Ion assisted deposition of thin films by substrate tuned radio frequency magnetron sputtering

The substrate tuning technique was applied to a radio frequency magnetron sputtering system to obtain a variable substrate bias without an additional source. The dependence of the substrate bias on the value of the external impedance was studied for different values of chamber pressure, gas composition and rf input power. A qualitative explanation of the results is given, based on a simple model, and the role of the stray capacitance is clearly disclosed. Langmuir probe measurements show that this system allows independent control of the ion flux and the ion energy bombarding the growing film. For an argon flow rate of 2.8 sccm and a radio frequency power of 300 W (intermediate values of the range studied) the ion flux incident on the substrate was 1.3 X 1020-m-2-s-1. The maximum ion energy available in these conditions can be varied in the range 30-150 eV. As a practical application of the technique, BN thin films were deposited under different ion bombardment conditions. An ion energy threshold of about 80 eV was found, below which only the hexagonal phase was present in the films, while for higher energies both hexagonal and cubic phase were present. A cubic content of about 60% was found for an ion energy of 120 V.

Hindleg muscle energy and substrate balances in cold-exposed rats

Rats chronically cannulated in the carotid artery and the muscular branch of the femoral vein were subjected to a cold (4 °C) environment for up to 2 h. The changes in blood flow (measured with 46Sc microspheres) and arterio-venous differences in the concentrations of glucose, lactate, triacylglycerols and amino acids allowed the estimation of substrate (and energy) balances across the hindleg. Mean glucose uptake was 0.28mmol min21, mean lactate release was 0.33mmol min21 and the free fatty acid basal release of 0.31mmol min21 was practically zero upon exposure to the cold; the initial uptake of triacylglycerols gave place to a massive release following exposure. The measurement of PO·, PCO· and pH also allowed the estimation of oxygen, CO2 and bicarbonate balances and respiratory quotient changes across the hindleg. The contribution of amino acids to the energy balance of the hindleg was assumed to be low. These data were used to determine the sources of energy used to maintain muscle shivering with time. Three distinct phases were observed in hindleg substrate utilization. (1) The onset of shivering, with the use of glucose/glycogen and an increase in lactate efflux. Lipid oxidation was practically zero (respiratory quotient near 1), but the uptake of triacylglycerols from the blood remained unchanged. (2) A substrate-energy shift, with drastically decreased use of glucose/glycogen, and of lactate efflux; utilization of triacylglycerol as practically the sole source of energy (respiratory quotient approximately 0.7); decreasing uptake of triacylglycerol and increased tissue lipid mobilization. (3) The onset of a new heat-homeostasis setting for prolonged cold-exposure, with maintenance of muscle energy and heat production based on triacylglycerol utilization and efflux from the hindleg (muscle plus skin and subcutaneous adipose masses) contributing energy to help sustain heat production by the core organs and surrounding brown adipose tissue.

Changes in sleeping and basal energy expenditure and substrate oxidation induced by short term thyroxin administration in man.

The present study was designed to explore the thermogenic effect of thyroid hormone administration and the resulting changes in nitrogen homeostasis. Normal male volunteers (n = 7) received thyroxin during 6 weeks. The first 3-week period served to suppress endogenous thyroid secretion (180 micrograms T4/day). This dose was doubled for the next 3 weeks. Sleeping energy expenditure (respiratory chamber) and BMR (hood) were measured by indirect calorimetry, under standardized conditions. Sleeping heart rate was continuously recorded and urine was collected during this 12-hour period to assess nitrogen excretion. The changes in energy expenditure, heart rate and nitrogen balance were then related to the excess thyroxin administered. After 3 weeks of treatment, serum TSH level fell to 0.15 mU/L, indicating an almost complete inhibition of the pituitary-thyroid axis. During this phase of treatment there was an increase in sleeping EE and sleeping heart rate, which increased further by doubling the T4 dose (delta EE: +8.5 +/- 2.3%, delta heart rate +16.1 +/- 2.2%). The T4 dose, which is currently used as a substitutive dose, lead to a borderline hyperthyroid state, with an increase in EE and heart rate. Exogenous T4 administration provoked a significant increase in urinary nitrogen excretion averaging 40%. It is concluded that T4 provokes an important stimulation of EE, which is mostly mediated by an excess protein oxidation.

