Quantitative control of Lettuce mosaic virus fitness and host defence inhibition by P1-HCPro

Two Lettuce mosaic virus isolates capable of overcoming the resistance afforded by the resistance gene mo1² in lettuce, LMV-AF199 from Brazil, and LMV-E, an European isolate, were evaluated for the rapidity and severity of symptoms induced on the lettuce variety Salinas 88 (mo1²). The mosaic symptoms on Salinas 88 plants inoculated with LMV-AF199 appeared 7 days post-inoculation (dpi) and 15 dpi for LMV-E. The symptoms induced by LMV-AF199 in this cultivar were also more severe than those induced by LMV-E. In order to identify the region of the viral genome responsible for this phenotype, recombinant viruses were constructed between these isolates and the phenotype of each recombinant was analysed. The region encoding proteins P1 and HcPro from LMV-AF199 was associated with the increased virulence in Salinas 88.

The effect of hydrostatic pressure on membrane-bound proteins

Many cellular proteins are bound to the surfaces of membranes and participate in various cell signaling responses. Interactions between this group of proteins are in part controlled by the membrane surface to which the proteins are bound. This review focuses on the effects of pressure on membrane-associated proteins. Initially, the effect of pressure on membrane surfaces and how pressure may perturb the membrane binding of proteins is discussed. Next, the effect of pressure on the activity and lateral association of proteins is considered. We then discuss how pressure can be used to gain insight into these types of proteins.

Viral nucleolar localisation signals determine dynamic trafficking within the nucleolus

Localisation of both viral and cellular proteins to the nucleolus is determined by a variety of factors including nucleolar localisation signals (NoLSs), but how these signals operate is not clearly understood. The nucleolar trafficking of wild type viral proteins and chimeric proteins, which contain altered NoLSs, were compared to investigate the role of NoLSs in dynamic nucleolar trafficking. Three viral proteins from diverse viruses were selected which localised to the nucleolus; the coronavirus infectious bronchitis virus nucleocapsid (N) protein, the herpesvirus saimiri ORF57 protein and the HIV-1 Rev protein. The chimeric proteins were N protein and ORF57 protein which had their own NoLS replaced with those from ORF57 and Rev proteins, respectively. By analysing the sub-cellular localisation and trafficking of these viral proteins and their chimeras within and between nucleoli using confocal microscopy and photo-bleaching we show that NoLSs are responsible for different nucleolar localisations and trafficking rates.

Long-Term Culture of Mouse Embryonic Stem Cell-Derived Adherent Neurospheres and Functional Neurons

Innumerous protocols, using the mouse embryonic stem (ES) cells as model for in vitro study of neurons functional properties and features, have been developed. Most of these protocols are short lasting, which, therefore, does not allow a careful analysis of the neurons maturation, aging, and death processes. We describe here a novel and efficient long-lasting protocol for in vitro ES cells differentiation into neuronal cells. It consists of obtaining embryoid bodies, followed by induction of neuronal differentiation with retinoic acid of nonadherent embryoid bodies (three-dimensional model), which further allows their adherence and formation of adherent neurospheres (AN, bi-dimensional model). The AN can be maintained for at least 12 weeks in culture under repetitive mechanical splitting, providing a constant microenvironment (in vitro niche) for the neuronal progenitor cells avoiding mechanical dissociation of AN. The expression of neuron-specific proteins, such as nestin, sox1, beta III-tubulin, microtubule-associated protein 2, neurofilament medium protein, Tau, neuronal nuclei marker, gamma-aminobutyric acid, and 5-hydroxytryptamine, were confirmed in these cells maintained during 3 months under several splitting. Additionally, expression pattern of microtubule-associated proteins, such as lissencephaly (Lis1) and nuclear distribution element-like (Ndel1), which were shown to be essential for differentiation and migration of neurons during embryogenesis, was also studied. As expected, both proteins were expressed in undifferentiated ES cells, AN, and nonrosette neurons, although presenting different spatial distribution in AN. In contrast to previous studies, using cultured neuronal cells derived from embryonic and adult tissues, only Ndel1 expression was observed in the centrosome region of early neuroblasts from AN. Mature neurons, obtained from ES cells in this work, display ionic channels and oscillations of membrane electrical potential typical of electrically excitable cells, which is a characteristic feature of the functional central nervous system (CNS) neurons. Taken together, our study demonstrated that AN are a long-term culture of neuronal cells that can be used to analyze the process of neuronal differentiation dynamics. Thus, the protocol described here provides a new experimental model for studying neurological diseases associated with neuronal differentiation during early development, as well as it represents a novel source of functional cells that can be used as tools for testing the effects of toxins and/or drugs on neuronal cells.

