Differences in essential oil production and leaf structure in pheno-types of damiana<Emphasis Type="Italic">(turnera diffusa</Emphasis> willd.

Plants of damiana(Turnera diffusa Willd.) are important to industry and traditional medicine in semi-arid climates. Although all populations are wild, no reports have been made previously of their different phenotypes. Here, we investigated various micromorphological characteristics and the levels of essential oils in two phenotypes. Oils were extracted from fresh leaves via hydrodistillation and analyzed by gas chromatography-mass spectrometry. Morphological analyses were conducted under a stereoscopic microscope and with a scanning electron microscope. In all, 56 compounds were identified, enabling us to distinguish separate phenotypes. DL1 plants mainly contained 1,8-cineole, 10-epi y eudesmol, and guaiol; whereas those of DL2 primarily constituted ß-pinene, ß-caryophyllene oxide, cadinene, and α-cadinol. These two phenotypes also differed in their morphologies, with DL1 leaves showing elevated essential oil concentrations, but lacking trichomes. In contrast, the DL2 plants had lower contents of essential oils but did possess trichomes on their abaxial and adaxial leaf surfaces. This documentation of individual damiana phenotypes is the initial process toward validating the quality of essential oils from this species as well as inherent structural variations.     

Modulation of the redox state of the copper sites of human ceruloplasmin by chloride

Incubation of human ceruloplasmin with physiological concentrations of chloride at neutral pH invariably caused dramatic changes of both the spectroscopic and the functional properties of the protein. The optical intensity at 610 nm increased up to 60%, with a concomitant decrease at 330 nm and the appearance of new bands between 410 and 500 nm. Signals previously undetectable appeared in the EPR spectrum. On the basis of computer simulations, they were interpreted as stemming from an oxidized type 1 copper site and from a half-reduced type 3 copper pair. Removal of chloride completely restored the original optical and EPR lineshapes. Hydrogen peroxide, added to ceruloplasmin in the presence of chloride, was able to capture the electron of the half-reduced type 3 site and to yield a protein insensitive to subsequent removal and readdition of the anion. As a whole, the spectroscopic data indicate that a blue site is partially reduced in the resting protein and that, upon binding of chloride, human ceruloplasmin undergoes a structural change leading to displacement of an electron from the reduced type 1 site to the type 3 site pair. Chloride dramatically affected the catalytic efficiency of human ceruloplasmin. At neutral pH, the anion was an activator of the oxidase activity, being able to enhance up to tenfold the catalytic rate. AtpH < 6, in line with all previous reports, chloride strongly inhibited the activity. At intermediate pH values, i.e., around 6, the effect was composite, with an activating effect at low concentration and an inhibitory effect at higher concentration. Since chloride is present at very high concentrations in the plasma, these results suggest that human ceruloplasmin is, in the plasma, under control of this anion.     

Expression of full-length human pro-urokinase in mammary glands of transgenic mice

Human pro-urokinase expressed in the mammary glands of transgenic animals is quickly activated and converted to urokinase by proteases that are present in the milk. Thus, it is nearly impossible to isolate full-sized pro-urokinase from the milk of transgenic animals. To solve this problem, we constructed transgenic mice that express human pro-urokinase and modified ecotin, which is a potent serine protease inhibitor from E. coli, in their mammary glands. The gene encoding ecotin was modified so as to enhance its specificity for the human urokinase-type plasminogen activator. Co-expression of modified ecotin and human pro-urokinase in the mammary glands allows for purification of full-length human pro-urokinase from these transgenic mice. The results described here suggest a general way of preventing the activation of zymogens that are expressed in the mammary glands of transgenic animals by co-expression of a zymogen along with a protease inhibitor.     

