Protein extraction and comparison of stain protocols for analysis of two-dimensional electrophoresis gels

Protein extraction and the proteome of Lactobacillus delbrueckii subsp. bulgaricus were studied using different stains. The reversible silver staining technique was shown to be more sensitive than the irreversible silver stain. Coomassie colloidal was demonstrated to be as sensitive as reversible silver stain; however, the Coomassie colloidal blue solution developed a higher background and for sample preparation was more time-consuming.     

Notch Signalling Is Required for the Formation of Structurally Stable Muscle Fibres in Zebrafish

Background

Accurate regulation of Notch signalling is central for developmental processes in a variety of tissues, but its function in pectoral fin development in zebrafish is still unknown.

Methodology/Principal Findings

Here we show that core elements necessary for a functional Notch pathway are expressed in developing pectoral fins in or near prospective muscle territories. Blocking Notch signalling at different levels of the pathway consistently leads to the formation of thin, wavy, fragmented and mechanically weak muscles fibres and loss of stress fibres in endoskeletal disc cells in pectoral fins. Although the structural muscle genes encoding Desmin and Vinculin are normally transcribed in Notch-disrupted pectoral fins, their proteins levels are severely reduced, suggesting that weak mechanical forces produced by the muscle fibres are unable to stabilize/localize these proteins. Moreover, in Notch signalling disrupted pectoral fins there is a decrease in the number of Pax7-positive cells indicative of a defect in myogenesis.

Conclusions/Significance

We propose that by controlling the differentiation of myogenic progenitor cells, Notch signalling might secure the formation of structurally stable muscle fibres in the zebrafish pectoral fin.     

Organisation of the lamprey (Lampetra fluviatilis) embryonic brain: insights from LIM-homeodomain, Pax and hedgehog genes

To investigate the embryonic development of the central nervous system of the lamprey Lampetra fluviatilis, we have isolated and analysed the expression patterns of members of the LIM-homeodomain, Pax, Hedgehog and Nkx2.1 families. Using degenerate RT-PCR, single representatives of Lhx1/Lhx5, Lhx2/Lhx9, Pax3/Pax7 and Hedgehog families could be isolated in L. fluviatilis. Expression analysis revealed that the lamprey forebrain presents a clear neuromeric pattern. We describe the existence of 4 embryonic diencephalic prosomeres whose boundaries can be identified by the combined and relative expressions of LfPax37, LfLhx15 and LfLhx29. This suggests that the embryonic lamprey and gnathostome forebrain are patterned in a highly similar manner. Moreover, analysis of the LfHh gene, which is expressed in the hypothalamus, zona limitans intrathalamica and floor plate, reveals the possible molecular origin of this neuromeric brain pattern. By contrast, LfHh and LfNkx2.1 expressions suggest major differences in patterning mechanisms of the ventral telencephalon when compared to gnathostomes. In summary, our findings highlight a neuromeric organisation of the embryonic agnathan forebrain and point to the possible origin of this organisation, which is thus a truly vertebrate character. They also suggest that Hh/Shh midline signalling might act as a driving force for forebrain evolution.     

Co-Inheritance of alpha-thalassemia and sickle cell disease in a cohort of Angolan pediatric patients

Molecular Biology Reports - The aim of this study was to explore the association between alpha-thalassemia, fetal hemoglobin, hematological indices, and clinical adverse events in Angolan sickle...     

Neuropeptide Y stimulates retinal neural cell proliferation--involvement of nitric oxide

Neuropeptide Y (NPY) is a 36 amino acid peptide widely present in the CNS, including the retina. Previous studies have demonstrated that NPY promotes cell proliferation of rat post-natal hippocampal and olfactory epithelium precursor cells. The aim of this work was to investigate the role of NPY on cell proliferation of rat retinal neural cells. For this purpose, primary retinal cell cultures expressing NPY, and NPY Y₁, Y₂, Y₄ and Y₅ receptors [Álvaro et al. , (2007) Neurochem. Int., 50, 757] were used. NPY (10–1000 nM) stimulated cell proliferation through the activation of NPY Y₁, Y₂ and Y₅ receptors. NPY also increased the number of proliferating neuronal progenitor cells (BrdU⁺/nestin⁺ cells). The intracellular mechanisms coupled to NPY receptors activation that mediate the increase in cell proliferation were also investigated. The stimulatory effect of NPY on cell proliferation was reduced by l -nitroarginine-methyl-esther ( l -NAME; 500 μM), a nitric oxide synthase inhibitor, 1H-[1,2,4]oxadiazolo-[4, 3-a]quinoxalin-1-one (ODQ; 20 μM), a soluble guanylyl cyclase inhibitor or U0126 (1 μM), an inhibitor of the extracellular signal-regulated kinase 1/2 (ERK 1/2). In conclusion, NPY stimulates retinal neural cell proliferation, and this effect is mediated through nitric oxide–cyclic GMP and ERK 1/2 pathways.     

Molecular characterization of the rostral-most somites in early somitic stages of the chick embryo

Segmentation consists on the progressive formation of repetitive embryonic structures, named somites, which are formed from the most rostral part of the presomitic mesoderm. Somites are subdivided into anterior and posterior compartments and several genes are differentially expressed in either compartment. This has provided evidence for the importance of establishing the anterior-posterior polarity within each somite, which is critical for the correct segmented pattern of the adult vertebrate body. Although all somites appear morphologically similar, fate map studies have shown that the first 4 somites do not give rise to segmented structures, in contrast to more posterior ones. Moreover, in several somitogenesis-related mutants the anterior somites are not affected while posterior somites present clear defects or do not form at all. Altogether these data suggest relevant differences between rostral and caudal somites. In order to check for molecular differences between anterior and posterior somites, we have performed a detailed expression pattern analysis of several Notch signalling related genes. For the first time, we show that the somitic expression pattern profile is not the same along the anterior-posterior axis and that the differences are not observed always at the same somite level.     

