Pim-1 kinase inhibits the activation of reporter gene expression in Elk-1 and c-Fos reporting systems but not the endogenous gene expression: an artifact of the reporter gene assay by transient co-transfection

We have studied the molecular mechanism and signal transduction of pim-1, an oncogene encoding a serine-threonine kinase. This is a true oncogene which prolongs survival and inhibits apoptosis of hematopoietic cells. In order to determine whether the effects of Pim-1 occur by regulation of the mitogen-activated protein kinase pathway, we used a transcriptional reporter assay by transient co-transfection as a screening method. In this study, we found that Pim-1 inhibited the Elk-1 and NFkappaB transcriptional activities induced by activation of the mitogen-activated protein kinase cascade in reporter gene assays. However, Western blots showed that the induction of Elk-1-regulated expression of endogenous c-Fos was not affected by Pim-1. The phosphorylation and activation of neither Erk1/2 nor Elk-1 was influenced by Pim-1. Also, in the gel shift assay, the pattern of endogenous NFkappaB binding to its probe was not changed in any manner by Pim-1. These data indicate that Pim-1 does not regulate the activation of Erk1/2, Elk-1 or NFkappaB. These contrasting results suggest a pitfall of the transient co-transfection reporter assay in analyzing the regulation of transcription factors outside of the chromosome context. It ensures that results from reporter gene expression assay should be verified by study of endogenous gene expression.

In vitro antifungal activities of leaf extracts of Lippia alba (Verbenaceae) against clinically important yeast species

Introduction There are few studies reporting the antifungal activities of Lippia alba extracts. Methods A broth microdilution assay was used to evaluate the antifungal effects of Lippia alba extracts against seven yeast species of Candida and Cryptococcus. The butanol fraction was investigated by gas chromatography-mass spectrometry. Results The butanol fraction showed the highest activity against Candida glabrata. The fraction also acted synergistically with itraconazole and fluconazole against C. glabrata. The dominant compounds in the butanol fraction were 2,2,5-trimethyl-3,4-hexanedione, 3,5-dimethyl-4-octanone and hexadecane. Conclusions The butanol fraction may be a good candidate in the search for new drugs from natural products with antifungal activity.

Genotyping of human parvovirus B19 in clinical samples from Brazil and Paraguay using heteroduplex mobility assay, single-stranded conformation polymorphism and nucleotide sequencing

Heteroduplex mobility assay, single-stranded conformation polymorphism and nucleotide sequencing were utilised to genotype human parvovirus B19 samples from Brazil and Paraguay. Ninety-seven serum samples were collected from individuals presenting with abortion or erythema infectiosum, arthropathies, severe anaemia and transient aplastic crisis; two additional skin samples were collected by biopsy. After the procedure, all clinical samples were classified as genotype 1.

Nitrate reductase activity and spad readings in leaf tissues of guinea grass submitted to nitrogen and potassium rates

Nitrogen and K deficiency are among the most yield limiting factors in Brazilian pastures. The lack of these nutrients can hamper the chlorophyll biosynthesis and N content in plant tissues. A greenhouse experiment was carried out to evaluate the relationship among N and K concentrations, the indirect determination of chlorophyll content (SPAD readings), nitrate reductase activity (RNO3-) in newly expanded leaf lamina (NL) and the dry matter yield for plant tops of Mombaça grass (Panicum maximum Jacq.). A fractionated 5² factorial design was used, with 13 combinations of N and K rates in the nutrient solution. The experimental units were arranged in a randomized block design, with four replications. Plants were harvested twice. The first harvest occurred 36 days after seedling transplanting and the second 29 days after the first. Significance occurred for the interaction between the N and K rates to SPAD readings and to RNO3- assessment taken on the NL during the first growth. Besides, RNO3- and SPAD readings increased only with the NL N concentration, reaching the highest values of both variables up to about 25 g kg-1, but were ratively constant at higher leaf N. Significant relationships either between SPAD readings or RNO3- activity and shoot dry mass weight were also observed. The critical levels of N concentration in the NL were, respectively, 22 and 17g kg-1 in the first and second harvest. Thus, SPAD instrument and RNO3- assessment can be used as complementary tools to evaluate the N status in forage grass.

