Cloning and characterization of transcripts differentially expressed in the pulp of ripening papaya

Papaya (Carica papaya) is a relevant tropical crop and physico-chemical changes take place very quickly, as a consequence of activation of biochemical pathways by de nova synthesis of several proteins. Thus, in order to have information on the changes in gene expression in ripening papaya, transcripts from the pulp of unripe and ripe fruit were profiled by differential-display RT-PCR (DDRT-PCR). Seventy transcript derived fragments (TDFs) isolated from gels were re-amplified by PCR and differential expression of 40 papaya genes was confirmed by reverse northern blotting. Twenty-nine positively cloned TDFs were sequenced, and 17 were putatively identified by homology search. Ten of these genes were downregulated during ripening and UDP-glucose glucosyltransferase, alpha-2 importin, RNase L inhibitor-like protein, and a syntaxin protein were identified. Among the up-regulated genes there was a carboxylesterase, an integral membrane Yip1 family protein, a glycosyl hydrolase family-like protein and an endopolygalacturonase. Considering their relatedness to papaya quality, the fragments of genes potentially implicated in carbohydrate metabolism and pulp softening may be considered of interest for further studies. According to the results, differential display was a feasible approach to investigate differences in gene expression during fruit ripening, and can provide interesting information about those fruits whose genomic data is scarce, as is the case of papayas. (c) 2009 Elsevier B.V. All rights reserved.

Biophysical characterization of the recombinant merozoite surface protein-3 of Plasmodium vivax

Plasmodium vivax Merozoite Surface Protein-3 alpha and 3 beta are members of a family of related merozoite surface proteins that contain a central alanine-rich domain with heptad repeats that is predicted to form alpha-helical secondary and coiled-coil tertiary structures. Seven recombinant proteins representing different regions of MSP-3 alpha and MSP-3 beta of P. vivax were generated to investigate their structure. Circular dichroism spectra analysis revealed that some proteins are folded with a high degree of alpha-helices as secondary structure, whereas other products contain a high content of random coil. Using size exclusion chromatography, we found that the two smaller fragments of the MSP-3 alpha, named CC4 and CC5, predicted to form coiled-coil (CC) structures, eluted at volumes corresponding to molecular weights larger than their monomeric masses. This result suggests that both proteins are oligomeric molecules. Analytical ultracentrifugation experiments showed that the CC5 oligomers are elongated molecules. Together, these data may help to understand important aspects of P. vivax biology. (C) 2008 Elsevier B.V. All rights reserved.

Transcript profiling of papaya fruit reveals differentially expressed genes associated with fruit ripening

Papaya (Carica papaya L) fruit has a short shelf life due to fast ripening induced by ethylene, but little is known about the genetic control of ripening and attributes of fruit quality. Therefore, we identified ripening-related genes affected by ethylene using cDNA-AFLP (Amplified Fragment Length Polymorphism of cDNA). Transcript profiling of non-induced and ethylene-induced fruit samples was performed, and 71 differentially expressed genes were identified. Among those genes some involved in ethylene biosynthesis, regulation of transcription, and stress responses or plant defence were found (heat shock proteins, polygalacturonase-inhibiting protein, and acyl-CoA oxidases). Several transcription factors were isolated, and except for a 14-3-3 protein, an AP2 domain-containing factor, a salt-tolerant zinc finger protein, and a suppressor of PhyA-105 1, most of them were negatively affected by ethylene, including fragments of transcripts similar to VRN1, and ethylene responsive factors (ERF). With respect to fruit quality, genes related to cell wall structure or metabolism, volatiles or pigment precursors, and vitamin biosynthesis were also found. (C) 2010 Elsevier Ireland Ltd. All rights reserved.

An everyday nostalgia: Memory and the fictions of belonging

In 1984, George Orwell presented the future as a dystopian vision, where everyday existence was governed and redefined by an oppressive regime. Winston Smith's daily duties at the Ministry of Truth involved the invention, rewriting and erasing of fragments of history as a means of perpetuating contentment, uniformity and control. History, as Orwell described it in the novel 'was a palimpsest, scraped clean and reinscribed exactly as often as was necessary.' More that a quarter of a century after the publication of 1984, Michel Foucault discussed the cinematic representation and misrepresentation of French history and identity in terms of what he called the manipulation of 'popular memory'. In what was tantamount to a diluted version of Orwell's palimpsestic histories, Foucault stated that 'people are not shown what they were, but what they must remember having been.' This paper will investigate notions of memory, identity and the everyday through a discussion of the community of Celebration in Florida. Conceived in the 1990s, Celebration was designed around a fictionalised representation of pre 1940s small town America, using nostalgia for a mythologised past to create a sense of comfort, community and conformity among its residents. Adapting issues raised by Orwell, Foucault and Baudrillard, this paper will discuss the way in which architecture, like film and literature, can participate in what Foucault discussed as the manipulation of popular memory, inducing and exploiting a nostalgia for an everyday past that that never really existed.

