Dye coupling and connexin expression by cortical radial glia in the early postnatal subventricular zone

In this study, we have analyzed the specific contribution of the cortical radial glia (RG) for gap junctional communication (GJC) within the postnatal subventricular zone (SVZ). To specifically target RG as source of dye‐coupling in situ, we have developed a new technique that involves direct cell loading through the processes that reach the pial surface, with a mix of gap junction permeant (Lucifer yellow, LY) and nonpermeant (rhodamine‐conjugated dextran 3 KDa, RD) fluorochromes, the latter used as a marker for direct loaded cells. Tissue sections were analyzed for identification of directly loaded (LY+RD+) and coupled cells (LY+RD–) in the SVZ. Directly loaded cells were restricted to the region underlying the pial loading surface area. Coupled cells were distributed in a bistratified manner, along the outer dorsal surface of the SVZ and aligning the ventricle, leaving the SVZ core relatively free. Blocking GJC prior to pial loading greatly reduced dye coupling. Phenotypic analysis indicated that coupling by RG excludes neuroblasts and is mostly restricted to cells of glial lineage. Notwithstanding, no corresponding restriction to specific cell phenotype was found for two connexin isotypes, Cx43 and Cx45, in the postnatal SVZ. The extensive homocellular cell coupling by RG suggests an important role in the regulation of neurogenesis and functional compartmentalization of the postnatal SVZ. © 2012 Wiley Periodicals, Inc. Develop Neurobiol 2012     

Eight years of ant‐exclusion from citrus canopies: effects on the arthropod assemblage and on fruit yield

• 1 Ants are important generalist predators in most terrestrial ecosystems. However, because many ant species are also hemipteran mutualists, their role in agriculture has generally been considered to be negative for plants.
• 2 In the present study, we report an experiment in ant‐exclusion from tree canopies in an organic citrus grove with two main objectives: (i) to examine whether the absence of ants increased the abundance of some beneficial arthropods and reduced the attack of some pests such as aphids and (ii) to examine whether ant‐exclusion increased the fruit yield of citrus trees.
• 3 The exclusion of ants from tree canopies had positive effects on the arthropod assemblage and on fruit yield. However, the 8‐year duration of the experiment can be divided into two periods with contrasting results. In the first period, the arthropod assemblage was only slightly affected, except for a greater density of aphids in ant‐excluded trees; in addition, fruit yield was higher in ant‐excluded trees than in the control ones. In the second period, ant‐exclusion increased the abundance of most arthropod groups, although the previous positive effect on fruit yield was no longer observed.
• 4 There are two main conclusions of the present study. First, from an applied perspective, ant‐exclusion from tree canopies is not a sound management alternative in citrus plantations in the Mediterranean. Second, the 8‐year duration of the experiment highlighted the importance of long‐term experiments in community ecology and biological control because the effects observed in the first 4 years of the experiment were very different from what occurred subsequently.

     

Basal adrenocorticotropin (ACTH) secretion is negatively modulated by protein phosphatase 5 in mouse pituitary corticotropin AtT20 cells

siRNA oligonucleotides for protein phosphatase 5 (PP5) were designed and transfected into mouse corticotroph AtT20 cells to induce lower PP5 expression levels. PP5-siRNA transfections (at 3 days) produced a approximately 50% down-regulation in targeted protein levels. PP5-underexpressing cells released significantly more ir-ACTH (10-12-fold) relative to baseline levels and promoted POMC release into the media. Neither CRF-mediated ACTH release nor dexamethasone-induced ACTH repression were affected in PP5-siRNA transfected cells. In summary, our observations suggest that endogenous PP5 can exert a negative modulatory effect on basal ACTH release in neurosecretion-competent AtT20 cells through a mechanism as yet unknown but which does not directly involve regulated CRF or glucocorticoid receptor-dependent pathways. However, PP5 may cause mis-sorting of POMC and POMC-derived peptides at the constitutive-like secretory pathway level in an unregulated manner. Such a missorting could lead to impaired processing of POMC.     

Domain 3 of NS5A protein from the hepatitis C virus has intrinsic alpha-helical propensity and is a substrate of cyclophilin A

Nonstructural protein 5A (NS5A) is essential for hepatitis C virus (HCV) replication and constitutes an attractive target for antiviral drug development. Although structural data for its in-plane membrane anchor and domain D1 are available, the structure of domains 2 (D2) and 3 (D3) remain poorly defined. We report here a comparative molecular characterization of the NS5A-D3 domains of the HCV JFH-1 (genotype 2a) and Con1 (genotype 1b) strains. Combining gel filtration, CD, and NMR spectroscopy analyses, we show that NS5A-D3 is natively unfolded. However, NS5A-D3 domains from both JFH-1 and Con1 strains exhibit a propensity to partially fold into an α-helix. NMR analysis identifies two putative α-helices, for which a molecular model could be obtained. The amphipathic nature of the first helix and its conservation in all genotypes suggest that it might correspond to a molecular recognition element and, as such, promote the interaction with relevant biological partner(s). Because mutations conferring resistance to cyclophilin inhibitors have been mapped into NS5A-D3, we also investigated the functional interaction between NS5A-D3 and cyclophilin A (CypA). CypA indeed interacts with NS5A-D3, and this interaction is completely abolished by cyclosporin A. NMR heteronuclear exchange experiments demonstrate that CypA has in vitro peptidyl-prolyl cis/trans-isomerase activity toward some, but not all, of the peptidyl-prolyl bonds in NS5A-D3. These studies lead to novel insights into the structural features of NS5A-D3 and its relationships with CypA.     

