Anfíbios Anuros do Distrito Federal

The frogs of the Federal District of Brazil are listed and discussed as to habit, biology and ecology. The F. D is situated 22° 54' 24" S. & 43° 10' 21" W Gr. and comprises 1.356 km². Its topography includes sea-shore, maritime scrub, lagoons, plains and marsh, open slopes, forested mountains and great heads of rock. Three thousand feet of altitude are attained at two points. Fifty two different frogs occur in the F.D. Three fifths of them live in open country. Two fifths of these have never been found above the plains; the others range higher but mostly in open country. Their environment offers conditions suitable for average tadpoles and adults. these frogs are more or less unspecialized. There are six genera and thirty species. Two thirds of the latter belong to the type genera of the large neotropical families Bufonidae, Leptodactylidae and Hylidae. Only in the maritime scrub formation are conditions somewhat different. Water for average tadpoles is provided by the lagoons. The xerophytism of the vegetation is, however, so marked that bromeliads growing on the ground provide almost the only appropriate shelter for adult tree-frogs used to sleeping upright on the vegetation. One large Hylid genus lives entirely in them. It is casque-headed and phragmotic, shutting the lumen of the leaf-cup with head used as a plug. Another large Hylid genus shows a lesser degree of the same specialization. (Lutz A & Lutz B, 1939 II). One genus with two species is entirely saxicolous; it lives on wet ledges of rock at all phases of its life history. (B. Lutz 1948). The other two fifths of the frogs from F. D. are montane forest forms. Their environment offers numerous and varied biotopes and is near optimum for adults. There is,however, hardly any standing water available for larvae. These frogs are ecologically diversified. They also show a general trend towards spawning in the adult biotipe, which leads to delayed hatching, semi-aquatic and terrestrial larvae and direct development. (B Lutz, 1948). The author interprets the morphological specialization of the casque-headed Hylids and the biological specialization of the montane forest forms as adaptive. Casque-headedness and phragmosis increase protection against blood-suckers and predators. The humidity of the rain forest permits eggs, embryos and larvae to develop, unharmed, outside their usual, aquatic, environment.

Ectodysplasin A1 promotes placodal cell fate during early morphogenesis of ectodermal appendages.

Organs developing as appendages of the ectoderm are initiated from epithelial thickenings called placodes. Their formation is regulated by interactions between the ectoderm and underlying mesenchyme, and several signalling molecules have been implicated as activators or inhibitors of placode formation. Ectodysplasin (Eda) is a unique signalling molecule in the tumour necrosis factor family that, together with its receptor Edar, is necessary for normal development of ectodermal organs both in humans and mice. We have shown previously that overexpression of the Eda-A1 isoform in transgenic mice stimulates the formation of several ectodermal organs. In the present study, we have analysed the formation and morphology of placodes using in vivo and in vitro models in which both the timing and amount of Eda-A1 applied could be varied. The hair and tooth placodes of K14-Eda-A1 transgenic embryos were enlarged, and extra placodes developed from the dental lamina and mammary line. Exposure of embryonic skin to Eda-A1 recombinant protein in vitro stimulated the growth and fusion of placodes. However, it did not accelerate the initiation of the first wave of hair follicles giving rise to the guard hairs. Hence, the function of Eda-A1 appears to be downstream of the primary inductive signal required for placode initiation during skin patterning. Analysis of BrdU incorporation indicated that the formation of the epithelial thickening in early placodes does not involve increased cell proliferation and also that the positive effect of Eda-A1 on placode expansion is not a result of increased cell proliferation. Taken together, our results suggest that Eda-A1 signalling promotes placodal cell fate during early development of ectodermal organs.

Influence de deux milieux séquentiels pour la culture d'embryons sur la production d'hormone gonadotrophine chorionique et de progestérone par des cellules JEG-3 et BeWo [Effects of different embryo development media on hCG and progesterone release by JEG-3 and BeWo cells]

Current in vitro fertilisation (IVF) practice requires synchronisation between the¦environment of cultured oocytes and embryos and the surroundings to what they would have¦been exposed to in vivo. Commercial, sequential media follow this requirement but their exact¦composition is not available. We have compared two widely used IVF culture media systems using¦the two choriocarcinoma cell lines JEG-3 and BeWo. The two hormones hCG and progesterone¦were determined in the culture supernatants as endpoints. In both cell lines, but in a more¦pronounced way in JEG-3, progesterone rather than hCG production was stimulated, and a¦higher hormone release was observed in the fertilisation than in the cleavage media. Differences¦between manufacturers were small and did not favour one system over the other. We conclude¦that both sequential media systems can be equally well used in current IVF laboratory practice.¦© 2012 Elsevier Masson SAS. All rights reserved.

