Central nitrergic system regulation of neuroendocrine secretion, fluid intake and blood pressure induced by angiotensin-II

Background: Nitric oxide (NO) synthesis has been described in several circumventricular and hypothalamic structures in the central nervous system that are implicated in mediating central angiotensin-II (ANG-II) actions during water deprivation and hypovolemia. Neuroendocrine and cardiovascular responses, drinking behavior, and urinary excretions were examined following central angiotensinergic stimulation in awake freely-moving rats pretreated with intracerebroventricular injections of N omega-nitro-L-arginine methyl ester (L-NAME, 40 mu g), an inhibitor of NO synthase, and L-arginine (20 ug), a precursor of NO. Results: Injections of L-NAME or ANG-II produced an increase in plasma vasopressin (VP), oxytocin (OT) and atrial natriuretic peptide (ANP) levels, an increase in water and sodium intake, mean arterial blood pressure and sodium excretion, and a reduction of urinary volume. L-NAME pretreatment enhanced the ANG-II response, while L-arginine attenuated VP and OT release, thirst, appetite for sodium, antidiuresis, and natriuresis, as well as pressor responses induced by ANG-II. Discussion and conclusion: Thus, the central nitrergic system participates in the angiotensinergic responses evoked by water deprivation and hypovolemia to refrain neurohypophysial secretion, hydromineral balance, and blood pressure homeostasis.

Predictive factors of non-deterioration of glucose tolerance following a 2-year behavioral intervention

Aim: To identify predictive factors associated with non-deterioration of glucose metabolism following a 2-year behavioral intervention in Japanese-Brazilians. Methods: 295 adults (59.7% women) without diabetes completed 2-year intervention program. Characteristics of those who maintained/improved glucose tolerance status (non-progressors) were compared with those who worsened (progressors) after the intervention. In logistic regression analysis, the condition of non-progressor was used as dependent variable. Results: Baseline characteristics of non-progressors (71.7%) and progressors were similar, except for the former being younger and having higher frequency of disturbed glucose tolerance and lower C-reactive protein (CRP). In logistic regression, non-deterioration of glucose metabolism was associated with disturbed glucose tolerance impaired fasting glucose or impaired glucose tolerance - (p < 0.001) and CRP levels <= 0.04 mg/dL (p = 0.01), adjusted for age and anthropometric variables. Changes in anthropometry and physical activity and achievement of weight and dietary goals after intervention were similar in subsets that worsened or not the glucose tolerance status. Conclusion: The whole sample presented a homogeneous behavior during the intervention. Lower CRP levels and diagnosis of glucose intolerance at baseline were predictors of non-deterioration of the glucose metabolism after a relatively simple intervention, independent of body adiposity.

Consequences for the Bovine Embryo of Being Derived from a Spermatozoon Subjected to Post-Ejaculatory Aging and Heat Shock: Development to the Blastocyst Stage and Sex Ratio

The objective was to determine whether aging of sperm caused by incubation at normothermic (38.5 C) or heat shock (40 C) temperatures for 4 h prior to oocyte insemination affects sperm motility, fertilizing ability, competence of the resultant embryo to develop to the blastocyst stage and blastocyst sex ratio. In the first experiment, the percent of sperm that were motile was reduced by aging (P<0.001) and the reduction in motility was greater for sperm at 40 C compared to sperm at 38.5 C (P<0.01). In the second experiment, oocytes were inseminated with aged sperm. A smaller percent of oocytes fertilized with sperm aged at either temperature cleaved by Day 3 after insemination than oocytes fertilized with fresh sperm (P<0.05). There was no effect of sperm aging on the percent of oocytes or cleaved embryos that developed to the blastocyst stage. Aging of sperm before fertilization at 38.5 C reduced the percent of blastocysts that were male (P=0.08). In the third experiment, incubation of sperm at 38.5 C or 40 C for 4 h did not reduce fertilizing ability of sperm as determined by pronuclear formation at 18 h post insemination. In conclusion, aging of sperm reduced cleavage rate and the percent of blastocysts that were males but had no effect on the developmental capacity of the. embryo. The effect of aging on cleavage rate may represent reduced motility and errors occurring after fertilization and pronuclear formation. Aging at a temperature characteristic of maternal hyperthermia had little additional effect except that polyspermy was reduced. Results indicate that embryo competence for development to the blastocyst stage is independent of sperm damage as a result of aging for 4 h at normothermic or hyperthermic temperatures.

