Identification of a Novel, Putative Rho-specific GDP/GTP Exchange Factor and a RhoA-binding Protein: Control of Neuronal Morphology

The small GTP-binding protein Rho has been implicated in the control of neuronal morphology. In N1E-115 neuronal cells, the Rho-inactivating C3 toxin stimulates neurite outgrowth and prevents actomyosin-based neurite retraction and cell rounding induced by lysophosphatidic acid (LPA), sphingosine-1-phosphate, or thrombin acting on their cognate G protein–coupled receptors. We have identified a novel putative GDP/GTP exchange factor, RhoGEF (190 kD), that interacts with both wild-type and activated RhoA, but not with Rac or Cdc42. RhoGEF, like activated RhoA, mimics receptor stimulation in inducing cell rounding and in preventing neurite outgrowth. Furthermore, we have identified a 116-kD protein, p116^Rip, that interacts with both the GDP- and GTP-bound forms of RhoA in N1E-115 cells. Overexpression of p116^Rip stimulates cell flattening and neurite outgrowth in a similar way to dominant-negative RhoA and C3 toxin. Cells overexpressing p116^Rip fail to change their shape in response to LPA, as is observed after Rho inactivation. Our results indicate that (a) RhoGEF may link G protein–coupled receptors to RhoA activation and ensuing neurite retraction and cell rounding; and (b) p116^Rip inhibits RhoA-stimulated contractility and promotes neurite outgrowth.     

Rapid effects of nerve growth factor on the Na,K-pump in rat pheochromocytoma cells

Nerve growth factor (NGF) induces neuronal differentiation of rat pheochromocytoma cells (PC12). Here we show that NGF causes a stimulation of Na⁺,K⁺-pump mediated K⁺ influx, with a maximum at 30 min after addition of NGF. The stimulation of the Na⁺,K⁺-pump is completely blocked by the Na⁺-flux inhibitor amiloride (0.2 mM) and can be mimicked by the Na⁺ ionophore monensin. These results suggest that NGF causes a rapid enhancement of Na⁺ influx leading to an activation of the Na⁺,K⁺-pump, a mechanism similar to the action of other growth factors.     

Red:far-red light conditions affect the emission of volatile organic compounds from barley (Hordeum vulgare), leading to altered biomass allocation in neighbouring plants

Background and Aims Volatile organic compounds (VOCs) play various roles in plant–plant interactions, and constitutively produced VOCs might act as a cue to sense neighbouring plants. Previous studies have shown that VOCs emitted from the barley (Hordeum vulgare) cultivar ‘Alva’ cause changes in biomass allocation in plants of the cultivar ‘Kara’. Other studies have shown that shading and the low red:far-red (R:FR) conditions that prevail at high plant densities can reduce the quantity and alter the composition of the VOCs emitted by Arabidopsis thaliana, but whether this affects plant–plant signalling remains unknown. This study therefore examines the effects of far-red light enrichment on VOC emissions and plant–plant signalling between ‘Alva’ and ‘Kara’.Methods The proximity of neighbouring plants was mimicked by supplemental far-red light treatment of VOC emitter plants of barley grown in growth chambers. Volatiles emitted by ‘Alva’ under control and far-red light-enriched conditions were analysed using gas chromatography–mass spectrometry (GC-MS). ‘Kara’ plants were exposed to the VOC blend emitted by the ‘Alva’ plants that were subjected to either of the light treatments. Dry matter partitioning, leaf area, stem and total root length were determined for ‘Kara’ plants exposed to ‘Alva’ VOCs, and also for ‘Alva’ plants exposed to either control or far-red-enriched light treatments.Key Results Total VOC emissions by ‘Alva’ were reduced under low R:FR conditions compared with control light conditions, although individual volatile compounds were found to be either suppressed, induced or not affected by R:FR. The altered composition of the VOC blend emitted by ‘Alva’ plants exposed to low R:FR was found to affect carbon allocation in receiver plants of ‘Kara’.Conclusions The results indicate that changes in R:FR light conditions influence the emissions of VOCs in barley, and that these altered emissions affect VOC-mediated plant–plant interactions.     