Depot- and gender-related differences in the lipolytic pathway of adipose tissue from severely obese patients.

The present study was performed to analyze in detail gender- and site-related alterations in the adrenergic signal transduction pathway of lipolysis in fat cells isolated from subcutaneous abdominal and visceral fat depots from severely obese patients. The study group consisted of 30 morbidly obese subjects (9 men and 21 women) aged 41.1+/-1.9 years, with a body mass index (BMI) of 54.7+/-1.7 kg/m2, who had undergone abdominal surgery. Protein levels of hormone-sensitive lipase (HSL) and adrenergic receptors (AR), as well as HSL activity and the lipolytic response to adrenergic agents were analyzed. Both fat depots had similar basal lipolysis, but the capacity of catecholamines to activate lipolysis was greater in visceral fat, both at AR and postreceptor levels. Basal lipolysis and lipolytic activity induced by dibutyryl cyclic AMP were higher in men than in women. However, the visceral depot of women showed a higher maximal stimulation by noradrenaline than that of men, in accordance with higher beta1- and beta3-AR protein levels. In conclusion, the main gender-related differences were located in the visceral depot, with women exhibiting a higher sensitivity to catecholamines associated with an increased provision of beta-AR, while men showed an enhanced lipolytic capacity at the postreceptor level.     

Effects of Growth in the Presence of Subinhibitory Concentrations of Dicloxacillin on Staphylococcus epidermidis and Staphylococcus haemolyticus Biofilms

Low concentrations of antibiotics can inhibit microbial adherence to medical device surfaces. However, little is known about the changes that occur in the physiology of bacteria within biofilms formed in the presence of subinhibitory (sub-MIC) concentrations of antibiotics. In this study, the densities and matrix compositions ofbiofilms formed by two coagulase-negative Staphylococcus species in the absence and in the presence of sub-MIC concentrations of dicloxacillin were evaluated. Biofilms formed in the presence of sub-MIC concentrations of dicloxacillin contained less biomass, and there were notable changes in the composition of the biofilm matrix. Changes in the spatial structure were also verified by confocal scanning laser microscopy, indicating that biofilms grown in the presence of sub-MIC concentrations of dicloxicilln had a lower cell density. Physiological alterations in the bacteria within biofilms grown in the presence of subinhibitory concentrations of the antibiotic were also evaluated. The results showed that there were differences in bacterial surface characteristics when cultures were grown in the presence of sub-MIC concentrations of dicloxacillin, including decreased hydrophobicity and decreased expression of the exopolysaccharide poly-N-acetylglucosamine. The elemental composition of the cell surface was also analyzed, and whereas in Staphylococcus epidermidis there were decreases in the oxygen and nitrogen contents, in Staphylococcus haemolyticus there were increases in these two parameters. Additionally, increases in resistance to several antibiotics were observed for the cells within biofilms formed in the presence of dicloxacillin.     

What drives invasibility? A multi‐model inference test and spatial modelling of alien plant species richness patterns in northern Portugal

Understanding and predicting biological invasions can focus either on traits that favour species invasiveness or on features of the receiving communities, habitats or landscapes that promote their invasibility. Here, we address invasibility at the regional scale, testing whether some habitats and landscapes are more invasible than others by fitting models that relate alien plant species richness to various environmental predictors. We use a multi‐model information‐theoretic approach to assess invasibility by modelling spatial and ecological patterns of alien invasion in landscape mosaics, and by testing competing hypotheses of environmental factors that may control invasibility. Because invasibility may be mediated by particular characteristics of invasiveness, we classified alien species according to their C‐S‐R plant strategies. We illustrate this approach with a set of 86 alien species in northern Portugal. We first focus on predictors influencing species richness and expressing invasibility, and then evaluate whether distinct plant strategies respond to the same or different groups of environmental predictors. We confirmed climate as a primary determinant of alien invasions, and as a primary environmental gradient determining landscape invasibility. The effects of secondary gradients were detected only when the area was sub‐sampled according to predictions based on the primary gradient. Then, multiple predictor types influenced patterns of alien species richness, with some types (landscape composition, topography and fire regime) prevailing over others. Alien species richness responded most strongly to extreme land management regimes, suggesting that intermediate disturbance induces biotic resistance by favouring native species richness. Land‐use intensification facilitated alien invasion, whereas conservation areas hosted few invaders, highlighting the importance of ecosystem stability in preventing invasions. Plants with different strategies exhibited different responses to environmental gradients, particularly when the variations of the primary gradient were narrowed by sub‐sampling. Such differential responses of plant strategies suggest using distinct control and eradication approaches for different areas and alien plant groups.     

Regulation of Hfq mRNA and Protein Levels in Escherichia coli and Pseudomonas aeruginosa by the Burkholderia cenocepacia MtvR sRNA

Small non-coding RNAs (sRNAs) are important players of gene expression regulation in bacterial pathogens. MtvR is a 136-nucleotide long sRNA previously identified in the human pathogen Burkholderia cenocepacia J2315 and with homologues restricted to bacteria of the Burkholderia cepacia complex. In this work we have investigated the effects of expressing MtvR in Escherichia coli and Pseudomonas aeruginosa. Results are presented showing that MtvR negatively regulates the hfq mRNA levels in both bacterial species. In the case of E. coli, this negative regulation is shown to involve binding of MtvR to the 5′-UTR region of the hfq_Ec mRNA. Results presented also show that expression of MtvR in E. coli and P. aeruginosa originates multiple phenotypes, including reduced resistance to selected stresses, biofilm formation ability, and increased susceptibility to various antibiotics.