Identification of suitable internal control genes for expression studies in Coffea arabica under different experimental conditions

Background: Quantitative data from gene expression experiments are often normalized by transcription levels of reference or housekeeping genes. An inherent assumption for their use is that the expression of these genes is highly uniform in living organisms during various phases of development, in different cell types and under diverse environmental conditions. To date, the validation of reference genes in plants has received very little attention and suitable reference genes have not been defined for a great number of crop species including Coffea arabica. The aim of the research reported herein was to compare the relative expression of a set of potential reference genes across different types of tissue/organ samples of coffee. We also validated the expression profiles of the selected reference genes at various stages of development and under a specific biotic stress.Results: The expression levels of five frequently used housekeeping genes (reference genes), namely alcohol dehydrogenase (adh), 14-3-3, polyubiquitin (poly), beta-actin (actin) and glyceraldehyde-3-phosphate dehydrogenase (gapdh) was assessed by quantitative real-time RT-PCR over a set of five tissue/organ samples (root, stem, leaf, flower, and fruits) of Coffea arabica plants. In addition to these commonly used internal controls, three other genes encoding a cysteine proteinase (cys), a caffeine synthase (ccs) and the 60S ribosomal protein L7 (rpl7) were also tested. Their stability and suitability as reference genes were validated by geNorm, NormFinder and BestKeeper programs. The obtained results revealed significantly variable expression levels of all reference genes analyzed, with the exception of gapdh, which showed no significant changes in expression among the investigated experimental conditions.Conclusion: Our data suggests that the expression of housekeeping genes is not completely stable in coffee. Based on our results, gapdh, followed by 14-3-3 and rpl7 were found to be homogeneously expressed and are therefore adequate for normalization purposes, showing equivalent transcript levels in different tissue/ organ samples. Gapdh is therefore the recommended reference gene for measuring gene expression in Coffea arabica. Its use will enable more accurate and reliable normalization of tissue/organ-specific gene expression studies in this important cherry crop plant.

In vitro effects of propolis on Giardia duodenalis trophozoites

In order to improve the current chemotherapy of Giardia infection, potential antigiardial agents have been screened, including natural products. Propolis, a resinous hive product collected by bees, has attracted attention as a useful and popular substance with several therapeutic activities. The present study was carried out aiming to evaluate the in vitro effects of an ethanolic extract of propolis on the growth and adherence of Giardia duodenalis trophozoites. Propolis inhibited the growth of trophozoites and the level of inhibition varied according to the extract concentration and incubation times. The highest reduction of parasite growth was observed in cultures exposed to 125, 250 and 500 mu g/ml of propolis, in all incubation periods (24, 48, 72 and 96 h). Growth reduction by 50% was observed in 125 mu g/ml propolis-treated cultures, while the concentrations of 250 and 500 mu g/ml were able to inhibit growth by more than 60%. Propolis also inhibited parasite adherence and all assayed propolis concentrations promoted the detachment of trophozoites. Light microscope observations revealed changes of the pear-shaped aspect of the cell and reduction of flagellar beating frequency in the great part of the trophozoites. Our results hold the perspective for the utilization of propolis as an antigiardial agent. (c) 2005 Elsevier GmbH. All rights reserved.

Chromosome Mapping of Retrotransposable Elements Rex1 and Rex3 in Three Fish Species in the Subfamily Hypoptopomatinae (Teleostei, Siluriformes, Loricariidae)

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

Morphological and enzymatic analysis of the midgut of Anopheles darlingi during blood digestion

The midgut of adult female Anopheles darlingi is comprised of narrow anterior and dilated posterior regions, with a single layered epithelium composed by cuboidal digestive cells. Densely packed apical microvilli and an intricate basal labyrinth characterize each cell pole. Before blood feeding, apical cytoplasm contains numerous round granules and whorled profiles of rough endoplasmic reticulum. Engorgement causes a great distension of midgut. This provokes the flattening of digestive cells and their nuclei. Simultaneously, apical granules disappear, the whorls of endoplasmic reticulum disassemble and 3 h post bloodmeal (PBM), nucleoli enlarge manyfold. An intense absorptive process takes place during the first 24h PBM, with the formation of large glycogen inclusions, which persist after the end of the digestive process. Endoproteases activities are induced after bloodmeal and attain their maximum values between 10 and 36 h PBM. At least two different aminopeptidases seem to participate in the digestive process, with their maximum activity values at 36 and 48 h PBM, respectively. Coarse electrondense aggregates, possibly debris from digested erythrocytes, begin to appear on the luminal face of the peritrophic membrane from 18 h PBM and persist during all the digestive process, and are excreted at its end. We suggest that these aggregates could contain some kind of insoluble form of haem, in order of neutralize its toxicity. (c) 2005 Elsevier Ltd. All rights reserved.

