Influence of Atlantic Rain Forest remnants on the biological control of Euselasia apisaon (Dahman) (Lepidoptera: Riodinidae) by Trichogramma maxacalii (Voegelé e Pointel) (Hymenoptera: Trichogrammatidae)

This study evaluated the effects of Atlantic Rain Forest remnants on the natural biological control of Euselasia apisaon (Dahman) by the parasitoid Trichogramma maxacalii (Voegelé e Pointel) in Eucalyptus plantations. The number of E. apisaon eggs/leaf was higher in the center than in the edge of the plantations (23.5 +/- 7.61 vs. 14.8 +/- 3.14), but parasitism showed the reversed pattern (72.4% in the center and 80.5% in the edge). The results indicated that natural regulation exerted by T. maxacalii on populations of E. apisaon may be enhanced by the preservation of fragments of native vegetation surrounding Eucalyptus plantations.     

Preservation of bovine preantral follicle viability and ultra-structure after cooling and freezing of ovarian tissue

Bovine preantral follicles within ovarian fragments were exposed and cryopreserved in absence or presence of 1.5 M glycerol (GLY), ethylene glycol (EG), propanediol (PROH) or dimethyl sulfoxide (DMSO), undergoing a previous cooling at 20 °C for 1 h (protocol 1) or at 4 °C for 24 h (protocol 2) in 0.9% saline solution. At the end of each treatment, preantral follicles were classified as non-viable/viable when they were stained/not stained with trypan blue, respectively. To confirm viability staining, ultra-structure of the follicles was evaluated by transmission electronic microscopy (TEM). Data were compared by Chi-square test (P < 0.05). The storage of the ovaries at 20 °C for 1 h (78%) and 4 °C for 24 h (80%) did not reduce significantly the percentage of viable preantral follicles when compared to the control (75%). Similar results were obtained when ovarian fragments, respectively, for protocols 1 and 2, were exposed to MEM (78 and 77%), 1.5 M EG (78 and 71%), as well as frozen in 1.5 M EG (74 and 77%). Percentages of viable follicles in control were similar to those observed after exposure (75%) and freezing (76%) in presence of 1.5 M DMSO only when protocol 1 was used. The increase of the concentration from 1.5 to 3.0 M, for all cryoprotectants, reduced significantly the percentage of viable preantral follicles after freezing. Ultra-structural analysis has confirmed trypan blue results, showing that not only basement membrane, but also organelles, were intact in viable preantral follicles. In conclusion, ovarian tissue cooling at 4 °C for 24 h before cryopreservation (protocol 2) does not affect the viability of bovine preantral follicles when 1.5 M EG is present in the cryopreservation medium.     

Persistent and remodeling hepatic preneoplastic lesions present differences in cell proliferation and apoptosis, as well as in p53, Bcl-2 and NF-kappaB pathways

During rat hepatocarcinogenesis preneoplastic lesions (PNL) emerge which may persist (pPNL) and be sites of progress to cancer or suffer remodeling (rPNL) tending to disappear. Cellular and molecular mechanisms involved in both phenotypes are not sufficiently elucidated. pPNL and rPNL cellular proliferation and apoptosis were evaluated in rats submitted to the resistant hepatocyte (RH) model, and an adjusted growth index (AGI) was established. p53, Bcl-2, and NF-kappaB p65 subunit expression was evaluated by immunohistochemistry in pPNL and rPNL. p65 expression and NF-kappaB activation was evaluated by Western blot assays in whole livers. A lower number of BrdU-stained hepatocyte nuclei/mm(2) and higher number of apoptotic bodies (AB) per mm(2) were observed in remodeling compared to pPNL. Cytoplasmic p53 accumulation is related to increased hepatocarcinoma malignancy. We observed that 71.3% pPNL and 25.4% rPNL (P < 0.05) presented p53 staining in the cytoplasm. Similarly, 67.7% pPNL and 23.1 % rPNL (P < 0.05) presented increased Bcl-2 staining. Thirty-two percent pPNL and 15.6% rPNL (P < 0.05) presented p65 staining. Compared to normal rats, increase (P < 0.05) of hepatic p65 expression and NF-kappaB activation in rats submitted to the RH model was observed. In agreement to previous studies hepatic pPNL and rPNL differ regarding cell proliferation and apoptosis. Moreover, persistence and remodeling involve differences in p53, Bcl-2, and NF-kappaB pathways. These data point to molecular pathways that may direct preneoplastic lesions to spontaneously regress or to progress to cancer. J. Cell. Biochem. 103: 538-546, 2008. (c) 2007 Wiley-Liss, Inc.     