Identification of GPx8 as a novel cellular substrate of the hepatitis C virus NS3-4A protease

Background: The hepatitis C virus (HCV) NS3-4A protease is not only an essential component of the viral replication complex and a prime target for a ntiviral intervention but also a key player i n the persistence and pathogenesis of HCV. It cleaves and thereby inactivates two crucial adaptor proteins in viral RNA sensing and innate immunity (MAVS and TRIF) as well as a phosphatase involved in growth factor signaling (TCPTP). T he aim of this study was to identify novel cellular substrates o f the N S3-4A protease and to investigate their role in the replication and pathogenesis of HCV. Methods: Cell lines inducibly expressing t he NS3-4A protease were analyzed in basal as well as interferon-α-stimulated states by stable isotopic l abeling using amino acids in cell culture (SILAC) coupled with protein separation and mass spectrometry. Candidates fulfilling stringent criteria for potential substrates or products of the NS3-4A protease were further i nvestigated in different experimental systems as well a s in liver biopsies from patients with chronic hepatitis C. Results: SILAC coupled with protein separation and mass spectrometry yielded > 5000 proteins of which 18 candidates were selected for further analyses. These allowed us to identify GPx8, a membrane-associated peroxidase involved in disulfide bond formation in the endoplasmic reticulum, as a n ovel cellular substrate of the H CV NS3-4A protease. Cleavage occurs at cysteine in position 11, removing the cytosolic tip of GPx8, and was observed in different experimental systems as well as in liver biopsies from patients with chronic hepatitis C. Further functional studies, involving overexpression and RNA silencing, revealed that GPx8 is a p roviral factor involved in viral particle production but not in HCV entry or HCV RNA replication. Conclusions: GPx8 is a proviral host factor cleaved by the HCV NS3-4A protease. Studies investigating the consequences of GPx8 cleavage for protein function are underway. The identification of novel cellular substrates o f the HCV N S3-4A protease should yield new insights i nto the HCV life cycle and the pathogenesis of hepatitis C and may reveal novel targets for antiviral intervention.

A fluorescent peptide substrate for the surface metalloprotease of Leishmania.

A fluorescent oligopeptide substrate for the promastigote surface protease (PSP) of Leishmania was designed using the data reported for the substrate specificity of the enzyme (Bouvier, J., Schneider, P., Etges, R. J., and Bordier, C. 1990. Biochemistry 29, 10113-10119). The indole fluorescence of the tryptophan residue was efficiently quenched through resonance energy transfer by an N-terminal dansyl group located five amino acid residues away. The heptapeptide, dansyl-A-Y-L-K-K-W-V-NH2, was cleaved by PSP between the tyrosine and leucine residues with a kcat/Km ratio of 8.8 x 10(6) M-1sec-1. Hydrolysis by the enzyme results in a time-dependent increase of fluorescence intensity of 3.7-fold. Assays can be designed based on the tryptophan fluorescence at 360 nm or by individual product analyses using thin-layer chromatography. The synthetic substrate is readily cleaved by the metalloprotease at the surface of fixed promastigotes. The specificity and sensitivity of such internally quenched fluorescent peptide substrate will facilitate the identification of novel inhibitors for the enzyme and aid in detailed studies on its enzymology.

The glucose transporter 4 FQQI motif is necessary for Akt Substrate of 160-Kilodalton-dependent plasma membrane translocation but not Golgi-localized g-ear-containing Arf-binding protein-dependent entry into the insulin-responsive storage compartment.

