Transgenic animal models for the functional analysis of vasoactive peptides

The interplay of vasoactive peptide systems is an essential determinant of blood pressure regulation in mammals. While the endothelin and the renin-angiotensin systems raise blood pressure by inducing vasoconstriction and sodium retention, the kallikrein-kinin and the natriuretic-peptide systems reduce arterial pressure by eliciting vasodilatation and natriuresis. Transgenic technology has proven to be very useful for the functional analysis of vasoactive peptide systems. As an outstanding example, transgenic rats overexpressing the mouse Ren-2 renin gene in several tissues become extremely hypertensive. Several other transgenic rat and mouse strains with genetic modifications of components of the renin-angiotensin system have been developed in the past decade. Moreover, in recent years gene-targeting technology was employed to produce mouse strains lacking these proteins. The established animal models as well as the main insights gained by their analysis are summarized in this review.

The DNA puff BhB10-1 gene is differentially expressed in various tissues of Bradysia hygida late larvae and constitutively transcribed in transgenic Drosophila

We extended the characterization of the DNA puff BhB10-1 gene of Bradysia hygida by showing that, although its mRNA is detected only at the end of the fourth larval instar, BhB10-1 expression is not restricted to the salivary gland, the tissue in which this gene is amplified. Different amounts of BhB10-1 mRNA were detected in other larval tissues such as gut, Malpighian tubules, fat body, brain and cuticle, suggesting that this gene is expressed differentially in the various tissues analyzed. Analysis of transgenic Drosophila carrying the BhB10-1 transcription unit and flanking sequences revealed that the tested fragment promotes transcription in a constitutive manner. We suggest that either cis-regulatory elements are missing in the transgene or factors that temporally regulate the BhB10-1 gene in B. hygida are not conserved in Drosophila.

Can organic and transgenic soy be used as a substitute for animal protein by rats?

We evaluated the protein quality of organic and transgenic soy fed to rats throughout life. Thirty female Wistar rats were divided into three groups (N = 10): organic soy group (OSG) receiving organic soy-based diet, genetically modified soy group (GMSG) receiving transgenic soy-based diet, and a control group (CG) receiving casein-based diet. All animals received water and isocaloric diet (10% protein), ad libitum for 291 days. After this, the weight of GMSG animals (290.9 ± 9.1 g) was significantly lower (P <= 0.04) than CG (323.2 ± 7.9 g). The weight of OSG (302.2 ± 8.7 g) was between that of the GMSG and the CG. Protein intake was similar for OSG (308.4 ± 6.8 g) and GMSG (301.5 ± 2.5 g), and significantly lower (P <= 0.0005) than the CG (358.4 ± 8.1 g). Growth rate was similar for all groups: OSG (0.80 ± 0.02 g), GMSG (0.81 ± 0.03 g) and CG (0.75 ± 0.02 g). In addition to providing a good protein intake and inducing less weight gain, both types of soy were utilized in a manner similar to that of casein, suggesting that the protein quality of soy is similar to that of the standard protein casein. The groups fed soy-based diet gained less weight, which may be considered to be beneficial for health. We conclude that organic and transgenic soy can be fed throughout life to rats in place of animal protein, because contain high quality protein and do not cause a marked increase in body weight.

Asynchronous expression of myeloid antigens in leukemic cells in a PML/RARalpha transgenic mouse model

Acute promyelocytic leukemia (APL) is characterized by the expansion of blasts that resemble morphologically promyelocytes and harbor a chromosomal translocation involving the retinoic acid receptor a (RARa) and the promyelocytic leukemia (PML) genes on chromosomes 17 and 15, respectively. The expression of the PML/RARa fusion gene is essential for APL genesis. In fact, transgenic mice (TM) expressing PML/RARa develop a form of leukemia that mimics the hematological findings of human APL. Leukemia is diagnosed after a long latency (approximately 12 months) during which no hematological abnormality is detected in peripheral blood (pre-leukemic phase). In humans, immunophenotypic analysis of APL blasts revealed distinct features; however, the precise immunophenotype of leukemic cells in the TM model has not been established. Our aim was to characterize the expression of myeloid antigens by leukemic cells from hCG-PML/RARa TM. In this study, TM (N = 12) developed leukemia at the mean age of 13.1 months. Morphological analysis of bone marrow revealed an increase of the percentage of immature myeloid cells in leukemic TM compared to pre-leukemic TM and wild-type controls (48.63 ± 16.68, 10.83 ± 8.11, 7.4 ± 5.46%, respectively; P < 0.05). Flow cytometry analysis of bone marrow and spleen from leukemic TM identified the asynchronous co-expression of CD34, CD117, and CD11b. This abnormal phenotype was rarely detected prior to the diagnosis of leukemia and was present at similar frequencies in hematologically normal TM and wild-type controls of different ages. The present results demonstrate that, similarly to human APL, leukemic cells from hCG-PML/RARa TM present a specific immunophenotype.

