Exact formula for the horizontal-to-vertical displacement ratio of Rayleigh waves in layered orthotropic half-spaces.

This paper is concerned with the propagation of Rayleigh waves in a compressible orthotropic half-space coated by a compressible orthotropic layer. The main purpose of the paper is to establish an explicit exact formula for the H/V ratio (the horizontal-to-vertical displacement ratio) of the Rayleigh wave. This formula is derived by using the transfer matrix for an orthotropic elastic layer and by the effective boundary condition technique. The formula recovers the H/V formula by Malischewsky and Scherbaum [(2004). Wave Motion 40, 57-67] for the compressible isotropic case and the H/V formula by Love [(1911). Some Problems of Geodynamics (Cambridge University Press, Cambridge)] for the incompressible isotropic case. The effect of anisotropy on the H/V ratio's properties is examined and it is numerically shown that the anisotropy strongly affects the peak and zero frequency of the H/V ratio and its prograde domain as well. As the H/V ratio is a dimensionless quantity and it is easily experimentally measured, the obtained formula will be a useful tool for the nondestructive evaluation of mechanical properties of thin films deposited onto half-spaces. Two numerical examples are performed to show how the obtained formula can be used to extract the material properties of deposited layers from measured values of the H/V ratio.

Real-time mobile teledermoscopy for skin cancer screening targeting an agricultural population: an experiment on 289 patients in France.

BACKGROUND: The incidence of skin cancer has reached epidemic proportions in the white population and is significantly elevated in agricultural populations, who are exposed to ultraviolet radiation during their professional activities. In 2014, the Agricultural Social Insurance Mutual Benefit Fund (MSA) offered its customers who work in agriculture and live in rural areas with reduced access to dermatologists the ability to participate in a 1-day teledermoscopic (TDS) screening event. OBJECTIVE: This study's aim was to assess the feasibility of real-time mobile TDS triage of a large number of agricultural workers by trained medical officers and occupational physicians. METHODS: Fifteen TDS screening centres were located in different areas of France. Individuals older than 18 years who worked in agriculture and lived in rural area near a TDS screening centre were invited to participate in a 1-day screening event and were examined by an MSA physician. In cases of suspicious skin lesions, clinical and dermoscopic images were obtained and transferred immediately to four dermatologists who were simultaneously present at the tele-platform for diagnosis and decision-making. Low-quality images were retaken. RESULTS: Two-hundred eighty-nine patients underwent skin cancer screening. Among 199 patients (69%), 390 suspicious lesions were identified and generated 412 pictures. All lesions were analysed by dermatologists. For 105 patients (53%), no follow-up was required. Seventeen patients were referred to local dermatologists for rapid examination, including 12 cases of suspected malignant melanocytic lesions. Among the 12 patients with suspected melanoma, face-to-face visits were conducted within 10 days for 11 of them, and 1 case of melanoma was confirmed by histopathology. CONCLUSIONS: Our study suggests that teledermoscopy performed in the context of occupational medicine and targeted to agricultural populations is feasible and could be useful for improving skin cancer screening in at-risk populations while avoiding face-to-face examinations by a dermatologist in 53% of cases.

Crosstalk reduction in a piezoelectric phononic plate induced by electrical boundary conditions: Application to multi-element transducers.

This paper proposes a method of passive electrical decoupling which aimed at found application in reducing the crosstalk phenomenon in multi-element ultrasonic transducers. A homogeneous piezoelectric plate, covered on one side by a 1D periodic arrangement of thin metallic electrodes and on the other side by a full electrode, is considered. Finite element analysis and experimental measurements are performed to obtain the dispersion curves and normal displacements at the surface of the structure. It is shown that applying inductive shunts at the electrodes, band gaps can be created in the first Brillouin zone, which can prevent from the establishment of the first thickness mode in the plate. In that way the mechanical inter-element coupling can be lowered. Thus, the acoustic radiation in water from one sector excited at the resonance frequency is found to be closer to that of a piston mode. The transposition of this principle to the situation of a transducer including a rear medium and a front matching layer confirms the possibility of reducing the inter-element coupling. However, the physical effects at the origin of this reduction are different from those inherent to the cutting of a piezocomposite ceramic as it is done in most probes available in the market. As a result, we show that taking advantage of the electrical boundary conditions upon the passive elements in a transducer gives real opportunities for crosstalk reduction that may be implemented in ultrasonic systems for imaging in the medical field and in NDT.