Babesia bovis: Genome size, number of chromosomes and telomeric probe hybridisation

Pulsed field gel electrophoresis of intact chromosomes of Babesia bovis revealed four chromosomes in the haploid genome. A telomere probe, derived from Plasmodium berghei, hybridised to eight SfiI restriction fragments of genomic B. bovis DNA digests indicating the presence of four chromosomes. A small subunit (18S) ribosomal RNA gene probe hybridised to the third chromosome only. The genome size of B. bovis is estimated to be 9.4 million base pairs. The sizes of chromosomes 1, 2, 3 and 4 are estimated to be 1.4, 2.0, 2.8 and 3.2 million base pairs, respectively. (C) 1997 Australian Society for Parasitology. Published by Elsevier Science Ltd.

Structure of a putative ancestral protein encoded by a single sequence repeat from a multidomain proteinase inhibitor gene from Nicotiana alata

Background: The ornamental tobacco Nicotiana alata produces a series of proteinase inhibitors (Pls) that are derived from a 43 kDa precursor protein, NaProPl. NaProPl contains six highly homologous repeats that fold to generate six separate structural domains, each corresponding to one of the native Pls. An unusual feature of NaProPl is that the structural domains lie across adjacent repeats and that the sixth Pl domain is generated from fragments of the first and sixth repeats. Although the homology of the repeats suggests that they may have arisen from gene duplication, the observed folding does not appear to support this. This study of the solution structure of a single NaProPl repeat (aPl1) forms a basis for unravelling the mechanism by which this protein may have evolved, Results: The three-dimensional structure of aPl1 closely resembles the triple-stranded antiparallel beta sheet observed in each of the native Pls. The five-residue sequence Glu-Glu-Lys-Lys-Asn, which forms the linker between the six structural domains in NaProPl, exists as a disordered loop in aPl1. The presence of this loop in aPl1 results in a loss of the characteristically flat and disc-like topography of the native inhibitors. Conclusions: A single repeat from NaProPl is capable of folding into a compact globular domain that displays native-like Pl activity. Consequently, it is possible that a similar single-domain inhibitor represents the ancestral protein from which NaProPl evolved.

Petrographic and isotope constraints on the origin of authigenic carbonate minerals and the associated fluid evolution in Late Permian coal measures, Bowen Basin (Queensland), Australia

Authigenic carbonate minerals are ubiquitous throughout the Late Permian coal measures of the Bowen Basin, Queensland, Australia. In the northern Bowen Basin, carbonates include the following assemblages: siderite I (delta O-18(SMOW) = +11.4 to + 17%, delta C-13(PDB) = - 5.3 to + 120), Fe-Mg calcite-ankerite-siderite II mineral association (delta O-18(SMOW) = +7.2 to + 10.20, delta C-13(PDB) = 10.9 to - 1.80 for ankerite) and a later calcite (delta O-18(SMOW) = +5.9 to + 14.60, delta C-13(PDB) = -11.4 to + 4.40). In the southern Bowen Basin, the carbonate phase consists only of calcite (delta O-18(SMOW) = +12.5 to + 14.80, delta C-13(PDB) = -19.4 to + 0.80), where it occurs extensively throughout all stratigraphic levels. Siderite I occurs in mudrocks and sandstones and predates all other carbonate minerals. This carbonate phase is interpreted to have formed as an early diagenetic mineral from meteoric waters under cold climate and reducing conditions. Fe-Mg calcite-ankerite-siderite Il occur in sandstones as replacement of volcanic rock fragments. Clay minerals (illite-smectite, chlorite and kaolinite) postdate Ca-Fe-Mg carbonates, and precipitation of the later calcite is associated with clay mineral formation. The Ca-Fe-Mg carbonates and later calcite of the northern Bowen Basin are regarded as having formed as a result of hydrothermal activity during the latest Triassic extensional tectonic event which affected this part of the basin, rather than deep burial diagenesis during the Middle to Late Triassic as previously reported. This hypothesis is based on the timing relationships of the authigenic mineral phases and the low delta O-18 values of ankerite and calcite, together with radiometric dating of illitic clays and recently published regional geological evidence. Following the precipitation of the Ca-Fe-Mg carbonates from strongly O-18-depleted meteoric-hydrothermal fluids, continuing fluid circulation and water-rock interaction resulted in dissolution of these carbonate phases as well as labile fragments of volcaniclastic rocks. Subsequently, the later calcite and day minerals precipitated from relatively evolved (O-18-enriched) fluids. The nearly uniform delta O-18 values of the southern Bowen Basin calcite have been attributed to very low water/rock ratio in the system, where the fluid isotropic composition was buffered by the delta O-18 values of rocks. (C) 2000 Elsevier Science B.V. All rights reserved.