Gene silencing during development of in vitro-produced female bovine embryos

In early development, female embryos (XX) produce twice the transcripts of X-linked genes compared with male embryos (XY). During the course of development, inactivation of the X chromosome equilibrates gene dosage, making the development of female embryos viable. Moreover, the biotechnologies used for producing embryos in vitro seem to work better with male embryos, making it easier for them to reach the blastocyst stage and allow for complete gestation. We investigated the expression of three X-linked genes that are involved in development, XIST, G6PD, and HPRT, and of the transcript interferon-tau, in male and female bovine blastocysts produced by nuclear transfer (NT) and by in vitro fertilization (IVF). Oocytes that had been matured in vitro were enucleated and reconstructed with somatic cells from adult animals at 18 h post-maturation. After fusion (two pulses of 2.25 kv/cm) and chemical activation (5.0 mu M ionomycin for 5 min and 2.0 mM 6-DMAP for 3 h), the oocytesomatic cell units were cultivated in CR2 with a monolayer of granulosa cells at 38.8 degrees C, in a humidified 5% CO(2) atmosphere. IVF embryos were inseminated, after centrifugation in a Percoll gradient, with 2 x 10(6) sperm/mL TALP medium supplemented with BSA and PHE and cultivated under the same conditions as the cloned embryos. We used real-time PCR to analyze the gene expression of individual blastocysts compared to expression of the housekeeping gene, GAPDH. The gene XIST was expressed in female embryos and not in male embryos produced by IVF, though it was expressed at low levels in male embryos produced by NT. Unlike previous reports, we found lower levels of the transcript of G6PD in females than in males, suggesting double silencing or other mechanisms of control of this gene. Female embryos produced by IVF expressed the HPRT gene at a higher level than female embryos produced by NT, suggesting that gene silencing proceeds faster in NT-produced female embryos due to ""inactivation memory"" from the nucleus donor. In conclusion, male and female embryos express different levels of X-chromosome genes and failures of these genes that are essential for development could reduce the viability of females. Nuclear transfer can modify this relation, possibly due to epigenetic memory, leading to frequent failures in nuclear reprogramming.

In vitro development of cloned bovine embryos produced by handmade cloning using somatic cells from distinct levels of cell culture confluence

The relationship between the level of cell confluence near the plateau phase of growth and blastocyst yield following somatic cell cloning is not well understood. We examined the effect of distinct cell culture confluence levels on in vitro development of cloned bovine embryos. In vitro-matured bovine oocytes were manually bisected and selected by DNA staining. One or two enucleated hemi-cytoplasts were paired and fused with an adult skin somatic cell. Cultured skin cells from an adult Nellore cow harvested at three distinct culture confluence levels (70-80, 80-90, and > 95%) were used for construction of embryos and hemi-embryos. After activation, structures were cultured in vitro as one embryo (1 x 100%) or as aggregates of two hemi-embryos (2 x 50%) per microwell. Fusion, cleavage and blastocyst rates were compared using the chi(2) test. The fusion rate for hemi-embryos (51.4%) was lower than for embryos (67.6%), with no influence of degree of cell confluence. However, blastocyst rates improved linearly (7.0, 17.5, and 29.4%) with increases in cell confluence. We conclude that degree of cell culture confluence significantly influences subsequent embryo development; use of a cell population in high confluence (> 90%) for nuclear transfer significantly improved blastocyst yield after cloning.