Optimization of invertase assay conditions in rubber tree plants (Hevea brasiliensis Muell. Arg.)

The objective of this work was to define the optimal conditions for invertase assay, seeking to determine the ideal parameters for the different isoenzymes of leaf and bark tissues in adult rubber trees. Assays of varying pH, sucrose concentration and temperature of the reaction medium were conducted for the two investigated isoenzymes. The results pointed out the existence of two different pH related isoforms for the two analyzed tissues, with an isoenzyme being more active at pH 5,5 and the other at neutral/alkaline pH. Leaf blade isoenzymes presented similar values for substrate concentration, whereas the bark isoenzyme presented maximum values below those previously reported. The assays at different temperatures presented similar values for leaf isoenzymes, though they have differed significantly among the obtained values.

Transient expression of a reporter gene introduced by bioballistic bombardment into Racosperma mangium (Leguminosae family) tissues

We report on an assay of direct transfer of DNA into calli and seeds of Racosperma (ex-Acacia) mangium, using a bioballistic method. We observed transient expression of the GUS gene in the treated tissues

Cytotoxicity of Marchantia convoluta leaf extracts to human liver and lung cancer cells

The cytotoxicity of three extracts (petroleum ether, ethyl acetate and n-butanol) from a plant used in folk medicine, Marchantia convoluta, to human non-small cell lung carcinoma (H1299) and liver carcinoma (HepG2) cell lines was tested. After 72-h incubation of lung and liver cancer cell cultures with varying concentrations of extracts (15 to 200 µg/mL), cytotoxicity was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and reported in terms of cell viability. The extracts that showed a significant cytotoxicity were subjected to gas chromatography-mass spectrometry analysis to identify the components. The ethyl acetate, but not the petroleum ether or n-butanol extract, had a significant cytotoxicity against lung and liver carcinoma cells with IC50 values of 100 and 30 µg/mL, respectively. A high concentration of ethyl acetate extract (100 µg/mL) rapidly reduced the number of H1299 cells. At lower concentrations of ethyl acetate extract (15, 30, and 40 µg/mL), the numbers of HepG2 cells started to decrease markedly. Gas chromatography-mass spectrometry analysis of the ethyl acetate extract revealed the presence of several compounds such as phytol (23.42%), 1,2,4-tripropylbenzene (13.09%), 9-cedranone (12.75%), ledene oxide (7.22%), caryophyllene (1.82%), and caryophyllene oxide (1.15%). HPLC analysis result showed that there were no flavonoids in ethyl acetate extract, but flavonoids are abundant in n-butanol extract. Further studies are needed regarding the identification, toxicity, and mechanism of action of active compounds.

Transient high-level expression of ß-galactosidase after transfection of fibroblasts from GM1 gangliosidosis patients with plasmid DNA

GM1 gangliosidosis is an autosomal recessive disorder caused by the deficiency of lysosomal acid hydrolase ß-galactosidase (ß-Gal). It is one of the most frequent lysosomal storage disorders in Brazil, with an estimated frequency of 1:17,000. The enzyme is secreted and can be captured by deficient cells and targeted to the lysosomes. There is no effective treatment for GM1 gangliosidosis. To determine the efficiency of an expression vector for correcting the genetic defect of GM1 gangliosidosis, we tested transfer of the ß-Gal gene (Glb1) to fibroblasts in culture using liposomes. ß-Gal cDNA was cloned into the expression vectors pSCTOP and pREP9. Transfection was performed using 4 µL lipofectamine 2000 and 1.5-2.0 µg DNA. Cells (2 x 10(5)/well) were harvested 24 h, 48 h, and 7 days after transfection. Enzyme specific activity was measured in cell lysate and supernatant by fluorometric assay. Twenty-four hours after transfection, treated cells showed a higher enzyme specific activity (pREP9-ß-Gal: 621.5 ± 323.0, pSCTOP-ß-Gal: 714.5 ± 349.5, pREP9-ß-Gal + pSCTOP-ß-Gal: 1859.0 ± 182.4, and pREP9-ß-Gal + pTRACER: 979.5 ± 254.9 nmol·h-1·mg-1 protein) compared to untreated cells (18.0 ± 3.1 for cell and 32.2 ± 22.2 nmol·h-1·mg-1 protein for supernatant). However, cells maintained in culture for 7 days showed values similar to those of untreated patients. In the present study, we were able to transfect primary patients' skin fibroblasts in culture using a non-viral vector which overexpresses the ß-Gal gene for 24 h. This is the first attempt to correct fibroblasts from patients with GM1 gangliosidosis by gene therapy using a non-viral vector.

