The synthetic peptide RPRAATF allows specific assay of Akt activity in cell lysates

The Akt protein kinase is a critical signaling molecule in a range of cellular processes. A key to identifying the role of this pleiotropic kinase in any particular process is the ability to quantitate its activity. In this study we show that the synthetic peptide RPRAATF is a specific substrate for the kinase in crude cell extracts, thus enabling rapid, convenient, and sensitive assay of Akt activity. Peptide kinase activity was confined to a single peak upon sequential ion-exchange chromatography of whole-cell extracts of Balb/c 3T3 fibroblasts. This activity was stimulated by both platelet-derived growth factor and pervanadate, phosphatidyl inositol 3-kinase dependent, and inhibited by specific immunodepletion with anti-Akt antisera. Furthermore, direct assays of crude extracts from a range of cell types using this peptide were consistent with the results obtained using specific immunoprecipitation assays.     

Reduction of 1,4-quinone and ubiquinones by hydrogen atom transfer under UVA irradiation

1,4-Benzoquinone, coenzyme Q 0 and Q 10 were reacted with a series of hydrogen donors in the ESR cavity in the presence or absence of UVA irradiation. The signals of the radicals generated from the hydrogen donors or of those of the semiquinones were detected. The reaction mechanism was interpreted by a hydrogen atom transfer instead of the usual electron transfer mechanism on the basis of the redox potentials of the reactants and the Marcus theory. The hydrogen atom transfer is explained by the excited triplet state of quinones, which, on the basis of quantum mechanic calculations, may be reached even under visible light. In some cases, hydrogen atom transfer was also observed without irradiation, although to a lesser extent.     

Direct identification of tyrosine 474 as a regulatory phosphorylation site for the Akt protein kinase

Understanding the regulation of Akt has been of major interest for elucidating the control of normal cellular physiology as well as malignant transformation. The paradigm for activation of Akt involves phosphatidylinositol 3-kinase-dependent membrane localization followed by activating phosphorylation of Thr-308 and Ser-473. Many of the activating signals for Akt involve the stimulation of receptor and non-receptor tyrosine kinases, and the most potent activator known is the tyrosine phosphatase inhibitor pervanadate, highlighting a possible role for tyrosine phosphorylation in the regulation of the enzyme. In this study we show that activation of Akt by pervanadate or serum is associated with tyrosine phosphorylation of Akt. In addition, in SKOV3 ovarian carcinoma cells that exhibit high basal levels of Akt activity, Akt was tyrosine-phosphorylated in the basal state, and this phosphorylation was further enhanced by both pervanadate and insulin-like growth factor-1. We have used NH(2)-terminal sequencing and phosphate release analysis to directly identify Tyr-474 as the site of tyrosine phosphorylation. Substitution of Tyr-474 with phenylalanine abolished tyrosine phosphorylation of Akt and resulted in up to 55% inhibition of Akt activation, indicating phosphorylation at Tyr-474 is required for full activation of the kinase. Our data identifies a novel regulatory mechanism for this pleiotropic enzyme that may be applicable to the AGC family of protein kinases given the conserved nature of the COOH-terminal hydrophobic motif containing Tyr-474.     

Effects of mercury on microbial biomass and enzyme activities in soil

Mercury (Hg) is a persistent soil pollutant that affects soil microbial activity. We monitored the changes in soil microbial biomass and activity of enzymes, including alkaline phosphatase, arylsulfatase, fluorescein diacetate (FDA) hydrolytic activity, and o-diphenol oxidase (o-DPO) in three soils contaminated with different concentrations of Hg. Increasing levels of Hg, from 0.5 to 10 μmol/g of dried soil, generally depressed microbial activity; however, the effects of Hg on soil microbial activity depended on soil type and composition, particularly organic matter content. o-DPO was less affected by Hg than the other three enzymes tested. Our results indicate that the analysis of microbial biomass content and soil-enzyme activities may be used to predict the soil quality contaminated with Hg.     