Metformin activates AMP-activated protein kinase in primary human hepatocytes by decreasing cellular energy status.

AIM/HYPOTHESIS: The glucose-lowering drug metformin has been shown to activate hepatic AMP-activated protein kinase (AMPK), a master kinase regulating cellular energy homeostasis. However, the underlying mechanisms remain controversial and have never been investigated in primary human hepatocytes. METHODS: Hepatocytes isolated from rat, mouse and human livers were treated with various concentrations of metformin. Isoform-specific AMPKα abundance and activity, as well as intracellular adenine nucleotide levels and mitochondrial oxygen consumption rates were determined at different time points. RESULTS: Metformin dose- and time-dependently increased AMPK activity in rat and human hepatocytes, an effect associated with a significant rise in cellular AMP:ATP ratio. Surprisingly, we found that AMPKα2 activity was undetectable in human compared with rat hepatocytes, while AMPKα1 activities were comparable. Accordingly, metformin only increased AMPKα1 activity in human hepatocytes, although both AMPKα isoforms were activated in rat hepatocytes. Analysis of mRNA expression and protein levels confirmed that only AMPKα1 is present in human hepatocytes; it also showed that the distribution of ß and γ regulatory subunits differed between species. Finally, we demonstrated that the increase in AMP:ATP ratio in hepatocytes from liver-specific Ampkα1/2 (also known as Prkaa1/2) knockout mice and humans is due to a similar and specific inhibition of the mitochondrial respiratory-chain complex 1 by metformin. CONCLUSIONS/INTERPRETATION: Activation of hepatic AMPK by metformin results from a decrease in cellular energy status owing to metformin's AMPK-independent inhibition of the mitochondrial respiratory-chain complex 1. The unique profile of AMPK subunits found in human hepatocytes should be considered when developing new pharmacological agents to target the kinase.

Symmetric Reflector Ultrasonic Transducer Modeling and Characterization: Role of the Matching Layer on Electroacoustic Performance.

Symmetric reflector ultrasonic transducers (SRUTs) are a new type of ultrasonic transducer that take advantage of the ultrasonic wave emitted on the rear face of the active element. In this work, the electroacoustic modeling and characterization of such a structure is reported. Using the well-known Krimholtz, Leedom, et Matthaei (KLM) model, the electroacoustic response of a SRUT based on a piezoelectric ceramic with one matching layer is calculated. Simulations show that the optimal acoustic impedance of the matching layer should be lower than for conventional transducers, leading to a relative bandwidth of approximately 50%. The characterization of such a transducer based on a piezoceramic plate has been carried out. Bandwidth and sensitivity are reported. They are found to be close to the simulation results and demonstrate that these new types of transducers need to be designed according to new rules compared to conventional ones.

Antimicrobial Resistance Pilot Surveillance of Pigs and Chickens in Vietnam, 2017-2019.

Antimicrobial use (AMU) and antimicrobial resistance (AMR) are a growing public health and economic threat in Vietnam. We conducted a pilot surveillance programme in five provinces of Vietnam, two in the south and three in the north, to identify antimicrobial resistance (AMR) in rectal swab samples from pigs and fecal samples from chickens at slaughter points during three different points in time from 2017 to 2019. Escherichia coli (E. coli) and non-typhoidal Salmonella (NTS) isolates were tested for antimicrobial susceptibility using disk diffusion assay for 19 antimicrobial agents belonging to nine antimicrobial classes and Etest for colistin (polymyxin). Almost all E. coli (99%; 1029/1042) and NTS (96%; 208/216) isolates were resistant to at least one antimicrobial agent; 94% (981/1042) of E. coli and 89% (193/216) of NTS isolates were multidrug-resistant (MDR). Higher proportions of E. coli and NTS isolated from chickens were resistant to all antimicrobial classes than those isolates from pigs. There was a significantly higher proportion of MDR NTS isolates from the southern provinces of Ho Chi Minh City and Long An (p = 0.008). Although there were increasing trends of NTS in proportion of resistance to fluoroquinolone over the three surveillance rounds, there was a significant decreasing trend of NTS in proportion of resistance to polymyxin (p = 0.002). It is important to establish an annual AMR surveillance program for livestock in Vietnam to assess the impact of interventions, observe trends and drive decision making that ultimately contributes to reducing AMR public health threat.