Instar-specific mortalities of coexisting Daphnia species in relation to food and invertebrate predation

Instar-specific mortalities of Daphnia hyalina and D.cucullatawere studied from May 19 to September 29, 1988 in combinationwith invertebrate predator and phytoplankton abundance. Simultaneouslife-table experiments were conducted under semi-natural conditionsin the laboratory to estimate juvenile mortality in a predator-freeenvironment. Juvenile mortality by predation was calculatedas the difference between juvenile mortality in the field andin the experiments and was the most important factor for thedifferences in abundance of the two species. For D.hyalina juvenilemortality was higher in early summer and probably caused byselective predation by Chaoborus flavicans. Predation by Leptodorakindtii was probably more important during the rest of the summer.Estimated mortality by predation adequately explained juvenilemortality, except for a 3-week period in August. Decreasingflagellate densities in July were accompanied by increased juvenilemortalities of D.hyalina and D.cucullata in the life-table experimentsin August and coincided with a Daphnia population decrease.     

Production of 6-Phenylacetylene Picolinic Acid from Diphenylacetylene by a Toluene-Degrading Acinetobacter Strain

Several strategies for using enzymes to catalyze reactions leading to the synthesis of relatively simple substituted picolinic acids have been described. The goal of the work described here was to synthesize a more complex molecule, 6-phenylacetylene picolinic acid [6-(2-phenylethynyl)pyridine-2-carboxylic acid], for use as a potential endcapping agent for aerospace polymers. We screened 139 toluene-degrading strains that use a variety of catabolic pathways for the ability to catalyze oxidative transformation of diphenylacetylene. Acinetobacter sp. strain F4 catalyzed the overall conversion of diphenylacetylene to a yellow metabolite, which was identified as a putative meta ring fission product (2-hydroxy-8-phenyl-6-oxoocta-2,4-dien-7-ynoic acid [RFP]). The activity could be sustained by addition of toluene at a flow rate determined empirically so that the transformations were sustained in spite of the fact that toluene is a competitive inhibitor of the enzymes. The overall rate of transformation was limited by the instability of RFP. The RFP was chemically converted to 6-phenylacetylene picolinic acid by treatment with ammonium hydroxide. The results show the potential for using the normal growth substrate to provide energy and to maintain induction of the enzymes involved in biotransformation during preliminary stages of biocatalyst development.     

Increased Cardiac Myocyte PDE5 Levels in Human and Murine Pressure Overload Hypertrophy Contribute to Adverse LV Remodeling

Background

The intracellular second messenger cGMP protects the heart under pathological conditions. We examined expression of phosphodiesterase 5 (PDE5), an enzyme that hydrolyzes cGMP, in human and mouse hearts subjected to sustained left ventricular (LV) pressure overload. We also determined the role of cardiac myocyte-specific PDE5 expression in adverse LV remodeling in mice after transverse aortic constriction (TAC).

Methodology/Principal Findings

In patients with severe aortic stenosis (AS) undergoing valve replacement, we detected greater myocardial PDE5 expression than in control hearts. We observed robust expression in scattered cardiac myocytes of those AS patients with higher LV filling pressures and BNP serum levels. Following TAC, we detected similar, focal PDE5 expression in cardiac myocytes of C57BL/6NTac mice exhibiting the most pronounced LV remodeling. To examine the effect of cell-specific PDE5 expression, we subjected transgenic mice with cardiac myocyte-specific PDE5 overexpression (PDE5-TG) to TAC. LV hypertrophy and fibrosis were similar as in WT, but PDE5-TG had increased cardiac dimensions, and decreased dP/dt_max and dP/dt_min with prolonged tau (P<0.05 for all). Greater cardiac dysfunction in PDE5-TG was associated with reduced myocardial cGMP and SERCA2 levels, and higher passive force in cardiac myocytes in vitro.

Conclusions/Significance

Myocardial PDE5 expression is increased in the hearts of humans and mice with chronic pressure overload. Increased cardiac myocyte-specific PDE5 expression is a molecular hallmark in hypertrophic hearts with contractile failure, and represents an important therapeutic target.     

