Cucurbitacins from Ecballium elaterium juice increase the binding of bilirubin and ibuprofen to albumin in human plasma

Ecballium elaterium, a medicinal plant, whose fruit juice is used for the treatment of jaundice in folk medicine, has been reported as being capable of decreasing bilirubinemia in animals with jaundice [H.H. Elayan, M.N. Garaibeh, S.M. Zmeili, S.A. Salhab, Effects of Ecballium elaterium juice on serum bilirubin concentration in male rats, Int. J. Crude Drug Res. 27 (1989) 227-234]. The aim of this study is to identify the Ecballium elaterium components, which are able to modify the binding of bilirubin to albumin. The juice is fiber-free but contains proteins, lipids, sugars, and minerals. The extract of the juice, analyzed by liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS), contains cucurbitacins (Cuc) B, D, E, and I as well as several glycosylated compounds. Human plasma containing no or serial concentrations of Ecballium elaterium components were prepared and the direct bilirubin (DB) and total bilirubin (TB) were determined by the Jendrassik and Grof method. Our results showed that Cuc D, E, and B decreased the levels of DB and TB in plasma, while Cuc I, glycosyl derivatives, and proteins of the juice did not modify the bilirubin levels. The binding of domain specific ligands to HSA, bilirubin (domain IIA), and ibuprofen (domain IIIA), were studied in the absence and presence of Cuc D, E, and I, by fluorescence spectroscopy. The values of binding constant K(a) and binding site number n, determined by Scatchard method, increased for the both ligands only in the presence of Cuc E and D. Cuc I decreased slightly the K(a) of ibuprofen, suggesting an interaction with the domain IIIA of the protein. As a conclusion, Cuc E, D, and B produce rearrangement in the structure of albumin leading to increase the binding of domain specific ligands, ibuprofen and bilirubin.     

Selective antibacterial activity of the cationic peptide PaDBS1R6 against Gram-negative bacteria

Infections caused by Gram-negative bacteria, Escherichia coli and Pseudomonas aeruginosa foremost among them, constitute a major worldwide health problem. Bioinformatics methodologies are being used to rationally design new antimicrobial peptides, a potential alternative for treating these infections. One of the algorithms used to develop antimicrobial peptides is the Joker, which was used to design the peptide PaDBS1R6. This study evaluates the antibacterial activities of PaDBS1R6 in vitro and in vivo, characterizes the peptide interaction to target membranes, and investigates the PaDBS1R6 structure in contact with mimetic vesicles. Moreover, we demonstrate that PaDBS1R6 exhibits selective antimicrobial activity against Gram-negative bacteria. In the presence of negatively charged and zwitterionic lipids the structural arrangement of PaDBS1R6 transits from random coil to α-helix, as characterized by circular dichroism. The tertiary structure of PaDBS1R6 was determined by NMR in zwitterionic dodecylphosphocholine (DPC) micelles. In conclusion, PaDBS1R6 is a candidate for the treatment of nosocomial infections caused by Gram-negative bacteria, as template for producing other antimicrobial agents.     

The ovarian and uterine responses of Baixadeiro mares to prostaglandin synchronization during the dry and rainy seasons

This study aimed to evaluate the effect of synchronization with prostaglandin F2α in Baixadeiro mares during the rainy and dry seasons. Fourteen mares were synchronized by administering two doses of 1 mL prostaglandin PGF 2α and monitored by rectal palpation and ultrasound for the assessment of follicular development and uterine echotexture. Of this total, nine mares allowed the collection of blood, in which the blood was collected by venipuncture of the jugular vein to determine progesterone (P4) by ELISA. Mares showed no differences (P > 0.05) in weight, body score condition (BSC), tone, uterine edema, frequency of ovulation, synchronization interval, estrus, and the total number of follicles between periods. However, there was a difference in large increased follicle diameter (P < 0.05) during the dry season. The average concentrations of P4 in mares differed (P < 0.05) between the pre- and post-ovulatory phases for both seasons and after ovulation, with higher concentrations in the rainy season. Furthermore, statistical differences in daily light (P < 0.05) were observed between the dry and rainy periods. Thus, we conclude that mares from the genetic grouping Baixadeiro showed no reproductive seasonality, though there was a difference in luminosity between the rainy and dry seasons. The treatment with two doses of PGF 2α was effective in synchronizing the mares, promoting the return of estrus in the dry and rainy periods. The mares remaining cyclically active throughout the year provided there were appropriate forage availability and quality levels to allow for normal values of body weight and condition.     