Patterns of oxygen consumption during establishment of cephalocaudal polarity in the early chick embryo.

Oxygen uptake was studied during the establishment of cephalocaudal polarity in the very early chick embryo, i.e., 10 hr before (stage VI) and at laying (stage X). Oxygen fluxes in minute regions of the intact blastoderms were measured in vitro by scanning microspectrophotometry in the presence or absence of glucose. The oxygen consumption of the whole blastoderm remained constant (6 nmol O2 X hr-1) throughout the period studied, although the number of cells increased more than twofold. The regional oxygen fluxes varied from 0.41 to 1.13 nmol O2 X hr-1 X mm-2 at stage VI and from 0.42 to 0.70 nmol O2 X hr-1 X mm-2 at stage X. At stage VI, the oxygen flux in the center of the blastoderm was significantly higher than that in its periphery. This pattern remained evident when the values were corrected for cell number or for cytoplasmic volume. At stage X, there was a tendency for the oxygen fluxes to decrease from the posterior to the anterior regions of the area pellucida. Thus the pattern of oxidative metabolism in the late uterine embryos seems to change from radial to bilateral. This change of symmetry probably reflects the process of formation of the embryonic axis. In addition, the fact that the oxygen uptake was similar in the presence or absence of glucose suggests that early chick embryos metabolize essentially intracellular stores.

Effects of Laurus nobilis (Lauraceae) on Biomphalaria glabrata (Say, 1818)

Experiments were carried out using aqueous extracts from leaves and flowers of Laurus nobilis on Biomphalaria glabrata. Treatments were performed on blastula stage (± 15 h after first cleavage) and on adult snails (11-18 mm). In both instances they were exposed for 24 h to different concentrations of the extracts on snails (200 to 2500 ppm) and embryos (20 to 300 ppm) at 25 ± 1ºC. The embryos were observed for a period of 20 days after treatment and the snails for 10 days. Results obtained with leaf aqueous extracts have shown a degree of toxicity on embryos starting at a concentration of 125 ppm, the flower extract being effective at 35 ppm. The malformation obtained with the different concentrations falls into the unespecific type category, however some cephalic and shell malformations were found in embryos treated with concentrations over 50 ppm (leaves) and 25 ppm (flowers). The LD90 on adult snails obtained by treatments with flower and leaf extract was observed at concentrations of 340 ppm and 1900 ppm respectively.

Ear acupuncture and fMRI: a pilot study for assessing the specificity of auricular points.

In recent years research explored different acupuncture stimulation techniques but interest has focused primarily on somatic acupuncture and on a limited number of acupoints. As regards ear Acupuncture (EA) there is still some criticism about the clinical specificity of auricular points/areas representing organs or structures of the body. The aim of this study was to verify through (Functional magnetic resonance imaging) fMRI the hypothesis of EA point specificity using two auricular points having different topographical locations and clinical significance. Six healthy volunteers underwent two experimental fMRI sessions: the first was dedicated to the stimulation of Thumb Auricular Acupoint (TAA) and the second to the stimulation of Brain Stem Auricular Acupoint (BSAA). The stimulation of the needle placed in the TAA of the left ear produced an increase in activation bilaterally in the parietal operculum, region of the secondary somatosensory area SII. Stimulation of the needle placed in the BSAA of the left ear showed a pattern that largely overlapped regions belonging to the pain matrix, as shown to be involved in previous somatic acupuncture studies but with local differences in the left amygdala, anterior cingulate cortex, and cerebellum. The differences in activation patterns between TAA and BSAA stimulation support the specificity of the two acupoints. Moreover, the peculiarity of the regions involved in BSAA stimulation compared to those involved in the pain matrix, is in accordance with the therapeutic indications of this acupoint that include head pain, dizziness and vertigo. Our results provide preliminary evidence on the specificity of two auricular acupoints; further research is warranted by means of fMRI both in healthy volunteers and in patients carrying neurological/psychiatric syndromes.