Effects of Triton X-100 and Quillaya Saponin on the ex situ bioremediation of a chronically polychlorobiphenyl-contaminated soil

The possibility of enhancing the ex situ bioremediation of a chronically polychlorinated biphenyl (PCB)-contaminated soil by using Triton X-100 or Quillaya Saponin, a synthetic and a biogenic surfactant, respectively, was studied. The soil, which contained about 350 mg/kg of PCBs and indigenous aerobic bacteria capable of growing on biphenyl or on monochlorobenzoic acids, was amended with inorganic nutrients and biphenyl, saturated with water and treated in aerobic batch slurry- and fixed-phase reactors. Triton X-100 and Quillaya Saponin were added to the reactors at a final concentration of 10 g/l at the 42nd day of treatment, and at the 43rd and 100th day, respectively. Triton X-100 was not metabolised by the soil microflora and it exerted inhibitory effects on the indigenous bacteria. Quillaya Saponin, on the contrary, was readily metabolised by the soil microflora. Under slurry-phase conditions, Triton X-100 negatively influenced the soil bioremediation process by affecting the availability of the chlorobenzoic acid degrading indigenous bacteria, whereas Quillaya Saponin slightly enhanced the biological degradation and dechlorination of the soil PCBs. In the fixed-phase reactors, where both the surfactant availability and the mixing of the soil were lower, Triton X-100 did not exert inhibitory effects on the soil biomass and enhanced significantly the soil PCB depletion, whereas Quillaya Saponin did not influence the bioremediation process. Received: 28 April 1998 / Received last revision: 15 July 1998 / Accepted: 29 July 1998     

Involvement of potassium channels in the antidepressant-like effect of venlafaxine in mice

AimsStudies have shown that the acute administration of venlafaxine elicits an antidepressant-like effect in the mouse forced swim test (FST) by a mechanism dependent on the l-arginine–nitric oxide (NO)–cyclic guanosine monophosphate (cGMP) pathway. Because it has been reported that NO activates different types of potassium (K⁺) channels in the brain, this study investigated the involvement of K⁺ channels in the antidepressant-like effect of venlafaxine in the mouse FST.Main methodsMale adult Swiss mice were pretreated with different K⁺ channel inhibitors or openers 15 min before venlafaxine administration. After 30 min, the open-field test (OFT) and FST were carried out.Key findingsIntracerebroventricular (i.c.v.) pretreatment of mice with subeffective doses of tetraethylammonium (TEA, a non-specific inhibitor of K⁺ channels, 25 pg/site), glibenclamide (an ATP-sensitive K⁺ channel inhibitor, 0.5 pg/site), charybdotoxin (a large- and intermediate-conductance calcium-activated K⁺ channel inhibitor, 25 pg/site) or apamin (a small-conductance calcium-activated K⁺ channel inhibitor, 10 pg/site) was able to potentiate the action of a subeffective dose of venlafaxine (2 mg/kg, i.p.). Moreover, the reduction in the immobility time elicited by an effective dose of venlafaxine (8 mg/kg, i.p.) in the FST was prevented by the pretreatment of mice with the K⁺ channel openers cromakalim (10 µg/site, i.c.v.) and minoxidil (10 µg/site, i.c.v.). The drugs used in this study did not produce any change in locomotor activity.SignificanceThe results demonstrate that the neuromodulatory effects of venlafaxine, via the inhibition of K⁺ channels, are possibly involved in its anti-immobility activity in the mouse FST.     

Perioperative Myocardial Injury after Adult Heart Transplant: Determinants and Prognostic Value

