Brain aromatase mRNA expression in two populations of the peacock blenny Salaria pavo with divergent mating systems

Aromatase, the key enzyme in the conversion of androgens to estrogens, regulates the availability of these hormones in tissues and controls many physiological and behavioral processes. In fish and other vertebrates, the regulation of aromatase expression in the brain has been implicated in the modulation of male sexual and aggressive behaviors. Here, the pattern of mRNA expression of the brain aromatase isoform (encoded by the CYP19A2 gene also referred as CYP19b) was quantified at the peak of spawning season in brain macroareas from males and females of the blenny Salaria pavo originated from two populations displaying male alternative reproductive tactics but differing in their mating systems. In Trieste (Adriatic) nesting males aggressively defend nests and take the initiative in courtship and perform sexual displays more often than females while in Ria Formosa (Southern Portugal) the pattern is reversed as a result of shortage of appropriate nesting sites. Nesting males from Ria Formosa had overall higher levels of brain aromatase mRNA expression than nesting males from Trieste, suggesting a higher brain estrogen synthesis in these males. Since in some fish species exogenous estradiol administration has been shown to decrease sexual and agonistic behaviors, the higher levels of brain aromatase in Ria Formosa nesting males may explain their reduced expression of sexual and aggressive displays when compared with nesting males from Trieste. Alternatively, the higher brain aromatase levels in nesting males from Ria Formosa could be a mechanism to decrease the putative androgen-induced activation of aggressive and sexual displays by reducing the local availability of androgens through their metabolization into estrogens. Although females and parasitic female-like males also differ in their displays between populations, the interpopulational pattern of brain aromatase mRNA expression was similar, suggesting that other neuroendocrine agents mediate the expression of female and female-like behaviors. In conclusion, brain aromatase availability seems like a probable mechanism to regulate the effects of steroids on the brain circuits underlying the expression of sexual and agonistic displays in S. pavo.     

Settlement of the diazotrophic,phytoeffective bacterial strain Pantoea agglomerans on and within winter wheat: An investigation using ELISA and transmission electron microscopy

The aim of this study was to investigate the ability of Pantoea agglomerans, a plant growth-promoting bacterium, to colonize various regions and tissues of the wheat plant (Triticum aestivum L.) by using different inoculation methods and inoculum concentrations. In addition, the enzyme-linked immunosorbent assay (ELISA) and transmission electron microscopy (TEM) were used to determine: (a) the ability of the bacterial cells to grow and survive both on the surface and within internal tissue of the plant and (b) the response of the plant to bacterial infection. After inoculation, cells of the diazotrophic bacterial strain P. agglomerans were found to be located in roots, stems and leaves. Colony development of bacterial cells was only detected within intercellular spaces of the root and on the root surface. However, single bacterial cells were observed in leaves and stems on the surface of the epidermis, in the vicinity to stomatal cells, within intercellular spaces of the mesophyll and within xylem vessels. Inoculated bacterial cells were found to be able to enter host tissues, to multiply in the plant and to maintain a delicate relationship between endophyte and host. The density of bacterial settlement in the plant in all experiments was about 10⁶ to 10⁷ cells per mL root or shoot sap. Establishment was confirmed by a low coefficient of variation of ELISA means at these concentrations.     

Plant-type dependent changes in arbuscular mycorrhizal communities as soil quality indicator in semi-arid Brazil

A large remaining of dry deciduous forest (woody Caatinga) in semi-arid Brazil has been reached by successive fires and exploratory actions what leads to the invasion of low load trees and shrub mesh, called “Carrasco vegetation”. As it restrains the sprouting of woody species, land recuperation was performed using a mixed plantation of native and Eucalyptus species to both preservation and to supply the demand for wood. In order to evaluate the recuperation, a study of microbial communities was proposed. In addition to the highest soil phosphorus content found in the Carrasco area, the greatest spore density of arbuscular mycorrhizal fungi (AMF) communities occurred in the rhizosphere of the both pioneer species: Carrasco and Eucalyptus. In contrast to the DGGE bacteria profile, it was possible to group AMF species of the preserved and experimental sites which were not clustered with Carrasco species through the DGGE of Glomales DNA and also by the principal component analysis (PCA) based on diversity index. Glomus and Acaulospora were the dominant genera at both the preserved site and Carrasco. Nevertheless, Gigaspora species were preferentially found in Dry Forest, while Scutellospora were absent. In contrast, Carrasco favoured the genus Scutellospora and the species Acaulospora scrobiculata. Our results allow one to conclude that vegetation type modifies the AMF communities, which may be used as good indicator of soil quality. Based on AMF communities as soil quality indicator, the mixed forest plantation appears to be underway towards the preserved site two years after transplantation.     

Ubiquitin over-expression promotes E6AP autodegradation and reactivation of the p53/MDM2 pathway in HeLa cells

It has been established that intracellular ubiquitin pools are subject to regulatory constrains. Less certain is the mechanism by which the pool of conjugated ubiquitin shift in parallel with total ubiquitin, and how this type of regulation affects the flux of substrates through the pathway. In this study we demonstrate that ubiquitin over-expression promotes the destabilization of the ubiquitin protein ligase E6AP, by a mechanism involving self-ubiquitination, and the stabilization of p53. These results represent the very first evidence that the levels of a ubiquitin ligase can be regulated in vivo by ubiquitin abundance, supporting the idea that a strict interrelationship between pathway component activities and ubiquitin pool size exists. Interestingly, ubiquitin-induced p53 accumulation did not induce cell-cycle arrest, suggesting that although fluctuations of the intracellular ubiquitin content may actively modulate the level of regulatory proteins, this event is not per se sufficient to elicit a cellular response in terms of proliferation.     