Transient stimulation of glucose metabolism by insulin in the 1-day chick embryo.

Effects of insulin upon glucose metabolism were investigated in chick embryos explanted in vitro during the first 30 h of incubation. Insulin stimulated the glucose consumption of the chick gastrula (18 h) and neurula (24 h), but had no effect on the late blastula (0 h:laying) and on the stage of six to eight somites (30 h). The increase in glucose consumption concerned both the embryonic area pellucida (AP) and extraembryonic area opaca (AO). AP responded to a greater extent (50%) and at a lower range of concentrations (0.1-1.0 ng/ml) than AO (30%; 1-100 ng/ml). Insulin had no effect on the oxygen consumption of blastoderms, whereas it stimulated the aerobic lactate production (approximately 70% of the additional glucose consumption was converted to lactate). The nanomolar range of stimulating concentrations suggests that insulin has a specific effect in the chick embryo, and that it could modulate glucose metabolism in ovo as well. The transient sensitivity of the embryo to insulin is discussed in relation to behavior of mesodermal cells.

Caracterització estructural i funcional del gen de la miogenia en l’orada (Sparus aurata )

Report for the scientific sojourn at the University of Maryland Biotechnology Institute from February to August 2007. Myogenesis of skeletal muscles in vertebrates is controlled by extracellular signalling molecules together with intracellular transcription factors. Among the transcriptional factors, the members of the myogenic regulatory family play important roles regulating skeletal muscle development and growth. To characterize the gene structure and expression of fish myogenin, we have isolated the myogenin genomic gene and cDNA from gilthead seabream (Sparus aurata) and analyzed the genomic structure, pattern of expression and the regulation of musclespecific expression. Sequence analysis revealed that the seabream myogenin shares a similar gene structure with other fish myogenins, with three exons, two introns and the highly conserved bHLH domain. Expression studies demonstrated that myogenin is expressed in both slow and fast muscles as well as in muscle cells in primary culture. In situ hybridization showed that myogenin was specifically expressed in developing somites of seabream embryos. Promoter activity analysis demonstrated that the myogenin promoter could drive green fluorescence protein expression in muscle cells of zebrafish embryos, as well as in myofibers of adult zebrafish and juvenile seabream.

Methylation profile of TP53 regulatory pathway and mtDNA alterations in breast cancer patients lacking TP53 mutations.

The present study investigated promoter hypermethylation of TP53 regulatory pathways providing a potential link between epigenetic changes and mitochondrial DNA (mtDNA) alterations in breast cancer patients lacking a TP53 mutation. The possibility of using the cancer-specific alterations in serum samples as a blood-based test was also explored. Triple-matched samples (cancerous tissues, matched adjacent normal tissues and serum samples) from breast cancer patients were screened for TP53 mutations, and the promoter methylation profile of P14(ARF), MDM2, TP53 and PTEN genes was analyzed as well as mtDNA alterations, including D-loop mutations and mtDNA content. In the studied cohort, no mutation was found in TP53 (DNA-binding domain). Comparison of P14(ARF) and PTEN methylation patterns showed significant hypermethylation levels in tumor tissues (P < 0.05 and <0.01, respectively) whereas the TP53 tumor suppressor gene was not hypermethylated (P < 0.511). The proportion of PTEN methylation was significantly higher in serum than in the normal tissues and it has a significant correlation to tumor tissues (P < 0.05). mtDNA analysis revealed 36.36% somatic and 90.91% germline mutations in the D-loop region and also significant mtDNA depletion in tumor tissues (P < 0.01). In addition, the mtDNA content in matched serum was significantly lower than in the normal tissues (P < 0.05). These data can provide an insight into the management of a therapeutic approach based on the reversal of epigenetic silencing of the crucial genes involved in regulatory pathways of the tumor suppressor TP53. Additionally, release of significant aberrant methylated PTEN in matched serum samples might represent a promising biomarker for breast cancer.