In vitro effects of Crotalus durissus terrificus and Bothrops jararaca venoms on Giardia duodenalis trophozoites

Considering the snake venoms' pharmacological properties and chemotherapeutic potential as well as the need for new alternatives for Giardia infection treatment, the present study was carried out aiming to evaluate the in vitro effects of crude Crotalus durissus terrificus and Bothrops jararaca venoms on the growth and adherence of Giardia duodenalis trophozoites. Trophozoites (10(6)) were exposed to serial twofold dilutions of C. durissus terrificus and B. jararaca venoms that ranged from 3.125 to 200 mu g/ml and from 5 to 320 mu g/ml, respectively. The two venoms inhibited the growth of trophozoites, and the level of inhibition varied according to the assayed concentrations and incubation times. The highest reduction of parasite growth was observed with increasing concentrations of the venoms. No effect on parasite adherence was observed. Light microscope observations revealed changes of the pear-shape aspect of the cell and reduction of flagellar beating frequency in the great part of the trophozoites. This is the first attempt to evaluate the in vitro effects of snake venoms on Giardia trophozoites. The findings stress the need for more investigations and prompt us to perform further studies to identify active principles with antigiardial effect.

Bouchardia rosea, a vanishing brachiopod species of the Brazilian platform: taphonomy, historical ecology and conservation paleobiology

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

TAXONOMIC POSITION of GUIRATINGIA MENDESI (MEGADESMIDAE) and THE EVOLUTION of PERMIAN ENDEMIC BIVALVE FAUNA of THE PARANA BASIN, BRAZIL

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

Biodiversity of brachyuran crabs (Crustacea : Decapoda) from non-consolidated sublittoral bottom on the northern coast of São Paulo State, Brazil

The brachyuran community of the coast of São Paulo State is represented by about 188 species of crabs inhabiting different kinds of coastal marine environments. The biodiversity of brachyurans found on non-consolidated sublittoral bottom was investigated. The Ubatuba region (Ubatumirim, Ubatuba and Mar Virado bays, Couves and Mar Virado Islands, offshore region) was sampled for 3 years (1998-2000), at depths of 2-40 m. All sampling was performed using a fishing boat equipped with two double-rig nets. We collected 79 brachyuran species representing 9 superfamilies (4 Dromioidea, 1 Homoloidea, 2 Calappoidea, 5 Leucosioidea, 20 Majoidea, 7 Parthenopoidea, 17 Portunoidea, 18 Xanthoidea, and 5 Pinnotheroidea) and 41 genera. Ubatuba bay showed the greatest species richness with 50 species, followed by Ubatumirim with 45 and Mar Virado with 29. The number of species collected represents about 57% of the known species of crabs already reported for the shore of São Paulo State. It is worth noticing that this percentage is restricted only to non-consolidated sublittoral bottom. This fact indicates a great biodiversity of the habitat in this studied region, probably one to the diversity of habitat types present in the bays.

Diet of the ichthyofauna associated with marginal vegetation of a mangrove forest in southeastern Brazil

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

Diet of invertebrates sampled in leaf-bags incubated in a tropical headwater stream

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Diet and trophic groups of an aquatic insect community in a tropical stream

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

Effect of narcosis with CO2 on the ovarian development in queens of Apis mellifera (Hymenoptera, Apini)

