Medusins: A new class of antimicrobial peptides from the skin secretions of phyllomedusine frogs

Natural drug discovery represents an area of research with vast potential. The investigation into the use of naturally-occurring peptides as potential therapeutic agents provides a new “chemical space” for the procurement of drug leads. Intensive and systematic studies on the broad-spectrum antimicrobial peptides found in amphibian skin secretions are of particular interest in the quest for new antibiotics to treat multiple drug-resistant bacterial infections. Here we report the molecular cloning of the biosynthetic precursor-encoding cDNAs and respective mature peptides representing a novel group of antimicrobial peptides from the skin secretions of representative species of phyllomedusine leaf frogs: the Central American red-eyed leaf frog (Agalychnis callidryas), the South American orange-legged leaf frog (Phyllomedusa hypochondrialis) and the Giant Mexican leaf frog (Pachymedusa dacnicolor). Each novel peptide possessed the highly-conserved sequence, LGMIPL/VAISAISA/SLSKLamide, and each exhibited activity against the Gram-positive bacterium, Staphylococcus aureus and the yeast, Candida albicans, but all were devoid of haemolytic effects at concentrations up to and including the MICs for both organisms. The novel peptide group was named medusins, derived from the name of the hylid frog sub-family, Phyllomedusinae, to which all species investigated belong. These data clearly demonstrate that comparative studies of the skin secretions of phyllomedusine frogs can continue to produce novel peptides that have the potential to be leads in the development of new and effective antimicrobials.     

AM representation in green treefrog auditory nerve fibers: neuroethological implications for pattern recognition and sound localization

In addition to spectral call components, temporal patterns in the advertisement-call envelope of green treefrog males (Hyla cinerea) provide important cues for female mate choice. Rapid amplitude modulation (AM) with rates of 250–300 Hz is typical for this species advertisement calls. Here we report data on the encoding of these rapid call modulations by studying the responses of single auditory nerve fibers to two-tone stimuli with envelope periodicities close to those of the natural call. The free-field response properties of 86 nerve fibers were studied from 32 anesthetized males. The accuracy of stimulus envelope coding was quantified using both a Gaussian function fit to the interspike interval histograms derived from the first seven 20-ms stimulus segments, and the vector-strength metric applied to the phase-locked responses. Often, AM encoding in the initial stimulus segment was more faithful than that in its second half. This result may explain why conspecific females prefer calls in which the initial segment is unmasked rather than masked. Both the questions of pattern recognition and localization are discussed, and the data are related to behavioral observations of female choice and localization performance in this species.     

Interactions of bullfrog tadpole predators and an insecticide: predation release and facilitation

The effect of a contaminant on a community may not be easily predicted, given that complex changes in food resources and predator-prey dynamics may result. The objectives of our study were to determine the interactive effects of the insecticide carbaryl and predators on body size, development, survival, and activity of tadpoles of the bullfrog (Rana catesbeiana). We conducted the study in cattle tank mesocosm ponds exposed to 0, 3.5, or 7.0 mg/l carbaryl, and no predators or two red-spotted newts (Notophthalmus viridescens), bluegill sunfish (Lepomis macrochirus), or crayfish (Orconectes sp.). Carbaryl negatively affected predator survival by eliminating crayfish from all ponds, and by eliminating bluegill sunfish from ponds exposed to the highest concentration of carbaryl; carbaryl exposure did not effect survival of red-spotted newts. Because crayfish were eliminated by carbaryl, bullfrogs were released from predation and survival was near that of predator controls at low concentrations of carbaryl exposure. High concentrations of carbaryl reduced tadpole survival regardless of whether predators survived carbaryl exposure or not. Presence of crayfish and newts reduced tadpole survival, while bluegill sunfish appeared to facilitate bullfrog tadpole survival. Presence of carbaryl stimulated bullfrog tadpole mass and development. Our study demonstrates that the presence of a contaminant stress can alter community regulation by releasing prey from predators that are vulnerable to contaminants in some exposure scenarios.Due to an error in the citation line, this revised PDF (published in December 2003) deviates from the printed version, and is the correct and authoritative version of the paper.     