Serum-Starved Apoptotic Fibroblasts Reduce Blastocyst Production but Enable Development to Term after SCNT in Cattle

Cell cycle synchronization by serum starvation (SS) induces apoptosis in somatic cells. This side effect of SS is hypothesized to negatively affect the outcome of somatic cell nuclear transfer (SCNT). We determined whether apoptotic fibroblasts affect SCNT yields. Serum-starved, adult, bovine fibroblasts were stained with annexin V-FITC/propidium iodide to allow apoptosis detection by flow cytometry. Positive and negative cells sorted by fluorescence activated cell sorting (FACS) and an unsorted control group were used as nuclear donors for SCNT. Reconstructed embryos were cultured in vitro and transferred to synchronized recipients. Apoptosis had no effect on fusion and cleavage rates; however, it resulted in reductions in blastocyst production and quality measured by apoptotic index. However, reconstructed embryos with apoptotic cells resulted in pregnancy rates similar to that of the control on day 30, and generated one live female calf. In conclusion, we showed that apoptotic cells present in serum-starved cultures negatively affect embryo production after SCNT without compromising full-term development. Further studies will evaluate the ability of the oocyte to reprogram cells in specific phases of apoptosis.

Morphological and growth alterations on early development stages of Iridaea cordata (Rhodophyta) under different intensities of UVB radiation

This study describes the effects of different intensities of UVB radiation on growth and morphology of early development stages of Iridaea cordata in germlings, young gametophytes originated in the laboratory and young fronds collected in the Magellan Strait, Chile. The experiments were carried out during four weeks in controlled conditions of temperature and photoperiod and the results were compared with a control treatment (without UVB). All UVB irradiation treatments caused bleaching and decrease in growth rates of germlings. Additionally, initial upright fronds were not observed in any of the UVB treatments, where as those cultivated in UVB absence developed erect ones in the second week of culture. The young gametophytes exhibited morphological alteration (small number and size of basal ramifications, curling of tips, bleaching and necrosis) and decrease in growth when exposed to UVB radiation. Young fronds collected from the field showed mainly morphological alterations (curling of frond). Morphological alterations in young gametophytes and young fronds of I. cordata could be interpreted as a defense against UVB by reducing the area exposed to radiation. However, high level of UVB radiation can produce irreparable damage, such as necrosis, observed in young gametophytes originated in the laboratory. Finally, the UVB effects on early developmental stages of I. cordata depend on the UVB irradiance and time of exposition.

Artificial Lighting as a Vector Attractant and Cause of Disease Diffusion

BACKGROUND: Traditionally, epidemiologists have considered electrification to be a positive factor. In fact, electrification and plumbing are typical initiatives that represent the integration of an isolated population into modern society, ensuring the control of pathogens and promoting public health. Nonetheless, electrification is always accompanied by night lighting that attracts insect vectors and changes people's behavior. Although this may lead to new modes of infection and increased transmission of insect-borne diseases, epidemiologists rarely consider the role of night lighting in their surveys. OBJECTIVE: We reviewed the epidemiological evidence concerning the role of lighting in the spread of vector-borne diseases to encourage other researchers to consider it in future studies. DISCUSSION: We present three infectious vector-borne diseases-Chagas, leishmaniasis, and malaria-and discuss evidence that suggests that the use of artificial lighting results in behavioral changes among human populations and changes in the prevalence of vector species and in the modes of transmission. CONCLUSION: Despite a surprising lack of studies, existing evidence supports our hypothesis that artificial lighting leads to a higher risk of infection from vector-borne diseases. We believe that this is related not only to the simple attraction of traditional vectors to light sources but also to changes in the behavior of both humans and insects that result in new modes of disease transmission. Considering the ongoing expansion of night lighting in developing countries, additional research on this subject is urgently needed.