Lack of evidence for regulation of cardiac P-type ATPases and MAP kinases in transgenic mice with cardiac-specific overexpression of constitutively active α1B-adrenoceptors

The regulatory function of α1B-adrenoceptors in mammalian heart homeostasis is controversial. The objective of the present study was to characterize the expression/activity of key proteins implicated in cardiac calcium handling (Na+/K+-ATPase and Ca2+-ATPases) and growth (ERK1/2, JNK1/2 and p38) in mice with cardiac-selective overexpression of constitutively active mutant α1B-adrenoceptor (CAMα1B-AR), which present a mild cardiac hypertrophy phenotype. Immunoblot assays showed that myocardial plasma membrane Ca2+-ATPase (PMCA) expression was increased by 30% in CAMα1B-AR mice (N = 6, P < 0.05), although there was no change in sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2) expression. Moreover, total Ca2+-ATPase activity was not modified, but a significant increase in the activity of the thapsigargin-resistant (PMCA) to thapsigargin-sensitive (SERCA) ratio was detected. Neither Na+/K+-ATPase activity nor the expression of α1 and α2 subunit isoforms was changed in CAMα1B-AR mouse hearts. Moreover, immunoblot assays did not provide evidence for an enhanced activation of the three mitogen-activated protein kinases studied in this stage of hypertrophy. Therefore, these findings indicate that chronic cardiac α1B-AR activation in vivo led to mild hypertrophy devoid of significant signs of adaptive modifications concerning primary intracellular calcium control and growth-related proteins, suggesting a minor pathophysiological role of this adrenergic receptor in mouse heart at this stage of development.

Hydration kinetics, physicochemical composition, and textural changes of transgenic corn kernels of flint, semi-flint, and dent varieties

The hydration kinetics of transgenic corn types flint DKB 245PRO, semi-flint DKB 390PRO, and dent DKB 240PRO was studied at temperatures of 30, 40, 50, and 67 °C. The concentrated parameters model was used, and it fits the experimental data well for all three cultivars. The chemical composition of the corn kernels was also evaluated. The corn cultivar influenced the initial rate of absorption and the water equilibrium concentration, and the dent corn absorbed more water than the other cultivars at the four temperatures analyzed. The effect of hydration on the kernel texture was also studied, and it was observed that there was no significant difference in the deformation force required for all three corn types analyzed with longer hydration period.

Effect of storage period on isoflavone content and physiological quality of conventional and transgenic soybean seeds

The objective in this research was to evaluate the isoflavone content and the physiological quality of seed from conventional and transgenic soybean cultivars before and after 180 days of storage. Twenty one soybean cultivars: CD 202, CD 206, CD 208, CD 213RR, CD 214RR, CD 215, CD 216, CD 217, CD 218, CD 221, BRS 184, BRS 185, BRS 214, BRS 244RR, BRS 245RR, BRS 246RR, BRS 255, BRS 257, BRS 258, BRS 261 and BRS 262, grown in the 2005/2006 crop season, were assayed. The seeds were packed in Kraft paper bags and stored at room temperature under laboratory conditions. Seeds were evaluated with respect to their germination and vigor (first germination count, accelerated aging and tetrazolium test) and their total isoflavone contents and respective aglycon forms (daidzein, genistein and glycitein),glycosides (daidzine, genistine and glycitine) and malonyl conjugates. A completely randomized block design with six replications with the treatments set out within a subplot scheme (21 cultivars x 2 storage periods) was used. The F-test was used to compare means between storage periods and the Scott-Knott test to compare cultivars for each storage period, both with a 95% probability. It was concluded that isoflavone contents differ between cultivars and show a distinct behavior throughout storage.