Metabolomic Profiling Reveals Biochemical Pathways and Biomarkers Associated with Pathogenesis in Cystic Fibrosis Cells

Cystic fibrosis (CF) is a life-shortening disease caused by a mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. To gain an understanding of the epithelial dysfunction associated with CF mutations and discover biomarkers for therapeutics development, untargeted metabolomic analysis was performed on primary human airway epithelial cell cultures from three separate cohorts of CF patients and non-CF subjects. Statistical analysis revealed a set of reproducible and significant metabolic differences between the CF and non-CF cells. Aside from changes that were consistent with known CF effects, such as diminished cellular regulation against oxidative stress and osmotic stress, new observations on the cellular metabolism in the disease were generated. In the CF cells, the levels of various purine nucleotides, which may function to regulate cellular responses via purinergic signaling, were significantly decreased. Furthermore, CF cells exhibited reduced glucose metabolism in glycolysis, pentose phosphate pathway, and sorbitol pathway, which may further exacerbate oxidative stress and limit the epithelial cell response to environmental pressure. Taken together, these findings reveal novel metabolic abnormalities associated with the CF pathological process and identify a panel of potential biomarkers for therapeutic development using this model system.     

Sperm competition selects for increased testes mass in Australian frogs

Game theory predicts that investment in spermatogenesis will increase with the risk and intensity of sperm competition. Widespread support for this prediction has come from comparative studies of internal fertilizing species reporting positive associations between testes mass and the probability that females mate with more than one male. Data for external fertilizers have generated conflicting results. We investigated how risk of sperm competition affects testes size in two families of Australian frogs: the Myobatrachidae and the Hylidae. We also examined effects of clutch size, egg size and oviposition location as alternative factors that might influence sperm production. Species were ranked according to probability of group spawning, and hence risk of sperm competition. Controlling for body size and phylogenetic relationships, we demonstrated that within the Myobatrachidae, the risk of sperm competition explained a significant amount of variation in testes mass. Oviposition location had a weak influence, with species ovipositing into foam having smaller testes. No significant effects of clutch size or egg size were detected. In hylids, the relationship between testes mass and risk of sperm competition was positive but not significant, again with no predictable effects related to egg size or number. These data provide an important test of sperm competition theory for externally fertilizing taxa.     

Water loss, cutaneous resistance, and effects of dehydration on locomotion of Eleutherodactylus frogs

Water loss, cutaneous resistance, and the effects of dehydration on jumping ability were measured in two neotropical frogs, the common coquí (Eleutherodactylus coqui) and the cave coquí (Eleutherodactylus cooki). In both species jumping performance declined with an increase in water loss and a greater duration of exposure to dehydrating conditions. The arboreal species, E. coqui, had a slightly higher rate of water loss and lower cutaneous resistance than the non-arboreal species, E. cooki. However, differences in cutaneous resistance and water loss were too small to explain differences in geographic distributions of these species. In both species, a decline in boundary layer resistance contributed to an increased rate of water loss at higher temperature. Accepted: 4 January 1999     

Mechanics of the exceptional anuran ear

The anuran ear is frequently used for studying fundamental properties of vertebrate auditory systems. This is due to its unique anatomical features, most prominently the lack of a basilar membrane and the presence of two dedicated acoustic end organs, the basilar papilla and the amphibian papilla. Our current anatomical and functional knowledge implies that three distinct regions can be identified within these two organs. The basilar papilla functions as a single auditory filter. The low-frequency portion of the amphibian papilla is an electrically tuned, tonotopically organized auditory end organ. The high-frequency portion of the amphibian papilla is mechanically tuned and tonotopically organized, and it emits spontaneous otoacoustic emissions. This high-frequency portion of the amphibian papilla shows a remarkable, functional resemblance to the mammalian cochlea.     