Role of Repeat I in the fast inactivation kinetics of the Ca(V)2.3 channel

The molecular basis for inactivation in Ca(V)2.3 (alpha 1E) channels was studied after expression of alpha 1E/alpha 1C (Ca(V)2.3/Ca(V)1.2) chimeras in Xenopus oocytes. In the presence of 10 mM Ba(2+), the CEEE chimera (Repeat I+part of the I-II linker from Ca(V)1.2) displayed inactivation properties similar to Ca(V)1.2 despite being more than 90% homologous to Ca(V)2.3. The transmembrane segments of Repeat I did not appear to be crucial as inactivation of EC(IS1-6)EEE was not significantly different than Ca(V)2.3. In contrast, EC(AID)EEE, with the beta-subunit binding domain from Ca(V)1.2, tended to behave like Ca(V)1.2 in terms of inactivation kinetics and voltage dependence. A detailed kinetic analysis revealed nonetheless that CEEE and EC(AID)EEE retained the fast inactivation time constant (tau(fast) approximately equal to 20-30 ms) that is a distinctive feature of Ca(V)2.3. Altogether, these data suggest that the region surrounding the AID binding site plays a pivotal albeit not exclusive role in determining the inactivation properties of Ca(V)2.3.     

A new heptasubstituted (E)-aurone glucoside and other aromatic compounds of Gomphrena agrestis with biological activity

A new aurone 1 and two known substances, aurantiamide acetate (2) and tiliroside (3), were isolated from the ethanolic extract of Gomphrena agrestis. The structural determination of 1 was based on spectroscopic and spectrometric data. The substance was defined as (E)-3'-O-beta-D-glucopyranosyl-4,5,6,4'-tetrahydroxy-7,2'-dimethoxyaurone. Biological activity of the ethanolic crude extract and isolated compounds against bacteria, fungi and Leishmania amazonensis amastigotes was evaluated. This appears to be the first report documenting aurone and aurantiamide compounds in the Amaranthaceae family. In the evaluation of biological activity the ethanolic extract of G. agrestis and compounds 1, 2, and 3 were shown to be active mainly against Staphylococcus aureus, Staphylococcus epidermidis and Pseudomonas aeruginosa.     

A proteomic approach to studying plant response to crenate broomrape (Orobanche crenata) in pea (Pisum sativum)

Crenate broomrape (Orobanche crenata) is a parasitic plant that threatens legume production in Mediterranean areas. Pea (Pisum sativum) is severely affected, and only moderate levels of genetic resistance have so far been identified. In the present work we selected the most resistant accession available (Ps 624) and compared it with a susceptible (Messire) cultivar. Experiments were performed by using pot and Petri dish bioassays, showing little differences in the percentage of broomrape seed germination induced by both genotypes, but a significant hamper in the number of successfully installed tubercles and their developmental stage in the Ps 624 compared to Messire. The protein profile of healthy and infected P. sativum root tissue were analysed by two-dimensional electrophoresis. Approximately 500 individual protein spots could be detected on silver stained gels. At least 22 different protein spots differentiated control, non-infected, Messire and Ps 624 accessions. Some of them were identified by MALDI-TOF mass spectrometry and database searching as cysteine proteinase, beta-1,3-glucanase, endochitinase, profucosidase, and ABA-responsive protein. Both qualitative and quantitative differences have been found among infected and non-infected root extracts. Thus, in the infected susceptible Messire genotype 34 spots were decreased, one increased and three newly detected, while in Ps 624, 15 spots were increased, three decreased and one newly detected. In response to the inoculation, proteins that correspond to enzymes of the carbohydrate metabolism (fructokinase, fructose-bisphosphate aldolase), nitrogen metabolism (ferredoxin-NADP reductase) and mitochondrial electronic chain transport (alternative oxidase 2) decreased in the susceptible check, while proteins that correspond to enzymes of the nitrogen assimilation pathway (glutamine synthetase) or typical pathogen defence, PR proteins, including beta-1,3-glucanase and peroxidases, increased in Ps 624. Results are discussed in terms of changes in the carbohydrate and nitrogen metabolism an induction of defence proteins in response to broomrape parasitism.     

