Application of the differential display RT-PCR strategy for the identification of inflammation-related mouse genes

The inflammatory response elicited by various stimuli such as microbial products or cytokines is determined by differences in the pattern of cellular gene expression. We have used the differential display RT-PCR (DDRT-PCR) strategy to identify mRNAs that are differentially expressed in various murine cell types stimulated with pro-inflammatory cytokines, microbial products or anti-inflammatory drugs. Mouse embryonic fibroblasts (MEFs) were treated with IFNs, TNF, or sodium salicylate. Also, peritoneal macrophages from C3H/Hej mice were stimulated with T. cruzi-derived GPI-mucin and/or IFN-g. After DDRT-PCR, various cDNA fragments that were differentially represented on the sequencing gel were recovered, cloned and sequenced. Here, we describe a summary of several experiments and show that, when 16 of a total of 28 recovered fragments were tested for differential expression, 5 (31%) were found to represent mRNAs whose steady-state levels are indeed modulated by the original stimuli. Some of the identified cDNAs encode for known proteins that were not previously associated with the inflammatory process triggered by the original stimuli. Other cDNA fragments (8 of 21 sequences, or 38%) showed no significant homology with known sequences and represent new mouse genes whose characterization might contribute to our understanding of inflammation. In conclusion, DDRT-PCR has proven to be a potent technology that will allow us to identify genes that are differentially expressed when cells are subjected to changes in culture conditions or isolated from different organs.

Gap junctions in isolated rat aorta: evidence for contractile responses that exhibit a differential dependence on intercellular communication

Connexin43 (Cx43) is a major gap junction protein present in the Fischer-344 rat aorta. Previous studies have identified conditions under which selective disruption of intercellular communication with heptanol caused a significant, readily reversible and time-dependent diminution in the magnitude of a1-adrenergic contractions in isolated rat aorta. These observations have indentified a significant role for gap junctions in modulating vascular smooth muscle tone. The goal of these steady-state studies was to utilize isolated rat aortic rings to further evaluate the contribution of intercellular junctions to contractions elicited by cellular activation in response to several other vascular spasmogens. The effects of heptanol were examined (0.2-2.0 mM) on equivalent submaximal (»75% of the phenylephrine maximum) aortic contractions elicited by 5-hydroxytryptamine (5-HT; 1-2 µM), prostaglandin F2a (PGF2a; 1 µM) and endothelin-1 (ET-1; 20 nM). Statistical analysis revealed that 200 µM and 500 µM heptanol diminished the maximal amplitude of the steady-state contractile responses for 5-HT from a control response of 75 ± 6% (N = 26 rings) to 57 ± 7% (N = 26 rings) and 34.9 ± 6% (N = 13 rings), respectively (P<0.05), and for PGF2a from a control response of 75 ± 10% (N = 16 rings) to 52 ± 8% (N = 19 rings) and 25.9 ± 6% (N = 18 rings), respectively (P<0.05). In contrast, 200 µM and 500 µM heptanol had no detectable effect on the magnitude of ET-1-induced contractile responses, which were 76 ± 5.0% for the control response (N = 38 rings), 59 ± 6.0% in the presence of 200 µM heptanol (N = 17 rings), and 70 ± 6.0% in the presence of 500 µM heptanol (N = 23 rings) (P<0.13). Increasing the heptanol concentration to 1 mM was associated with a significant decrease in the magnitude of the steady-state ET-1-induced contractile response to 32 ± 5% (21 rings; P<0.01); further increasing the heptanol concentration to 2 mM had no additional effect. In rat aorta then, junctional modulation of tissue contractility appears to be agonist-dependent.

A brain microdialysis study on 5-HT release in freely moving rat lines selectively bred for differential 5-HT1A receptor function

