Selection of a Streptomyces strain able to produce cell wall degrading enzymes and active against Sclerotinia sclerotiorum

Control of plant pathogen Sclerotinia sclerotiorum is an ongoing challenge because of its wide host range and the persistence of its sclerotia in soil. Fungicides are the most commonly used method to control this fungus but these can have ecotoxicity impacts. Chitinolytic Streptomyces strains isolated from Brazilian tropical soils were capable of inhibiting S. sclerotiorum growth in vitro, offering new possibilities for integrated pest management and biocontrol, with a new approach to dealing with an old problem. Strain Streptomyces sp. 80 was capable of irreversibly inhibiting fungal growth. Compared to other strains, its crude enzymes had the highest chitinolytic levels when measured at 25°C and strongly inhibited sclerotia from S. sclerotiorum. It produced four hydrolytic enzymes involved in fungal cell wall degradation when cultured in presence of the fungal mycelium. The best production, obtained after three days, was 0.75 U/ml for exochitinase, 0.9 U/ml for endochitinase, 0.16 U/ml for glucanase, and 1.78 U/ml for peptidase. Zymogram analysis confirmed two hydrolytic bands of chitinolytic activity with apparent molecular masses of 45.8 and 206.8 kDa. One glucanase activity with an apparent molecular mass of 55 kDa was also recorded, as well as seven bands of peptidase activity with apparent molecular masses ranging from 15.5 to 108.4 kDa. Differential interference contrast microscopy also showed alterations of hyphal morphology after co-culture. Streptomyces sp. 80 seems to be promising as a biocontrol agent against S. sclerotiorum, contributing to the development of new methods for controlling plant diseases and reducing the negative impact of using fungicides.     

Bolus administration of obestatin does not change glucose and insulin levels neither in the systemic nor in the portal circulation of the rat

Obestatin is a second peptide derived from the preproghrelin polypeptide. It was originally thought to have anorexigenic effects, thereby functioning as an antagonist of ghrelin. However, this has been a subject of debate ever since. Since acylated ghrelin strongly induces insulin resistance, it could be hypothesized that obestatin plays a role in glucose homeostasis as well. In the present study we evaluated the effect of obestatin on glucose and insulin metabolism in the systemic and portal circulation. Obestatin 200 nmol/kg was administered systemically as a single intravenous bolus injection to fasted pentobarbital anesthetized adult male Wistar rats. Up to 50 min after administration, blood samples were taken to measure glucose and insulin concentrations, both in the portal and in the systemic circulation. The effect of obestatin was evaluated in fasted and in glucose-stimulated conditions (IVGTT) and compared to control groups treated with saline or IVGTT, respectively. Intravenous administration of obestatin did not have any effect on glucose and insulin concentrations, neither systemic nor portal, when compared to the control groups. Only the glucose peak 1 min after administration of IVGTT was slightly higher in the obestatin treated rats: 605.8 ± 106.3% vs. 522.2 ± 47.1% in the portal circulation, respectively (NS), and 800.7 ± 78.7% vs. 549.6 ± 37.0% in the systemic circulation, respectively (P < 0.02), but it can be debated whether this has any clinical relevance. In the present study, we demonstrated that intravenously administered obestatin does not influence glucose and insulin concentrations, neither in the portal nor in the systemic circulation.     

Neural and cardiovascular responses to emotional stress in humans

Sympathetic neural responses to mental stress are well documented but controversial, whereas sympathetic neural responses to emotional stress are unknown. The purpose of this study was to investigate neural and cardiovascular responses to emotional stress evoked by negative pictures and reexamine the relationship between muscle sympathetic nerve activity (MSNA) and perceived stress. Mean arterial pressure (MAP), heart rate (HR), MSNA, and perceived stress levels were recorded in 18 men during three randomized trials: 1) neutral pictures, 2) negative pictures, and 3) mental stress. MAP and HR increased during mental stress (Delta14 +/- 2 mmHg and Delta15 +/- 2 beats/min, P < 0.001) but did not change during viewing of negative or neutral pictures. MSNA did not change during viewing of neutral (Delta1 +/- 1 burst/min, n = 16) or negative (Delta0 +/- 1 burst/min, n = 16) pictures or during mental stress (Delta1 +/- 2 burst/min, n = 13). Perceived stress levels were higher during mental stress (3 +/- 0 arbitrary units) than during viewing negative pictures (2 +/- 0 arbitrary units, P < 0.001). Perceived stress levels were not correlated to changes in MSNA during negative pictures (r = 0.10, P = 0.84) or mental stress (r = 0.36, P = 0.23). In conclusion, our results demonstrate robust increases in MAP and HR during mental stress, but not during emotional stress evoked by negative pictures. Although the influence of mental stress on MSNA remains unresolved, our findings challenge the concept that perceived stress levels modulate MSNA during mental stress.     

