NtWBC1, an ABC transporter gene specifically expressed in tobacco reproductive organs

To identify genes specifically or predominantly expressed in the stigmas/styles and to establish their possible function in the reproductive process of plants, a tobacco stigma/style cDNA library was constructed and differentially screened, resulting in the isolation of several cDNA clones. The molecular characterization of one of these clones is described here. After sequencing the cDNA and the isolated genomic clone, it was determined that the corresponding gene encodes a protein containing an ATP-binding cassette, characteristic of ABC transporters. This gene, designated as NtWBC1 (Nicotiana tabacum ABC transporter of the White-Brown Complex subfamily), encodes a protein that contains the typical structure of the 'half-transporters' of the White subfamily. To establish the spatial expression pattern of the NtWBC1 gene, northern blot and real-time RT-PCR analyses with total RNA from roots, stems, leaves, sepals, petals, stamens, stigmas/styles, ovaries, and seeds were performed. The result revealed a transcript of 2.5 kb present at high levels in stigmas and styles and a smaller transcript (2.3 kb) present at a lower level in stamens. NtWBC1 expression is developmentally regulated in stigmas/styles, with mRNA accumulation increasing toward anthesis. In situ hybridization experiments demonstrated that NtWBC1 is expressed in the stigmatic secretory zone and in anthers, at the stomium region and at the vascular bundle. NtWBC1 is the first ABC transporter gene with specific expression in plant reproductive organs to be identified and its expression pattern suggests important role(s) in the reproductive process.

Downregulation of the RECK-tumor and metastasis suppressor gene in glioma invasiveness

Invasive behavior is the pathological hallmark of malignant gliomas, being responsible for the failure of surgery, radiation, and chemotherapy. Matrix metalloproteinases (MMPs) are essential for proper ECM remodeling and invasion. The tumor and metastasis suppressor RECK protein regulates at least three members of the MMPs family: MMP-2, MMP-9, and MT1-MMP. In order to mimic the in vivo invasion process, A172 and T98G, respectively, non-invasive and invasive human glioblastoma cell lines, were cultured onto uncoated (control) or type I collagen gel-coated surface, and maintained for up to 7 days to allow establishment of the invasive process. We show that the collagen substrate causes decreased growth rates and morphological alterations correlated with the invasive phenotype. Electronic transmission microscopy of T98G cells revealed membrane invaginations resembling podosomes, which are typically found in cells in the process of crossing tissue boundaries, since they constitute sites of ECM degradation. Real time PCR revealed higher RECK mRNA expression in A172 cells, when compared to T98G cells and, also, in samples obtained from cultures where the invasive process was fully established. Interestingly, the collagen substrate increases RECK expression in A172 cells and the same tendency is displayed by T98G cells. MMPs-2 and -9 displayed higher levels of expression and activity in T98G cells, and their activities are also upregulated by collagen. Therefore, we suggest that: (1) RECK down regulation is critical for the invasiveness process displayed by T98G cells; (2) type 1 collagen could be employed to modulate RECK expression in glioblastoma cell lines. Since a positive correlation between RECK expression and patients survival has been noted in several types of tumors, our results may contribute to elucidate the complex mechanisms of malignant gliomas invasiveness.

Analysis of the association of polymorphism in the osteoprotegerin gene with susceptibility to chronic kidney disease and periodontitis

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Wolbachia endosymbiont in a species of the Anastrepha fraterculus complex (Diptera: Tephritidae)

In Anastrepha sp.2 aff. fraterculus, the egg-cell harbours a large population of endosymbionts. The bacteria were identified as belonging to genus Wolbachia by PCR assay using primers of the ftsZ gene followed by sequencing of the amplified band. Newly deposited eggs stained in toto by Hoechst show that the bacteria are unevenly dispersed throughout the egg-cell, with a higher accumulation at the posterior pole, and that the degree of infestation varies from egg to egg. Analysis by transmission electron microscopy shows that bacteria are present in the female germ line of embryonic and larval stages, as well as in the different cell types of the ovaries at the adult stage. Mature ova within the follicles harbour a large population of the symbionts. The results indicate the existence of a transovarian transmission of the endosymbionts in this fly.

Strong selection against hybrids at a hybrid zone in the Ensatina ring species complex and its evolutionary implications

The analysis of interactions between lineages at varying levels of genetic divergence can provide insights into the process of speciation through the accumulation of incompatible mutations. Ring species, and especially the Ensatina eschscholtzii system exemplify this approach. The plethodontid salamanders E. eschscholtzii xanthoptica and E. eschscholtzii platensis hybridize in the central Sierran foothills of California. We compared the genetic structure across two transects (southern and northern Calaveras Co.), one of which was resampled over 20 years, and examined diagnostic molecular markers (eight allozyme loci and mitochondrial DNA) and a diagnostic quantitative trait (color pattern). Key results across all studies were: (1) cline centers for all markers were coincident and the zones were narrow, with width estimates of 730 m to 2000 m; (2) cline centers at the northern Calaveras transect were coincident between 1981 and 2001, demonstrating repeatability over five generations; (3) there were very few if any putative F1s, but a relatively high number of backcrossed individuals in the central portion of transects: and (4) we found substantial linkage disequilibrium in all three studies and strong heterozygote deficit both in northern Calaveras, in 2001, and southern Calaveras. Both linkage disequilibrium and heterozygote deficit showed maximum values near the center of the zones. Using estimates of cline width and dispersal, we infer strong selection against hybrids. This is sufficient to promote accumulation of differences at loci that are neutral or under divergent selection, but would still allow for introgression of adaptive alleles. The evidence for strong but incomplete isolation across this centrally located contact is consistent with theory suggesting a gradual increase in postzygotic incompatibility between allopatric populations subject to divergent selection and reinforces the value of Ensatina as a system for the study of divergence and speciation at multiple stages. © 2005 The Society for the Study of Evolution. All rights reserved.