Clarice Lispector - the hour of the star: the speech in the exhibition pamphlet

This work attempts to discuss, in the light of the French Analysis of the Discourse, how the concept of memory and heterogeneity in language actions can contribute to a reflection on information and documentation studies. Starting from cuttings of Clarice Lispector - the hour of the star exhibition pamphlet, accomplished in the second semester of 2007 by the Portuguese Language Museum (Luz train station, Sao Paulo), we interpreted the several voices that surround and sustain the subject and the sense.

Diagnostic model based on Raman spectra of normal, hyperplasia and prostate adenocarcinoma tissues in vitro

This study evaluated the use of Raman spectroscopy to identify the spectral differences between normal (N), benign hyperplasia (BPH) and adenocarcinoma (CaP) in fragments of prostate biopsies in vitro with the aim of developing a spectral diagnostic model for tissue classification. A dispersive Raman spectrometer was used with 830 nm wavelength and 80 mW excitation. Following Raman data collection and tissue histopathology (48 fragments diagnosed as N, 43 as BPH and 14 as CaP), two diagnostic models were developed in order to extract diagnostic information: the first using PCA and Mahalanobis analysis techniques and the second one a simplified biochemical model based on spectral features of cholesterol, collagen, smooth muscle cell and adipocyte. Spectral differences between N, BPH and CaP tissues, were observed mainly in the Raman bands associated with proteins, lipids, nucleic and amino acids. The PCA diagnostic model showed a sensitivity and specificity of 100%, which indicates the ability of PCA and Mahalanobis distance techniques to classify tissue changes in vitro. Also, it was found that the relative amount of collagen decreased while the amount of cholesterol and adipocyte increased with severity of the disease. Smooth muscle cell increased in BPH tissue. These characteristics were used for diagnostic purposes.

Use of cholesterol-rich nanoparticles that bind to lipoprotein receptors as a vehicle to paclitaxel in the treatment of breast cancer: pharmacokinetics, tumor uptake and a pilot clinical study

Purpose In animal experiments paclitaxel oleate associated with a cholesterol-rich nanoemulsion concentrated in the neoplastic tissues and showed reduced toxicity and increased antitumor activity compared with paclitaxel-Cremophor EL. Here, a clinical study was performed in breast cancer patients to evaluate the tumoral uptake, pharmacokinetics and toxicity of paclitaxel associated to nanoemulsions. Methods Twenty-four hours before mastectomy [(3)H]paclitaxel oleate associated with [(14)C]-cholesteryl oleatenanoemulsion or [(3)H]- paclitaxel in Cremophor EL were injected into five patients for collection of blood samples and fragments of tumor and normal breast tissue. A pilot clinical study of paclitaxel-nanoemulsion administered at 3-week intervals was performed in four breast cancer patients with refractory advanced disease at 175 and 220 mg/m(2) dose levels. Results T(1/2) of paclitaxel oleate associated to the nanoemulsion was greater than that of paclitaxel (t(1/2) = 15.4 +/- 4.7 and 3.5 +/- 0.80 h). Uptake of the [(14)C]-cholesteryl ester nanoemulsion and [(3)H]- paclitaxel oleate by breast malignant tissue was threefold greater than the normal breast tissue and toxicity was minimal at the two dose levels. Conclusions Our results suggest that the paclitaxel-nanoemulsion preparation can be advantageous for use in the treatment of breast cancer because the pharmacokinetic parameters are improved, the drug is concentrated in the neoplastic tissue and the toxicity of paclitaxel is reduced.

Relationship of adhesion molecules expression with epithelial differentiation markers during fetal skin development

Background: Cadherins and integrins are important for maintenance of tissue integrity and in signal transduction during skin development. Distribution of these molecules in human skin development was investigated and associated with markers of differentiation, cytokeratins (CK) and involucrin (INV). Methods: Using immunohistochemistry expression of E- and P-cadherins, integrins beta-1 and -4, CK10, CK14 and INV was assessed in skin fragments of 10 human fetuses (gestational weeks ranged from 4 to 24, all weighing up to 500 g). Results: At initial phases of development, integrins beta-1 and -4 and E- and P-cadherins were present on epithelial cell membranes in all layers. CK14 and CK10 were expressed in all epithelial layers and INV weakly detected in the superficial layer. In more advanced stages, integrins were detected in all layers, but a marked polarized expression was seen in basal layer. E-cadherin was detected in all layers, but the cornified stratum and P-cadherin were observed in the lower layers. CK14 was expressed in basal layer, CK10 in suprabasal stratum and INV was observed in cornified layer. Conclusions: Cadherins and integrins are essential for skin development, being spatially and temporally regulated. Their expression is related with the expression of maturation markers of the epidermis.