Inhibition of monoamine oxidase A promotes secretory differentiation in basal prostatic epithelial cells

Abstract Monoamine oxidase A (MAO-A) expression is associated with high-grade prostate cancer. Immunohistochemistry showed that MAO-A is also expressed in the basal epithelial cells of normal prostate glands. Using cultured primary prostatic epithelial cells as a model, we showed that MAO-A prevents basal epithelial cells from differentiating into secretory cells. Under differentiation-promoting conditions, clorgyline, an irreversible MAO-A inhibitor, induced secretory cell-like morphology and repressed expression of cytokeratin 14, a basal cell marker. More importantly, clorgyline induced mRNA and protein expression of androgen receptor (AR), a hallmark of secretory epithelial cells. In clorgyline-treated cells, androgen induced luciferase activity controlled by the promoter of prostate-specific antigen, an AR target gene, in a dose-dependent manner. This activity was blocked by the AR antagonist Casodex, showing that AR is functional. In turn, androgen decreased MAO-A expression in clorgyline-treated, secretory-like cells. Our results demonstrated that cultured basal epithelial cells have the potential to differentiate into secretory cells, and that inhibition of MAO-A is a key factor in promoting this process. Increased expression of MAO-A in high-grade prostate cancer may be an important contributor to its de-differentiated phenotype, raising the possibility that MAO-A inhibition may restore differentiation and reverse the aggressive behavior of high-grade cancer.     

A biophysical model for plant cell plate maturation based on the contribution of a spreading force

Plant cytokinesis, a fundamental process of plant life, involves de novo formation of a “cell plate” partitioning the cytoplasm of dividing cells. Cell plate formation is directed by orchestrated delivery, fusion of cytokinetic vesicles, and membrane maturation to form a nascent cell wall by timely deposition of polysaccharides. During cell plate maturation, the fragile membrane network transitions to a fenestrated sheet and finally a young cell wall. Here, we approximated cell plate sub-structures with testable shapes and adopted the Helfrich-free energy model for membranes, including a stabilizing and spreading force, to understand the transition from a vesicular network to a fenestrated sheet and mature cell plate. Regular cell plate development in the model was possible, with suitable bending modulus, for a two-dimensional late stage spreading force of 2–6 pN/nm, an osmotic pressure difference of 2–10 kPa, and spontaneous curvature between 0 and 0.04 nm⁻¹. With these conditions, stable membrane conformation sizes and morphologies emerged in concordance with stages of cell plate development. To reach a mature cell plate, our model required the late-stage onset of a spreading/stabilizing force coupled with a concurrent loss of spontaneous curvature. Absence of a spreading/stabilizing force predicts failure of maturation. The proposed model provides a framework to interrogate different players in late cytokinesis and potentially other membrane networks that undergo such transitions. Callose, is a polysaccharide that accumulates transiently during cell plate maturation. Callose-related observations were consistent with the proposed model’s concept, suggesting that it is one of the factors involved in establishing the spreading force.

The late-stage onset of an “areal” spreading and stabilizing force is essential for regular plant cell plate development and maturation.     