Yeast species utilizing uric acid,adenine,n-alkylamines or diamines as sole source of carbon and energy

Yeast strains utilizing uric acid, adenine, monoamines or diamines as sole source of carbon and energy were isolated from several soil samples by the enrichment culture method. The most common species wasTrichosporon cutaneum. Strains ofCandida catenulata, C. famata, C. parapsilosis, C. rugosa, Cryptococcus laurentii, Stephanoascus ciferrii andTr. adeninovorans were also isolated. All strains utilizing uric acid as sole carbon source utilized some primaryn-alkyl-l-amines hydroxyamines or diamines as well. The ascomycetous yeast strains showing these characteristics all belonged to species known to assimilate hydrocarbons. Type strains of hydrocarbon-positive yeast species which were not found in the enrichment cultures generally assimilated putrescine, some type strains also butylamine or pentylamine, but none assimilated uric acid. Methanol-positive species were not isolated. Type strains of methanol-positive and of hydrocarbon-negative species did not assimilate uric acid, butylamine or putrescine. Assimilation of putrescine as sole source of carbon and energy may be a valuable diagnostic criterion in yeast taxonomy.     

Serotype distribution of invasive Streptococcus pneumoniae in Canada during the introduction of the 13-valent pneumococcal conjugate vaccine, 2010

A baseline serotype distribution was established by age and region for 2058 invasive Streptococcus pneumoniae isolates collected during the implementation period of the 13-valent pneumococcal conjugate vaccine (PCV13) program in many parts of Canada in 2010. Serotypes 19A, 7F, and 3 were the most prevalent in all age groups, accounting for 57% in <2 year olds, 62% in 2-4 year olds, 45% in 5-14?year olds, 44% in 15-49?year olds, 41% in 50-64?year olds, and 36% in ≥65?year olds. Serotype 19A was most predominant in Western and Central Canada representing 15% and 22%, respectively, of the isolates from those regions, whereas 7F was most common in Eastern Canada with 20% of the isolates. Other prevalent serotypes include 15A, 23B, 12F, 22F, and 6C. PCV13 serotypes represented 65% of the pneumococci isolated from?<2 year olds, 71% of 2-4 year olds, 61% of 5-14 year olds, 60% of 15-49 year olds, 53% of 50-64 year olds, and 49% of the?≥65?year olds. Continued monitoring of invasive pneumococcal serotypes in Canada is important to identify epidemiological trends and assess the impact of the newly introduced PCV13 vaccine on public health.     

Cytokine profiles in the joint depend on pathology,but are different between synovial fluid,cartilage tissue and cultured chondrocytes

Introduction

This study aimed to evaluate whether profiles of several soluble mediators in synovial fluid and cartilage tissue are pathology-dependent and how their production is related to in vitro tissue formation by chondrocytes from diseased and healthy tissue.

Methods

Samples were obtained from donors without joint pathology (n = 39), with focal defects (n = 65) and osteoarthritis (n = 61). A multiplex bead assay (Luminex) was performed measuring up to 21 cytokines: Interleukin (IL)-1α, IL-1β, IL-1RA, IL-4, IL-6, IL-6Rα, IL-7, IL-8, IL-10, IL-13, tumor necrosis factor (TNF)α, Interferon (IFN)γ, oncostatin M (OSM), leukemia inhibitory factor (LIF), adiponectin, leptin, monocyte chemotactic factor (MCP)1, RANTES, basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), vascular growth factor (VEGF).

Results

In synovial fluid of patients with cartilage pathology, IL-6, IL-13, IFNγ and OSM levels were higher than in donors without joint pathology (P ≤0.001). IL-13, IFNγ and OSM were also different between donors with cartilage defects and OA (P <0.05). In cartilage tissue from debrided defects, VEGF was higher than in non-pathological or osteoarthritic joints (P ≤0.001). IL-1α, IL-6, TNFα and OSM concentrations (in ng/ml) were markedly higher in cartilage tissue than in synovial fluid (P <0.01). Culture of chondrocytes generally led to a massive induction of most cytokines (P <0.001). Although the release of inflammatory cytokines was also here dependent on the pathological condition (P <0.001) the actual profiles were different from tissue or synovial fluid and between non-expanded and expanded chondrocytes. Cartilage formation was lower by healthy unexpanded chondrocytes than by osteoarthritic or defect chondrocytes.

Conclusions

Several pro-inflammatory, pro-angiogenic and pro-repair cytokines were elevated in joints with symptomatic cartilage defects and/or osteoarthritis, although different cytokines were elevated in synovial fluid compared to tissue or cells. Hence a clear molecular profile was evident dependent on disease status of the joint, which however changed in composition depending on the biological sample analysed. These alterations did not affect in vitro tissue formation with these chondrocytes, as this was at least as effective or even better compared to healthy chondrocytes.