The role of exon shuffling in shaping protein-protein interaction networks

Background: Physical protein-protein interaction (PPI) is a critical phenomenon for the function of most proteins in living organisms and a significant fraction of PPIs are the result of domain-domain interactions. Exon shuffling, intron-mediated recombination of exons from existing genes, is known to have been a major mechanism of domain shuffling in metazoans. Thus, we hypothesized that exon shuffling could have a significant influence in shaping the topology of PPI networks. Results: We tested our hypothesis by compiling exon shuffling and PPI data from six eukaryotic species: Homo sapiens, Mus musculus, Drosophila melanogaster, Caenorhabditis elegans, Cryptococcus neoformans and Arabidopsis thaliana. For all four metazoan species, genes enriched in exon shuffling events presented on average higher vertex degree (number of interacting partners) in PPI networks. Furthermore, we verified that a set of protein domains that are simultaneously promiscuous (known to interact to multiple types of other domains), self-interacting (able to interact with another copy of themselves) and abundant in the genomes presents a stronger signal for exon shuffling. Conclusions: Exon shuffling appears to have been a recurrent mechanism for the emergence of new PPIs along metazoan evolution. In metazoan genomes, exon shuffling also promoted the expansion of some protein domains. We speculate that their promiscuous and self-interacting properties may have been decisive for that expansion.

Solution structure of the human signaling protein RACK1

Background: The adaptor protein RACK1 (receptor of activated kinase 1) was originally identified as an anchoring protein for protein kinase C. RACK1 is a 36 kDa protein, and is composed of seven WD repeats which mediate its protein-protein interactions. RACK1 is ubiquitously expressed and has been implicated in diverse cellular processes involving: protein translation regulation, neuropathological processes, cellular stress, and tissue development. Results: In this study we performed a biophysical analysis of human RACK1 with the aim of obtaining low resolution structural information. Small angle X-ray scattering (SAXS) experiments demonstrated that human RACK1 is globular and monomeric in solution and its low resolution structure is strikingly similar to that of an homology model previously calculated by us and to the crystallographic structure of RACK1 isoform A from Arabidopsis thaliana. Both sedimentation velocity and sedimentation equilibrium analytical ultracentrifugation techniques showed that RACK1 is predominantly a monomer of around 37 kDa in solution, but also presents small amounts of oligomeric species. Moreover, hydrodynamic data suggested that RACK1 has a slightly asymmetric shape. The interaction of RACK1 and Ki1/57 was tested by sedimentation equilibrium. The results suggested that the association between RACK1 and Ki-1/57(122-413) follows a stoichiometry of 1:1. The binding constant (KB) observed for RACK1-Ki-1/57(122-413) interaction was of around (1.5 +/- 0.2) x 10(6) M(-1) and resulted in a dissociation constant (KD) of (0.7 +/- 0.1) x 10(-6) M. Moreover, the fluorescence data also suggests that the interaction may occur in a cooperative fashion. Conclusion: Our SAXS and analytical ultracentrifugation experiments indicated that RACK1 is predominantly a monomer in solution. RACK1 and Ki-1/57(122-413) interact strongly under the tested conditions.

Source populations in coastal crabs: parameters affecting egg production

Benthic marine invertebrates may form metapopulations connected via propagule dispersal. Conservation efforts often target potential source coastlines to indirectly benefit areas depending on allochthonous offspring production. Besides population density, adult size structure, sex ratio, brooding frequency and the proportion of breeding individuals may significantly influence the reproductive output of benthic populations, but these effects have seldom been tested. We used rocky shore crabs to assess the spatial variability of such parameters at relevant scales for conservation purposes and to test their consistency over 2 consecutive years; we then used the data to address whether bottom-up processes or biological interactions might explain the patterns observed. We decomposed egg production rates into their components for the 2 most abundant brachyuran species inhabiting the intertidal rocky habitat. Adult density and brooding frequency varied consistently among shores for both species and largely explained the overall spatial trends of egg production. Temporally consistent patterns also included among-shore differences in the size of ovigerous females of the grapsid Pachygrapsus transversus and between-bay differences in the fecundity of the spider crab Epialtus brasiliensis. Sex ratio was remarkably constant in both. We found no positive or negative correlations between adult density and brooding frequency to support either the existence of a component Allee effect (lack of mate encounters) or an effect of intra-specific competition. Likewise, shore-specific potential growth in P. transversus does not negatively correlate with frequency of ovigerous individuals, as would be expected under a critical balance between these 2 processes. The patterns observed suggest that bottom-up drivers may best explain spatial trends in the reproductive output of these species.