Ultrastructure of the ovary of Dermatobia hominis (Diptera: cuterebridae). I. Development during the 3rd larval instar

The ultrastructure and distribution of gonial and somatic cells in the ovary of Dermatobia hominis was studied during the 3rd larval instar. In larvae weighing between 400 and 500 mg, the ovary is partially divided into basal and apical regions by oblong somatic cells that penetrate from the periphery; these cells show ovoid nucleus and cytoplasm full of microtubules. In both regions, gonial cells with regular outlines, large nucleus and low electron-density cytoplasm are scattered among the interstitial somatic cells. These later cells have small nucleus and electrodense cytoplasm. Clear somatic cells with small nucleus and cytoplasm of very low electron-density are restrict to the apical region of the gonad. Degenerating interstitial somatic cells are seen in the basal portion close to the ovary peduncle. During all this larval period the morphological features of the ovary remain almost the same. At the end of the period there is a gradual deposition of glycogen in the cytoplasm of the somatic cells, increase in the number and density of their mitochondria plus nuclear modification as membrane wrinkling and chromatin condensation in masses.

Ultrastructure of the ovary of Dermatobia hominis (Diptera: cuterebridae). II. Origin of the tunica propria in ovarioles

Ovaries up to the 8th day pupae of Dermatobia hominis were studied by transmission electron microscopy. Ovarioles were recognized in ovaries of 4-day old pre-pupae, surrounded by a thin tunica propria of acellular fibrilar material similar in structure to the internal portion of the external tunica of the ovary. There is continuity of the tunica propria and the ovarian tunica, indicating that the former structure originates from the tunica externa. In 5 to 7-day pupae the interstitial somatic cells from the apical region of the ovary, close to the ovarioles, show delicate filamentous material inside of their rough endoplasmic reticulum cisternae; similar material is seem among these cells. Our observations suggest that interstitial somatic cells do not originate the tunica propria but contribute to its final composition.

Reversal of the silencing of tetracycline-controlled genes requires the coordinate action of distinctly acting transcription factors.

BACKGROUND: Regulation of genes transferred to eukaryotic organisms is often limited by the lack of consistent expression levels in all transduced cells, which may result in part from epigenetic gene silencing effects. This reduces the efficacy of ligand-controlled gene switches designed for somatic gene transfers such as gene therapy. METHODS: A doxycycline-controlled transgene was stably introduced in human cells, and clones were screened for epigenetic silencing of the transgene. Various regulatory proteins were targeted to the silent transgene, to identify those that would mediate regulation by doxycycline. RESULTS: A doxycycline-controlled minimal promoter was found to be prone to gene silencing, which prevents activation by a fusion of the bacterial TetR DNA-binding domain with the VP16 activator. DNA modification studies indicated that the silenced transgene adopts a poorly accessible chromatin structure. Several cellular transcriptional activators were found to restore an accessible DNA structure when targeted to the silent transgene, and they cooperated with Tet-VP16 to mediate regulation by doxycycline. CONCLUSIONS: Reversal of the silencing of a tetracycline-regulated minimal promoter requires a chromatin-remodeling activity for subsequent promoter activation by the Tet-VP16 fusion protein. Thus, distinct regulatory elements may be combined to obtain long-term regulation and persistent expression of exogenous genes in eukaryotic cells.

Effects of different mating scenarios on embryo viability in brown trout.

Mating with attractive or dominant males is often predicted to offer indirect genetic benefits to females, but it is still largely unclear how important such non-random mating can be with regard to embryo viability. We sampled a natural population of adult migratory brown trout (Salmo trutta), bred them in vitro in a half-sib breeding design to separate genetic from maternal environmental effects, raised 2098 embryos singly until hatching, and exposed them experimentally to different levels of pathogen stress at a late embryonic stage. We found that the embryos' tolerance to the induced pathogen stress was linked to the major histocompatibility complex (MHC) of their parents, i.e. certain MHC genotypes appeared to provide better protection against infection than others. We also found significant additive genetic variance for stress tolerance. Melanin-based dark skin patterns revealed males with 'good genes', i.e. embryos fathered by dark coloured males had a high tolerance to infection. Mating with large and dominant males would, however, not improve embryo viability when compared to random mating. We used simulations to provide estimates of how mate choice based on MHC or melanin-based skin patterns would influence embryos' tolerance to the experimentally induced pathogen stress.