Structural transition and magnetic properties of Mn doped Bi0.88Sm0.12FeO3 ceramics.

We investigated the effects of Mn doping on the crystal structure, phonon vibration, and magnetic properties of Bi0.88Sm0.12FeO3 ceramics. Mn doping effectively modified the rhombohedral symmetry and induced a structural transition from an R3c rhombohedral to Pnam orthorhombic structure. Magnetic measurements revealed a weak ferromagnetic behavior, which was related to the canted antiferromagnetic order of the Pnam structure. The cycloidal spin structure of the R3c phase could not be suppressed by substitution of Mn at the Fe site. Studies on the self-phase transition and electric field-induced structural transition revealed many changes in coercivity and remanent magnetization, which are believed to originate from the R3c/Pnam phase switching along with spin frustration. Observations of the field step-dependent hysteresis loop and the ferromagnetic-like hysteresis loop after poling in an electric field provided direct evidence of phase boundary (PB) ferromagnetism and magnetic coupling at the PB.

Free carrier enhanced depletion in ZnO nanorods decorated with bimetallic AuPt nanoclusters.

The decoration of semiconductor nanostructures with small metallic clusters usually leads to an improvement of their properties in sensing or catalysis. Bimetallic cluster decoration typically is claimed to be even more effective. Here, we report a detailed investigation of the effects of Au, Pt or AuPt nanocluster decoration of ZnO nanorods on charge transport, photoluminescence and UV sensitivity. ZnO nanorods were synthesized by chemical bath deposition while decoration with small nanoclusters (2-3 nm in size) was achieved by a laser-ablation based cluster beam deposition technology. The structural properties were investigated by scanning electron microscopy, high resolution transmission electron microscopy, X-ray photoelectron spectroscopy and Rutherford backscattering spectrometry, and the optoelectronic properties by current-voltage and photoluminescence measurements. The extent of band bending at the cluster-ZnO interface was quantitatively modeled through numerical simulations. The decoration of ZnO nanorods with monometallic Au or Pt nanoclusters causes a significant depletion of free electrons below the surface, leading to a reduction of UV photoluminescence, an increase of ZnO nanorod dark resistance (up to 200 times) and, as a consequence, an improved sensitivity (up to 6 times) to UV light. These effects are strongly enhanced (up to 450 and 10 times, respectively) when ZnO nanorods are decorated with bimetallic AuPt nanoclusters that substantially augment the depletion of free carriers likely due to a more efficient absorption of the gas molecules on the surface of the bimetallic AuPt nanoclusters than on that of their monometallic counterparts. The depletion of free carriers in cluster decorated ZnO nanorods is quantitatively investigated and modelled, allowing the application of these composite materials in UV sensing and light induced catalysis.

Comparative Fe and Zn K-edge X-ray absorption spectroscopic study of the ferroxidase centres of human H-chain ferritin and bacterioferritin from Desulfovibrio desulfuricans.

Iron uptake by the ubiquitous iron-storage protein ferritin involves the oxidation of two Fe(II) ions located at the highly conserved dinuclear "ferroxidase centre" in individual subunits. We have measured X-ray absorption spectra of four mutants (K86Q, K86Q/E27D, K86Q/E107D, and K86Q/E27D/E107D, involving variations of Glu to Asp on either or both sides of the dinuclear ferroxidase site) of recombinant human H-chain ferritin (rHuHF) in their complexes with reactive Fe(II) and redox-inactive Zn(II). The results for Fe-rHuHf are compared with those for recombinant Desulfovibrio desulfuricans bacterioferritin (DdBfr) in three states: oxidised, reduced, and oxidised/Chelex-treated. The X-ray absorption near-edge region of the spectrum allows the oxidation state of the iron ions to be assessed. Extended X-ray absorption fine structure simulations have yielded accurate geometric information that represents an important refinement of the crystal structure of DdBfr; most metal-ligand bonds are shortened and there is a decrease in ionic radius going from the Fe(II) to the Fe(III) state. The Chelex-treated sample is found to be partly mineralised, giving an indication of the state of iron in the cycled-oxidised (reduced, then oxidised) form of DdBfr, where the crystal structure shows the dinuclear site to be only half occupied. In the case of rHuHF the complexes with Zn(II) reveal a surprising similarity between the variants, indicating that the rHuHf dinuclear site is rigid. In spite of this, the rHuHf complexes with Fe(II) show a variation in reactivity that is reflected in the iron oxidation states and coordination geometries.

