible frog have been reported. The implicated allergens have not been defined so far. The frog material and the patient's serum from a case of severe food-induced anaphylaxis were used to define the implicated allergen at the protein and DNA level. METHODS: Immunoblotting techniques and N-terminal protein microsequencing were used to define the allergen recognized by the patient's serum. Back translation from the identified protein sequence was used to design degenerated primers to amplify the allergen's cDNA by polymerase chain reaction (PCR). We defined the nucleotide sequence of the allergen from the frog of Indonesian origin that was consumed by the patient, and the homologous cDNA from Rana esculenta. RESULTS: Protein microsequencing revealed that the implicated frog allergen belonged to the parvalbumin family. cDNAs coding for alpha- and beta-parvalbumin of R. esculenta and Rana species were cloned. Recombinant proteins were expressed in Escherichia coli. The patient's serum IgE antibodies recognized parvalbumin prepared from frog muscle and recombinant alpha-parvalbumin from R. species but not from R. esculenta. Recombinant beta-parvalbumin was not recognized by the IgE antibodies. CONCLUSION: This work defines at the protein and DNA levels alpha-parvalbumin as the allergen implicated in a case of IgE-mediated anaphylaxis to frog muscle. It also shows that a protein belonging to the parvalbumin family is implicated in type I allergies outside the fish species.

Allergens↗

Formicine ants: An arthropod source for the pumiliotoxin alkaloids of dendrobatid poison frogs.

A remarkable diversity of bioactive lipophilic alkaloids is present in the skin of poison frogs and toads worldwide. Originally discovered in neotropical dendrobatid frogs, these alkaloids are now known from mantellid frogs of Madagascar, certain myobatrachid frogs of Australia, and certain bufonid toads of South America. Presumably serving as a passive chemical defense, these alkaloids appear to be sequestered from a variety of alkaloid-containing arthropods. The pumiliotoxins represent a major, widespread, group of alkaloids that are found in virtually all anurans that are chemically defended by the presence of lipophilic alkaloids. Identifying an arthropod source for these alkaloids has been a considerable challenge for chemical ecologists. However, an extensive collection of neotropical forest arthropods has now revealed a putative arthropod source of the pumiliotoxins. Here we report on the presence of pumiliotoxins in formicine ants of the genera Brachymyrmex and Paratrechina, as well as the presence of these ants in the stomach contents of the microsympatric pumiliotoxin-containing dendrobatid frog, Dendrobates pumilio. These pumiliotoxins are major alkaloids in D. pumilio, and Brachymyrmex and Paratrechina ants now represent the only known dietary sources of these toxic alkaloids. These findings further support the significance of ant-specialization and alkaloid sequestration in the evolution of bright warning coloration in poison frogs and toads.

Alkaloids↗

A novel molecular determinant for cAMP-dependent regulation of the frog heart Na+-Ca2+ exchanger.

Na+-Ca2+ exchanger is one of the major sarcolemmal Ca2+ transporters of cardiac myocytes. In frog ventricular myocytes the exchanger is regulated by isoproterenol via a beta-adrenoreceptor/adenylate-cyclase/cAMPdependent signaling pathway providing a molecular mechanism for the relaxant effect of the hormone. Here, we report on the presence of a novel exon of 27-base pair insertion, which generates a nucleotide binding motif (P-loop) in the frog cardiac Na+-Ca2+ exchanger. To examine the functional role of this motif, we constructed a full-length frog heart Na+-Ca2+ exchanger cDNA (fNCX1a) containing this exon. The functional expression of fNCX1a in oocytes showed characteristic voltage dependence, divalent (Ni2+, Cd2+) inhibition, and sensitivity to cAMP in a manner similar to that of native exchanger in frog myocytes. In oocytes expressing the dog heart NCX1 or the frog mutant (DeltafNCX1a) lacking the 9-amino acid exon, cAMP failed to regulate Na+-dependent Ca2+ uptake. We suggest that this motif is responsible for the observed cAMP-dependent functional differences between the frog and the mammalian hearts.