Endothelin-1 decreases gap junctional intercellular communication by inducing phosphorylation of connexin 43 in human ovarian carcinoma cells

Endothelin-1 (ET-1) is overexpressed in ovarian carcinoma and acts as an autocrine factor selectively through the ETA receptor (ETAR) to promote tumor cell proliferation, survival, neovascularization, and invasiveness. Loss of gap junctional intercellular communication (GJIC) is critical for tumor progression by allowing the cells to escape growth control. Exposure of HEY and OVCA 433 ovarian carcinoma cell lines to ET-1 led to a 50-75% inhibition in intercellular communication and to a decrease in the connexin 43 (Cx43)-based gap junction plaques. To investigate the phosphorylation state of Cx43, ovarian carcinoma cell lysates were immunoprecipitated and transient tyrosine phosphorylation of Cx43 was detected in ET-1-treated cells. BQ 123, a selective ETAR antagonist, blocked the ET-1-induced Cx43 phosphorylation and cellular uncoupling. Gap junction closure was prevented by tyrphostin 25 and by the selective c-Src inhibitor, PP2. Furthermore, the increased Cx43 tyrosine phosphorylation was correlated with ET-1-induced increase of c-Src activity, and PP2 suppressed the ET-1-induced Cx43 tyrosine phosphorylation, indicating that inhibition of Cx43-based GJIC is mainly mediated by the Src tyrosine kinase pathway. In vivo, the inhibition of human ovarian tumor growth in nude mice induced by the potent ETAR antagonist, ABT-627, was associated with a reduction of Cx43 phosphorylation. These findings indicate that the signaling mechanisms involved in GJIC disruption on ovarian carcinoma cells depend on ETAR activation, which leads to the Cx43 tyrosine phosphorylation mediated by c-Src, suggesting that ETAR blockade may contribute to the control of ovarian carcinoma growth and progression also by preventing the loss of GJIC.     

Use of a novel triple-tracer approach to assess postprandial glucose metabolism

Numerous studies have used the dual-tracer method to assess postprandial glucose metabolism. The present experiments were undertaken to determine whether the marked tracer nonsteady state that occurs with the dual-tracer approach after food ingestion introduces error when it is used to simultaneously measure both meal glucose appearance (R(a meal)) and endogenous glucose production (EGP). To do so, a novel triple-tracer approach was designed: 12 subjects ingested a mixed meal containing [1-(13)C]glucose while [6-(3)H]glucose and [6,6-(2)H(2)]glucose were infused intravenously in patterns that minimized the change in the plasma ratios of [6-(3)H]glucose to [1-(13)C]glucose and of [6,6-(2)H(2)]glucose to endogenous glucose, respectively. R(a meal) and EGP measured with this approach were essentially model independent, since non-steady-state error was minimized by the protocol. Initial splanchnic glucose extraction (ISE) was 12.9% +/- 3.4%, and suppression of EGP (EGPS) was 40.3% +/- 4.1%. In contrast, when calculated with the dual-tracer one-compartment model, ISE was higher (P < 0.05) and EGPS was lower (P < 0.005) than observed with the triple-tracer approach. These errors could only be prevented by using time-varying volumes different for R(a meal) and EGP. Analysis of the dual-tracer data with a two-compartment model reduced but did not totally avoid the problems associated with marked postprandial changes in the tracer-to-tracee ratios. We conclude that results from previous studies that have used the dual-tracer one-compartment model to measure postprandial carbohydrate metabolism need to be reevaluated and that the triple-tracer technique may provide a useful approach for doing so.     

Properties of Air Dried Radiation Processed Amniotic Membranes under Different Storage Conditions

Amniotic membranes collected from the placentae of screened donors were processed, air dried and sterilized by gamma irradiation at 25 kGy. Effect of storage under different temperature and humidity conditions (10 degrees C, RH 80-90%; 10 degrees C, RH 40-50%; 40 degrees C, RH 50-60% and 40 degrees C, RH 10-20%) on the properties of the membrane were examined. Infrared (IR) spectral scanning was carried out to examine degradation or change if any in the tissue under different storage conditions. The degradation of amnion on irradiation with gamma rays or during storage after irradiation would tend to produce the relative variation in IR absorption troughs. This kind of addendum was absent in all the samples indicating no qualitative change in the material property of amnion. Water absorption and water vapour transmission rate (WVTR) of the membrane remained unchanged even after 6 months. No effect on the microbial permeability of membrane was observed during storage. The amniotic membranes were found to be impermeable to different strains of bacteria - Bacillus, Escherichia coli, Pseudomonas, Citrobacter, Flavimonas and Staphylococcus. The results indicate that amniotic membranes processed by air-drying are stable and can be stored under different environmental conditions without compromise to their clinical performance.     

StCDPK1 is expressed in potato stolon tips and is induced by high sucrose concentration

StCDPK1 encodes a calcium-dependent protein kinase (CDPK) from Solanum tuberosum, which is transiently induced upon tuberization in swelling stolons. In situ hybridization determined that StCDPK1 mRNA is localized in the apical dome of tuberizing stolon tips, close to the region where sucrose was reported to accumulate. The expression of StCDPK1, and other tuber-specific genes was enhanced when in vitro-cultured potato plants were transferred to high sucrose or high sorbitol containing media. Glucose, fructose or a mixture of both showed no effect on CDPK expression. Okadaic acid blocked sucrose-inducible gene expression, suggesting that phosphatases from the PP1/PP2A family could also participate in the regulation of StCDPK1 and other tuberization-related genes.