Basal Flux Research Articles

Numerical modeling of gas hydrate emplacements in oceanic sediments

We have implemented a 2-dimensional numerical model for simulating gas hydrate and free gas accumulation in marine sediments. The starting equations are those of the conservation of the transport of momentum, energy, and mass, as well as those of the thermodynamics of methane hydrate stability and methane solubility in the pore-fluid. These constitutive equations are then integrated into a finite element in space, finite-difference in time scheme. We are then able to examine the formation and distribution of methane hydrate and free gas in a simple geologic framework, with respect to the geothermal heat flow, fluid flow, the methane in-situ production and basal flux. Three simulations are performed, leading to the build up of hydrate emplacements largely linear through time. Models act primarily as free gas accumulators and are relatively inefficient with respect to hydrate emplacements: 26–33% of formed methane are converted to hydrate. Seepage of methane across the sea-floor is negligible for fluid flow below 2. 10 −11 kg/m 2/s. At 5.625 10 −11 kg/m 2/s however, 9.7% of the formed methane seeps out of the model. Moreover, along strike variation arising in the 2-dimensional model are outlined. In the absence of focused flow, the thermodynamics of hydrate accumulation are primarily one-dimensional. However, changes in free methane compressibility (density) and methane solubility (the intrinsic dissolved methane flux) subtlety impact on the formation of a free gas zone and the distribution of the hydrate emplacements in our 2-dimensional simulations.

The basal chromospheric Mg ii h+k flux of evolved stars: probing the energy dissipation of giant chromospheres

Of a total of 177 cool G, K, and M giants and supergiants, we measured the Mg II h+k line emission of extended chromospheres in high-resolution (LWR) IUE spectra by using the IUE final data archive at STScI, and derived the respective stellar surface fluxes. They represent the chromospheric radiative energy losses presumably related to basal heating by the dissipation of acoustic waves, plus a highly variable contribution due to magnetic activity. Thanks to the large sample size, we find a very well defined lower limit, the basal chromospheric Mg II h+k line flux of cool giant chromospheres, as a function of T_eff. A total of 16 giants were observed several times, over a period of up to 20 years. Their respective minimal Mg II h+k line fluxes confirm the basal flux limit very well because none of their emissions dip beneath the empirically deduced basal flux line representative for the overall sample. Based on a total of 15 to 22 objects with very low Mg II h+k emission, we find as limit: log F_Mg_h+k = 7.33 log T_eff - 21.75 (cgs units; based on B-V). Within its uncertainties, this is almost the same relation as has been found in the past for the geometrically much thinner chromospheres of main sequence stars. Concerning any residual dependence of the basal flux on the surface gravity it can be stated that over a gravity range of more than four orders of magnitude (main-sequence stars to supergiants), the basal flux does not appear to vary by more than a factor of 2. These findings are in good agreement with the predictions by previous hydrodynamic models of acoustic wave propagation and energy dissipation, as well as with earlier empirical determinations. Finally, we discuss the idea that the ample energy flux of the chromospheric acoustic waves in a cool giant may yield, as a by-product, the energy flux required by its cool wind (i.e., non-dust-driven, "Reimers-type" mass-loss).

Impact of a Glycogen Phosphorylase Inhibitor and Metformin on Basal and Glucagon-Stimulated Hepatic Glucose Flux in Conscious Dogs

The effects of a glycogen phosphorylase inhibitor (GPI) and metformin (MT) on hepatic glucose fluxes (μmol · kg(-1) · min(-1)) in the presence of basal and 4-fold basal levels of plasma glucagon were investigated in 18-h fasted conscious dogs. Compared with the vehicle treatment, GPI infusion suppressed net hepatic glucose output (NHGO) completely (-3.8 ± 1.3 versus 9.9 ± 2.8) despite increased glucose 6-phosphate (G-6-P) neogenesis from gluconeogenic precursors (8.1 ± 1.1 versus 5.5 ± 1.1). MT infusion did not alter those parameters. In response to a 4-fold rise in plasma glucagon levels, in the vehicle group, plasma glucose levels were increased 2-fold, and NHGO was increased (43.9 ± 5.7 at 10 min and 22.7 ± 3.4 at steady state) without altering G-6-P neogenesis (3.7 ± 1.5 and 5.5 ± 0.5, respectively). In the GPI group, there was no increase in NHGO due to decreased glucose-6-phosphatase flux associated with reduced G-6-P concentration. A lower G-6-P concentration was the result of increased net glycogenesis without altering G-6-P neogenesis. In the MT group, the increment in NHGO (22.2 ± 4.4 at 10 min and 12.1 ± 3.6 at steady state) was approximately half of that of the vehicle group. The lesser NHGO was associated with reduced glucose-6-phosphatase flux but a rise in G-6-P concentration and only a small incorporation of plasma glucose into glycogen. In conclusion, the inhibition of glycogen phosphorylase a activity decreases basal and glucagon-induced NHGO via redirecting glucose 6-phosphate flux from glucose toward glycogen, and MT decreases glucagon-induced NHGO by inhibiting glucose-6-phosphatase flux and thereby reducing glycogen breakdown.