MethodsData on 362 consecutive recipients (mean age: 47.8±13.7, 20.2% female, 18.2% diabetics, 22.1% with previous cardiac operations, 27.6% hospitalized, 84.9±29.4 ml/min preoperative glomerular filtration rate) were analyzed using multivariable logistic regression modeling. Target outcomes were determinants of troponin release, early graft failure (EGF), acute kidney injury (AKI) and operative death.ResultsMean cTnI release measured 24 hours after transplant was 10.9±11.6 μg/L. Overall hospital mortality was 10.8%, EGF 10.5%, and AKI was 12.2%. cTnI release>10 μg/L proved an independent predictor of EGF (OR 2.2; 95% CI, 1.06–4.6) and AKI (OR 1.031; 95% CI, 1.001-1.064). EGF, in turn, proved a determinant of hospital mortality. Risk factors for cTnI>10 μg/L release were: status 2B (OR 0.35; 95% CI, 0.18-0.69, protective), duration of the ischemic period (OR 1.006; 95% CI, 1.001-1.011), previous cardiac operation (OR 2.9; 95% CI, 1.67-5.0), and left ventricular hypertrophy (OR 3.3; 95% CI, 1.9-5.6).ConclusionsMyocardial enzyme leakage clearly emerged as an epiphenomenon of more complicated clinical course. The complex interplay between surgical procedure features, graft characteristics and recipient end-organ function highlights cTnI release as a risk marker of graft failure and acute kidney injury. The search for optimal myocardial preservation is still an issue.     

D1 and D2 Inhibitions of the Soleus H-Reflex Are Differentially Modulated during Plantarflexion Force and Position Tasks

Presynaptic inhibition (PSI) has been shown to modulate several neuronal pathways of functional relevance by selectively gating the connections between sensory inputs and spinal motoneurons, thereby regulating the contribution of the stretch reflex circuitry to the ongoing motor activity. In this study, we investigated whether a differential regulation of Ia afferent inflow by PSI may be associated with the performance of two types of plantarflexion sensoriomotor tasks. The subjects (in a seated position) controlled either: 1) the force level exerted by the foot against a rigid restraint (force task, FT); or 2) the angular position of the ankle when sustaining inertial loads (position task, PT) that required the same level of muscle activation observed in FT. Subjects were instructed to maintain their force/position at target levels set at ~10% of maximum isometric voluntary contraction for FT and 90° for PT, while visual feedback of the corresponding force/position signals were provided. Unconditioned H-reflexes (i.e. control reflexes) and H-reflexes conditioned by electrical pulses applied to the common peroneal nerve with conditioning-to-test intervals of 21 ms and 100 ms (corresponding to D1 and D2 inhibitions, respectively) were evoked in a random fashion. A significant main effect for the type of the motor task (FT vs PT) (p = 0.005, η² _p = 0.603) indicated that PTs were undertaken with lower levels of Ia PSI converging onto the soleus motoneuron pool. Additionally, a significant interaction between the type of inhibition (D1 vs D2) and the type of motor task (FT vs PT) (p = 0.038, η² _p = 0.395) indicated that D1 inhibition was associated with a significant reduction in PSI levels from TF to TP (p = 0.001, η² _p = 0.731), whereas no significant difference between the tasks was observed for D2 inhibition (p = 0.078, η² _p = 0.305). These results suggest that D1 and D2 inhibitions of the soleus H-reflex are differentially modulated during the performance of plantarflexion FT and PT. The reduced level of ongoing PSI during PT suggests that, in comparison to FT, there is a larger reliance on inputs from muscle spindles primary afferents when the neuromuscular system is required to maintain position-controlled plantarflexion contractions.     

A novel marine mesocosm facility to study global warming,water quality,and ocean acidification

We describe a completely randomizable flow‐through outdoor mesocosm for climate change and ecotoxicology studies that was built with inexpensive materials. The 16 raceway tanks allow up to 6× water renewal per hour, avoiding changes in natural abiotic seawater conditions. We use an open‐source hardware board (Arduino) that was adapted to control heaters and an innovative CO₂ injection system. This system reduced seawater pH up to ?0.9 units and increased temperature up to +6°C in three treatments and a control. Treatments can be continuously compared with the control and vary according to diel fluctuations, thus following the diel range observed in the sea. The mesocosm facility also includes an integrated secondary system of 48 aquaria for ecotoxicology studies. We validated the reproducibility and relevance of our experimental system by analyzing the variation of the total DNA of the microbial community extracted from corals in three elevated temperature scenarios during a 40‐day experiment. We also present data from temperature, acidification, and copper contamination trials, which allowed continuous, reliable, and consistent treatment manipulations.