Anthelmintic effects of condensed tannins on Trichostrongylus colubriformis in experimentally infected sheep

Recent surveys have identified anthelmintic effects from many bioactive substances particularly from condensed tannin (CT) sources. The aims of the present study were to investigate the potential anthelmintic effects of condensed tannins (CT) on Trichostrongylus colubriformis in experimentally infected sheep and the nutritional consequences on animals. Twenty helminth-free lambs were divided into five groups of four animals. Groups I to IV were artificially infected with 6,000 third stage larvae (L3) of T. colubriformis. Group I was the infected control and group V was the uninfected control. Twenty-eight days post-infection (p.i.) lambs from GII were supplemented with tanniniferous sorghum (350 g/animal/day, during seven days); GIII were drenched with Acacia mearnsii extract (15% CT) for just one day and GIV during two days (1.6 g extract/kg BW). At day 36 p.i., animals from infected group (GI to GIV) were slaughtered. Faecal egg counts (FEC) values present a reduction on GII when compared with GI at day 29 p.i. (P < 0.05) and between GIII and GI at day 35 and 36 p.i. (P < 0.05). The values of egg hatchability and number of L3 recovered from the faeces were not statistical analyzed (there was no duplicate data), however there was a considerable reduction between the values from treated and control group. The use of CT on diet did not cause significant difference on blood parameters, body-weight and carcass-weight (P > 0.05). No difference was related on total worm burden between treatments; however, GIV presented lower number of females than GI (P < 0.05). The use of CT could be a promising alternative source to reduce the pasture contamination and to control T. colubriformis infection in sheep.

Effects of Molybdenum, Nickel, and Nitrogen Sources on the Mineral Nutrition and Growth of Rice Plants

Upland rice plants, cultivar `IAC 202,` were grown in nutrient solution until full tillering. Treatments consisted of ammonium nitrate (AN) or urea (UR) as nitrogen (N) source plus molybdenum (Mo) and/or nickel (Ni): AN + Mo + Ni, AN + Mo - Ni, AN - Mo + Ni, UR + Mo + Ni, UR + Mo - Ni, and UR - Mo + Ni. The experiment was carried out to better understand the effect of these treatments on dry-matter yield, chlorophyll, net photosynthesis rate, nitrate (NO3 --N), total N, in vitro activities of urease and nitrate reductase (NR), and Mo and Ni concentrations. In UR-grown plants, Mo and Ni addition increased yield of dry matter. Regardless of the N source, chlorophyll concentration and net photosynthesis rate were reduced when Mo or Ni were omitted, although not always significantly. The omission of either Mo or Ni led to a decrease in urease activity, independent of N source. Nitrate reductase activity increased in nutrient solutions without Mo, although NO3 --N increased. There was not a consistent variation in total N concentration. Molybdenum and Ni concentration in roots and shoots were influenced by their supply in the nutrient solution. Molybdenum concentration was not influenced by N sources, whereas Ni content in both root and shoots was greater in ammonium nitrate-grown plants. In conclusion, it can be hypothesized that there is a relationship between Mo and Ni acting on photosynthesis, although is an indirect one. This is the first evidence for a beneficial effect of Mo and Ni interaction on plant growth.

A NOVEL EPIPHYTIC CYANOBACTERIAL SPECIES FROM THE GENUS BRASILONEMA CAUSING DAMAGE TO EUCALYPTUS LEAVES

A cyanobacterial mat colonizing the leaves of Eucalyptus grandis was determined to be responsible for serious damage affecting the growth and development of whole plants under the clonal hybrid nursery conditions. The dominant cyanobacterial species was isolated in BG-11 medium lacking a source of combined nitrogen and identified by cell morphology characters and molecular phylogenetic analysis (16S rRNA gene and cpcBA-IGS sequences). The isolated strain represents a novel species of the genus Brasilonema and is designated Brasilonema octagenarum strain UFV-E1. Thin sections of E. grandis leaves analyzed by light and electron microscopy showed that the B. octagenarum UFV-E1 filaments penetrate into the leaf mesophyll. The depth of infection and the mechanism by which the cyanobacterium invades leaf tissue were not determined. A major consequence of colonization by this cyanobacterium is a reduction in photosynthesis in the host since the cyanobacterial mats decrease the amount of light incident on leaf surfaces. Moreover, the cyanobacteria also interfere with stomatal gas exchange, decreasing CO2 assimilation. To our knowledge, this is the first report of an epiphytic cyanobacterial species causing damage to E. grandis leaves.