Cytotoxic turrianes of Kermadecia elliptica from the New Caledonian rainforest

In the course of an automated screening for small molecules presenting cytotoxic activity, eight new cyclophanes named kermadecins A-H (1-8), have been isolated from the bark of a New Caledonian plant, Kermadecia elliptica, Proteaceae. A LC/APCI-MS study performed on kermadecins A, B and C, provided a reliable method for the detection of other analogues existing in small quantities in the extract. This led to the isolation of five other members of this chemical series. The structures were elucidated by extensive mono- and bi-dimensional spectroscopy and mass spectrometry. The cytotoxic activity of four of them was evaluated on various cell lines.     

Effects of crude oil on survival,morphology, and anatomy of two aquatic macrophytes from the Amazon floodplains

Aquatic herbaceous macrophytes grow in profusion in the Amazon fertile varzea floodplains. A large number of species occur but only a few are particularly abundant, supporting food chains, contributing substantially to carbon and nutrient cycles. Their growth and role in the ecosystem depend, among other, on its life cycles and habits, floating or semi-aquatic. Although in the last decades, petroliferous activity intensified in the Central Amazon region and so did oil spills, the effect of petroleum on the native aquatic plants is unknown. The present study was designed to test experimentally the survival and morpho-anatomical modifications of the free floating water hyacinth Eichhornia crassipes and the semi-aquatic grass Echinochloa polystachya to 10 different concentrations of crude oil. Higher concentrations of crude oil caused the mortality in both species; however, lethal dose (LD₅₀) values showed that E. polystachya was more sensitive than E. crassipes. Despite the higher tolerance of E. crassipes, the inhibition of root and leaf growth as well as anatomical modifications in leaves were registered in higher concentrations. Additionally, the oil caused a reduction in leaf numbers in both species. Although mortality of the floating species was lower, it may increases over time, since important alterations in morphology and anatomy occurred. These results show that oil spills in the Amazon varzea can cause severe alterations in the aquatic flora and in the floodplain dynamics.     

Identification and characterization of <Emphasis Type="Italic">Gluconacetobacter diazotrophicus</Emphasis> mutants defective in the solubilization of phosphorus and zinc

Gluconacetobacter diazotrophicus is a plant-growth-promoting bacterium, which is able to colonize sugarcane and other plant species of economic importance. The potentially beneficial effects promoted by this bacterium on plants are nitrogen-fixation, production of phythormones, action against pathogens and mineral nutrient solubilization. In this study, the molecular mechanisms associated with phosphorus and zinc solubilization were analyzed. A transposon mutant library was constructed and screened to select for mutants defective for phosphorous [Ca₅(PO₄)₃OH] and zinc (ZnO) solubilization. A total of five mutants were identified in each screen. Both screenings, performed independently, allowed to select the same mutants. The interrupted gene in each mutant was identified by sequencing and the results demonstrate that the production of gluconic acid is a required pathway for solubilization of such nutrients in G. diazotrophicus. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. A. C. Intorne and M. V. V. de Oliveira contributed equally to this work.     

Trans-Generational Influence of Human Disturbances in Japanese Quail: Egg Quality Influences Male Social and Sexual Behaviour

In many bird species prenatal exposure to yolk androgens of maternal origin has been found to influence offspring behavioural phenotype. In contrast to altricial birds, far less is known about maternal effects in precocial birds. In a previous experiment we found that female quail ( Coturnix japonica ) that were not previously habituated to humans (NH) produced eggs with less androgens (testosterone, androstenedione) and more progesterone when exposed to human disturbances than females habituated to humans (H). Here, we analysed social motivation and sexual behaviour of the male offspring of NH and H females. Classical behavioural test procedures were applied including separation, runway, partner choice and female encounter tests. As chicks, offspring of the NH females spent more time far from conspecifics than offspring of the H females. As adults, the same NH males showed less crowing and courtship behaviour (ritual preening) in female encounter tests than H males. Thus, maternal environment and egg quality may be key factors in the emergence of individual variability of appetitive behaviour, such as social proximity and courtship behaviour. Human disturbance of the mother seems to have triggered trans-generational effects resulting in consistently reduced social and sexual motivation in offspring until adulthood.     