Newly synthesized glucose transporter 4 (GLUT4) enters into the insulin-responsive storage compartment in a process that is Golgi-localized γ-ear-containing Arf-binding protein (GGA) dependent, whereas insulin-stimulated translocation is regulated by Akt substrate of 160 kDa (AS160). In the present study, using a variety of GLUT4/GLUT1 chimeras, we have analyzed the specific motifs of GLUT4 that are important for GGA and AS160 regulation of GLUT4 trafficking. Substitution of the amino terminus and the large intracellular loop of GLUT4 into GLUT1 (chimera 1-441) fully recapitulated the basal state retention, insulin-stimulated translocation, and GGA and AS160 sensitivity of wild-type GLUT4 (GLUT4-WT). GLUT4 point mutation (GLUT4-F5A) resulted in loss of GLUT4 intracellular retention in the basal state when coexpressed with both wild-type GGA and AS160. Nevertheless, similar to GLUT4-WT, the insulin-stimulated plasma membrane localization of GLUT4-F5A was significantly inhibited by coexpression of dominant-interfering GGA. In addition, coexpression with a dominant-interfering AS160 (AS160-4P) abolished insulin-stimulated GLUT4-WT but not GLUT4-F5A translocation. GLUT4 endocytosis and intracellular sequestration also required both the amino terminus and large cytoplasmic loop of GLUT4. Furthermore, both the FQQI and the SLL motifs participate in the initial endocytosis from the plasma membrane; however, once internalized, unlike the FQQI motif, the SLL motif is not responsible for intracellular recycling of GLUT4 back to the specialized compartment. Together, we have demonstrated that the FQQI motif within the amino terminus of GLUT4 is essential for GLUT4 endocytosis and AS160-dependent intracellular retention but not for the GGA-dependent sorting of GLUT4 into the insulin-responsive storage compartment.

Phytochrome Kinase Substrate 4 is phosphorylated by the phototropin 1 photoreceptor.

Phototropism allows plants to redirect their growth towards the light to optimize photosynthesis under reduced light conditions. Phototropin 1 (phot1) is the primary low blue light-sensing receptor triggering phototropism in Arabidopsis. Light-induced autophosphorylation of phot1, an AGC-class protein kinase, constitutes an essential step for phototropism. However, apart from the receptor itself, substrates of phot1 kinase activity are less clearly established. Phototropism is also influenced by the cryptochromes and phytochromes photoreceptors that do not provide directional information but influence the process through incompletely characterized mechanisms. Here, we show that Phytochrome Kinase Substrate 4 (PKS4), a known element of phot1 signalling, is a substrate of phot1 kinase activity in vitro that is phosphorylated in a phot1-dependent manner in vivo. PKS4 phosphorylation is transient and regulated by a type 2-protein phosphatase. Moreover, phytochromes repress the accumulation of the light-induced phosphorylated form of PKS4 showing a convergence of photoreceptor activity on this signalling element. Our physiological analyses suggest that PKS4 phosphorylation is not essential for phototropism but is part of a negative feedback mechanism.

Quantitative proteomics identifies the membrane-associated peroxidase GPx8 as a cellular substrate of the hepatitis C virus NS3-4A protease.

The hepatitis C virus (HCV) NS3-4A protease is not only an essential component of the viral replication complex and a prime target for antiviral intervention but also a key player in the persistence and pathogenesis of HCV. It cleaves and thereby inactivates two crucial adaptor proteins in viral RNA sensing and innate immunity, mitochondrial antiviral signaling protein (MAVS) and TRIF, a phosphatase involved in growth factor signaling, T-cell protein tyrosine phosphatase (TC-PTP), and the E3 ubiquitin ligase component UV-damaged DNA-binding protein 1 (DDB1). Here we explored quantitative proteomics to identify novel cellular substrates of the NS3-4A protease. Cell lines inducibly expressing the NS3-4A protease were analyzed by stable isotopic labeling using amino acids in cell culture (SILAC) coupled with protein separation and mass spectrometry. This approach identified the membrane-associated peroxidase GPx8 as a bona fide cellular substrate of the HCV NS3-4A protease. Cleavage by NS3-4A occurs at Cys 11, removing the cytosolic tip of GPx8, and was observed in different experimental systems as well as in liver biopsies from patients with chronic HCV. Overexpression and RNA silencing studies revealed that GPx8 is involved in viral particle production but not in HCV entry or RNA replication. Conclusion: We provide proof-of-concept for the use of quantitative proteomics to identify cellular substrates of a viral protease and describe GPx8 as a novel proviral host factor targeted by the HCV NS3-4A protease. (Hepatology 2014;59:423-433).