Detection of adventitious presence of genetically modified seeds in lots of non transgenic soybean

The difficulty on identifying, lack of segregation systems and absence of suitable standards for coexistence of non trangenic and transgenic soybean are contributing for contaminations that occur during productive system. The objective of this study was to evaluate the efficiency of two methods for detecting mixtures of seeds genetically modified (GM) into samples of non-GM soybean, in a way that seed lots can be assessed within the standards established by seed legislation. Two sizes of soybean samples (200 and 400 seeds), cv. BRSMG 810C (non-GM) and BRSMG 850GRR (GM), were assessed with four contamination levels (addition of GM seeds, for obtaining 0.0%, 0.5%, 1.0%, and 1.5% contamination), and two detection methods: immunoassay of lateral flux (ILF) and bioassay (pre-imbibition into 0.6% herbicide solution; 25 ºC; 16 h). The bioassay is efficient in detecting presence of GM seeds in seed samples of non-GM soybean, even for contamination lower than 1.0%, provided that seeds have high physiological quality. The ILF was positive, detecting the presence of target protein in contaminated samples, indicating test effectiveness. There was significant correlation between the two detection methods (r = 0.82; p < 0.0001). Sample size did not influence efficiency of the two methods in detecting presence of GM seeds.

Investigating the environmental risks of transgenic crops

Legitimation of public policies that support the widespread plantings of transgenic crops presuppose, among other conditions, that (1) evidence supports that there are no unmanageable environmental risks and (2) there are no better ways to produce enough nourishing food that can dispense with the transgenics-oriented ways. This paper discusses: (a) the kinds of scientific inquiry that are needed to address (1) adequately, (b) the connections between investigations of (1) and (2), and (c) how these investigations are related with controversial social values.

Lignification of the plant and seed quality of RR soybeans sprayed with herbicide glyphosate

Differences in levels of lignin in the plant between conventional and transgenic cultivars RR has been reported by several authors, however, there are few studies evaluating the influence of spraying of glyphosate on the lignin in the plant and RR soybean seeds. The aim of this study was to evaluate the physiological quality of RR transgenic soybean seeds and the lignin contents of plants sprayed with the herbicide glyphosate. The assays were conducted both in greenhouse and field in the municipality of Lavras, MG, in the agricultural year 2007/08. The experiment was arranged in a splitplot design with four replicates, considering the treatments hand weeding and herbicide glyphosate as plots, and five RR soybean cultivars (BRS 245 RR, BRS 247 RR, Valiosa RR, Silvânia RR and Baliza RR) as splitplots. In the greenhouse, the cultivars tested were BRS 245 RR and Valiosa RR in a randomized block design with four replicates. The sprayings were carried out at stages V3, V7 and early R5 (3L/ha). The 1000 seed weight, mechanical injury, germination and germination velocity index, emergence velocity index, accelerated aging, electrical conductivity and water soaking seed test, lignin content in the seed coat, in the stem and legumes were determined. The spraying of glyphosate herbicide, in greenhouse and field, did not alter the physiological quality of seeds and the lignin contents in the plant.

Rootstocks resistant to Meloidogyne incognita and compatibility of grafting in net melon

Due to the few studies about grafting in net melon, in order to obtain better control of soil pathogens, the aim of the present study was to evaluate 16 genotypes of Cucurbitaceae: Benincasa hispida, Luffa cylindrica, pumpkin 'Jacarezinho', pumpkin 'Menina Brasileira', squash 'Exposição', squash 'Coroa', pumpkin 'Canhão Seca', pumpkin 'Squash', pumpkin 'Enrrugado Verde', pumpkin 'Mini Paulista', pumpkin 'Goianinha', watermelon 'Charleston Gray', melon 'Rendondo Gaucho', melon 'Redondo Amarelo', cucumber 'Caipira HS' and cucumber 'Caipira Rubi', regarding to compatibility of grafting in net melon and resistance to Meloidogyne incognita, based on the reproduction factor (RF), according to Oostenbrink (1966). To assess resistance, the seedlings were transplanted to ceramic pots and inoculated with 300/mL eggs and/or second stage juveniles of M. incognita. At 50 days after transplanting, the plants were removed from the pots and the resistance was evaluated. The compatibility between resistant rootstock and grafts of net melon was determined by performing simple cleft grafting, in a commercial net melon hybrid of great market acceptance and susceptible to M. incognita (Bonus no. 2). The genotypes Luffa cylindrica, pumpkin 'Goianinha', pumpkin 'Mini-Paulista', melon 'Redondo Amarelo', watermelon 'Charleston Gray' are resistant to the nematode M. incognita. The better compatibilities occurred with the rootstocks melon 'Amarelo', which presented 100% of success, followed by pumpkin 'Mini-Paulista' with 94%. On the other hand, Sponge gourd, watermelon 'Charleston Gray' and pumpkin 'Goianinha' showed low graft take percentages of 66%, 62% and 50%, respectively.