Response of mantle heat flux to plate evolution

By analyzing convection characteristics and surface and basal heat flux we investigate the influence on mantle convection of finite thickness plates with dynamically determined velocities and mobile boundaries. Our 6 × 6 × 1 Cartesian geometry numerical model features periodic sidewall boundary conditions, a lower mantle that increases in viscosity and a Rayleigh number of 5 × 106 (based on the uniform viscosity upper mantle). A calculation with 9 plates and fixed plate boundaries is compared with a second calculation incorporating evolving plate boundaries. The number of plates in the latter case fluctuates between 6 and 9. We find that mean heat flux output decreases during periods characterized by the presence of larger plates and that evolving plate boundaries cause surface (and basal) heat flux output to vary much more than the heat flux observed with either fixed plate boundaries or a free‐slip surface.

The Saccharomyces cerevisiae zinc factor protein Stb5p is required as a basal regulator of the pentose phosphate pathway

In Saccharomyces cerevisiae, the oxidative stress-activated zinc cluster protein Stb5p activates genes involved in NADPH production and most genes of the pentose phosphate (PP) pathway. To gain insight into the role of Stb5p, we studied the behaviour of stb5 deletion mutants during aerobic and anaerobic growth on glucose. stb5 mutants were auxotrophic for methionine and pyrimidine nucleotides. The methionine auxotrophy phenotype was air dependent, suggesting an impaired aerobic NADPH status. Consistent with this, the acetate level was reduced and the α-ketoglutarate level was increased in the stb5 mutant. stb5 cells also required pyrimidine nucleotides for aerobic and anaerobic growth, consistent with a reduction in 5-phosphoribosyl-1-pyrophosphate production caused by a reduced flux through the PP pathway. Strains overexpressing STB5 could not grow on glucose. This growth defect was restored by overproduction of an NADPH-butanediol dehydrogenase, which reoxidizes the excess NADPH in the oxidative PP pathway. These findings suggest a major role for the transcription factor Stb5p in maintaining a basal flux through the PP pathway to meet the NADPH requirements for aerobic growth, and to provide the nucleotide precursors. Our data also demonstrate the potential use of a system based on overproduction of this transcription factor to increase flux through the PP pathway.

Physiological and pharmacological characterizations of the larval Anopheles albimanus rectum support a change in protein distribution and/or function in varying salinities

Ion regulation is a biological process crucial to the survival of mosquito larvae and a major organ responsible for this regulation is the rectum. The recta of anopheline larvae are distinct from other subfamilies of mosquitoes in several ways, yet have not yet been characterized extensively. Here we characterize the two major cell types of the anopheline rectum, DAR and non-DAR cells, using histological, physiological, and pharmacological analyses. Proton flux was measured at the basal membrane of 2%- and 50%-artificial sea water-reared An. albimanus larvae using self-referencing ion-selective microelectrodes, and the two cell types were found to differ in basal membrane proton flux. Additionally, differences in the response of that flux to pharmacological inhibitors in larvae reared in 2% versus 50% ASW indicate changes in protein function between the two rearing conditions. Finally, histological analyses suggest that the non-DAR cells are structurally suited for mediating ion transport. These data support a model of rectal ion regulation in which the non-DAR cells have a resorptive function in freshwater-reared larvae and a secretive function in saline water-reared larvae. In this way, anopheline larvae may adapt to varying salinities.

Role of Inositol Trisphosphate Receptors in Autophagy in DT40 Cells

Previous studies have shown that small interfering RNA knockdown and pharmacological inhibition of inositol 1,4,5-trisphosphate receptors (IP(3)Rs) stimulate autophagy. We have investigated autophagy in chicken DT40 cell lines containing targeted deletions of all three IP(3)R isoforms (triple knock-out (TKO) cells). Using gel shifts of microtubule-associated protein 1 light chain 3 as a marker of autophagy, we find that TKO cells have enhanced basal autophagic flux even under nutrient-replete conditions. Stable DT40 cell lines derived from TKO cells containing the functionally inactive D2550A IP(3)R mutant did not suppress autophagy in the same manner as wild-type receptors. This suggests that the channel function of the receptor is important in its regulatory role in autophagy. There were no marked differences in the phosphorylation state of AMP-activated protein kinase, Akt, or mammalian target of rapamycin between wild-type and TKO cells. The amount of immunoprecipitated complexes of Bcl-2-Beclin-1 and Beclin-1-Vps34 were also not different between the two cell lines. The major difference noted was a substantially decreased mTORC1 kinase activity in TKO cells based on decreased phosphorylation of S6 kinase and 4E-BP1. The discharge of intracellular stores with thapsigargin stimulated mTORC1 activity (measured as S6 kinase phosphorylation) to a greater extent in wild-type than in TKO cells. We suggest that basal autophagic flux may be negatively regulated by IP(3)R-dependent Ca(2+) signals acting to maintain an elevated mTORC1 activity in wild-type cells and that Ca(2+) regulation of this enzyme is defective in TKO cells. The protective effect of a higher autophagic flux in cells lacking IP(3)Rs may play a role in the delayed apoptotic response observed in these cells.