Lack of starvation-induced activation of AMP-activated protein kinase in the hypothalamus of the Lou/C rats resistant to obesity.

OBJECTIVE: The AMP-activated protein kinase (AMPK) is involved in the control of food intake by the hypothalamus. The aim of this work was to investigate if modification of hypothalamic AMPK regulation could be related to the spontaneous food restriction of Lou/C rats, a strain resistant to obesity exhibiting a 40% reduction in caloric intake compared with their lean Wistar counterparts. DESIGN: Three-month-old male Lou/C rats were compared with age-matched male Wistar rats in both fed ad libitum and 24-h food deprivation state. MEASUREMENTS AND RESULTS: We first confirmed that starvation activated both isoforms of AMPK catalytic alpha subunits and enhanced the phosphorylation state of its downstream targets acetyl-CoA carboxylase and elongation factor 2 in the hypothalamus of Wistar rats. These changes were not observed in the hypothalamus of Lou/C rats. Interestingly, the starvation-induced changes in hypothalamic mRNA levels of the main orexigenic and anorexigenic neuropeptides were also blunted in the Lou/C rats. Analysis of the concentrations of circulating substrates and hormones known to regulate hypothalamic AMPK indicated that the starvation-induced changes in ghrelin, adiponectin and leptin were not observed in Lou/C rats. Furthermore, an increased phosphorylation state of signal transducer and activator of transcription 3 (STAT3), which admittedly mediates leptin signaling, was evidenced in the hypothalamus of the starved Lou/C rats, as well as modifications of expression of the leptin-sensitive genes suppressor of cytokine signaling-3 and stearoyl-coenzyme A desaturase 1. In addition, despite reduced leptin level in fed Lou/C rats, the phosphorylation state of hypothalamic STAT3 remained similar to that found in fed Wistar rats, an adaptation that could be explained by the concomitant increase in ObRb leptin receptor mRNA expression. CONCLUSION: Activation of hypothalamic AMPK by starvation, which stimulates food intake through changes in (an)orexigenic neuropeptides in the normal rats, was not observed in the spontaneously hypophagic Lou/C rats.

Antagonistic effects of leucine and glutamine on the mTOR pathway in myogenic C2C12 cells.

This study compared the effects of leucine and glutamine on the mTOR pathway, on protein synthesis and on muscle-specific gene expression in myogenic C(2)C(12) cells. Leucine increased the phosphorylation state of mTOR, on both Ser2448 and Ser2481, and its downstream effectors, p70(S6k), S6 and 4E-BP1. By contrast, glutamine decreased the phosphorylation state of mTOR on Ser2448, p70(S6k) and 4E-BP1, but did not modify the phosphorylation state of mTOR on Ser2481 and S6. Whilst the phosphorylation state of the mTOR pathway is usually related to protein synthesis, the incorporation of labelled methionine/cysteine was only transiently modified by leucine and was unaltered by glutamine. However, these two amino acids affected the mRNA levels of desmin, myogenin and myosin heavy chain in a time-dependant manner. In conclusion, leucine and glutamine have opposite effects on the mTOR pathway. Moreover, they induce modification of muscle-specific gene expression, unrelated to their effects on the mTOR/p70(S6k) pathway.

Phosphorylation and activation of heart PFK-2 by AMPK has a role in the stimulation of glycolysis during ischaemia.