Adenylyl Cyclases↗

Isolation, structural characterization, and bioactivity of a novel neuromedin U analog from the defensive skin secretion of the Australasian tree frog, Litoria caerulea.

We report the isolation of a novel bioactive peptide, neuromedin U-23 (NmU-23), from the defensive skin secretion of the Australasian tree frog, Litoria caerulea. The primary structure of the peptide was established by a combination of microsequencing, mass spectroscopy and site-directed antiserum immunoreactivity as SDEEVQVPGGVISNGYFLFRPRN-amide (M(r) 2580.6). A synthetic replicate of frog NmU-23 displaced monoradioiodinated rat NmU-23 from uterine membranes in a dose-dependent fashion indistinguishable from nonisotopically labeled rat NmU-23. In a rat uterine smooth muscle strip preparation, synthetic frog NmU-23 produced dose-dependent contractions identical to porcine NmU-25. However, in a preparation of human urinary bladder muscle strip, the synthetic frog peptide was more potent than porcine NmU-25 in eliciting contraction and produced desensitization of the preparation to the latter peptide. This report demonstrates that the defensive skin secretion of a frog contains a novel peptide exhibiting a high degree of primary structural similarity to the endogenous vertebrate peptide, NmU, and that this frog skin analog displays biological activity in mammalian tissues.

Amino Acid Sequence↗

Parathion accumulation in cricket frogs and its effect on American kestrels.

Adult cricket frogs (Acris crepitans) were held individually for 96 h in static systems containing initial concentrations of either 0, 0.1, 1.0, or 10 ppm parathion in 10 ml water. Mortality of cricket frogs was directly related to the parathion concentration in the water. Frogs from the 1.0- and 10-ppm groups accumulated 0.08 and 4.6 ppm parathion, respectively. One of four American kestrels (Falco sparverius) fed frogs from the 10-ppm group died from organophosphate poisoning less than 3 h after consuming five frogs. Mortality did not occur in kestrels fed frogs from the other treatment groups, which represented more environmentally realistic levels of exposure.

Animals↗

Catalase activity of liver and kidney in frogs with spontaneous renal carcinoma.

The kidney carcinoma of the leopard frog has served for various studies on catalase activity, as a first step in gaining information on enzymatic properties of neoplasms in cold blooded animals. It was found that the activity level of this tumor is reduced to approximately 13 per cent of that of the normal frog's kidney. Systemic effects of the tumor on catalase activity of liver and kidney are evident. Liver catalase in tumor-bearing frogs is diminished to about 50 per cent of the normal, and kidney catalase to an even greater degree; i.e., to about 34 per cent. A positive correlation exists between levels of catalase in tumor and in liver. Frogs kept at 3 different temperatures, 8, 18, and 26.5 degrees C., for upward of 29 days exhibited no significant change in activity levels either of tumor or of livers from normal or from tumor-bearing animals. It is suggested that a seasonal variation may occur in catalase activity of the frog. During the winter months catalase activity of the tumors was found to be higher than during the summer, whereas liver catalase was below the level of normal frogs examined during the summer. Intracoelomic injections of homogenates of tumors promptly lead to diminution of liver catalase lasting for several days. Injection of normal kidney has no such effect. These results with a spontaneous tumor of a cold blooded animal are in essential agreement with the many observations made with transplanted tumors of warm blooded animals. They lend support to the view that neoplasia is a ubiquitous biological process with similar characteristics in all species or classes of vertebrates.

Animals↗

The evolution of development: two portraits of skull ossification in pipoid frogs.

