Transgenic passionfruit expressing RNA derived from Cowpea aphid-borne mosaic virus is resistant to passionfruit woodiness disease

Sixteen transgenic yellow passionfruit (Passiflora spp.) plants (R0) were obtained which express a non-translatable transgenic RNA corresponding to the 3' region of the NIb gene and the 5' region of the CP gene, derived from the genome of a Brazilian isolate of Cowpea aphid-borne mosaic virus (CABMV). The transgenic plants were propagated by stem cuttings and challenged by sap inoculation with isolates CABMV-MG1 and CABMV-PE1. One transgenic plant (TE5-10) was resistant to the isolate CABMV-MG1, but susceptible to CABMV-PE1. The remaining transgenic plants developed systemic symptoms, equal to non-transformed plants, when inoculated with either isolate. The absence of virus in TE5-10 plants was confirmed by indirect ELISA. Transcription analysis of the transgene demonstrated that the TE5-10 plant did not accumulate transgenic mRNA, even before inoculation. After inoculation, viral RNA was only detected in plants inoculated with CABMV-PE1. These results confirm that the transgenic plant TE5-10 is resistant to isolate CABMV-MG1, and suggest that the resistance mechanism is post-transcriptional gene silencing, which is already activated in the transgenic plants before virus inoculation.

Viróides e virusóides: relíquias do mundo de RNA

Até meados do século XX, os vírus eram considerados os representantes mais simples da escala biológica. A descoberta dos RNAs satélites e dos viróides por volta de 1970 foi surpreendente, pois comprovou-se a existência de uma nova classe de moléculas auto-replicativas ainda mais simples, denominada agentes sub-virais. Há indícios de que os viróides e virusóides (que formam uma classe de RNAs satélites), teriam feito parte do "Mundo de RNA" (que precedeu o mundo atual baseado no DNA e proteínas), podendo ser considerados fósseis moleculares dessa era antiga. A simplicidade desses agentes sub-virais e o fato de que a molécula de RNA deve interagir diretamente com fatores do hospedeiro para o desenvolvimento do seu ciclo infeccioso colocam esses patógenos como um modelo para o estudo de processos metabólicos celulares. Nos últimos anos, tem-se observado um volume grande de publicações visando elucidar aspectos da interação viróide/hospedeiro, como os mecanismos da patogênese, movimento dos viróides nas plantas hospedeiras, silenciamento gênico e atividades das ribozimas. Mudanças recentes ocorridas na taxonomia desses patógenos com a criação de famílias, gêneros e espécies, além da descoberta de novos viróides, também têm sido verificadas. A presente revisão visa atualizar o leitor quanto aos recentes avanços nas pesquisas com viróides, principalmente na taxonomia, filogenia e em vários aspectos moleculares da interação viróide/hospedeiro. Estão incluídas também algumas características dos virusóides e sua relação evolutiva com os viróides.

Localization of metastasis within the sentinel lymph node biopsies: a predictor of additional axillary spread of breast cancer?

PURPOSE: To explore the relationship between morphological characteristics and histologic localization of metastasis within sentinel lymph nodes (SLN) and axillary spread in women with breast cancer. METHODS: We selected 119 patients with positive SLN submitted to complete axillary lymph node dissection from July 2002 to March 2007. We retrieved the age of patients and the primary tumor size. In the primary tumor, we evaluated histologic and nuclear grade, and peritumoral vascular invasion (PVI). In SLNs we evaluated the size of metastasis, their localization in the lymph node, number of foci, number of involved lymph nodes, and extranodal extension. RESULTS: Fifty-one (42.8%) patients had confirmed additional metastasis in non-sentinel lymph nodes (NLSN). High histologic grade, PVI, intraparenchymatous metastasis, extranodal neoplastic extension and size of metastasis were associated with positive NLSN. SLN metastasis affecting the capsule were associated to low risk incidence of additional metastasis. After multivariate analysis, PVI and metastasis size in the SLN remained as the most important risk factors for additional metastasis. CONCLUSIONS:The risk of additional involvement of NSLN is higher in patients with PVI and it increases progressively according the histologic localization in the lymph node, from capsule, where the afferent lymphatic channel arrives, to the opposite side of capsule promoting the extranodal extension. Size of metastasis greater than 6.0 mm presents higher risk of additional lymph node metastasis.