Purification and partial characterization of camel (Camelus Dromedarius) ceruloplasmin

Adult and young camel ceruloplasmin (Cp) were isolated and purified using the single-step chromatography on amino ethyl-activated sepharose. There are no differences between the adult and the young camel protein. The molecular mass of the protein, as estimated by SDS-PAGE (denaturant conditions), was approximately 130000 Da. The electrophoretic mobility of camel Cp is slightly higher as compared to human and sheep protein suggesting that the camel Cp is homogeneous, compact and more acid. The copper content was estimated to be 5.8+/-0.3 atoms per molecule. The spectroscopic feature includes an absorption maximum at 610 nm, which could be attributed to type 1 copper. The EPR spectrum was completely devoid of any typical signal of the type 2 copper. The kinetic parameters of the adult camel Cp for the specific activity as p-phenylendiamine oxidase were determined as K(m)=0.42 mM and V(max)=0.93 microM NADH/mn/mg Cp. The optimum pH for the activity was 5.7.     

Fusarium oxysporum G-protein beta subunit Fgb1 regulates hyphal growth, development, and virulence through multiple signalling pathways

The vascular wilt fungus Fusarium oxysporum causes disease in a wide variety of crops. A signalling cascade controlled by the extracellular-regulated mitogen-activated protein kinase (MAPK) Fmk1 was previously found to be required for plant infection. To investigate the role of the heterotrimeric G-protein beta subunit Fgb1 as a putative upstream component of the Fmk1 signalling cascade, we generated F. oxysporum strains carrying either a Deltafgb1 loss-of-function allele or an fgb1(W115G) allele that mimicks the yeast STE4(W136G) mutation resulting in insensitivity to the cognate G-protein alpha subunit. Both types of mutants showed reduced virulence on tomato plants, similar to Deltafmk1 strains. However, in contrast to the latter, Deltafgb1 mutants displayed an abnormal hyphal growth phenotype with highly elongated cells, increased tip growth, a completely straight hyphal growth axis, and reduced subapical branching. Exogenous cAMP reversed part but not all of the Deltafgb1 growth phenotypes. Likewise, expression of the fgb1(W115G) allele only partly reversed growth phenotypes and failed to restore virulence on plants, whereas reintroduction of a functional fgb1 allele fully restored the wild type phenotype. Immunoblot analysis showed that levels of Fmk1 phosphorylation in fgb1 mutants were comparable to those in the wild type strain. Our results support a model in which Fgb1 controls hyphal growth, development and virulence in F. oxysporum both through cAMP-dependent and -independent pathways.     

Testosterone and intrasexual competition in men: is there any relation with digit ratio (2D:4D)?

Digit ratio 2D:4D is a sexually dimorphic characteristic and it is believed that this difference is related to high levels of prenatal testosterone and circulating testosterone in men. High levels of testosterone are also associated with traits related to competitiveness. Therefore, this study aimed to examine the relation between intrasexual competition and 2D:4D in men. One hundred thirteen college men answered a questionnaire to measure their scores of intrasexual competition and donated a saliva sample to measure their testosterone levels; finally, the finger length from both hands was measured. It was found a positive correlation between testosterone levels and intrasexual competition scores, and a negative correlation between testosterone levels and left 2D:4D. Finally, we did not find a significant association between digit ratios 2D:4D and intrasexual competition scores. Our study shows that men with higher testosterone levels also have higher intrasexual competition scores and lower values of left digit ratio 2D:4D. Further studies will have to take into account fluctuations in testosterone over the time to observe if the relation between competitiveness scores and digit ratios 2D:4D becomes significant.     

Biochemical and NMR study on the competition between proteins SC35, SRp40, and heterogeneous nuclear ribonucleoprotein A1 at the HIV-1 Tat exon 2 splicing site