Development creates morphology, and the study of developmental processes has repeatedly shed light on patterns of morphological evolution. However, development itself evolves as well, often concomitantly with changes in life history or in morphology. In this paper, two approaches are used to examine the evolution of skull development in pipoid frogs. Pipoids have highly unusual morphologies and life histories compared to other frogs, and their development also proves to be remarkable. First, a phylogenetic examination of skull bone ossification sequences reveals that jaw ossification occurs significantly earlier in pipoids than in other frogs; this represents a reversal to the primitive vertebrate condition. Early jaw ossification in pipoids is hypothesized to result from the absence of certain larval specializations possessed by other frogs, combined with unusual larval feeding behaviors. Second, thin-plate spline morphometric studies of ontogenetic shape change reveal important differences between pipoid skull development and that of other frogs. In the course of frog evolution, there has been a shift away from salamander-like patterns of ontogenetic shape change. The pipoids represent the culmination of this trend, and their morphologies are highly derived in numerous respects. This study represents the first detailed examination of the evolution of skull development in a diverse vertebrate clade within a phylogenetic framework. It is also the first study to examine ossification sequences across vertebrates, and the first to use thin-plate spline morphometrics to quantitatively describe ontogenetic trajectories.

Animals↗

Effective population sizes and temporal stability of genetic structure in Rana pipiens, the northern leopard frog.

Although studies of population genetic structure are very common, whether genetic structure is stable over time has been assessed for very few taxa. The question of stability over time is particularly interesting for frogs because it is not clear to what extent frogs exist in dynamic metapopulations with frequent extinction and recolonization, or in stable patches at equilibrium between drift and gene flow. In this study we collected tissue samples from the same five populations of leopard frogs, Rana pipiens, over a 22-30 year time interval (11-15 generations). Genetic structure among the populations was very stable, suggesting that these populations were not undergoing frequent extinction and colonization. We also estimated the effective size of each population from the change in allele frequencies over time. There exist few estimates of effective size for frog populations, but the data available suggest that ranid frogs may have much larger ratios of effective size (Ne) to census size (Nc) than toads (bufonidae). Our results indicate that R. pipiens populations have effective sizes on the order of hundreds to at most a few thousand frogs, and Ne/Nc ratios in the range of 0.1-1.0. These estimates of Ne/Nc are consistent with those estimated for other Rana species. Finally, we compared the results of three temporal methods for estimating Ne. Moment and pseudolikelihood methods that assume a closed population gave the most similar point estimates, although the moment estimates were consistently two to four times larger. Wang and Whitlock's new method that jointly estimates Ne and the rate of immigration into a population (m) gave much smaller estimates of Ne and implausibly large estimates of m. This method requires knowing allele frequencies in the source of immigrants, but was thought to be insensitive to inexact estimates. In our case the method may have failed because we did not know the true source of immigrants for each population. The method may be more sensitive to choice of source frequencies than was previously appreciated, and so should be used with caution if the most likely source of immigrants cannot be identified clearly.

Animals↗

Neuroanatomical and physiological evidence for the involvement of pituitary adenylate cyclase-activating polypeptide in the regulation of the distal lobe of the frog pituitary.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a 38 amino-acid peptide which belongs to the glucagon/secretin/vasoactive intestinal peptide superfamily. The sequence of PACAP is identical in all mammalian species studied so far but frog PACAP differs by one amino-acid from mammalian PACAP. The aim of the present study was to investigate the presence of PACAP in the hypothalamo-pituitary complex of the frog Rana ribibunda and to determine the biological activity of frog PACAP on homologous pituitary cells. The distribution of PACAP-containing neurons and fibers was examined by the indirect immunofluorescence method using an antiserum raised against the N-terminal region of the peptide. In the hypothalamus, PACAP-immunoreactive perikarya were localized in the preoptic nucleus and the dorsal and ventral infundibular nuclei. Beaded nerve fibers were observed coursing from the ventral infundibular nucleus to the external vascular layer of the median eminence. A dense network of immunoreactive axons terminated in the vicinity of the capillaries of the hypophysial portal system. The neurointermediate lobe and the distal lobe of the pituitary were devoid of immunoreactive elements. The amount of PACAP-like immunoreactive material in hypothalamus extracts was measured by radioimmunoassay; the apparent concentration of PACAP was 4.5 ng/mg protein. Synthetic frog PACAP38 and PACAP27 induced a similar dose-dependent stimulation of cAMP production in isolated frog distal lobe pituitary fragments (ED50 = 2 x 10(-8) M). At the maximum dose tested (5 x 10(-6) M), both frog PACAP38 and PACAP27 produced a 4-fold increase in cAMP production.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Biochemical characterization of crystallins from frog lenses.