Breeding for high and low hypothermic responses to systemic administration of a serotonin1A (5-HT1A) receptor agonist (8-hydroxy-2-(di-n-propylamino)tetralin, 8-OH-DPAT) has resulted in high DPAT-sensitive (HDS) and low DPAT-sensitive (LDS) lines of rats, respectively. These lines also differ in several behavioral measures associated with stress. In the present microdialysis study we observed that basal 5-HT concentrations in the prefrontal cortex and dorsal hippocampus did not differ significantly between HDS and LDS rats. Thus, behavioral differences between the HDS and LDS lines might not be attributed to differences in basal 5-HT release. However, both lines had lower basal levels of 5-HT release than their randomly bred control group (random DPAT-sensitive, RDS) in the prefrontal cortex (mean ± SEM, pg/20 µl, was 3.0 ± 0.4 for LDS, 3.8 ± 0.3 for HDS and 6.4 ± 0.6 for RDS; F(2,59) = 5.8, P<0.005). The administration of (±)-fenfluramine (10 mg/kg) induced a greater increase in hippocampal 5-HT levels in HDS rats (500%) as compared with LDS (248%) or RDS (243%) rats (P<0.0001). There were no significant differences in the prefrontal cortex among lines, with a fenfluramine-induced 5-HT increase of about 900% in the three groups. This differential response to fenfluramine may be due to functional alterations of hippocampal 5-HT reuptake sites in the HDS line.

Normal skin and hypertrophic scar fibroblasts differentially regulate collagen and fibronectin expression as well as mitochondrial membrane potential in response to basic fibroblast growth factor

Basic fibroblast growth factor (bFGF) regulates skin wound healing; however, the underlying mechanism remains to be defined. In the present study, we determined the effects of bFGF on the regulation of cell growth as well as collagen and fibronectin expression in fibroblasts from normal human skin and from hypertrophic scars. We then explored the involvement of mitochondria in mediating bFGF-inducedeffects on the fibroblasts. We isolated and cultivated normal and hypertrophic scar fibroblasts from tissue biopsies of patients who underwent plastic surgery for repairing hypertrophic scars. The fibroblasts were then treated with different concentrations of bFGF (ranging from 0.1 to 1000 ng/mL). The growth of hypertrophic scar fibroblasts became slower with selective inhibition of type I collagen production after exposure to bFGF. However, type III collagen expression was affected in both normal and hypertrophic scar fibroblasts. Moreover, fibronectin expression in the normal fibroblasts was up-regulated after bFGF treatment. bFGF (1000 ng/mL) also induced mitochondrial depolarization in hypertrophic scar fibroblasts (P < 0.01). The cellular ATP level decreased in hypertrophic scar fibroblasts (P < 0.05), while it increased in the normal fibroblasts following treatment with bFGF (P < 0.01). These data suggest that bFGF has differential effects and mechanisms on fibroblasts of the normal skin and hypertrophic scars, indicating that bFGF may play a role in the early phase of skin wound healing and post-burn scar formation.

Mapping the Obama administration's response to the Arab Spring

When the Arab Spring broke out, the United States was in a quandary over how to handle the crisis in its attempt to balance its moral obligations and ideals without undercutting its strategic interests and those of its close allies. Flaws in US diplomatic approach have contributed to one of the most serious foreign policy crisis for a US administration to date with consequential upheaval and erosion of the US-built balance of power. The reactions and policy responses of the Obama administration highlight the difficulties in grasping with the new reality in the Middle East and in enunciating a policy platform that could combine American interests and values.

Dilemma of choice: China's response to climate change

In recent years, erratic global climate conditions have generated an incessant series of natural disasters in China. This article seeks to explore China's climate change policies. This article addresses the impacts of climate change on China's environment and China's perception, principle, objective and policy actions in response to climate change.

SPAD-502 readings in response to photon fluence in leaves with different chlorophyll content

The chlorophyll meter (SPAD-502) is widely used to estimate chlorophyll content, but non-uniform chloroplast distribution can affect its accuracy. This study aimed to assess the effect of photon fluence (F, irradiance x time of illumination) in leaves with different chlorophyll content and determine the effect of chlorophyll a/b on SPAD values of four tropical tree species (Croton draconoides Müll. Arg., Hevea guianensis Aubl., Hymenaea courbaril L. and Matisia cordata H.B.K.). There were also determined calibration equations for the chlorophyll meter and assessed the effect of F on SPAD values between 07:00 h and 17:00 h. Calibration equations were obtained after determining leaf chlorophyll content in the laboratory. Increases in F with time caused a reduction in SPAD values in species with a high chlorophyll content, with reductions of 20% in M. cordata and 10% in H. guianensis. Leaves of C. draconoides and H. courbaril had lower chlorophyll content and showed no changes in SPAD values with increase in F. The chlorophyll a/b ratio increased with SPAD values and the SPAD/chlorophyll relationship was best described by an exponential equation. It seems that F may affect SPAD values in leaves with high chlorophyll content, probably due to non-uniform chloroplast distribution at high irradiance. This indicates that SPAD values tend to be more accurate if recorded early in morning when irradiance is low.