Detection of rabies virus nucleoprotein-RNA in several organs outside the Central Nervous System in naturally-infected vampire bats

Rabies is a neurological disease, but the rabies virus spread to several organs outside the central nervous system (CNS). The rabies virus antigen or RNA has been identified from the salivary glands, the lungs, the kidneys, the heart and the liver. This work aimed to identify the presence of the rabies virus in non-neuronal organs from naturally-infected vampire bats and to study the rabies virus in the salivary glands of healthy vampire bats. Out of the five bats that were positive for rabies in the CNS, by fluorescent antibody test (FAT), viral isolation in N2A cells and reverse transcription - polymerase chain reaction (RT-PCR), 100% (5/5) were positive for rabies in samples of the tongue and the heart, 80% (4/5) in the kidneys, 40% (2/5) in samples of the salivary glands and the lungs, and 20% (1/5) in the liver by RT-PCR test. All the nine bats that were negative for rabies in the CNS, by FAT, viral isolation and RT-PCR were negative for rabies in the salivary glands by RT-PCR test. Possible consequences for rabies epidemiology and pathogenesis are discussed in this work.

Localization of the cotyledon reserves of Theobroma grandiflorum (Willd. ex Spreng.) K. Schum., T. subincanum Mart., T. bicolor Bonpl. and their analogies with T. cacao L.

Cotyledon mesophyll cell morphology and lipid and protein synthesis of T. grandiflorum, T. subincanum and T. bicolor were analyzed and compared with T. cacao. These species possess foliar cotyledons folded around the hypocotyl radicle axis, typical of Sterculiaceae. Fruit size, morphology and weight are very distinct amongst the four species and so are the respective seeds. The main axis of the T. grandiflorum and T. bicolor seeds measured about 30 mm, while T. subincanum and T. cacao seeds measured 17 mm and 26 mm respectively. The seed weights of T. grandiflorum, T. bicolor, T. subincanum and T. cacao were 11.6 g, 9.4 g, 2.1 g and 3.0 g, respectively. The cotyledon mesophylls of the four species contained mainly polysaccharides and lipid-protein reserve cells. Theobroma cacao, T. grandiflorum and T. subincanum were composed of greater than 50% lipids. For the four species, lipid globules gradually accumulated adjacent to the cell wall, and these globules measured from 1 to 3 µm. TEM showed low-density proteins inside the central vacuole of the young mesophyll cells of T. cacao. The protein reserves of the mature cells were densely scattered amongst the lipid bodies, and a few starch granules occurred together with the cotyledon mesophyll of the four species. Polyphenolic cells were found throughout the mesophyll cells or aligned with the respective vascular bundles. Immature cells demonstrated the capacity to synthesize all these reserves, but gradually the pre-determined cells produced mainly lipid-protein reserves. Besides the unique characteristics of the T. cacao products, the lipid-protein synthesis capacities of T. grandiflorum, T. subincanum and T. bicolor suggest various possibilities for new industrialized food, pharmaceutical and cosmetic products.

Localization of NMDA receptors in the cerebral cortex: a schematic overview

The fundamental role of N-methyl-D-aspartate (NMDA) receptors in many cortical functions has been firmly defined, as has its involvement in a number of neurological and psychiatric diseases. However, until recently very little was known about the anatomical localization of NMDA receptors in the cerebral cortex of mammals. The recent application of molecular biological techniques to the study of NMDA receptors has provided specific tools which have greatly expanded our understanding of the localization of NMDA receptors in the cerebral cortex. In particular, immunocytochemical studies on the distribution of cortical NMDA receptors have shown that NMDA receptors are preferentially localized on dendritic spines, have disclosed an unknown fraction of presynaptic NMDA receptors on both excitatory and inhibitory axon terminals, and demonstrated that cortical astrocytes do express NMDA receptors. These studies suggest that the effects induced by the activation of NMDA receptors are not due solely to the opening of NMDA channels on neuronal postsynaptic membranes, as previously assumed, but that the activation of presynaptic and glial NMDA receptors may mediate part of these effects

Receptor localization of steroid hormones and drugs: discoveries through the use of thaw-mount and dry-mount autoradiography

The history of receptor autoradiography, its development and applications, testify to the utility of this histochemical technique for localizing radiolabeled hormones and drugs at cellular and subcellular sites of action in intact tissues. Localization of diffusible compounds has been a challenge that was met through the introduction of the "thaw-mount" and "dry-mount" autoradiographic techniques thirty years ago. With this cellular receptor autoradiography, used alone or combined with other histochemical techniques, sites of specific binding and deposition in vivo and in vitro have been characterized. Numerous discoveries, some reviewed in this article, provided information that led to new concepts and opened new areas of research. As an example, in recent years more than fifty target tissues for vitamin D have been specified, challenging the conventional view about the main biological role of vitamin D. The functions of most of these vitamin D target tissues are unrelated to the regulation of systemic calcium homeostasis, but pertain to the (seasonal) regulation of endo- and exocrine secretion, cell proliferation, reproduction, neural, immune and cardiovascular responses, and adaptation to stress. Receptor autoradiography with cellular resolution has become an indispensable tool in drug research and development, since information can be obtained that is difficult or impossible to gain otherwise