The relative effects of severe burn injury and pre- and post-natal protein deprivation on mandibular condyle morphology

The mandible has a mixed embryological origin, and its growth is associated with the secondary cartilage of the condyle process (CP). In this area, growth depends on an array of intrinsic and extrinsic factors that influence protein metabolism. In the present study, we used an adolescent rat model to evaluate the growth and development of the CP under conditions of pre- and postnatal protein deficiency, combined with or without the stress of severe burn injury (BI). We found that protein deficiency severely undermined the growth of the CP, by altering the thickness of its constituent layers. BI is also capable of affecting CP growth, although the effect is less severe than protein deficiency. Interestingly, the summed effect of protein deficiency and BI on the CP is less severe than protein deficiency alone. A possible explanation is that the increased carbohydrates in a hypoproteic diet stimulate the production of endogenous insulin and protein synthesis, which partially compensates for the loss of lean body mass caused by BI.

Development of a DNA-dosimeter system for monitoring the effects of solar-ultraviolet radiation

Solar radiation sustains and affects all life forms on Earth. In recent years, the increase in environmental levels of solar-UV radiation due to depletion of the stratospheric ozone layer, as a result of anthropogenic emission of destructive chemicals, has highlighted serious issues of social concern. This becomes still more dramatic in tropical and subtropical regions, where the intensity of solar radiation is higher. To better understand the impact of the harmful effects of solar-UV radiation on the DNA molecule, we developed a reliable biological monitoring system based on the exposure of plasmid DNA to artificial UV lamps and sunlight. The determination and quanti. cation of different types of UV photoproducts were performed through the use of specific DNA repair enzymes and antibodies. As expected, a significant number of CPDs and 6-4PPs was observed when the DNA-dosimeter system was exposed to increasing doses of UVB radiation. Moreover, CPDs could also be clearly detected in plasmid DNA when this system was exposed to either UVA or directly to sunlight. Interestingly, although less abundant, 6-4PPs and oxidative DNA damage were also generated after exposure to both UVA and sunlight. These results confirm the genotoxic potential of sunlight, reveal that UVA may also produce CPDs and 6-4PPs directly in naked DNA and demonstrate the applicability of a DNA-dosimeter system for monitoring the biological effects of solar-UV radiation.

Development and validation of a fast RP-HPLC method to determine the analogue of the thyroid hormone, 3,5,3 '-triiodothyroacetic acid (TRIAC), in polymeric nanoparticles

The objective of this work was to develop and validate a rapid Reversed-Phase High-Performance Liquid Chromatography method for the quantification of 3,5,3 '-triiodothyroacetic acid (TRIAC) in nanoparticles delivery system prepared in different polymeric matrices. Special attention was given to developing a reliable reproductive technique for the pretreatment of the samples. Chromatographic runs were performed on an Agilent 1200 Series HPLC with a RP Phenomenex (R) Gemini C18 (150 x 4, 6 mm i.d., 5 mu m) column using acetonitrile and triethylamine buffer 0.1% (TEA) (40 : 60 v/v) as a mobile phase in an isocratic elution, pH 5.6 at a flow rate of 1 ml min(-1). TRIAC was detected at a wavelength of 220 nm. The injection volume was 20 mu l and the column temperature was maintained at 35 degrees C. The validation characteristics included accuracy, precision, specificity, linearity, recovery, and robustness. The standard curve was found to have a linear relationship (r(2) - 0.9996) over the analytical range of 5-100 mu g ml(-1) . The detection and quantitation limits were 1.3 and 3.8 mu g ml(-1), respectively. The recovery and loaded TRIAC in colloidal system delivery was nearly 100% and 98%, respectively. The method was successfully applied in polycaprolactone, polyhydroxybutyrate, and polymethylmethacrylate nanoparticles.