A hot knot of toads: Aggregation provides thermal benefits to metamorphic Andean toads

We tested the thermal benefits of aggregation (“knotting”) for a high-elevation (>4300 m) population of metamorphic Rhinella spinulosa. Fewer knots were recorded when the sun was low on the horizon and aggregating toads dispersed under cloudy skies or when experimentally shaded. Body temperatures of sun-exposed toads averaged 2.3 °C higher when in knots than when solitary. Proximity to water and similarity in body size between aggregating and solitary toads suggest that grouping is not imposed by a need for reducing evaporative water loss. Aggregation apparently provides thermal benefits, which should translate into faster growth.     

Fictive respiratory rhythm in the isolated brainstem of frogs

Spontaneous rhythmically bursting activity was recorded from the trigeminal, vagal and hypoglossal nerve roots of the isolated brainstem from the frogsRana catesbeiana andRana pipiens superfused with a bicarbonate-free HEPES-buffer solution. Burst frequency, burst duration and the activity profile of the spontaneous neural discharges in vitro resembled those of a less radical preparation, the decerebrate, fictively breathing frog. After complete midsagittal section, each half of the isolated brainstem generated its own rhythmic neural activity which resembled that of the intact isolated brainstem. The spontaneous activity generated within each half of the brainstem is probably coordinated by decussating axons or by groups of neurons located along the midline of the brainstem. Our results suggest that these coordinating entities extend the length of the brainstem (in a rostro-caudal dimension) and the degree of contact rather than the location of the contact between the two halves of the brainstem determines the synchronization of the right and left halves. Burst frequency of both the intact and hemisected brainstem preparation was decreased by alkaline challenge and increased by acid challenge. We conclude that this endogeneous rhythmic activity represents the efferent motor output underlying lung ventilation in these animals.Abbreviations EMG electromyogram - ENG electroneurogram - V trigeminal nerve - Vmd mandibular branch of trigeminal nerve - X vagal nerve - X1 laryngeal branch of vagal nerve - H hypoglossal nerve - Hsh sternohyoid branch of hypoglossal nerve - Hm main branch of hypoglossal nerve     

Changes in oxidative stress parameters and acid phosphatase activity in the pre-regressing and regressing tail of Indian jumping frog Polypedates maculatus (Anura,Rhacophoridae)

Activities of acid phosphatase (normal and Co²⁺-sensitive), superoxide dismutase and catalase and levels of lipid peroxidation, hydrogen peroxide were compared in the tails of tadpoles of stage III, XVIII, XXI and XXIII, respectively, of the Indian Jumping frog Polypedates maculatus. It is noticed that acid phosphatase activity (normal and Co²⁺-sensitive), and levels of lipid peroxidation and hydrogen peroxide increased during tail regression. There is also an increase in the level of superoxide dismutase and catalase in the regressing tail. A positive correlation between activity of acid phosphatase and lipid peroxidation, hydrogen peroxide and lipid peroxidation, acid phosphatase and hydrogen peroxide was noticed in the tail of tadpoles during different developmental stages, suggesting a critical interaction between reactive oxygen species and lysosomal activity during metamorphosis.     

Ultrasonic communication in concave-eared torrent frogs (Amolops tormotus)

The concave-eared torrent frogs (Amolops tormotus) have highly unusual ear morphology--in males the eardrums are embedded deep inside ear cavities. In collaboration with our colleagues we investigated the functional significance of this morphological feature in hearing. Sound recordings in the field showed that males of A. tormotus produce diverse bird-like melodic calls with pronounced frequency modulations and non-linear phenomena (e.g., frequency jumps, different orders of subharmonics, and chaos) that often contain spectral energy in the ultrasonic range. The audible as well as the ultrasonic components of the species call could effectively evoke males' vocal responses, demonstrating that they can hear and respond to ultrasound. Electrophysiological recordings from the auditory midbrain confirmed the ultrasonic hearing capacity of these frogs. The recessed tympana and extremely thin tympanic membranes are adaptations for hearing ultrasound--this sensitivity may have evolved in response to the intense, predominately low-frequency ambient noise from local streams. Finally, results from the isolated laryngeal preparation in euthanized frogs revealed that the origin of call complexity and diversity lies with having a vocal system with nonlinear properties.     