Mutational analysis of three <Emphasis Type="Italic">bchH</Emphasis> paralogs in (bacterio-)chlorophyll biosynthesis in <Emphasis Type="Italic">Chlorobaculum tepidum</Emphasis>

The first committed step in the biosynthesis of (bacterio-)chlorophyll is the insertion of Mg²⁺ into protoporphyrin IX by Mg-chelatase. In all known (B)Chl-synthesizing organisms, Mg-chelatase is encoded by three genes that are homologous to bchH, bchD, and bchI of Rhodobacter spp. The genomes of all sequenced strains of green sulfur bacteria (Chlorobi) encode multiple bchH paralogs, and in the genome of Chlorobaculum tepidum, there are three bchH paralogs, denoted CT1295 (bchT), CT1955 (bchS), and CT1957 (bchH). Cba. tepidum mutants lacking one or two of these paralogs were constructed and characterized. All of the mutants lacking only one of these BchH homologs, as well as bchS bchT and bchH bchT double mutants, which can only produce BchH or BchS, respectively, were viable. However, attempts to construct a bchH bchS double mutant, in which only BchT was functional, were consistently unsuccessful. This result suggested that BchT alone is unable to support the minimal (B)Chl synthesis requirements of cells required for viability. The pigment compositions of the various mutant strains varied significantly. The BChl c content of the bchS mutant was only ~10% of that of the wild type, and this mutant excreted large amounts of protoporphyrin IX into the growth medium. The observed differences in BChl c production of the mutant strains were consistent with the hypothesis that the three BchH homologs function in end product regulation and/or substrate channeling of intermediates in the BChl c biosynthetic pathway. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The genetic status of the violet copper Lycaena helle– a relict of the cold past in times of global warming

Rising temperatures and agricultural changes (intensification and succession on fallow land) during the last few decades have caused a strong decline of moist and cool sites on nutrient-poor grasslands and species depending on these habitats. We tested the effects of habitat deterioration on a local and regional scale in such a species, the highly endangered butterfly Lycaena helle , which was more widely distributed over central Europe during the postglacial period, but has recently become restricted to some remnants. We analysed five polymorphic microsatellite loci in 220 individuals sampled at ten different localities. The study sites in Germany, Luxembourg and Belgium are geographically split into three mountain regions: the Ardennes, the Eifel and the Westerwald; the latter is separated from the other two by the river Rhine. A comparatively high genetic diversity was detected in all local populations and genetic differentiation was found among the Ardennes, the Eifel and the Westerwald (F_CT: 0.084). The genetic differentiation among all populations (F_ST: 0.137) underlines natural and anthropogenic habitat fragmentation. While ongoing gene flow seems to exist among the Eifel populations indicating the only intact metapopulation, a high genetic differentiation in the Ardennes and the Westerwald indicates a disruption of population connectivity. Our genetic data obtained on different spatial scales show the genetic consequence of long-term isolation and should trigger necessary conservation measures at the metapopulation level.     

Camelid nanobodies raised against an integral membrane enzyme,nitric oxide reductase

Nitric Oxide Reductase (NOR) is an integral membrane protein performing the reduction of NO to N₂O. NOR is composed of two subunits: the large one (NorB) is a bundle of 12 transmembrane helices (TMH). It contains a b type heme and a binuclear iron site, which is believed to be the catalytic site, comprising a heme b and a non-hemic iron. The small subunit (NorC) harbors a cytochrome c and is attached to the membrane through a unique TMH. With the aim to perform structural and functional studies of NOR, we have immunized dromedaries with NOR and produced several antibody fragments of the heavy chain (VHHs, also known as nanobodies™). These fragments have been used to develop a faster NOR purification procedure, to proceed to crystallization assays and to analyze the electron transfer of electron donors. BIAcore experiments have revealed that up to three VHHs can bind concomitantly to NOR with affinities in the nanomolar range. This is the first example of the use of VHHs with an integral membrane protein. Our results indicate that VHHs are able to recognize with high affinity distinct epitopes on this class of proteins, and can be used as versatile and valuable tool for purification, functional study and crystallization of integral membrane proteins.