KeaA, a Dictyostelium kelch-domain protein that regulates the response to stress and development

Background: The protein kinase YakA is responsible for the growth arrest and induction of developmental processes that occur upon starvation of Dictyostelium cells. yakA-cells are aggregation deficient, have a faster cell cycle and are hypersensitive to oxidative and nitrosoative stress. With the aim of isolating members of the YakA pathway, suppressors of the death induced by nitrosoative stress in the yakA-cells were identified. One of the suppressor mutations occurred in keaA, a gene identical to DG1106 and similar to Keap1 from mice and the Kelch protein from Drosophila, among others that contain Kelch domains. Results: A mutation in keaA suppresses the hypersensitivity to oxidative and nitrosoative stresses but not the faster growth phenotype of yakA-cells. The growth profile of keaA deficient cells indicates that this gene is necessary for growth. keaA deficient cells are more resistant to nitrosoative and oxidative stress and keaA is necessary for the production and detection of cAMP. A morphological analysis of keaA deficient cells during multicellular development indicated that, although the mutant is not absolutely deficient in aggregation, cells do not efficiently participate in the process. Gene expression analysis using cDNA microarrays of wild-type and keaA deficient cells indicated a role for KeaA in the regulation of the cell cycle and pre-starvation responses. Conclusions: KeaA is required for cAMP signaling following stress. Our studies indicate a role for kelch proteins in the signaling that regulates the cell cycle and development in response to changes in the environmental conditions.

The P450 oxidoreductase, RedA, controls development beyond the mound stage in Dictyostelium discoideum

Background: NADPH-cytochrome- P450 oxidoreductase (CPR) is a ubiquitous enzyme that belongs to a family of diflavin oxidoreductases and is required for activity of the microsomal cytochrome-P450 monooxygenase system. CPR gene-disruption experiments have demonstrated that absence of this enzyme causes developmental defects both in mouse and insect. Results: Annotation of the sequenced genome of D. discoideum revealed the presence of three genes (redA, redB and redC) that encode putative members of the diflavin oxidoreductase protein family. redA transcripts are present during growth and early development but then decline, reaching undetectable levels after the mound stage. redB transcripts are present in the same levels during growth and development while redC expression was detected only in vegetative growing cells. We isolated a mutant strain of Dictyostelium discoideum following restriction enzyme-mediated integration (REMI) mutagenesis in which redA was disrupted. This mutant develops only to the mound stage and accumulates a bright yellow pigment. The mound-arrest phenotype is cell-autonomous suggesting that the defect occurs within the cells rather than in intercellular signaling. Conclusion: The developmental arrest due to disruption of redA implicates CPR in the metabolism of compounds that control cell differentiation.

Nitrogen dynamics during ecosystem development in tropical forest restoration

We considered whether ecological restoration using high diversity of native tree species serves to restore nitrogen dynamics in the Brazilian Atlantic Forest. We measured delta(15)N and N content in green foliage and soil; vegetation N:P ratio; and soil N mineralization in a preserved natural forest and restored forests of ages 21 and 52 years. Green foliage delta(15)N values, N content, N:P ratio, inorganic N and net mineralization and nitrification rates were all higher, the older the forest. Our findings indicate that the recuperation of N cycling has not been achieved yet in the restored forests even after 52 years, but show that they are following a trajectory of development that is characterized by their N cycling intensity becoming similar to a natural mature forest of the same original forest formation. This study demonstrated that some young restored forests are more limited by N compared to mature natural forests. We document that the recuperation of N cycling in tropical forests can be achieved through ecological restoration actions. (C) 2011 Elsevier B.V. All rights reserved.

Development of novel microsatellites from Moniliophthora perniciosa, causal agent of the witches` broom disease of Theobroma cacao

Moniliophthora perniciosa is the causal agent of the witches` broom disease of cacao. Based on available genomic sequences, we identified 30 new microsatellite loci, which were analysed using 50 isolates from four populations sampled over a wide geographical area in Brazil, including three populations from the Amazon, the fungal putative centre of diversity, plus one from Bahia. Nine loci were polymorphic, with an average of 2.9 alleles per locus. The level of polymorphism observed was low, but these markers may allow the evaluation of pathogen diversity and the establishment of molecular standards for isolate fingerprinting to support cacao breeding.