The human immunodeficiency virus, type 1, Tat protein plays a key role in virus multiplication. Because of its apoptotic property, its production is highly controlled. It depends upon the A3 splicing site utilization. A key control of site A3 activity is the ESS2 splicing silencer, which is located within the long stem-loop structure 3 (SLS3), far downstream from site A3. Here, by enzymatic footprints, we demonstrate the presence of several heterogeneous nuclear ribonucleoprotein (hnRNP) A1-binding sites on SLS3 and show the importance of the C-terminal Gly domain of hnRNP A1 in the formation of stable complexes containing several hnRNP A1 molecules bound on SLS3. Mutations in each of the UAG triplets in ESS2 strongly reduce the overall hnRNP A1 binding, showing the central role of ESS2 in hnRNP A1 assembly on SLS2-SLS3. Using NMR spectroscopy, we demonstrate the direct interaction of ESS2 with the RNA recognition motifs domains of hnRNP A1. This interaction has limited effect on the RNA two-dimensional structure. The SR proteins SC35 and SRp40 were found previously to be strong activators of site A3 utilization. By enzymatic and chemical footprints, we delineate their respective binding sites on SLS2 and SLS3 and find a strong similarity between the hnRNP A1-, SC35-, and SRp40-binding sites. The strongest SC35-binding site only has a modest contribution to site A3 activation. Hence, the main role of SR proteins at site A3 is to counteract hnRNP A1 binding on ESS2 and ESE2. Indeed, we found that ESE2 has inhibitory properties because of its ability to bind hnRNP A1.     

Corticosteroids inhibit prostaglandin release from perfused mesenteric blood vessels of rabbit and from perfused lungs of sensitized guinea pig

Infusion of norephinephrine (NE) (1 – 3 μg/ml/min) into the isolated mesenteric vascular preparation of rabbit resulted in a rise in perfusion pressure, which was associated with the release of a prostaglandin E-like substance (PGE) at a concentration of 2.81 ± 0.65 ng/ml in terms of PGE₂. Indomethacin (3 μg/ml) abolished the NE-induced release of PGE. Arachidonic acid (0.2 μg/ml) in the presence of indomethacin did not restore the NE-induced release of PGE. Hydrocortisone (10 – 30 μg/ml) and dexamethasone (2 – 5 μg/ml) also inhibited the NE-induced release of PGE. The inhibitory action of both corticosteroids was abolished by arachidonic acid (0.2 μg/ml). Antigen-induced release of a prostaglandin-like substance (PGs) (43.1 ± 3.8 ng/ml in terms of PGE₂ and a rabbit aorta contracting substance (RCS) from perfused lungs of sensitized guinea pigs was completely abolished by indomethacin (5 μg/ml) or by hydrocortisone (100 μg/ml). Indomethacin, however, increased histamine release up to 280% of the control level, which was 470 ± 54 ng/ml, while hydrocortisone diminished histamine release down to 30% of the control level. A superimposed infusion of arachidonic acid (1 μg/ml) into the pulmonary artery reversed the hydrocortisone-induced blockade of the release of RCS and PGs. It may be concluded that corticosteroids neither inhibit prostaglandin synthetase nor influence prostaglandin transport through the membranes but they do impair the availability of the substrate for the enzyme.     

Targeted screening strategies to detect Trypanosoma cruzi infection in children

Background

Millions of people are infected with Trypanosoma cruzi, the causative agent of Chagas disease in Latin America. Anti-trypanosomal drug therapy can cure infected individuals, but treatment efficacy is highest early in infection. Vector control campaigns disrupt transmission of T. cruzi, but without timely diagnosis, children infected prior to vector control often miss the window of opportunity for effective chemotherapy.

Methods and Findings

We performed a serological survey in children 2–18 years old living in a peri-urban community of Arequipa, Peru, and linked the results to entomologic, spatial and census data gathered during a vector control campaign. 23 of 433 (5.3% [95% CI 3.4–7.9]) children were confirmed seropositive for T. cruzi infection by two methods. Spatial analysis revealed that households with infected children were very tightly clustered within looser clusters of households with parasite-infected vectors. Bayesian hierarchical mixed models, which controlled for clustering of infection, showed that a child''s risk of being seropositive increased by 20% per year of age and 4% per vector captured within the child''s house. Receiver operator characteristic (ROC) plots of best-fit models suggest that more than 83% of infected children could be identified while testing only 22% of eligible children.

Conclusions

We found evidence of spatially-focal vector-borne T. cruzi transmission in peri-urban Arequipa. Ongoing vector control campaigns, in addition to preventing further parasite transmission, facilitate the collection of data essential to identifying children at high risk of T. cruzi infection. Targeted screening strategies could make integration of diagnosis and treatment of children into Chagas disease control programs feasible in lower-resource settings.