Cytogenetic analysis of the neotropical spider Nephilengys cruentata (Araneomorphae, tetragnathidae): standard staining, NORs, C-bands and base-specific fluorochromes

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Quantitative control of Lettuce mosaic virus fitness and host defence inhibition by P1-HCPro

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

Cytogenetic markers as diagnoses in the identification of the hybrid between Piauçu (Leporinus macrocephalus) and Piapara (Leporinus elongatus)

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

Evaluation of Different Retention Systems on a Distal Extension Removable Partial Denture Associated With an Osseointegrated Implant

The aim of this study was to evaluate the biomechanical behavior of a mandibular distal extension removable partial denture (DERPD) associated with an implant and different retention system, by bidimensional finite element method. Five hemimandible models with a canine and external hexagon implant at second molar region associated with DERPD were simulated: model A, hemimandible with a canine and a DERPD; model B, hemimandible with a canine and implant with a healing abutment associated to a DERPD; model C, hemimandible with a canine and implant with an ERA attachment associated to a DERPD; model D, hemimandible with a canine and implant with an O'ring attachment associated to a DERPD; and model E, hemimandible with a canine and implant-supported prosthesis associated to a DERPD. Cusp tips were loaded with 50 N of axial or oblique force (45 degrees). Finite element analysis was performed in ANSYS 9.0. model E showed the higher displacement and overload in the supporting tissues; the patterns of stress distribution around the dental apex of models B, C, and D were similar. The association between a DERPD and an osseointegrated implant using the ERA or O'ring systems shows lower stress values. Oblique forces showed higher stress values and displacement. Oblique forces increased the displacement and stress levels in all models; model C displayed the best stress distribution in the supporting structures; healing abutment, ERA, and O'ring systems were viable with RPD, but DERPD association with a single implant-supported prosthesis was nonviable.

Cytogenetic characterization of Brycon amazonicus (Spix et Agassiz, 1829) (Teleostei: Characidae) from Caicara del Orinoco, Venezuela

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Identification of Rv3852 as a nucleoid-associated protein in Mycobacterium tuberculosis

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Study of the nucleolar cycle and ribosomal RNA distribution during meiosis in triatomines (Triatominae, Heteroptera)

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

Photoacoustic spectroscopy of aromatic amino acids in proteins

This paper concerns the use of photoacoustic spectroscopy (PAS) to study the presence of aromatic amino acid in proteins. We examined the aromatic amino acids in six proteins with well-known structures using absorption spectra of near ultraviolet PAS over the wavelength range 240-320 nm. The fundamental understanding of the physical and chemical properties that govern the absorption of light and a subsequent release of heat to generate a transient pressure wave was used to test the concept of monitoring aromatic amino acids with this method. Second derivative spectroscopy in the ultraviolet region of proteins was also used to study the regions surrounding the aromatics and the percentage area in each band was related in order to determine the contribution in function of the respective molar extinction coefficients for each residue. Further investigation was conducted into the interaction between sodium dodecyl sulphate (SDS) and bothropstoxin-I (BthTx-I), with the purpose of identifying the aromatics that participate in the interaction. The clear changes in the second derivative and curve-fitting procedures suggest that initial SDS binding to the tryptophan located in the dimer interface and above 10 SDS an increased intensity between 260 and 320 nm, demonstrating that the more widespread tyrosine and phenylalanine residues contribute to the SDS/BthTx-I interactions. These results demonstrate the potential of near UV-PAS for the investigation of membrane proteins/detergent complexes in which light scattering is significant.

Cytogenetic analyses of two Curimatidae species (Pisces; Characiformes) from the Paranapanema and Tietê Rivers

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

Astylus variegatus (Coleoptera, Melyridae): Cytogenetic study of a population exposed to agrochemical products

In this work we describe the cytogenetic analyses performed in specimens of Astylus variegatus (Germar, 1824) collected in two localities: one area of natural vegetation and one of agricultural crops, where agrochemical products were used. Astylus variegatus had karyotypes with 2n(male) = 16+Xy p and 2n (female) = 16+XXp, with exclusively metacentric chromosomes. Pachytene spermatocytes showed synapsed autosomal bivalents and non-associated sex chromosomes. In diplotene, the autosomal bivalents exhibited one or two terminal chiasmata and the Xy p had a typical parachute configuration. In meiotic cells of some specimens, an extra chromosome, interpreted as a B chromosome, was observed. C-banding showed constitutive heterochromatin in the pericentromeric region of all chromosomes, with the exception of the y p. Silver nitrate staining revealed one nucleolus organizer region (NOR) on the terminal region of the short arm of the second autosome pair. Silver staining of meiotic cells confirmed the NOR pattern detected in mitotic cells and revealed an argentophilous material on the Xy p. A cytogenetic comparison between the two populations of A. variegatus showed a statistically significant divergence (chi2 = 117.10; df = 1) in the number of aneuploid cells and a higher frequency of B chromosome in the population exposed to agrochemicals.

Pattern of nucleolar activity during spermiogenesis in triatomines (Heteroptera, Reduviidae) as analysed by silver staining

Nucleoli are the sites of biosynthesis of ribosomal precursors. In this work the nucleolar activity at interphase and the meiotic cells of the testis in five species of triatomines were analysed by means of silver staining. Several nucleolar blocks in the polyploid nuclei of testicular tubules were observed, whereas only one nucleolar body could be seen in the spermatogonial nuclei of all five species. A single nucleolar body was evident in the 'confused stage' of Triatoma brasiliensis, T. delpontei, T. lecticularia and T. rubrovaria, while T. sordida presented two nucleolar dots. The existence of small, silver-stained dots in some metaphase I chromosomes of T. brasiliensis and T. sordida is reported. The number of nucleolar dots present in spermatids of each species varied within and among species. It is suggested that in addition to providing information on rRNA biosynthesis, studies of nucleolar organizing activity can also be important sources of data on differentiation patterns and species development.