Sequential injections as an alternative to gradient exploitation for implementing differential kinetic analysis in a flow injection system

A novel flow-based strategy for implementing simultaneous determinations of different chemical species reacting with the same reagent(s) at different rates is proposed and applied to the spectrophotometric catalytic determination of iron and vanadium in Fe-V alloys. The method relies on the influence of Fe(II) and V(IV) on the rate of the iodide oxidation by Cr(VI) under acidic conditions, the Jones reducing agent is then needed Three different plugs of the sample are sequentially inserted into an acidic KI reagent carrier stream, and a confluent Cr(VI) solution is added downstream Overlap between the inserted plugs leads to a complex sample zone with several regions of maximal and minimal absorbance values. Measurements performed on these regions reveal the different degrees of reaction development and tend to be more precise Data are treated by multivariate calibration involving the PLS algorithm The proposed system is very simple and rugged Two latent variables carried out ca 95% of the analytical information and the results are in agreement with ICP-OES. (C) 2010 Elsevier B V. All rights reserved.

Building a Open Source Framework for Virtual Medical Training

This paper presents a framework to build medical training applications by using virtual reality and a tool that helps the class instantiation of this framework. The main purpose is to make easier the building of virtual reality applications in the medical training area, considering systems to simulate biopsy exams and make available deformation, collision detection, and stereoscopy functionalities. The instantiation of the classes allows quick implementation of the tools for such a purpose, thus reducing errors and offering low cost due to the use of open source tools. Using the instantiation tool, the process of building applications is fast and easy. Therefore, computer programmers can obtain an initial application and adapt it to their needs. This tool allows the user to include, delete, and edit parameters in the functionalities chosen as well as storing these parameters for future use. In order to verify the efficiency of the framework, some case studies are presented.

Recombinant human prolactin promotes human beta cell survival via inhibition of extrinsic and intrinsic apoptosis pathways

Transplantation of pancreatic islets constitutes a promising alternative treatment for type 1 diabetes. However, it is limited by the shortage of organ donors. Previous results from our laboratory have demonstrated beneficial effects of recombinant human prolactin (rhPRL) treatment on beta cell cultures. We therefore investigated the role of rhPRL action in human beta cell survival, focusing on the molecular mechanisms involved in this process. Human pancreatic islets were isolated using an automated method. Islet cultures were pre-treated in the absence or presence of rhPRL and then subjected to serum starvation or cytokine treatment. Beta cells were labelled with Newport green and apoptosis was evaluated using flow cytometry analysis. Levels of BCL2 gene family members were studied by quantitative RT-PCR and western blot. Caspase-8, -9 and -3 activity, as well as nitric oxide production, were evaluated by fluorimetric assays. The proportion of apoptotic beta cells was significantly lowered in the presence of rhPRL under both cell death-induced conditions. We also demonstrated that cytoprotection may involve an increase of BCL2/BAX ratio, as well as inhibition of caspase-8, -9 and -3. Our study provides relevant evidence for a protective effect of lactogens on human beta cell apoptosis. The results also suggest that the improvement of cell survival may involve, at least in part, inhibition of cell death pathways controlled by the BCL2 gene family members. These findings are highly relevant for improvement of the islet isolation procedure and for clinical islet transplantation.