Reduced Prostasin (CAP1/PRSS8) Activity Eliminates HAI-1 and HAI-2 Deficiency-Associated Developmental Defects by Preventing Matriptase Activation.

Loss of either hepatocyte growth factor activator inhibitor (HAI)-1 or -2 is associated with embryonic lethality in mice, which can be rescued by the simultaneous inactivation of the membrane-anchored serine protease, matriptase, thereby demonstrating that a matriptase-dependent proteolytic pathway is a critical developmental target for both protease inhibitors. Here, we performed a genetic epistasis analysis to identify additional components of this pathway by generating mice with combined deficiency in either HAI-1 or HAI-2, along with genes encoding developmentally co-expressed candidate matriptase targets, and screening for the rescue of embryonic development. Hypomorphic mutations in Prss8, encoding the GPI-anchored serine protease, prostasin (CAP1, PRSS8), restored placentation and normal development of HAI-1-deficient embryos and prevented early embryonic lethality, mid-gestation lethality due to placental labyrinth failure, and neural tube defects in HAI-2-deficient embryos. Inactivation of genes encoding c-Met, protease-activated receptor-2 (PAR-2), or the epithelial sodium channel (ENaC) alpha subunit all failed to rescue embryonic lethality, suggesting that deregulated matriptase-prostasin activity causes developmental failure independent of aberrant c-Met and PAR-2 signaling or impaired epithelial sodium transport. Furthermore, phenotypic analysis of PAR-1 and matriptase double-deficient embryos suggests that the protease may not be critical for focal proteolytic activation of PAR-2 during neural tube closure. Paradoxically, although matriptase auto-activates and is a well-established upstream epidermal activator of prostasin, biochemical analysis of matriptase- and prostasin-deficient placental tissues revealed a requirement of prostasin for conversion of the matriptase zymogen to active matriptase, whereas prostasin zymogen activation was matriptase-independent.

Differential contribution of mitochondria, NADPH oxidases, and glycolysis to region-specific oxidant stress in the anoxic-reoxygenated embryonic heart.

The ability of the developing myocardium to tolerate oxidative stress during early gestation is an important issue with regard to possible detrimental consequences for the fetus. In the embryonic heart, antioxidant defences are low, whereas glycolytic flux is high. The pro- and antioxidant mechanisms and their dependency on glucose metabolism remain to be explored. Isolated hearts of 4-day-old chick embryos were exposed to normoxia (30 min), anoxia (30 min), and hyperoxic reoxygenation (60 min). The time course of ROS production in the whole heart and in the atria, ventricle, and outflow tract was established using lucigenin-enhanced chemiluminescence. Cardiac rhythm, conduction, and arrhythmias were determined. The activity of superoxide dismutase, catalase, gutathione reductase, and glutathione peroxidase as well as the content of reduced and oxidized glutathione were measured. The relative contribution of the ROS-generating systems was assessed by inhibition of mitochondrial complexes I and III (rotenone and myxothiazol), NADPH oxidases (diphenylene iodonium and apocynine), and nitric oxide synthases (N-monomethyl-l-arginine and N-iminoethyl-l-ornithine). The effects of glycolysis inhibition (iodoacetate), glucose deprivation, glycogen depletion, and lactate accumulation were also investigated. In untreated hearts, ROS production peaked at 10.8 ± 3.3, 9 ± 0.8, and 4.8 ± 0.4 min (means ± SD; n = 4) of reoxygenation in the atria, ventricle, and outflow tract, respectively, and was associated with arrhythmias. Functional recovery was complete after 30-40 min. At reoxygenation, 1) the respiratory chain and NADPH oxidases were the main sources of ROS in the atria and outflow tract, respectively; 2) glucose deprivation decreased, whereas glycogen depletion increased, oxidative stress; 3) lactate worsened oxidant stress via NADPH oxidase activation; 4) glycolysis blockade enhanced ROS production; 5) no nitrosative stress was detectable; and 6) the glutathione redox cycle appeared to be a major antioxidant system. Thus, the glycolytic pathway plays a predominant role in reoxygenation-induced oxidative stress during early cardiogenesis. The relative contribution of mitochondria and extramitochondrial systems to ROS generation varies from one region to another and throughout reoxygenation.