Improved gene targeting in Magnaporthe grisea by inactivation of MgKU80 required for non-homologous end joining

The ascomycete Magnaporthe grisea is a model species for the study of plant fungal interactions. As in many filamentous fungi, targeted gene replacement occurs at low frequency in M. grisea (average 7%). mus52/KU80 is a gene essential for non-homologous end joining (NHEJ) of DNA double-strand breaks. Its deletion increases the frequency of targeted gene replacement in fungi [Ninomiya, Y., Suzuki, K., Ishii, C., Inoue, H., 2004. Highly efficient gene replacements in Neurospora strains deficient for non-homologous end joining. Proc. Natl. Acad. Sci. USA 101(33), 12248-53]. M. grisea KU80 deletion mutants were constructed and displayed wild-type phenotypes regarding pathogenicity, growth, sporulation and mating. MgADE4 targeted gene replacement frequency was increased in Deltaku80 mutants (80% vs 5%) and high frequencies (>80%) were observed at seven other loci. However, the deletion of MgKU80 did not increase the frequency of ACE1 replacement indicating that this locus has an intrinsic reduced ability for gene replacement. These results open the way to large-scale reverse genetics experiments in M. grisea facilitating the study of the infection process.     

Spatial distribution patterns of interphase centromeres during retinoic acid-induced differentiation of promyelocytic leukemia cells

BACKGROUND: The pericentromeric heterochromatin is an important element for the regulation of gene silencing. Its spatial distribution during interphase appears to be cell-type specific. This study analyzes three-dimensional (3D) centromere distribution patterns during cellular differentiation along the neutrophil pathway. METHODS: Differentiation of the promyelocytic leukemia cell line NB4 was induced by retinoic acid. Centromeres in interphase nuclei were visualized by immunofluorescence staining of centromere-associated proteins with CREST serum. 3D images of nuclei were obtained by confocal microscopy. Automated methods for the segmentation of point-like objects in 3D images were implemented to detect the position of centromeres. Features of centromere localization patterns were determined by constructing the minimal spanning tree of the centromere distribution. RESULTS: In differentiated NB4 cells, the number of centromere conglomerates (chromocenters) was decreased and the distance between chromocenters was increased as compared with untreated controls. The nuclear volume did not differ between the two groups. CONCLUSIONS: The measured rearrangement of centromeres indicates a progressive clustering of heterochromatin and a global remodeling of interphase chromosome territories during differentiation of NB4 cells. The developed methods for the analysis of 3D centromere distribution patterns provide the opportunity for a fast and objective analysis of heterochromatin remodeling.     

High-Activity Radio-Iodine Labeling of Conventional and Stealth Liposomes

A new method to label preformed liposomes with high activities of radiohalogenated compounds has been developed. It uses activated esters of simple synthetic molecules that may be readily halogenated, such as Bolton-Hunter reagent (BH), and arginine-containing liposomes. BH, in the form of an activated ester, crosses the liposome membrane to react with arginine inside the liposomes, as demonstrated by thin-layer chromatography and by the fact that saline-containing liposomes, or hydrolyzed BH or the water soluble sulfo-BH afforded only marginal encapsulation yields. Under optimized conditions, between 37 and 55°C, 62 ± 4% (mean ± SD) of radiolabeled BH were consistently encapsulated in the liposomes within 30 min. In molar amounts, this corresponds to a mean of 56 nmol of BH per μmol of lipids. Based on achievable specific activity, up to 2.8 GBq of iodine-131 could be entrapped per μmol of lipids. Leakage of radioactivity was very low, with less than 5% of the encapsulated activity released within 6 days at 4°C in phosphate-buffered saline and less than 50% within 24h in human serum at 37°C. The labeling stability, and the fact that both conventional and PEGylated liposomes can be readily labeled with high doses of radioactivity, will make this technique useful for in vivo targeting applications, such as tumor detection (using iodine-123 or iodine-124) or therapy (with iodine-131 or astatine-211).     

Independence of the Sodium and Potassium Conductance Channels: A Kinetic Argument

Tightly coupled models for the sodium and potassium conductance changes, in which the potassium “on” process is intimately related to the sodium “on” and “off” processes, are studied. It is shown that such coupled models are incapable of simultaneously showing the observed effects of conditioning potentials on sodium inactivation and on the translation of the potassium conductance in time. It is concluded that the primary mechanisms for the sodium and potassium channels are probably independent.