Lens crystallins were isolated from the homogenate of frog (Rana catesbeiana) eye lenses by gel permeation chromatography and characterized by gel electrophoresis, amino acid analysis and circular dichroism. Four well-defined fractions corresponding to alpha/beta-, beta-, frog 39.5 kDa and gamma-crystallins comprising the relative weight percentages in the total soluble cytoplasmic proteins of 18%, 15%, 14% and 48% respectively were obtained. The native molecular masses for each purified fraction were determined to be 432, 207, 40 and 23 kDa, respectively. The polypeptide compositions as determined by SDS-gel electrophoresis revealed the typical subunit structures of mammalian crystallins with the exception of 39.5 kDa monomeric crystallin, which has not been shown in other classes of vertebrate lenses. The spectra of circular dichroism indicate a predominant beta-sheet structure in all four fractions, which also bears a resemblance to the secondary structure of mammalian crystallins. Comparison of the amino acid compositions of frog crystallins with those of mammalian and fish crystallins suggests that gamma-crystallin from the frog is more closely related to that of porcine than fish crystallins, and the frog 39.5 kDa, frog beta- and lamprey 48 kDa crystallins are probably mutually interrelated.

Amino Acids↗

Identification of cholecystokinin from frog and turtle. Divergence of cholecystokinin and gastrin occurred before the evolution of amphibia.

Cholecystokinins from brain and small intestine of the bullfrog (Rana catesbeiana) and red-eared slider turtle (Pseudomys scripta) were isolated. The purifications were monitored by an antiserum specific for the common C-terminus of mammalian cholecystokinin and gastrin. The peptide structures were identified by sequence analysis of the intact peptides and proteolytic fragments, mass spectrometry, and amino acid analysis. Brain and small intestine of both species contained cholecystokinin-8 and substantial amounts of cholecystokinin-7. Furthermore, the small intestine of both frog and turtle contained a major fraction of the immunoreactive material as large peptides consisting of 69 residues and 70 residues, respectively. The structure for frog cholecystokinin-69 is ASSSAQLKPFQRIDGTSDQKAVIGAMLAKYLQTRKAGSSTGRYAVLPNRPVIDPTHRINDRDYMGWMDF .NH2 and the structure for turtle cholecystokinin-70 is VPSSAGQLKPIQRLDGNVDQKANIGALLAKYLQQARKGPTGRISMMGNRVQNIDPTHRINDRDYMGWMD F.NH2. All the isolated peptides were tyrosine sulfated at the seventh last residue. The peptides are highly similar to each other and to mammalian cholecystokinins (70% mutual identity and more than 50% identity with human cholecystokinin). Thus, they are clearly related to the known mammalian cholecystokinins. Both peptides include the monobasic and dibasic cleavage sites giving rise to cholecystokinins-33, -39, and -58 in mammals. However, only a small amount of turtle cholecystokinin-40 (corresponding to mammalian cholecystokinin-39) was isolated. This confirms that post-translational processing is highly species dependent. Recently, we isolated peptides from frog and turtle antrum. Following their origin they were named gastrins in spite of their C-terminal cholecystokinin-like structure. Thus, two different cholecystokinin/gastrin peptides exist in frog and turtle justifying the choice of two names. This finding of two members of the cholecystokinin/gastrin family in frog shows that the divergence of cholecystokinin and gastrin occurred simultaneously with or earlier than the appearance of amphibia during phylogenesis. Frog cholecystokinin and gastrin show sufficient similarity along the whole sequence to support the notion of a gene duplication of a common ancestor.

Amino Acid Sequence↗

Modulation by substrate concentration of maximal shortening velocity and isometric force in single myofibrils from frog and rabbit fast skeletal muscle.