Functional characterization and localization of AQP3 in the human colon

Water channels or aquaporins (AQPs) have been identified in a large variety of tissues. Nevertheless, their role in the human gastrointestinal tract, where their action is essential for the reabsorption and secretion of water and electrolytes, is still unclear. The purpose of the present study was to investigate the structure and function of water channels expressed in the human colon. A cDNA fragment of about 420 bp with a 98% identity to human AQP3 was amplified from human stomach, small intestine and colon by reverse transcription polymerase chain reaction (RT-PCR) and a transcript of 2.2 kb was expressed more abundantly in colon than in jejunum, ileum and stomach as indicated by Northern blots. Expression of mRNA from the colon of adults and children but not from other gastrointestinal regions in Xenopus oocytes enhanced the osmotic water permeability, and the urea and glycerol transport in a manner sensitive to an antisense AQP3 oligonucleotide, indicating the presence of functional AQP3. Immunocytochemistry and immunofluorescence studies in human colon revealed that the AQP3 protein is restricted to the villus epithelial cells. The immunostaining within these cells was more intense in the apical than in the basolateral membranes. The presence of AQP3 in villus epithelial cells suggests that AQP3 is implicated in water absorption across human colonic surface cells.

RNA and DNA aptamers as potential tools to prevent cell adhesion in disease

Recent research has shown that receptor-ligand interactions between surfaces of communicating cells are necessary prerequisites for cell proliferation, cell differentiation and immune defense. Cell-adhesion events have also been proposed for pathological conditions such as cancer growth, metastasis, and host-cell invasion by parasites such as Trypanosoma cruzi. RNA and DNA aptamers (aptus = Latin, fit) that have been selected from combinatorial nucleic acid libraries are capable of binding to cell-adhesion receptors leading to a halt in cellular processes induced by outside signals as a consequence of blockage of receptor-ligand interactions. We outline here a novel approach using RNA aptamers that bind to T. cruzi receptors and interrupt host-cell invasion in analogy to existing procedures of blocking selectin adhesion and function in vitro and in vivo.

Ligand-induced endocytosis and nuclear localization of angiotensin II receptors expressed in CHO cells

A construct (AT1R-NF) containing a "Flag" sequence added to the N-terminus of the rat AT1 receptor was stably expressed in Chinese hamster ovary cells and quantified in the cell membrane by confocal microscopy after reaction with a fluorescein-labeled anti-Flag monoclonal antibody. Angiotensin II bound to AT1R-NF and induced endocytosis with a half-time of 2 min. After 60-90 min, fluorescence accumulated around the cell nucleus, suggesting migration of the ligand-receptor complex to the nuclear membrane. Angiotensin antagonists also induced endocytosis, suggesting that a common step in the transduction signal mechanism occurring after ligand binding may be responsible for the ligand-receptor complex internalization.

Molecular characterization of DDX26, a human DEAD-box RNA helicase, located on chromosome 7p12

DEAD-box proteins comprise a family of ATP-dependent RNA helicases involved in several aspects of RNA metabolism. Here we report the characterization of the human DEAD-box RNA helicase DDX26. The gene is composed of 14 exons distributed over an extension of 8,123 bp of genomic sequence and encodes a transcript of 1.8 kb that is expressed in all tissues evaluated. The predicted amino acid sequence shows a high similarity to a yeast DEAD-box RNA helicase (Dbp9b) involved in ribosome biogenesis. The new helicase maps to 7p12, a region of frequent chromosome amplifications in glioblastomas involving the epidermal growth factor receptor (EGFR) gene. Nevertheless, co-amplification of DDX26 with EGFR was not detected in nine tumors analyzed.

Colocalization of coilin and nucleolar proteins in Cajal body-like structures of micronucleated PtK2 cells

Cajal bodies (CB) are ubiquitous nuclear structures involved in the biogenesis of small nuclear ribonucleoproteins and show narrow association with the nucleolus. To identify possible relationships between CB and the nucleolus, the localization of coilin, a marker of CB, and of a set of nucleolar proteins was investigated in cultured PtK2 cells undergoing micronucleation. Nocodazol-induced micronucleated cells were examined by double indirect immunofluorescence with antibodies against coilin, fibrillarin, NOR-90/hUBF, RNA polymerase I, PM/Scl, and To/Th. Cells were imaged on a BioRad 1024-UV confocal system attached to a Zeiss Axiovert 100 microscope. Since PtK2 cells possess only one nucleolus organizer region, micronucleated cells presented only one or two micronuclei containing nucleolus. By confocal microscopy we showed that in most micronuclei lacking a typical nucleolus a variable number of round structures were stained by antibodies against fibrillarin, NOR-90/hUBF protein, and coilin. These bodies were regarded as CB-like structures and were not stained by anti-PM/Scl and anti-To/Th antibodies. Anti-RNA polymerase I antibodies also reacted with CB-like structures in some micronuclei lacking nucleolus. The demonstration that a set of proteins involved in RNA/RNP biogenesis, namely coilin, fibrillarin, NOR-90/hUBF, and RNA polymerase I gather in CB-like structures present in nucleoli-devoid micronuclei may contribute to shed some light into the understanding of CB function.