Spatial and temporal profiles for anti-inflammatory gene expression in leukocytes during a resolving model of peritonitis

The recent appreciation of the role played by endogenous counterregulatory mechanisms in controlling the outcome of the host inflammatory response requires specific analysis of their spatial and temporal profiles. In this study, we have focused on the glucocorticoid-regulated anti-inflammatory mediator annexin 1. Induction of peritonitis in wild-type mice rapidly (4 h) produced the expected signs of inflammation, including marked activation of resident cells (e.g., mast cells), migration of blood-borne leukocytes, mirrored by blood neutrophilia. These changes subsided after 48-96 h. In annexin 1null mice, the peritonitis response was exaggerated (∼40% at 4 h), with increased granulocyte migration and cytokine production. In blood leukocytes, annexin 1 gene expression was activated at 4, but not 24, h postzymosan, whereas protein levels were increased ai both time points. Locally, endothelial and mast cell annexin 1 gene expression was not detectable in basal conditions, whereas it was switched on during the inflammatory response. The significance of annexin 1 system plasticity in the anti-inflammatory properties of dexamethasone was assessed. Clear induction of annexin 1 gene in response to dexamethasone treatment was evident in the circulating and migrated leukocytes, and in connective tissue mast cells; this was associated with the steroid failure to inhibit leukocyte trafficking, cytokine synthesis, and mast cell degranulation in the annexin 1null mouse. In conclusion, understanding how inflammation is brought under control will help clarify the complex interplay between pro- and anti-inflammatory pathways operating during the host response to injury and infection. Copyright © 2006 by The American Association of Immunologists, Inc.

HFE gene mutations in Brazilian thalassemic patients

Hereditary hemochromatosis is a disorder of iron metabolism characterized by increased iron intake and progressive storage and is related to mutations in the HFE gene. Interactions between thalassemia and hemochromatosis may further increase iron overload. The ethnic background of the Brazilian population is heterogeneous and studies analyzing the simultaneous presence of HFE and thalassemia-related mutations have not been carried out. The aim of this study was to evaluate the prevalence of the H63D, S65C and C282Y mutations in the HFE gene among 102 individuals with alpha-thalassemia and 168 beta-thalassemia heterozygotes and to compare them with 173 control individuals without hemoglobinopathies. The allelic frequencies found in these three groups were 0.98, 2.38, and 0.29% for the C282Y mutation, 13.72, 13.70, and 9.54% for the H63D mutation, and 0, 0.60, and 0.87% for the S65C mutation, respectively. The chi-square test for multiple independent individuals indicated a significant difference among groups for the C282Y mutation, which was shown to be significant between the beta-thalassemia heterozygote and the control group by the Fisher exact test (P value = 0.009). The higher frequency of inheritance of the C282Y mutation in the HFE gene among beta-thalassemic patients may contribute to worsen the clinical picture of these individuals. In view of the characteristics of the Brazilian population, the present results emphasize the need to screen for HFE mutations in beta-thalassemia carriers.

Role of annexin 1 gene expression in mouse craniofacial bone development

BACKGROUND: Annexin 1 is a 37-kDa protein that has complex intra- and extracellular effects. To discover whether the absence of this protein alters bone development, we monitored this event in the annexin-A1 null mice in comparison with littermate wild-type controls. METHODS: Radiographic and densitometry methods were used for the assessment of bone in annexin-A1 null mice at a gross level. We used whole-skeleton staining, histological analysis, and Western blotting techniques to monitor changes at the tissue and cellular levels. RESULTS: There were no gross differences in the appendicular skeleton between the genotypes, but an anomalous development of the skull was observed in the annexin-A1 null mice. This was characterized in the newborn annexin-A1 null animals by a delayed intramembranous ossification of the skull, incomplete fusion of the interfrontal suture and palatine bone, and the presence of an abnormal suture structure. The annexin-A1 gene was shown to be active in osteocytes during this phase and COX-2 was abundantly expressed in cartilage and bone taken from annexin-A1 null mice. CONCLUSIONS: Expression of the annexin-A1 gene is important for the normal development of the skull in mice, possibly through the regulation of osteoblast differentiation and a secondary effect on the expression of components of the cPLA2-COX-2 system. © 2007 Wiley-Liss, Inc.