Broccoli yield in response to top-dressing fertilization with green manure and biofertilizer

The objective of this work was to evaluate the productive performance of broccoli under different top-dressing organic fertilizations. The experiment was conducted under protected cultivation, in a completely randomized design with four replications, with two plants per experimental unit. Broccoli seedlings were produced in a commercial substrate in styrofoam trays. The seedlings were transplanted to plastic pots containing 10.0 L of substrate made up of subsoil and organic compost at the ratio of 3:1 (v/v), respectively, which is equivalent to about 20.0 t ha-1 of organic compost at planting. After seedling establishment, the top-dressing fertilization treatments were applied: gliricidia biomass associated or not with liquid biofertilizer of cattle manure to the soil and bokashi. Two control treatments were established: one with mineral fertilization recommended for the crop and the other without top-dressing fertilization. The broccoli production was evaluated (commercial standard). Plants that received mineral fertilizer were more productive, however, they were not significantly different (p>0.05), by Dunnet test, from the plants fertilized with 2.5 t ha-1 gliricidiabiomass (dry mass) associated with liquid biofertilizer (2.0 L m-2) applied to soil. Top-dressing fertilizations with only gliricidia, at 2.5 and 5.0 t ha-1 of biomass (dry mass), resulted in no significant increase in production of broccoli inflorescence. The use of bokashi in addition to gliricidia biomass and liquid biofertilizer reduced the efficiency of the fertilization compared with plants that received only gliricidia and liquid biofertilizer.

Initial growth of maize in response to application of rock phosphate, vermicompost and endophytic bacteria

Due to the high energy requirement and demand for non-renewable resources for the production of chemical fertilizers, added also to the environmental impact caused by the use of such products, it is important to intensify research on bio-based agricultural inputs. The use of nitrogen-fixing endophytic and phosphate solubilizing bacteria can provide these nutrients to the plants from the air and poorly soluble phosphorus sources, such as phosphate rock. The objective of this study was to evaluate the nutrition and initial growth of maize (Zea mays L.) in response to the inoculation of nitrogen-fixing and rock phosphate solubilizing endophytic bacteria, in single or mixed formulation, applied with vermicompost. The treatments containing bacteria, both diazotrophic and phosphate solubilizing, when compared to controls, showed higher levels of leaf nitrogen and phosphorus in maize, as well as higher growth characteristics. The application of vermicompost showed synergistic effect when combined with endophytic bacteria. Thus, the innovation of the combination of the studied factors may contribute to the early development of maize.

Adventitious rooting in cuttings of croton and hibiscus in response to indolbutyric acid and humic acid

Adventitious rooting of ornamental plants can be accelerated by the application of growth regulators, such as auxin. Humic acids, organic matter in soil and organic compounds also have a biostimulant effect. This work evaluated the rooting in cuttings of croton (Codianeum variegatum L. Rumph) and hibiscus (Hibiscus rosa-sinensis L) in response to the application of different concentrations of indolbutyric acid (IBA) and humic acid (HA). The experiment was carried out in a greenhouse. Apical stem cuttings were treated with solutions at concentrations of: 0, 250, 500, 1000, 2000 mg L-1 IBA and 0, 10, 20, 30, 40 mmol L-1 HA carbon isolated from vermicomposting. Forty-five days after the applications, the cuttings were removed from the pots containing carbonized rice hull and the following variables were measured: rooting number, length and width of leaves, fresh and dry matter of root and aerial part and root area. The results were subjected to analysis of variance and the qualitative and quantitative effects of the treatments were compared by contrast and regression, respectively. Regression equations were used to determine the maximum efficiency level of root dry matter according to IBA and HA. Higher accumulation of root dry matter was recorded for the treatments with the doses 579 mg L-1 IBA and 14 mmol L-1 HA and 970 mg L-1 IBA and 50 mmol L-1 HA for root cuttings of croton and hibiscus, respectively. It was found that the application of eiher IBA or HA at the indicated doses accelerates rooting in cuttings of croton and hibiscus and contributes to the formation of vigorous plants.