Exposure of 1, 4, 7 & 10 day-old virgin queens of Apis mellifera Linne 1758 for 1 min to CO2 accelerated their ovarian development, having a similar effect as mating on the initial formation of the ovarian follicles. In 3 day-old queens the exposure to CO2 enhanced the initial stage of germ cell differentiation into oocytes and nurse cells in the ovarioles, a developmental stage only seen in 5 day-old untreated virgin queens, the age at which they are ready to mate. In 10 day-old untreated virgin queens, some regions of the ovarioles presented tissue disorganization and many cells with pycnotic nuclei. However, narcotized virgin queens of the same age did not present such ovary degeneration. Conversely, they showed nitid follicle formation, arising in the ovarioles' initial differentiation between nurse and oocytic chambers, although still without vitellogenesis. The accelerative effect of CO2 is limited to the ages near to those proper for mating, since 15 and 18 day-old treated virgin queens presented ovaries with extensive regions of high tissue disorganization and a great number of cells with pycnotic nuclei. According to the results, the narcosis presented three levels of effect on the ovary of honeybee virgin queens: 1) accelerated the germ cell differentiation, 2) preserved the tissue integrity even after the queen mating period and 3) stimulated the initial differentiation of a vitellarium. This later condition was only seen in untreated queens after mating. All these effects are not maintained if the queen is kept virgin beyond 15 days old.

A new type of sternal gland present in Oxaea flavescens (Hymenoptera, Oxaeinae): location and histology

The sternal glands of the abdomen of Oxaea flavescens consist of class ill glandular cells, with the excretory canaliculus linking to the lateral intersegmental membrane of segments ill, TV and V. The intersegmental membrane is augmented and folded into several lobes forming a reservoir covered by secretory cells. The intersegmental membrane is then transformed into an intima that lines a reservoir space containing secretions of a type of mucus which is periodic acid-Schiff positive. The storage of a great amount of secretion suggests that it is not used continuously. These glands are absent from males, indicating that their products must have a specific function linked to the female sex.

The use of biodiversity as source of new chemical entities against defined molecular targets for treatment of malaria, tuberculosis, and T-cell mediated diseases: a review

The modern approach to the development of new chemical entities against complex diseases, especially the neglected endemic diseases such as tuberculosis and malaria, is based on the use of defined molecular targets. Among the advantages, this approach allows (i) the search and identification of lead compounds with defined molecular mechanisms against a defined target (e.g. enzymes from defined pathways), (ii) the analysis of a great number of compounds with a favorable cost/benefit ratio, (iii) the development even in the initial stages of compounds with selective toxicity (the fundamental principle of chemotherapy), (iv) the evaluation of plant extracts as well as of pure substances. The current use of such technology, unfortunately, is concentrated in developed countries, especially in the big pharma. This fact contributes in a significant way to hamper the development of innovative new compounds to treat neglected diseases. The large biodiversity within the territory of Brazil puts the country in a strategic position to develop the rational and sustained exploration of new metabolites of therapeutic value. The extension of the country covers a wide range of climates, soil types, and altitudes, providing a unique set of selective pressures for the adaptation of plant life in these scenarios. Chemical diversity is also driven by these forces, in an attempt to best fit the plant communities to the particular abiotic stresses, fauna, and microbes that co-exist with them. Certain areas of vegetation (Amazonian Forest, Atlantic Forest, Araucaria Forest, Cerrado-Brazilian Savanna, and Caatinga) are rich in species and types of environments to be used to search for natural compounds active against tuberculosis, malaria, and chronic-degenerative diseases. The present review describes some strategies to search for natural compounds, whose choice can be based on ethnobotanical and chemotaxonomical studies, and screen for their ability to bind to immobilized drug targets and to inhibit their activities. Molecular cloning, gene knockout, protein expression and purification, N-terminal sequencing, and mass spectrometry are the methods of choice to provide homogeneous drug targets for immobilization by optimized chemical reactions. Plant extract preparations, fractionation of promising plant extracts, propagation protocols and definition of in planta studies to maximize product yield of plant species producing active compounds have to be performed to provide a continuing supply of bioactive materials. Chemical characterization of natural compounds, determination of mode of action by kinetics and other spectroscopic methods (MS, X-ray, NMR), as well as in vitro and in vivo biological assays, chemical derivatization, and structure-activity relationships have to be carried out to provide a thorough knowledge on which to base the search for natural compounds or their derivatives with biological activity.

Effect of paprika on the ovarian development of Africanized honeybee workers (Apis mellifera L.) in queenless colonies

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)