(Nota n.º 3) Nematódeos da aboboreira

In the State of S. Paulo, Brazil, squash plants (Cucurbita spp.) are attacked by the Javanese nematode (Meloidogyne javanica) and by M. incognita acrita. Squash belongs to that group of plants in which the root-knot nematodes break through the root surface, so that the egg-producing females protrude from the root, showing yellowish or brow nish egg masses attached to them. Washed roots show numerous small dark spots, each corresponding to an ootheca, which is adhering to a mature female. A curious abnormal female of M. i. acrita was obtained from a sample of squash roots. This female's body showed two globular parts, separated by a deep constriction. The convoluted ovaries were found to fill both portions of the body.

Molecular biological approaches to the study of vectors in relation to malaria control

To a large extent, control of malaria vectors relies on the elimination of breeding sites and the application of chemical agents. There are increasing problems associated with the use of synthetic insecticides for vector control, including the evolution of resistance, the high cost of developing and registering new insecticides and an awareness of pollution from insecticide residues. These factors have stimulated interest in the application of molecular biology to the study of mosquito vectors of malaria; focussing primarily on two aspects. First, the improvement of existing control measures through the development of simplified DNA probe systems suitable for identification of vectors of malaria. The development of synthetic, non-radioactive DNA probes suitable for identification of species in the Anopheles gambiae complex is described with the aim of defining a simplified methodology wich is suitable for entomologist in the field. The second aspect to be considered is the development of completely novel strategies through the development of completely novel strategies through the genetic manipulation of insect vectors of malaria in order to alter their ability to transmit the disease. The major requirements for producing transgenic mosquitoes are outlined together with the progress wich has been made to date and discussed in relation to the prospects which this type of approach has for the future control of malaria.

Characterization of constitutive and putative differentially expressed mRNAs by means of expressed sequence tags, differential display reverse transcriptase-PCR and randomly amplified polymorphic DNA-PCR from the sand fly vector Lutzomyia longipalpis

Molecular studies of insect disease vectors are of paramount importance for understanding parasite-vector relationship. Advances in this area have led to important findings regarding changes in vectors' physiology upon blood feeding and parasite infection. Mechanisms for interfering with the vectorial capacity of insects responsible for the transmission of diseases such as malaria, Chagas disease and dengue fever are being devised with the ultimate goal of developing transgenic insects. A primary necessity for this goal is information on gene expression and control in the target insect. Our group is investigating molecular aspects of the interaction between Leishmania parasites and Lutzomyia sand flies. As an initial step in our studies we have used random sequencing of cDNA clones from two expression libraries made from head/thorax and abdomen of sugar fed L. longipalpis for the identification of expressed sequence tags (EST). We applied differential display reverse transcriptase-PCR and randomly amplified polymorphic DNA-PCR to characterize differentially expressed mRNA from sugar and blood fed insects, and, in one case, from a L. (V.) braziliensis-infected L. longipalpis. We identified 37 cDNAs that have shown homology to known sequences from GeneBank. Of these, 32 cDNAs code for constitutive proteins such as zinc finger protein, glutamine synthetase, G binding protein, ubiquitin conjugating enzyme. Three are putative differentially expressed cDNAs from blood fed and Leishmania-infected midgut, a chitinase, a V-ATPase and a MAP kinase. Finally, two sequences are homologous to Drosophila melanogaster gene products recently discovered through the Drosophila genome initiative.

Germline transformation of Aedes fluviatilis (Diptera:Culicidae) with the piggyBac transposable element

The technique to generate transgenic mosquitoes requires adaptation for each target species because of aspects related to species biology, sensitivity to manipulation and rearing conditions. Here we tested different parameters on the microinjection procedure in order to obtain a transgenic Neotropical mosquito species. By using a transposon-based strategy we were able to successfully transform Aedes fluviatilis (Lutz), which can be used as an avian malaria model. These results demonstrate the usefulness of the piggyBac transposable element as a transformation vector for Neotropical mosquito species and opens up new research frontiers for South American mosquito vectors.