Variability in leaf rust susceptibility among peach cultivars

The aim of this work was to estimate the susceptibility of thirty-six peach cultivars to leaf rust caused by Tranzschelia discolor f. sp. persica. The incidence and severity of the disease as well as defoliation in peach trees of an experimental orchard of Parana Federal University of Technology, Campus Dois Vizinhos were evaluated on the growing seasons 2004/2005 and 2005/2006. Immunity to this disease was not observed in the studied cultivars. There was difference in leaf rust intensity depending on the growing season conditions. Cultivars 'Pilcha', 'Sinuelo', 'Chirua', 'Sulina', 'Eldorado' and 'Pampeano' showed tolerance to leaf rust, whereas cultivars 'Vila Nova', 'Fla 1372', 'Coral 2', 'Chimarrita', 'Della Nona', 'BR-1 ' and 'Guaiaca' showed high susceptibility.

Influence of leaf retention on cutting propagation of Lavandula dentata L.

Vegetative propagation of lavender offers several advantages over sexual propagation, among them crop homogeneity and yield of higher quality essential oil. However, Lavandula species have been propagated mostly by seeds and are said to be recalcitrant to rooting when propagated by cuttings. During cutting propagation, one of the important variables that influence the rooting capacity of cuttings is the leaf retention. The objective of this work was to evaluate the influence of leaf retention on rooting of L. dentata cuttings. Apical cuttings of L. dentata of 10 cm in length, keeping approximately 1/3, 1/2 or 2/3 of their leaves were planted in commercial substrate Plantmax HT® under intermittent mist. After two months, averages of root number, length of the longest root, root fresh and dry weights, and the survival percentage were evaluated. Root length and fresh weight were statistically greater with 2/3 of leaf retention and when fewer leaves were kept on the cuttings, lower means of root dry weight was observed. Under the conditions applied in this study, greater leaf retention was better for rooting of L. dentata cuttings.

Initial performance of pineapple and utilization of rock phosphate applied in combination with organic compounds to leaf axils

Rock phosphates have low solubility in water, but good solubility in acid. The use of organic compounds together with these phosphorus sources applied to the basal leaf axils of pineapple can increase the solubility of this phosfate source and increase the P availability to the crop. A greenhouse experiment was conducted using Araxá rock phosphate (10 g) in combination or not with solutions containing increasing concentrations of humic acids (0 to 40 mmol L-1 of carbon), with or without citric acid (0.005 mmol L-1), applied to basal leaf axils of pineapple cv. Pérola. Growth and nutritional characteristics of aerial plant parts were assessed. Growth rates of aerial parts and N, P, K, Ca and Mg contents increased curvilinearly with increasing concentration of carbon in the form of humic acids. Maximum values were found for the concentration of 9.3 mmol L-1 of carbon combined with 0.005 mmol L-1 of citric acid and natural phosphate.

The effect of leaf area reduction on corn plants during the reproduction phase

Reduction in leaf area in corn plants during reproduction changes physiological metabolism and consequently the accumulation of dry matter in grains. The aim of this work was to study changes in agronomic characteristics caused by defoliation in corn during the reproduction phase. The experiment was carried out in Uberlândia, Minas Gerais state, in the agricultural year 2007/2008. The experiment was arranged in a randomized block design, consisting of seven treatments: control without defoliation, removal of two apical leaves, removal of four apical leaves, removal of all leaves above spike, removal of four intermediate leaves, removal of all leaves below spike, and removal of all plant leaves, with five repetitions. The genotype used for the evaluations was hybrid NB 7376. Defoliation was carried out when plants were at the growth stage R2. The variables assessed were: yield, density of spikes and corncobs, root resistance and stem integrity. When all leaves above the spike were removed, grain yield was reduced by 20%. Corncob density, stem integrity and root resistance to uprooting were also affected. Spike density was only affected when all plant leaves were removed. The leaf area remaining physiologically active above the spike was found to be most efficient in terms of grain yield.