Aging and calorie restriction modulate yeast redox state, oxidized protein removal, and the ubiquitin-proteasome system

The ubiquitin-proteasome system governs the half-life of most cellular proteins. Calorie restriction (CR) extends the maximum life span of a variety of species and prevents oxidized protein accumulation. We studied the effects of CR on the ubiquitin-proteasome system and protein turnover in aging Saccharomyces cerevisiae. CR increased chronological life span as well as proteasome activity compared to control cells. The levels of protein carbonyls, a marker of protein oxidation, and those of polyubiquitinated proteins were modulated by CR. Controls, but not CR cells, exhibited a significant increase in oxidized proteins. In keeping with decreased proteasome activity, polyubiquitinated proteins were increased in young control cells compared to time-matched CR cells, but were profoundly decreased in aged control cells despite decreased proteasomal activity. This finding is related to a decreased polyubiquitination ability due to the impairment of the ubiquitin-activating enzyme in aged control cells, probably related to a more oxidative microenvironment. CR preserves the ubiquitin-proteasome system activity. Overall, we found that aging and CR modulate many aspects of protein modification and turnover. (C) 2011 Elsevier Inc. All rights reserved.

Human multipotent adipose-derived stem cells restore dystrophin expression of Duchenne skeletal-muscle cells in vitro

Background information. DMD (Duchenne muscular dystrophy) is a devastating X-linked disorder characterized by progressive muscle degeneration and weakness. The use of cell therapy for the repair of defective muscle is being pursued as a possible treatment for DMD. Mesenchymal stem cells have the potential to differentiate and display a myogenic phenotype in vitro. Since liposuctioned human fat is available in large quantities, it may be an ideal source of stem cells for therapeutic applications. ASCs (adipose-derived stem cells) are able to restore dystrophin expression in the muscles of mdx (X-linked muscular dystrophy) mice. However, the outcome when these cells interact with human dystrophic muscle is still unknown. Results. We show here that ASCs participate in myotube formation when cultured together with differentiating human DMD myoblasts, resulting in the restoration of dystrophin expression. Similarly, dystrophin was induced when ASCs were co-cultivated with DMD myotubes. Experiments with GFP (green fluorescent protein)-positive ASCs and DAPI (4,6-diamidino-2-phenylindole)-stained DMD myoblasts indicated that ASCs participate in human myogenesis through cellular fusion. Conclusions. These results show that ASCs have the potential to interact with dystrophic muscle cells, restoring dystrophin expression of DMD cells in vitro. The possibility of using adipose tissue as a source of stem cell therapies for muscular diseases is extremely exciting.

IS ORDINARY ELECTRIC DRILLS` VENTING PORT A POTENTIAL SOURCE OF SURGICAL INFECTION?

Objective: To evaluate the potential risk of surgical contamination by the venting port of ordinary electric drills (ED) employed in orthopaedic surgeries. Materials and Methods: an experimental laboratory, randomized study was developed to analyze EDs in surgical practice and new cleaned and sterilized equipment, which were contaminated with Bacillus atrophaeus spores at a concentration of 84 X 10(6) UFC. The air generated by the engine of each drill was collected and cultivated on sterile agar plates. Results: Positive culture was identified in two ED in surgical practice, as well as a positive culture to Bacillus atrophaeus with 1 CFU growth (1, 19 X 10(-8)). Conclusion: In the conditions of the experiment, the air generated by the venting port of the ED`s engine does not consist of a source of contamination for the surgical site.

Exercise training and caloric restriction prevent reduction in cardiac Ca(2+)-handling protein profile in obese rats

Previous studies show that exercise training and caloric restriction improve cardiac function in obesity. However, the molecular mechanisms underlying this effect on cardiac function remain unknown. Thus, we studied the effect of exercise training and/or caloric restriction on cardiac function and Ca(2+) handling protein expression in obese rats. To accomplish this goal, male rats fed with a high-fat and sucrose diet for 25 weeks were randomly assigned into 4 groups: high-fat and sucrose diet, high-fat and sucrose diet and exercise training, caloric restriction, and exercise training and caloric restriction. An additional lean group was studied. The study was conducted for 10 weeks. Cardiac function was evaluated by echocardiography and Ca(2+) handling protein expression by Western blotting. Our results showed that visceral fat mass, circulating leptin, epinephrine, and norepinephrine levels were higher in rats on the high-fat and sucrose diet compared with the lean rats. Cardiac nitrate levels, reduced/oxidized glutathione, left ventricular fractional shortening, and protein expression of phosphorylated Ser(2808)-ryanodine receptor and Thr(17-)phospholamban were lower in rats on the high-fat and sucrose diet compared with lean rats. Exercise training and/or caloric restriction prevented increases in visceral fat mass, circulating leptin, epinephrine, and norepinephrine levels and prevented reduction in cardiac nitrate levels and reduced: oxidized glutathione ratio. Exercise training and/or caloric restriction prevented reduction in left ventricular fractional shortening and in phosphorylation of the Ser(2808)-ryanodine receptor and Thr(17)-phospholamban. These findings show that exercise training and/or caloric restriction prevent cardiac dysfunction in high-fat and sucrose diet rats, which seems to be attributed to decreased circulating neurohormone levels. In addition, this nonpharmacological paradigm prevents a reduction in the Ser(2808)-ryanodine receptor and Thr(17-)phospholamban phosphorylation and redox status. (Hypertension. 2010;56:629-635.)