The RNA interference revolution

The discovery of double-stranded RNA-mediated gene silencing has rapidly led to its use as a method of choice for blocking a gene, and has turned it into one of the most discussed topics in cell biology. Although still in its infancy, the field of RNA interference has already produced a vast array of results, mainly in Caenorhabditis elegans, but recently also in mammalian systems. Micro-RNAs are short hairpins of RNA capable of blocking translation, which are transcribed from genomic DNA and are implicated in several aspects from development to cell signaling. The present review discusses the main methods used for gene silencing in cell culture and animal models, including the selection of target sequences, delivery methods and strategies for a successful silencing. Expected developments are briefly discussed, ranging from reverse genetics to therapeutics. Thus, the development of the new paradigm of RNA-mediated gene silencing has produced two important advances: knowledge of a basic cellular mechanism present in the majority of eukaryotic cells and access to a potent and specific new method for gene silencing.

Enucleated L929 mouse fibroblasts support invasion and multiplication of Shigella flexneri 5a

Invasive bacteria can induce their own uptake and specify their intracellular localization; hence it is commonly assumed that proximate modulation of host cell transcription is not required for infection. However, bacteria can also modulate, directly or indirectly, the transcription of many host cell genes, whose role in the infection may be difficult to determine by global gene expression. Is the host cell nucleus proximately required for intracellular infection and, if so, for which pathogens and at what stages of infection? Enucleated cells were previously infected with Toxoplasma gondii, Chlamydia psittaci, C. trachomatis, or Rickettsia prowazekii. We enucleated L929 mouse fibroblasts by centrifugation in the presence of cytochalasin B, and compared the infection with Shigella flexneri M90T 5a of nucleated and enucleated cells. Percent infection and bacterial loads were estimated with a gentamicin suppression assay in cultures fixed and stained at different times after infection. Enucleation reduced by about half the percent of infected cells, a finding that may reflect the reduced endocytic ability of L929 cytoplasts. However, average numbers of bacteria and frequency distributions of bacterial numbers per cell at different times were similar in enucleated and nucleated cells. Bacteria with actin-rich tails were detected in both cytoplasts and nucleated cells. Lastly, cytoplasts were similarly infected 2 and 24 h after enucleation, suggesting that short-lived mRNAs were not involved in the infection. Productive S. flexneri infection could thus take place in cells unable to modulate gene transcription, RNA processing, or nucleus-dependent signaling cascades.

A search for RNA insertions and NS3 gene duplication in the genome of cytopathic isolates of bovine viral diarrhea virus

Calves born persistently infected with non-cytopathic bovine viral diarrhea virus (ncpBVDV) frequently develop a fatal gastroenteric illness called mucosal disease. Both the original virus (ncpBVDV) and an antigenically identical but cytopathic virus (cpBVDV) can be isolated from animals affected by mucosal disease. Cytopathic BVDVs originate from their ncp counterparts by diverse genetic mechanisms, all leading to the expression of the non-structural polypeptide NS3 as a discrete protein. In contrast, ncpBVDVs express only the large precursor polypeptide, NS2-3, which contains the NS3 sequence within its carboxy-terminal half. We report here the investigation of the mechanism leading to NS3 expression in 41 cpBVDV isolates. An RT-PCR strategy was employed to detect RNA insertions within the NS2-3 gene and/or duplication of the NS3 gene, two common mechanisms of NS3 expression. RT-PCR amplification revealed insertions in the NS2-3 gene of three cp isolates, with the inserts being similar in size to that present in the cpBVDV NADL strain. Sequencing of one such insert revealed a 296-nucleotide sequence with a central core of 270 nucleotides coding for an amino acid sequence highly homologous (98%) to the NADL insert, a sequence corresponding to part of the cellular J-Domain gene. One cpBVDV isolate contained a duplication of the NS3 gene downstream from the original locus. In contrast, no detectable NS2-3 insertions or NS3 gene duplications were observed in the genome of 37 cp isolates. These results demonstrate that processing of NS2-3 without bulk mRNA insertions or NS3 gene duplications seems to be a frequent mechanism leading to NS3 expression and BVDV cytopathology.