Effects of increased atmospheric CO2 and N supply on photosynthesis, growth and cell composition of the cyanobacterium Spirulina platensis (Arthrospira)

The consequences of the addition of CO2 (1%) in cultures of S. platensis are examined in terms of biomass yield, cell composition and external medium composition. CO2 enrichment was tested under nitrogen saturating and nitrogen limiting conditions. Increasing CO2 levels did not cause any change in maximum growth rate while it decreased maximum biomass yield. Protein and pigments were decreased and carbohydrate increased by high CO2, but the capability to store carbohydrates was saturated. C:N ratio remained unchanged while organic carbon released to the external medium was enhanced, suggesting that organic carbon release in S. platensis is an efficient mechanism for the maintenance of the metabolic integrity, balancing the cell C:N ratio in response to environmental CO2 changes. CO2 affected the pigment content: Phycocyanin, chlorophyll and carotenoids were reduced in around 50%, but the photosynthetic parameters were slightly changed. We propose that in S. platensis CO2 could act promoting degradation of pigments synthetised in excess in normal CO2 conditions, that are not necessary for light harvesting. Nitrogen assimilation was significantly not affected by CO2, and it is proposed that the inability to stimulate N assimilation by CO2 enrichment determined the lack of response in maximum growth rate. This revised version was published online in June 2006 with corrections to the Cover Date.     

Morbidity of Chagas heart disease in the microregion of Rio Negro,Amazonian Brazil: a case-control study

A case-control study on the morbidity of Chagas heart disease was carried out in the municipality of Barcelos in the microregion of the Rio Negro, state of Amazonas. One hundred and six individuals, who were serologically positive for Trypanosoma cruzi infection, as confirmed by at least two techniques with different principles, were matched according to age and sex with an equal number of seronegative individuals. The cases and controls were evaluated using an epidemiological questionnaire and clinical, electrocardiograph and echocardiograph examinations. In the seroepidemiological evaluation, 62% of the interviewees recognised triatomines and most of them confirmed that they had seen these insects in the piassava plantations of the riverside communities of the Negro River tributaries. Of the seropositive patients, 25.8% affirmed that they had been stung by the triatomines and 11.7% denied having been stung. The principal clinical manifestations of the seropositive individuals were palpitations, chest pain and dyspnoea upon effort. Cardiac auscultation revealed extrasystoles, bradycardia and systolic murmurs. The electrocardiographic alterations were ventricular extrasystoles, left and right bundle branch block, atrioventricular block and primary T wave alterations. The echocardiogram was altered in 22.6% of the seropositive individuals and in 8.5% of the seronegative individuals.     

Identification and characterization of human archaemetzincin-1 and -2, two novel members of a family of metalloproteases widely distributed in Archaea

Systematic analysis of degradomes, the complete protease repertoires of organisms, has demonstrated the large and growing complexity of proteolytic systems operating in all cells and tissues. We report here the identification of two new human metalloproteases that have been called archaemetzincin-1 (AMZ1) and archaemetzincin-2 (AMZ2) to emphasize their close relationship to putative proteases predicted by bioinformatic analysis of archaeal genomes. Both human proteins contain a catalytic domain with a core motif (HEXXHXXGX3CX4CXMX17CXXC) that includes an archetypal zinc-binding site, the methionine residue characteristic of metzincins, and four conserved cysteine residues that are not present at the equivalent positions of other human metalloproteases. Analysis of genome sequence databases revealed that AMZs are widely distributed in Archaea and vertebrates and contribute to the defining of a new metalloprotease family that has been called archaemetzincin. However, AMZ-like sequences are absent in a number of model organisms from bacteria to nematodes. Phylogenetic analysis showed that these enzymes have undergone a complex evolutionary process involving a series of lateral gene transfer, gene loss, and genetic duplication events that have shaped this novel family of metalloproteases. Northern blot analysis showed that AMZ1 and AMZ2 exhibit distinct expression patterns in human tissues. AMZ1 is mainly detected in liver and heart whereas AMZ2 is predominantly expressed in testis and heart, although both are also detectable at lower levels in other tissues. Both human enzymes were produced in Escherichia coli, and the purified recombinant proteins hydrolyzed synthetic substrates and bioactive peptides, demonstrating that they are functional proteases. Finally, these activities were abolished by inhibitors of metalloproteases, providing further evidence that AMZs belong to this catalytic class of proteolytic enzymes.     

Enhancement of growth rate and β-galactosidase activity, and variation in organic acid profile of Bifidobacterium animalis subsp. lactis Bb 12

The behavior of Bifidobacterium animalis subsp. lactis Bb 12 under batch cultivation, after continuous culturing for up to 12 d, was monitored in skim milk-based media. Previous continuous culture for longer than 6 d affected the physiology of said microorganism. The minimum inhibitory concentrations of lactic and acetic acids increased from 18 to 26 g/l, whereas the molar ratio of acetic to lactic acid increased from 0.8 to 1.55, when the previous continuous culture increased its duration from 1 to 12 d. The specific lactose consumption rate decreased from 0.94 to 0.77 g_lactose/g_{cell dry mass}/h within the batch culture timeframe; this was concomitant with greater amounts of acetic and formic acids, and lower amounts of lactic acid produced. The β-galactosidase activity increased as continuous culturing time increased, and reached 446 units/ml by 12 d; however, the rate of enzyme synthesis decreased concomitantly. Succinic acid was produced during the exponential growth and stationary phases of the batch culture, but the former at exponential growth phase was higher as the continuous culturing time was longer. For comparison purposes, batch cultivation of samples taken from continuous cultures by 1 and 12 d was done using a semi-synthetic medium with glucose as carbon source; a pattern similar to that observed when using skim milk-based media was observed.