BACKGROUND: The role of protein phosphorylation in the Pasteur effect--the phenomenon whereby anaerobic conditions stimulate glycolysis--has not been addressed. The AMP-activated protein kinase (AMPK) is activated when the oxygen supply is restricted. AMPK acts as an energy-state sensor and inhibits key biosynthetic pathways, thus conserving ATP. Here, we studied whether AMPK is involved in the Pasteur effect in the heart by phosphorylating and activating 6-phosphofructo-2-kinase (PFK-2), the enzyme responsible for the synthesis of fructose 2,6-bisphosphate, a potent stimulator of glycolysis. RESULTS: Heart PFK-2 was phosphorylated on Ser466 and activated by AMPK in vitro. In perfused rat hearts, anaerobic conditions or inhibitors of oxidative phosphorylation (oligomycin and antimycin) induced AMPK activation, which correlated with PFK-2 activation and with an increase in fructose 2,6-bisphosphate concentration. Moreover, in cultured cells transfected with heart PFK-2, oligomycin treatment resulted in a parallel activation of endogenous AMPK and PFK-2. In these cells, the activation of PFK-2 was due to the phosphorylation of Ser466. A dominant-negative construct of AMPK abolished the activation of endogenous and cotransfected AMPK, and prevented both the activation and phosphorylation of transfected PFK-2 by oligomycin. CONCLUSIONS: AMPK phosphorylates and activates heart PFK-2 in vitro and in intact cells. AMPK-mediated PFK-2 activation is likely to be involved in the stimulation of heart glycolysis during ischaemia.

Cyclic AMP suppresses the inhibition of glycolysis by alternative oxidizable substrates in the heart.

In normoxic conditions, myocardial glucose utilization is inhibited when alternative oxidizable substrates are available. In this work we show that this inhibition is relieved in the presence of cAMP, and we studied the mechanism of this effect. Working rat hearts were perfused with 5.5 mM glucose alone (controls) or together with 5 mM lactate, 5 mM beta-hydroxybutyrate, or 1 mM palmitate. The effects of 0.1 mM chlorophenylthio-cAMP (CPT-cAMP), a cAMP analogue, were studied in each group. Glucose uptake, flux through 6-phosphofructo-1-kinase, and pyruvate dehydrogenase activity were inhibited in hearts perfused with alternative substrates, and addition of CPT-cAMP completely relieved the inhibition. The mechanism by which CPT-cAMP induced a preferential utilization of glucose was related to an increased glucose uptake and glycolysis, and to an activation of phosphorylase, pyruvate dehydrogenase, and 6-phosphofructo-2-kinase, the enzyme responsible for the synthesis of fructose 2,6-bisphosphate, the well-known stimulator of 6-phosphofructo-1-kinase. In vitro phosphorylation of 6-phosphofructo-2-kinase by cAMP-dependent protein kinase increased the Vmax of the enzyme and decreased its sensitivity to the inhibitor citrate. Therefore, in hearts perfused with various oxidizable substrates, cAMP induces a preferential utilization of glucose by a concerted stimulation of glucose transport, glycolysis, glycogen breakdown, and glucose oxidation.

Enhancement of classification accuracy of a time-frequency approach for an EEG-based brain-computer interface.

OBJECTIVES: The aim of this paper is to develop a new algorithm to enhance the performance of EEG-based brain-computer interface (BCI). METHODS: We improved our time-frequency approach of classification of motor imagery (MI) tasks for BCI applications. The approach consists of Laplacian filtering, band-pass filtering and classification by correlation of time-frequency-spatial patterns. RESULTS AND CONCLUSIONS: Through off-line analysis of data collected during a "cursor control" experiment, we evaluated the capability of our new method to reveal major features of the EEG control for enhancement of MI classification accuracy. The pilot results in a human subject are promising, with an accuracy rate of 96.1%.

Direct and indirect activation of eukaryotic elongation factor 2 kinase by AMP-activated protein kinase.