Initial performance of maize in response to NPK fertilization combined with Herbaspirillum seropedicae

The inoculation with plant growth-promoting bacteria can be a technological approach useful for increasing the production of maize. The objective of this study was to evaluate the initial performance of maize in response to application of doses of NPK combined with the inoculation of the diazotrophic bacteria Herbaspirillum seropedicae in an greenhouse experiment. The experiment consisted of six fertilizer levels: 0, 25, 50, 75, 100 and 200% of the recommended dose of NPK applied to maize inoculated and non-inoculated with H. seropedicae. At 30 days after the treatment application, the growth characteristics and nutritional status of the plants were evaluated. Plant development was influenced by fertilization, but it was enhanced by combination with the bacteria, which resulted in significant increases in the dry mass of shoots (7%) and leaf area (9%) when compared with non-inoculated plants. The results showed increases in the concentration of N (11%), P (30%) and K (17%) of maize plants in response to bacterial inoculation together with NPK compared with plants that were applied fertilize only. The greater consistency and stability response of the host plant to bacterization in the presence of chemical fertilizer indicate a promissory biotechnological approach for improving the initial growth and adaptation of maize to the cultivation environment.

Response of rice cultivars to nitrogen in upland conditions

Against the background of a growing world population, rice (Oryza sativa L.) consumption is expected to grow faster than its production. Therefore, an appropriate question would be: how to increase productivity in the short-term? In this respect, it becomes important the implementation of modern agricultural production systems, such as upland rice with supplemental sprinkler irrigation. Additional information is needed to maximize the available resources, with special attention given to research on the use of nitrogen. This study aimed to evaluate the agronomic performance of commercial rice cultivars with different plant characteristics in upland conditions with supplemental sprinkler irrigation, when subjected to nitrogen in topdress application at the R1 stage (panicle differentiation). The experiment was arranged in a randomized block with split plot design, with 65 treatments, consisting of the combination of 13 cultivars in the plots, and five nitrogen levels in the subplots (0, 40, 80, 120 and 160 kg ha-1), with four replications. Genetic variability was detected among rice cultivars and the agronomic performance in response to the applied nitrogen. The topdressing application of nitrogen increases, in general, the production components and grain yield in rice. Cultivars BRS Primavera, Caiapó and IAC 202 stood out for grain yield, followed by Baldo, Carnaroli, BRS Curinga and IAC 500 with lower yields.

No-till corn performance in response to P and fertilization modes

No-tillage systems provide soil changes that affect nutrient dynamics, hence, changing rates and forms of fertilizer application. This study aimed to evaluate the effect of phosphorus (P) and modes of nitrogen (N) and P application in corn under long-term no-tillage in a clayey Oxisol. Two experiments were carried out in the same experimental area and in the same year, in a randomized blocks design with four replications. In experiment I, the treatments consisted of five doses of phosphorus (0, 40, 80, 120 and 160 kg ha-1 of P2O5) applied in the sowing furrow. In experiment II, the treatments consisted of the N and P application modes (topdressing, in the sowing furrow and control - without N and P). Experiment I evaluated the root length, P uptake and grain yield and, the Experiment II, the firing height and yield. The P rates provided linear increases in root length in the 0-10 cm layer, P uptake and grain production. The different modes of application provided differences in the firing height and corn yield. The control treatment (0 kg ha-1 of N and P) provided the highest firing height, superior than those of topdressing and application in the furrow, which were not significantly different. The topdress application of N and P provided an increase in corn yield that exceeded 16 and 42% of the application in the furrow and the control, respectively. Thus, the results confirmed that increasing rates of P2O5, in soil with high initial P content, influence positively corn production factors, but with little significant responses, and the topdress application of N and P on soil with high P content, without water restriction, provided increased grain yield in relation to the application in the furrow.

Gladiolus development in response to bulb treatment with different concentrations of humic acids

Gladiolus is an ornamental species produced for cut flowers and propagated by corms. The early flowering and increase in the number of flower buds, besides the production of commercial corms are constant challenges to be addressed in the crop improvement. Commercial production of ornamentals is technologically accelerated by means of growth regulators. Among them, the auxins stand out for their key role in the adventitious rooting and cell elongation. Alternatively, the humic substances present in the organic matter also have biostimulating effect, which is very similar to the auxinic effect. Therefore, this work aimed to study the growth and development of gladiolus in response to application of different concentrations of humic acids (HA) isolated from vermicompost. Corms were soaked for 24 hours in solutions containing 0, 10, 20, 30 and 40 mmol L-1 of C from HA. The corms were planted in 10-dm³ plastic bags filled with substrate and kept in a greenhouse. Growth of shoots and roots was evaluated. The results showed that the use of HA accelerates growth, and anticipates and increases flowering of Gladiolus.