Physical factors affecting plasmid DNA compaction in stearylamine-containing nanoemulsions intended for gene delivery

Cationic lipids have been used in the development of non-viral gene delivery systems as lipoplexes. Stearylamine, a cationic lipid that presents a primary amine group when in solution, is able to compact genetic material by electrostatic interactions. In dispersed systems such as nanoemulsions this lipid anchors on the oil/water interface confering a positive charge to them. The aim of this work was to evaluate factors that influence DNA compaction in cationic nanoemulsions containing stearylamine. The influence of the stearylamine incorporation phase (water or oil), time of complexation, and different incubation temperatures were studied. The complexation rate was assessed by electrophoresis migration on agarose gel 0.7%, and nanoemulsion and lipoplex characterization was done by Dynamic Light Scattering (DLS). The results demonstrate that the best DNA compaction process occurs after 120 min of complexation, at low temperature (4 ± 1 °C), and after incorporation of the cationic lipid into the aqueous phase. Although the zeta potential of lipoplexes was lower than the results found for basic nanoemulsions, the granulometry did not change. Moreover, it was demonstrated that lipoplexes are suitable vehicles for gene delivery. © 2012 by the authors; licensee MDPI, Basel, Switzerland.

At least two indigenous species of the Bemisia tabaci complex are present in Brazil

Bemisia tabaci is one of the most important global agricultural insect pests, being a vector of emerging plant viruses such as begomoviruses and criniviruses that cause serious problems in many countries. Although knowledge of the genetic diversity of B. tabaci populations is important for controlling this pest and understanding viral epidemics, limited information is available on this pest in Brazil. A survey was conducted in different locations of São Paulo and Mato Grosso states, and the phylogenetic relationships of B. tabaci individuals from 43 populations sampled from different hosts were analysed based on partial mitochondrial cytochrome oxidase 1 gene (mtCOI) sequences. According to the recently proposed classification of the B. tabaci complex, which employs the 3.5% mtCOI sequence divergence threshold for species demarcation, most of the specimens collected were found to belong to the Middle East-Asia Minor 1 species, which includes the invasive populations of the commonly known B biotype, within the Africa/Middle East/Asia Minor high-level group. Three specimens collected from Solanun gilo and Ipomoea sp. were grouped together and could be classified in the New World species that includes the commonly known A biotype. However, six specimens collected from Euphorbia heterophylla, Xanthium cavanillesii and Glycine maxima could not be classified into any of the 28 previously proposed species, although according to the 11% mtCOI sequence divergence threshold, they belong to the New World high-level group. These specimens were classified into a new recently proposed species named New World 2 that includes populations from Argentina. Middle East-Asia Minor 1, New World and New World 2 were differentiated by RFLP analysis of the mtCOI gene using TaqI enzyme. Taq I analysis in silico also differentiates these from Mediterranean species, thus making this method a convenient tool to determine population dynamics, especially critical for monitoring the presence of this exotic pest in Brazil. © 2012 Blackwell Verlag, GmbH.

Analysis of inteins in the Candida parapsilosis complex for simple and accurate species identification

Inteins are coding sequences that are transcribed and translated with flanking sequences and then are excised by an autocatalytic process. There are two types of inteins in fungi, mini-inteins and full-length inteins, both of which present a splicing domain containing well-conserved amino acid sequences. Full-length inteins also present a homing endonuclease domain that makes the intein a mobile genetic element. These parasitic genetic elements are located in highly conserved genes and may allow for the differentiation of closely related species of the Candida parapsilosis (psilosis) complex. The correct identification of the three psilosis complex species C. parapsilosis, Candida metapsilosis, and Candida orthopsilosis is very important in the clinical setting for improving antifungal therapy and patient care. In this work, we analyzed inteins that are present in the vacuolar ATPase gene VMA and in the threonyl-tRNA synthetase gene ThrRS in 85 strains of the Candida psilosis complex (46 C. parapsilosis, 17 C. metapsilosis, and 22 C. orthopsilosis). Here, we describe an accessible and accurate technique based on a single PCR that is able to differentiate the psilosis complex based on the VMA intein. Although the ThrRS intein does not distinguish the three species of the psilosis complex by PCR product size, it can differentiate them by sequencing and phylogenetic analysis. Furthermore, this intein is unusually present as both mini- and full-length forms in C. orthopsilosis. Additional population studies should be performed to address whether this represents a common intraspecific variability or the presence of subspecies within C. orthopsilosis. Copyright © 2013, American Society for Microbiology. All Rights Reserved.

Susceptibilidade antifúngica, produção de biofilme e caracterização do gene ALS3 em isolados de Candida albicans e não-albicans do hospital das clínicas, UNESP, Botucatu

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Evolução e organização genômica do gene FOXL2 em vertebrados

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Abordagem multivariada na seleção de progênies de soja superiores e portadoras do gene RR

Pós-graduação em Agronomia (Genética e Melhoramento de Plantas) - FCAV

Proteína capsidial do Rupestris stem pitting-associated vírus: seqüenciamento do gene, expressão em Escherichia coli, purificação e produção de anti-soro policlonal

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)