BACKGROUND: Eukaryotic elongation factor 2 (eEF2) kinase (eEF2K) is a key regulator of protein synthesis in mammalian cells. It phosphorylates and inhibits eEF2, the translation factor necessary for peptide translocation during the elongation phase of protein synthesis. When cellular energy demand outweighs energy supply, AMP-activated protein kinase (AMPK) and eEF2K become activated, leading to eEF2 phosphorylation, which reduces the rate of protein synthesis, a process that consumes a large proportion of cellular energy under optimal conditions. AIM: The goal of the present study was to elucidate the mechanisms by which AMPK activation leads to increased eEF2 phosphorylation to decrease protein synthesis. METHODS: Using genetically modified mouse embryo fibroblasts (MEFs), effects of treatments with commonly used AMPK activators to increase eEF2 phosphorylation were compared with that of the novel compound 991. Bacterially expressed recombinant eEF2K was phosphorylated in vitro by recombinant activated AMPK for phosphorylation site-identification by mass spectrometry followed by site-directed mutagenesis of the identified sites to alanine residues to study effects on the kinetic properties of eEF2K. Wild-type eEF2K and a Ser491/Ser492 mutant were retrovirally re-introduced in eEF2K-deficient MEFs and effects of 991 treatment on eEF2 phosphorylation and protein synthesis rates were studied in these cells. RESULTS & CONCLUSIONS: AMPK activation leads to increased eEF2 phosphorylation in MEFs mainly by direct activation of eEF2K and partly by inhibition of mammalian target of rapamycin complex 1 (mTORC1) signaling. Treatment of MEFs with AMPK activators can also lead to eEF2K activation independently of AMPK probably via a rise in intracellular Ca2+. AMPK activates eEF2K by multi-site phosphorylation and the newly identified Ser491/Ser492 is important for activation, leading to mTOR-independent inhibition of protein synthesis. Our study provides new insights into the control of eEF2K by AMPK, with implications for linking metabolic stress to decreased protein synthesis to conserve energy reserves, a pathway that is of major importance in cancer cell survival.

Thromboembolic Disease in Patients with Metastatic Femoral Lesions: A Comparison Between Prophylactic Fixation and Fracture Fixation.

BACKGROUND: We are not aware of any previous studies that have compared the rate of venous thromboembolic events in patients who underwent prophylactic intramedullary nailing because of an impending fracture with the rate in patients who underwent intramedullary nailing after a pathological fracture. The objective of the present study was to determine if the rate of venous thromboembolic events varies between patients who are managed with prophylactic fixation and those who are managed with fixation after a pathological fracture. METHODS: We performed a retrospective comparative study in which the Nationwide Inpatient Sample database was used to identify all patients who had undergone femoral stabilization, either for a pathological femoral fracture or for prophylactic fixation of femoral metastatic lesion, over a period of 10 consecutive years (between 2002 and 2011) in the United States. Demographic data, comorbidities, venous thromboembolic event rates, and other common postoperative complications were compared between the 2 groups. RESULTS: Patients who were managed with prophylactic fixation had significantly higher rates of pulmonary embolism (p < 0.001; adjusted odds ratio, 2.1) and deep-vein thrombosis (p = 0.03; adjusted odds ratio, 1.5). Patients who were managed with fixation after a pathological fracture had a significantly greater need for blood transfusion, higher rates of postoperative urinary tract infection, and a decreased likelihood of being discharged to home (p < 0.001 for all). CONCLUSIONS: Patients with metastatic disease who undergo prophylactic intramedullary nailing have higher observed rates of venous thromboembolic events than those who undergo nailing for the treatment of a pathological fracture and should be actively and vigilantly managed in the postoperative period. LEVEL OF EVIDENCE: Therapeutic Level III. See Instructions for Authors for a complete description of levels of evidence.

No-flow ischemia inhibits insulin signaling in heart by decreasing intracellular pH.

Glucose-insulin-potassium solutions exert beneficial effects on the ischemic heart by reducing infarct size and mortality and improving postischemic left ventricular function. Insulin could be the critical protective component of this mixture, although the insulin response of the ischemic and postischemic myocardium has not been systematically investigated. The aim of this work was to study the insulin response during ischemia by analyzing insulin signaling. This was evaluated by measuring changes in activity and/or phosphorylation state of insulin signaling elements in isolated perfused rat hearts submitted to no-flow ischemia. Intracellular pH (pH(i)) was measured by NMR. No-flow ischemia antagonized insulin signaling including insulin receptor, insulin receptor substrate-1, phosphatidylinositol 3-kinase, protein kinase B, p70 ribosomal S6 kinase, and glycogen synthase kinase-3. These changes were concomitant with intracellular acidosis. Perfusing hearts with ouabain and amiloride in normoxic conditions decreased pH(i) and insulin signaling, whereas perfusing at pH 8.2 counteracted the drop in pH(i) and the inhibition of insulin signaling by ischemia. Incubation of cardiomyocytes in normoxic conditions, but at pH values below 6.75, mimicked the effect of ischemia and also inhibited insulin-stimulated glucose uptake. Finally, the in vitro insulin receptor tyrosine kinase activity was progressively inhibited at pH values below physiological pH(i), being abolished at pH 6.0. Therefore, ischemic acidosis decreases kinase activity and tyrosine phosphorylation of the insulin receptor thereby preventing activation of the downstream components of the signaling pathway. We conclude that severe ischemia inhibits insulin signaling by decreasing pH(i).