Heterochronic shifts during early cranial neural crest cell migration in two ranid frogs

We describe the development of the cranial neural crest cell streams relative to embryonic events such as neural tube formation and somite appearance in two Eurasian frog species belonging to the Ranidae, Rana temporaria and Sylvirana nigrovittata, and demonstrate developmental heterochronies. The mandibular stream appeared well developed in R. temporaria at a time when the embryo was still spherical, the neural folds were elevated, and the neural plate was wide open, thus earlier than known from any frog species so far. The appearance of the second stream and its division into hyoid and branchial portions was clearly accelerated in R. temporaria relative to other embryonic events when compared to S. nigrovittata. For example, in R. temporaria, the hyoid and branchial portions of the cranial neural crest cell streams were separated before the neural folds had started to fuse, whereas in S. nigrovittata this event took place only after the neural folds had fused completely. Such ostentatious heterochronies related to the characters used herein have formerly only been reported from comparisons between species belonging to different higher taxa. Our results re‐confirm that to understand the full dynamics of the evolution of development, studies need to implement comparative embryological approaches, and include phylogenetically relatively closely related taxa.     

Urinary Biomarkers of Brain Diseases

Biomarkers are the measurable changes associated with a physiological or pathophysiological process. Unlike blood, urine is not subject to homeostatic mechanisms. Therefore, greater fluctuations could occur in urine than in blood, better reflecting the changes in human body. The roadmap of urine biomarker era was proposed. Although urine analysis has been attempted for clinical diagnosis, and urine has been monitored during the progression of many diseases, particularly urinary system diseases, whether urine can reflect brain disease status remains uncertain. As some biomarkers of brain diseases can be detected in the body fluids such as cerebrospinal fluid and blood,there is a possibility that urine also contain biomarkers of brain diseases. This review summarizes the clues of brain diseases reflected in the urine proteome and metabolome.     

Biomarkers response to zinc exposure in the symbiont-bearing foraminifer Amphistegina lessonii (Amphisteginidae, Foraminifera)

The acute effects of zinc (Zn) were evaluated in the symbiont-bearing foraminifer Amphistegina lessonii from the Fernando de Noronha Archipelago (Northeastern Brazil). Foraminifers were acutely (48 h) exposed to dissolved Zn concentrations ranging from 9.5 to 93.4 μg Zn/l. Endpoints analyzed included mortality, visual alterations (white spots and dark-brown areas in the test), oxidative stress biomarkers (reactive oxygen species generation, lipid peroxidation and total superoxide dismutase activity), and concentration of metallothionein-like proteins in whole individuals after Zn exposure. No significant mortality was observed during the 48-h exposure period to waterborne Zn. However, a significant percentage of individuals showed visual alterations (white spots and/or dark-brown areas in the test) after 24 and 48 h of Zn exposure. In fact, a significant positive correlation between this endpoint and dissolved Zn concentrations was observed for both times of exposure. Based on this endpoint, the 24-h and 48-h EC₅₀ values and their corresponding 95% confidence intervals for total measured Zn concentrations were calculated as 112.2 (86.5 - 199.5) and 43.6 (34.9 - 57.3) μg Zn/l, respectively. Based on the dissolved Zn concentrations, they were 100.7 (75.3 - 175.9) and 38.2 (29.7 - 49.4) μg Zn/l, respectively. Therefore, a significant increase in Zn toxicity was observed with increasing time of exposure. After 48 h of Zn exposure, whole body antioxidant capacity was lower in normal-appearing individuals than in those at the initial stage of bleaching. Increases in lipid peroxidation, metallothionein-like protein concentration and total SOD activity was observed at a greater extent in pale/partly-bleached individuals associated with an increased Zn toxicity measured as visual alterations. These findings suggest that an activation of some components of the antioxidant system occurred in A. lessonii to counteract the oxidative stress induced by Zn exposure, and consequently avoid a possible complete loss of the symbiont.