1. The effects of magnesium adenosine triphosphate (MgATP; also referred to as 'substrate') concentration on maximal force and shortening velocity have been studied at 5 C in single and thin bundles of striated muscle myofibrils. The minute diameters of the preparations promote rapid diffusional equilibrium between the bathing medium and lattice space so that during contraction fine control of substrate and product concentrations is achieved. 2. Myofibrils from frog tibialis anterior and rabbit psoas fast skeletal muscles were activated maximally by rapidly (10 ms) exchanging a continuous flux of pCa 8.0 for one at pCa 4.75 at a range of substrate concentrations from 10 microM to 5 mM. At high substrate concentrations maximal isometric tension and shortening velocity of both frog and rabbit myofibrils were very close to those determined in whole fibre preparations from the same muscle types. 3. As in frog and rabbit skinned whole fibres, the maximal isometric force of the myofibril preparations decreases as MgATP concentration is increased. The maximal velocity of unloaded shortening (V0) depends hyperbolically on substrate concentration. V0 extrapolated to infinite MgATP (3.6 +/- 0.2 and 0.8 +/- 0.03 l0 s-1 in frog and rabbit myofibrils, respectively) is very close to that determined directly at high substrate concentration. The Km is 210 +/- 20 microM for frog tibialis anterior and 120 +/- 10 microM for rabbit psoas myofibrils, values about half those found in larger whole fibre preparations of the same muscle types. This implies that measurements in whole skinned fibres are perturbed by diffusional delays, even in the presence of MgATP regenerating systems. 4. In both frog and rabbit myofibrils, the Km for V0 is about one order of magnitude higher than the Km for myofibrillar MgATPase determined biochemically in the same experimental conditions. This confirms that the difference between the Km values for MgATPase and shortening velocity is a basic feature of the mechanism of chemomechanical transduction in muscle contraction.

Adenosine Triphosphate↗

Distribution of P1- and P2-purinoceptors in the guinea-pig and frog heart.

1 The effects of adenyl compounds were examined on the guinea-pig and frog heart in terms of the P(1)/P(2)-purinoceptor hypothesis.2 The effects of two slowly degradable adenosine 5'-triphosphate (ATP) analogues; beta,gamma-methylene adenosine 5'-triphosphate (APPCP) and alpha,beta-methylene adenosine 5'-triphosphate (APCPP) were also examined.3 Adenosine, adenosine 5'-monophosphate (AMP), adenosine 5'-diphosphate (ADP), ATP and APPCP produced inhibitory effects in guinea-pig atria. These inhibitory effects were antagonized competitively by theophylline and potentiated by dipyridamole. APCPP did not produce a similar inhibitory response.4 Guinea-pig ventricles were insensitive to adenyl compounds.5 ATP and ADP produced initial excitatory effects in frog atria which were followed by inhibitory effects. Adenosine and AMP produced inhibitory effects alone whereas APCPP produced excitatory effects only. The inhibitory effects were antagonized competitively by theophylline and potentiated by dipyridamole.6 ATP, ADP, APPCP and APCPP evoked excitatory responses in frog ventricles. These responses were not affected by theophylline or dipyridamole. Adenosine and AMP were inactive on frog ventricles.7 It is concluded that only P(1)-receptors are present in guinea-pig atria; that both P(1)- and P(2)-receptors are present in frog atria; and that only P(2)-receptors are present in frog ventricles. No evidence was found for the presence of either P(1)- or P(2)-purinoceptors in guinea-pig ventricles.

Adenine Nucleotides↗

Estimates of statistical release parameters from crayfish and frog neuromuscular junctions.