The love of a sage or the command of a superior: The natural law doctrines of Leibniz and Pufendorf

The study of natural law theories is presently one of the most fruitful areas of research in the studies of early modern intellectual history, and moral and political theory. Likewise the historical significance of the Enlightenment for the development of modernisation' in many different forms continues to be the subject of controversy. This collection therefore offers a timely opportunity to re-examine both the coherence of the concept of an early Enlightenment', and the specific contribution of natural law theories to its formation. The works of major thinkers such as Grotius, Hobbes, Locke, Malebranche, Pufendorf and Thomasius are reassessed, and the appeal and importance of the discourse of natural jurisprudence both to those working inside conventional educational and political structures and to those outside - such as in the Huguenot diaspora - is evaluated. This volume will therefore be of importance to all those readers concerned to study the character of the debates in the period 1650-1750 surrounding moral and political agency, sovereignty and obligation, and the legitimation of religious toleration in the divergent states and patriotic contexts of Europe.

Morphological comparison of different protocols of skeletal muscle remobilization in rats after hindlimb suspension

The aim of this study was to evaluate and compare the efficacy of different remobilization protocols in different skeletal muscles considering the changes induced by hindlimb suspension of the tail. Thirty-six female Wistar rats were divided into six groups: control I, control II, suspended, suspended free, suspended trained on a declined treadmill and suspended trained on a flat treadmill. Fragments of soleus and tibialis anterior (TA) muscle were frozen and processed by different histochemical methods. The suspended soleus showed a significant increase in the proportional number of intermediate/hybrid fibers and a decrease in the number of type I fibers. Some of these changes proved to be reversible after remobilization. The three remobilization programs led to the recovery of both the proportional number of fibers and their size. The TA muscle presented a significant increase in the number and size of type I fibers and a cell size reduction of type IIB fibers, which were recovered after training on a declined treadmill and free movement. Especially regarding the soleus, the present findings indicate that, among the protocols, training on a declined treadmill was found to induce changes of a more regenerative nature, seemingly indicating a better tissue restructuring after the suspension procedure.

Effects of eccentric and concentric training on capillarization and myosin heavy chain contents in rat skeletal muscles after hindlimb suspension

We studied the effects of different protocols of post-disuse rehabilitation on angiogenesis and myosin heavy chain (MHC) content in rat hindlimb muscles after caudal suspension. Thirty female Wistar rats were divided into five groups: (1) Control I, (2) Control II, (3) Suspended, (4) Suspended trained on declined treadmill, and (5) Suspended trained on flat treadmill. Fragments of the soleus and tibialis anterior (TA) muscles were frozen and processed by electrophoresis and immunohistochemistry (CD31 antibody). Hindlimb suspension caused reduction of capillary/fiber (C/F) ratios and contents of MHC type I (MHCI) in the soleus in parallel to increased capillary density. Flat treadmill protocols increased the content of the MHCI isoform. The C/F ratio was increased by concentric training after hypokinesis, but was not modified by eccentric training, which caused a greater reduction of capillary density compared to the other protocols. In the TA muscle, hindlimb suspension caused a non-significant increase in capillary density and C/F ratio with limited changes in MHC. The present data demonstrate that the different training protocols adopted and the functional performance of the muscles analyzed caused specific changes in capillarization and in the content of the various MHC types. (C) 2010 Published by Elsevier GmbH.

Genetic analysis of forest species Eugenia uniflora L. through of newly developed SSR markers

Nine microsatellite loci for genetic analysis of three populations of the tropical tree Eugenia uniflora L. (pitanga or Brazilian cherry) from fragments of semideciduous forest were developed. We used the technique of building a (GA)(n) and (CA)(n) microsatellite-enriched library by capture with streptavidin-coated magnetic beads. We assessed the polymorphism of seven microsatellites in 84 mature trees found in three areas (Ribeir (a) over tildeo Preto, Tambau and S (a) over tildeo Jose do Rio Pardo), highly impacted by the agricultural practices, in a large region among Pardo river and Mogi-Guacu river basins, in state of S (a) over tildeo Paulo, Brazil. All loci were polymorphic, and the number of alleles was high, ranging from 6 to 24, with a mean of 14.4. All stands showed the same high level of genetic diversity (mean H(E) = 0.83) and a low genetic differentiation (mean F(ST) = 0.031), indicating that genetic diversity was higher within rather than among populations. Seven of the nine loci were highly variable, and sufficiently informative for E. uniflora. It was concluded that these new SSR markers can be efficiently used for gene flow studies.