Aerobic exercise training improves skeletal muscle function and Ca(2+) handling-related protein expression in sympathetic hyperactivity-induced heart failure

Bueno CR Jr, Ferreira JC, Pereira MG, Bacurau AV, Brum PC. Aerobic exercise training improves skeletal muscle function and Ca(2+) handling-related protein expression in sympathetic hyperactivity-induced heart failure. J Appl Physiol 109: 702-709, 2010. First published July 1, 2010; doi: 10.1152/japplphysiol.00281.2010.-The cellular mechanisms of positive effects associated with aerobic exercise training on overall intrinsic skeletal muscle changes in heart failure (HF) remain unclear. We investigated potential Ca(2+) abnormalities in skeletal muscles comprising different fiber compositions and investigated whether aerobic exercise training would improve muscle function in a genetic model of sympathetic hyperactivity-induced HF. A cohort of male 5-mo-old wild-type (WT) and congenic alpha(2A)/alpha(2C) adrenoceptor knockout (ARKO) mice in a C57BL/6J genetic background were randomly assigned into untrained and trained groups. Exercise training consisted of a 8-wk running session of 60 min, 5 days/wk (from 5 to 7 mo of age). After completion of the exercise training protocol, exercise tolerance was determined by graded treadmill exercise test, muscle function test by Rotarod, ambulation and resistance to inclination tests, cardiac function by echocardiography, and Ca(2+) handling-related protein expression by Western blot. alpha(2A)/alpha(2C)ARKO mice displayed decreased ventricular function, exercise intolerance, and muscle weakness paralleled by decreased expression of sarcoplasmic Ca(2+) release-related proteins [alpha(1)-, alpha(2)-, and beta(1)-subunits of dihydropyridine receptor (DHPR) and ryanodine receptor (RyR)] and Ca(2+) reuptake-related proteins [sarco(endo) plasmic reticulum Ca(2+)-ATPase (SERCA) 1/2 and Na(+)/Ca(2+) exchanger (NCX)] in soleus and plantaris. Aerobic exercise training significantly improved exercise tolerance and muscle function and reestablished the expression of proteins involved in sarcoplasmic Ca(2+) handling toward WT levels. We provide evidence that Ca(2+) handling-related protein expression is decreased in this HF model and that exercise training improves skeletal muscle function associated with changes in the net balance of skeletal muscle Ca(2+) handling proteins.

Effects of different levels of protein intake and physical training on growth and nutritional status of young rats

This study aimed to investigate the effects of physical training, and different levels of protein intake in the diet, on the growth and nutritional status of growing rats. Newly-weaned Wistar rats (n=48) were distributed into six experimental groups: three of them were subjected to physical swim training (1 h per day. 5 d per week, for 4 wk, after 2 wk of familiarization) and the other three were considered as controls (non-trained). Each pair of groups, trained and non-trained, received diets with a different level of protein in their composition: 14%. 21% or 28%. The animals were euthanized at the end of the training period and the following analyses were performed: proteoglycan synthesis as a biomarker of bone and cartilage growth, IGF-I (insulin-like growth factor-I) assay as a biomarker of growth and nutritional status. total RNA and protein concentration and protein synthesis measured in vivo using a large-dose phenylalanine method. As a main finding, increased dietary protein, combined with physical training, was able to improve neither tissue protein synthesis nor muscle growth. In addition, cartilage and bone growth seem to be deteriorated by the lower and the higher levels of protein intake. Our data allow us to conclude that protein enhancement in the diet, combined with physical exercise, does not stimulate tissue protein synthesis or muscle mass growth. Furthermore, physical training, combined with low protein intake, was not favorable to bone development in growing animals.