Effects of starvation on haemolymphatic glucose levels, glycogen contents and nucleotidase activities in different tissues of Helix aspersa (Müller, 1774) (Mollusca, Gastropoda)

In the present study, the glucose concentration in the haemolymph and glycogen levels were determined in the various body parts of the Helix aspersa snail after feeding lettuce ad libitum and after various periods of starvation. To characterize the effect of starvation on nucleotidase activity, enzyme assays were performed on membranes of the nervous ganglia and digestive gland. Results demonstrated the maintenance of the haemolymph glucose concentration for up to 30 days of starvation, probably due to the consumption of glycogen from the mantle. In the nervous ganglia, depletion of glycogen occurs progressively during the different periods of starvation. No significant changes were observed on ATP and ADP hydrolysis in the membranes of nervous ganglia and no alterations in Ca2+ -ATPase and Mg2+ -ATPase occurred in the membranes of the digestive gland of H. aspersa during the different periods of starvation. Although there were no changes in the enzyme activities during starvation, they could be modulated by effectors in situ with concomitant changes in products/reactants during starvation.     

Molecular phylogeny of Demospongiae: implications for classification and scenarios of character evolution

An analysis of the phylogenetic relationships of the 13 orders of Demospongiae, based on 18S and C1, D1 and C2 domains of 28S rRNA (for, respectively, 26 and 32 taxa) has been performed. The class Demospongiae as traditionally defined is not found to be monophyletic. Instead, a clade comprising all demosponges except Homoscleromorpha is well-supported, and we define phylogenetically the name Demospongiae in this more restricted sense to preclude the possibility of drastic alterations of the meaning of Demospongiae in the future, depending on the position of Homoscleromorpha. Within this clade Demospongiae s.s., ceractinomorphs and tetractinomorphs are polyphyletic, implying homoplastic evolution of characters such as reproductive strategies (viviparity vs. oviparity) and skeleton architecture (reticulate vs. radiate). The topology derived from our molecular data provides a basis for proposing a new classification of Demospongiae s.s., and suggests a reverse polarity of some characters, with respect to traditional conceptions: viviparity, presence of monaxon spicules and of spongin appear to be ancestral, whereas oviparity, and presence of tetraxon spicules appear as derived characters.     

Pancreatic hepatocytes in hamsters (Mesocricetus auratus) infected with Trypanosoma cruzi

Hepatocytic metaplasia may be induced in hamsters by carcinogens, and associated with aging, diabetes or chronic pancreatitis. By means of histopathologic and immunohistochemic studies, we observed pancreatic hepatocytes in hamsters infected and reinfected with Trypanosoma cruzi. The change was seen in 18 (19%) out of 94 infected animals, and was not found among 53 controls, Normal islet cells were immunoreactive for neuron-specific enolase and not reactive for NCL-HAS. Metaplastic cells were immunoreactive for NCL-HAS and not reactive for islet hormones and enolase. No relationship was observed between number of inoculations and metaplasia; however, the intensity of the inflammatory process and sequels seems to favor the development of metaplastic cells. Hamsters infected with T. cruzi may be useful to study hepatocytic metaplasia, and contribute to clarify aspects of Chagas' disease and pancreatic changes. Our data indicate that aging, in addition to inflammation and atrophy, plays a role in this change.