1. Transmitter release at crayfish and frog neuromuscular junctions was studied by recording synaptic potentials with extracellular or intracellular glass micro-electrodes. 2. The binomial release parameters n and p were calculated using the experimental observations for the mean number of quanta (m) released in a series of trials, the variance (var) of the quantum content distribution and the number of transmission failures (n-o). 3. In one series of experiments on frog neuromuscular junction, action potentials were blocked by tetrodotoxin. Transmitter release was limited to a circumscribed part of the nerve terminal by focal stimulation with a glass micro-electrode. Values for m were between 0-6 and 5-7 and calculated values for n ranged from 3 to 23. p was between 0-05 and 0-48. In a second series of experiments on frog neuromuscular junction transmitter release was evoked by conventional stimulation of the nerve trunk in Mg-Ringer. m was mostly between 20 and 60 and in the same experiments n was calculated to be between 70 and 125. Values for p ranged from 0-25 to 0-48. 4. It can be concluded from the present results that transmitter release at frog neuromuscular junction is described by binomial statistics as is the case for neuromuscular junction of crayfish. 5. In comparing the values for n for the two series of experiments on frog neuromuscular junction it appears that n is dependent on the length of the activated synaptic contact. The numbers for n calculated from frog neuromuscular junction are of the same order of magnitude as the number of 'active zones' seen in the corresponding length of the synaptic nerve terminal.

Action Potentials↗

Effects of exposure to cold on metabolic characteristics in gastrocnemius muscle of frog (Rana pipiens).

1. Responses of enzymic characteristics of gastrocnemius muscle were studied when frogs (Rana pipiens) were exposed to cold environment (4 degrees C). 2. The content of adenosine triphosphate (ATP) decreased significantly after cold exposure. This decrease was greater in starved than in fed frogs. 3. Although the glycogen content did not change, lactate levels were lower in cold-exposed than room-temperature (control) frogs. No change was observed in glycogen and lactate between fed and unfed frogs kept at 4 degrees C for 2 months. Lactate dehydrogenase activity tended to increase during chronic cold exposure, but not significantly. 4. The activities of citrate synthase, cytochrome oxidase, and beta-hydroxyacyl CoA dehydrogenase were higher in gastrocnemius of chronically cold-exposed frogs than in room-temperature controls. This increase was statistically significant only in the muscles of starved frogs; these muscles had the greatest decrease in ATP. 5. It was suggested that chronic cold exposure decreases skeletal muscle ATP content but may not affect glycolysis. The data also suggested that the decrease in ATP content stimulates mitochondrial biogenesis which increases enzyme activities.

Adenosine Triphosphate↗

Volume status influences atrial peptide-induced water conductivity changes in leopard frog mesenteric capillaries.

1. Three sets of North American leopard frogs (Rana pipiens) were housed on damp sand (DS, n = 19), in fresh water (FW, n = 19), or with free access to both damp sand and fresh water (FA, n = 23) for a period of 5-6 days. At the end of this period pithed frogs were prepared for measurement of capillary hydraulic conductivity and samples of whole-blood, lymph and body cavity fluid were collected for measurement of haematocrit and protein content. 2. To measure hydraulic conductivity (Lp), individual capillaries of the mesenteries of the three groups were cannulated and perfused with a minimum of two, and a maximum of three, frog Ringer solutions. The first solution, common to all experiments, contained dialysed bovine serum albumin (BSA) at a concentration of 10 mg ml-1. The second and third solutions also contained either 10 nM-atrial peptide (AP) or 1 microM-sodium nitroprusside (SNP). Lp was determined from measurements of fluid filtration rate at a minimum of two different capillary pressures (Michel, Mason, Curry, Tooke & Hunter, 1974). 3. In vessels from damp sand-stored frogs (DS), the paired Lp in the presence of 10 nM-atrial peptide (Lp, AP) was 3.2 +/- 0.4-fold higher than Lp, BSA (n = 25). By contrast, in the FW group, Lp, AP did not differ significantly from Lp, BSA (Lp, AP-FW/Lp, BSA = 1.3 +/- 0.2; n = 23). In frogs given free access to water and dry terrain the response to atrial peptide was similar to that observed in the DS group (Lp, AP-FA/Lp, BSA = 3.6 +/- 07; n = 23). Thus, storage in fresh water appears to have inhibited the acute Lp response to atrial peptide observed in vessels from the FA or DS animals. 4. Atrial peptide and sodium nitroprusside elevate intracellular cyclic GMP levels by membrane receptor-dependent and -independent processes, respectively. No difference was observed in the Lp response to 1 microM-perfusate sodium nitroprusside (Lp, SNP) in DS (n = 7), FW (n = 8) or FA (n = 30) frogs (Lp, SNP/Lp, BSA ratios were 3.5 +/- 1.5-, 2.7 +/- 0.8- and 2.6 +/- 0.5-fold, respectively). Thus, the Lp response to sodium nitroprusside was not affected by storage conditions. 5. The mean haematocrit of skin blood samples from the DS groups was 26.5 +/- 1.0% (+/- S.E.M.) compared to 19.1 +/- 1.2% from the FW groups.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