Leaf area and epicuticular wax content of Sida spp

Morpho-physiological characteristics and chemical composition are directly related to superior competitive ability of crops. This study intended to make a comparative analysis of dry matter production, leaf area and amount of epicuticular wax of three species of Sida spp: S. urens L., S. rhombifolia L. and S. spinosa L. Plants were collected at three growth stages: V1: stage described as up to 10 fully expanded leaves; V2: between 11 leaves and flowering; and R: after flowering. At stages V2 and R, the highest number of leaves was recorded for S. rhombifolia, followed by S. spinosa at V2 and S. urens at R. These results were relatively proportional to leaf area for all species. S. spinosa at the vegetative stage produced the highest values of specific leaf area (SLA), with no significant differences between species at the stage R. The amount of wax per unit of leaf area between species at the same developmental stage was significantly different only at the reproductive stage, where S. spinosa produced 23.18 and 6.23 fold more wax than S. urens and S. rhombifolia respectively. Between the growth stages of each species, there was decrease in the amount of wax with plant age and increase in leaf area (AFE), number of leaves and dry matter. The leaves of the Sida species exhibit different characteristics and this information can be used to optimize the use of herbicides in the control of these weeds.

In vitro growth and leaf anatomy of Cattleya walkeriana (Gardner, 1839) grown in natural ventilation system

Natural ventilation system facilitates gaseous exchanges in in vitro plants promoting changes in the leaf tissue, which can be evaluated through the leaf anatomy, and it allows a cultivation closer to the photoautrophic micropropagation. The objective of this work was to evaluate the effects on in vitro growth and on the leaf anatomy of Cattleya walkeriana grown in natural and conventional ventilation system with different concentrations of sucrose (0; 15; 30 and 45 L-1) combined with different cultivation systems (conventional micropropagation and natural ventilation system). The culture medium was composed of MS salts, solidified with 7 g L-1 of agar and pH adjusted to 5.8. Forty milliliters of culture medium were distributed in 250 mL flasks, autoclaved at 120 ºC for 20 minutes. The greater plant growth, as well as the greater thickness of the mesophyll was observed with the use of 20 g L-1 sucrose in natural ventilation system. Plants grown in natural ventilation system showed a thicker leaf mesophyll, which is directly related to photoautotrophic crops. The natural ventilation system induced more elliptical stomata and probably more functional formats.

Juvenile tree growth correlates with photosynthesis and leaf phosphorus content in central Amazonia

Light and soil water availability may limit carbon uptake of trees in tropical rainforests. The objective of this work was to determine how photosynthetic traits of juvenile trees respond to variations in rainfall seasonality, leaf nutrient content, and opening of the forest canopy. The correlation between leaf nutrient content and annual growth rate of saplings was also assessed. In a terra firme rainforest of the central Amazon, leaf nutrient content and gas exchange parameters were measured in five sapling tree species in the dry and rainy season of 2008. Sapling growth was measured in 2008 and 2009. Rainfall seasonality led to variations in soil water content, but it did not affect leaf gas exchange parameters. Subtle changes in the canopy opening affected CO2 saturated photosynthesis (A pot, p = 0.04). Although A pot was affected by leaf nutrient content (as follows: P > Mg > Ca > N > K), the relative growth rate of saplings correlated solely with leaf P content (r = 0.52, p = 0.003). At present, reduction in soil water content during the dry season does not seem to be strong enough to cause any effect on photosynthesis of saplings in central Amazonia. This study shows that leaf P content is positively correlated with sapling growth in the central Amazon. Therefore, the positive effect of atmospheric CO2 fertilization on long-term tree growth will depend on the ability of trees to absorb additional amount of P