Fructose 2,6-bisphosphate and the control of glycolysis by glucocorticoids and by other agents in rat hepatoma cells.

The rate, key enzymes, and several metabolites of glycolysis in rat hepatoma (HTC) cells have been compared to those in rat hepatocytes. At 5 to 10 mM glucose, lactate release was greater in HTC cells. This could be explained in part by the absence of key gluconeogenic enzymes, by the substitution of glucokinase by hexokinase, and by an increase in phosphofructokinase 1 and pyruvate kinase activity. In addition, fructose 2,6-bisphosphate, the most potent stimulator of phosphofructokinase 1, was identified in HTC cells and shown to stimulate phosphofructokinase 1 partially purified from these cells. Dexamethasone increased the release of lactate in HTC cells. This glucocorticoid increased the concentration of fructose 2,6-bisphosphate and the Vmax of the enzyme that catalyzes its synthesis, phosphofructokinase 2. The data were consistent with an indirect effect at the gene level, mediated by glucocorticoid receptors. Dexamethasone had no effect on the other rate-limiting glycolytic enzymes. Several agents (adenosine, dibutyryl cyclic adenosine 3':5'-monophosphate, ethanol, antimycin) known to decrease fructose 2,6-bisphosphate in hepatocytes were without effect on this stimulator in HTC cells. DL-Glyceraldehyde inhibited glycolysis in HTC cells and eventually killed them. Although this substance decreased fructose 2,6-bisphosphate inhibition of glycolysis through an action at another level could not be ruled out.

AMPK antagonizes hepatic glucagon-stimulated cyclic AMP signalling via phosphorylation-induced activation of cyclic nucleotide phosphodiesterase 4B.

Biguanides such as metformin have previously been shown to antagonize hepatic glucagon-stimulated cyclic AMP (cAMP) signalling independently of AMP-activated protein kinase (AMPK) via direct inhibition of adenylate cyclase by AMP. Here we show that incubation of hepatocytes with the small-molecule AMPK activator 991 decreases glucagon-stimulated cAMP accumulation, cAMP-dependent protein kinase (PKA) activity and downstream PKA target phosphorylation. Moreover, incubation of hepatocytes with 991 increases the Vmax of cyclic nucleotide phosphodiesterase 4B (PDE4B) without affecting intracellular adenine nucleotide concentrations. The effects of 991 to decrease glucagon-stimulated cAMP concentrations and activate PDE4B are lost in hepatocytes deleted for both catalytic subunits of AMPK. PDE4B is phosphorylated by AMPK at three sites, and by site-directed mutagenesis, Ser304 phosphorylation is important for activation. In conclusion, we provide a new mechanism by which AMPK antagonizes hepatic glucagon signalling via phosphorylation-induced PDE4B activation.

Activation of nitric oxide synthase by ischaemia in the perfused heart.

OBJECTIVE: Recent data have indicated that the activity of nitric oxide synthase (NO synthase), the enzyme producing NO from L-arginine, could be modified by ischaemia. The aim of the present work was therefore to study whether ischaemia activated NO synthase. METHODS: NO synthase activity was measured by the conversion of radioactive arginine into citrulline in extracts of isolated perfused rabbit hearts submitted to low-flow ischaemia and reperfusion. RESULTS: When measured in heart homogenates, NO synthase activity was significantly increased during ischaemia. This activation was already detectable after 5 min of ischaemia and was maintained during the whole ischaemic period. After cell fractionation, NO synthase was recovered in cytosolic and membrane fractions. The increase in NO synthase activity by ischaemia was related to an activation of the cytosolic activity, while the membrane-bound NO synthase activity remained constant. CONCLUSION: NO synthase activity in the heart is rapidly stimulated by ischaemia and this stimulation is maintained during the whole ischaemic episode. This activation is found only in the cytosolic fraction, whereas the particulate activity is not affected by ischaemia.