Active and passive forces of isolated myofibrils from cardiac and fast skeletal muscle of the frog.

1. Force measurements in isolated myofibrils (15 degrees C; sarcomere length, 2.10 microns) were used in this study to determine whether sarcomeric proteins are responsible for the large differences in the amounts of active and passive tension of cardiac versus skeletal muscle. Single myofibrils and bundles of two to four myofibrils were prepared from glycerinated tibialis anterior and sartorius muscles of the frog. Skinned frog atrial myocytes were used as a model for cardiac myofibrils. 2. Electron microscope analysis of the preparations showed that: (i) frog atrial myocytes contained a small and variable number of individual myofibrils (from 1 to 7); (ii) the mean cross-sectional area and mean number of myosin filaments of individual cardiac myofibrils did not differ significantly from those of single skeletal myofibrils; and (iii) the total myofibril cross-sectional area of atrial myocytes was on average comparable to that of bundles of two to four skeletal myofibrils. 3. In maximally activated skeletal preparations, values of active force ranged from 0.45 +/- 0.03 microN for the single myofibrils (mean +/- S.E.M.; n = 16) to 1.44 +/- 0.24 microN for the bundles of two to four myofibrils (n = 9). Maximum active force values of forty-five cardiac myocytes averaged 1.47 +/- 0.10 microN and exhibited a non-continuous distribution with peaks at intervals of about 0.5 microN. The results suggest that variation in active force among cardiac preparations mainly reflects variability in the number of myofibrils inside the myocytes and that individual cardiac myofibrils develop the same average amount of force as single skeletal myofibrils. 4. The mean sarcomere length-resting force relation of atrial myocytes could be superimposed on that of bundles of two to four skeletal myofibrils. This suggests that, for any given amount of strain, individual cardiac and skeletal sarcomeres bear essentially the same passive force. 5. The length-passive tension data of all preparations could be fitted by an exponential equation. Equation parameters obtained for both types of myofibrils were in reasonable agreement with those reported for larger preparations of frog skeletal muscle but were very different from those estimated for multicellular frog atrial preparations. It is concluded that myofibrils are the major determinant of resting tension in skeletal muscle; structures other than the myofibrils are responsible for the high passive stiffness of frog cardiac muscle.

Animals↗

Response of intestinal flora of laboratory-reared leopard frogs (Rana pipiens) to cold and fasting.

The bacterial flora of the large intestine was examined in 35 laboratory-reared leopard frogs (Rana pipiens) subjected to one of the following four treatments: (i) normal feeding at 21 degrees C (10 frogs); (ii) fasting for 2 weeks at 21 degrees C (8 frogs); (iii) chilling for 1 week at 4 degrees C (9 frogs); and (iv) simulated hibernation for 3 weeks at 4 degrees C (8 frogs). Bacteria from the intestinal contents and mucosa were counted microscopically and by colony counting after strictly anaerobic culturing. The predominant bacteria were isolated and partially characterized. Fasting for 2 weeks produced no significant changes in total counts or in the types of bacteria cultured. Chilling, whether rapid or in the course of simulated hibernation, was associated with a decrease in the numbers and variety of bacteria. Thus it appears that the lowering of temperature rather than the absence of food is the important factor in the reduction of bacterial flora seen in hibernating frogs. However, the bacteria showed some adaptation to the low temperature, as the longer the host had been at 4 degrees C, the higher the proportion of bacteria which could grow when cultured at that temperature.

Aerobiosis↗