Optoelectronic properties of CuPc thin films deposited at different substrate temperatures

Structural and optical characterization of copper phthalocyanine thin film thermally deposited at different substrate temperatures was the aim of this work. The morphology of the films shows strong dependence on temperature, as can be observed by atomic force microscopy and x-ray diffraction spectroscopy, specifically in the grain size and features of the grains. The increase in the crystal phase with substrate temperature is shown by x-ray diffractometry. Optical absorption coefficient measured by photothermal deflection spectroscopy and optical transmittance reveal a weak dependence on the substrate temperature. Besides, the electro-optical response measured by the external quantum efficiency of Schottky ITO/CuPc/Al diodes shows an optimized response for samples deposited at a substrate temperature of 60 °C, in correspondence to the I-V diode characteristics.

Substrate-selective repair and restart of replication forks by DNA translocases

Stalled replication forks are sources of genetic instability. Multiple fork-remodeling enzymes are recruited to stalled forks, but how they work to promote fork restart is poorly understood. By combining ensemble biochemical assays and single-molecule studies with magnetic tweezers, we show that SMARCAL1 branch migration and DNA-annealing activities are directed by the single-stranded DNA-binding protein RPA to selectively regress stalled replication forks caused by blockage to the leading-strand polymerase and to restore normal replication forks with a lagging-strand gap. We unveil the molecular mechanisms by which RPA enforces SMARCAL1 substrate preference. E. coli RecG acts similarly to SMARCAL1 in the presence of E. coli SSB, whereas the highly related human protein ZRANB3 has different substrate preferences. Our findings identify the important substrates of SMARCAL1 in fork repair, suggest that RecG and SMARCAL1 are functional orthologs, and provide a comprehensive model of fork repair by these DNA translocases.

Liquid fructose down-regulates liver insulin receptor substrate 2 and gluconeogeneic enzymes by modifying nutrient sensing factors in rats

High consumption of fructose-sweetened beverages has been linked to a high prevalence of chronic metabolic diseases. We have previously shown that a short course of fructose supplementation as a liquid solution induces glucose intolerance in female rats. In the present work, we characterized the fructose-driven changes in the liver and the molecular pathways involved. To this end, female rats were supplemented or not with liquid fructose (10%, w/v) for 7 or 14 days. Glucose and pyruvate tolerance tests were performed, and the expression of genes related to insulin signaling, gluconeogenesis and nutrient sensing pathways was evaluated. Fructose-supplemented rats showed increased plasma glucose excursions in glucose and pyruvate tolerance tests and reduced hepatic expression of several genes related to insulin signaling, including insulin receptor substrate 2 (IRS-2). However, the expression of key gluconeogenic enzymes, glucose-6-phosphatase and phosphoenolpyruvate carboxykinase, was reduced. These effects were caused by an inactivation of hepatic forkhead box O1 (FoxO1) due to an increase in its acetylation state driven by a reduced expression and activity of sirtuin 1 (SIRT1). Further contributing to FoxO1 inactivation, fructose consumption elevated liver expression of the spliced form of X-box-binding-protein-1 as a consequence of an increase in the activity of the mammalian target of rapamycin 1 and protein 38-mitogen activated protein kinase (p38-MAPK). Liquid fructose affects both insulin signaling (IRS-2 and FoxO1) and nutrient sensing pathways (p38-MAPK, mTOR and SIRT1), thus disrupting hepatic insulin signaling without increasing the expression of key gluconeogenic enzymes.