Digital laser doppler flowmetry may discriminate “limited” from “diffuse” systemic sclerosis

Objectives To investigate skin blood flux and microvascular functional changes by laser Doppler flowmetry (LD) in patients with systemic sclerosis (SSc) at baseline and following dynamic stimulations. Methods Skin blood flux of the dorsal hands was recorded by LD at baseline and after the cold test and the post-occlusive hyperemia test in 59 SSc patients (49 limited cutaneous, 10 diffuse cutaneous). Twenty-five patients with primary Raynaud's phenomenon (PRP), and 31 healthy donors (HD) were studied as controls. Results After the cold test, SSc patients had a significantly higher reduction of the blood flux (− 38.4% ± 28) than PRP (− 21.1% ± 37) and HD (− 22.1% ± 23) subjects ( p < 0.05). Within the SSc group, the cold test flux was significantly reduced in limited-SSc (− 399% ± 28, p < 0.05), but not in diffuse-SSc (− 31.2% ± 29), whereas, the time needed to recover the basal flux after the occlusive/ischemic test was significantly longer in diffuse-SSc (18.8 s ± 21)than in limited-SSc (4.5 s ± 4, p < 0.01) or HD (2.2 s ± 2, p < 0.01) or PRP (0.4 s ± 0.7, p < 0.01). Conclusions These data clearly indicate an impairment of vascular tone regulatory mechanisms in SSc and suggest that a peculiar pathogenic mechanism may be involved in different SSc subset. Nevertheless, it has clear that PRP and SSc-associated RP have a distinct pattern at LD evaluation, and monitoring patients with PRP could be helpful to understand whether a change in the LD pattern might predict the development of SSc.

Distribution of thermal conductivities in the Groß Schönebeck (Germany) test site based on 3D inversion of deep borehole data

The main objective of the study was to develop and calibrate the thermal model of the Groß Schönebeck test site in Northeastern Germany, and to perform sensitivity analyses based on the availability of high-quality temperature data from deep wells. A 3D geological model of the area was constructed and then discretized into a finite element mesh that included 13 different lithologic units of variable thickness. Forward numerical calculations of the temperature field were performed based on measured and assumed data and properties. Finally, inverse modeling was used to improve parameter estimation and to obtain comprehensive statistic information. The results of the computations indicate some spatial thermal conductivity inhomogeneities in the different geologic layers and imply that the basal heat flux in the area is about 60 mW m −2.

Modeling ice sheets from the bottom up

Three facts should guide ice-sheet modeling. (1) Ice height above the bed is controlled by the strength of ice-bed coupling, reducing ice thickness by some 90 percent when coupling vanishes. (2) Ice-bed coupling vanishes along ice streams that end as floating ice shelves and drain up to 90 percent of an ice sheet. (3) Because of (1) and (2), ice sheets can rapidly collapse and disintegrate, thereby removing ice sheets from Earth's climate system and forcing abrupt climate change. The first model of ice-sheet dynamics was developed in Australia and applied to the present Antarctic Ice Sheet in 1970. It treated slow sheet flow, which prevails over some 90 percent of the ice sheet, but is the least dynamic component. The model made top-down calculations of ice velocities and temperatures, based on known surface conditions and an assumed basal geothermal heat flux. In 1972, Joseph Fletcher proposed a six-step research strategy for studying dynamic systems. The first step was identifying the most dynamic components, which for Antarctica are fast ice streams that discharge up to 90 percent of the ice. Ice-sheet models developed at the University of Maine in the 1970s were based on the Fletcher strategy and focused on ice streams, including calving dynamics when ice streams end in water. These models calculated the elevation of ice sheets based in the strength of ice-bed coupling. This was a bottom-up approach that lowered ice elevations some 90 percent when ice-bed coupling vanished. Top-down modeling is able to simulate changes in the size and shape of ice sheets through a whole glaciation cycle, provided the mass balance is treated correctly. Bottom-up modeling is able to produce accurate changes in ice elevations based on changes in ice-bed coupling, provided the force balance is treated correctly. Truly holistic ice-sheet models should synthesize top-down and bottom-up approaches by combining the mass balance with the force balance in ways that merge abrupt changes in stream flow with slow changes in sheet flow. Then discharging 90 percent of the ice by ice streams mobilizes 90 percent of the area so ice sheets can self-destruct, and thereby terminate a glaciation cycle.

Transepidermal water loss for probing full-thickness skin barrier function: Correlation with tritiated water flux, sensitivity to punctures and diverse surfactant exposures

Skin barrier function is a key parameter to consider when performing in vitro percutaneous absorption studies. Whilst tritiated water flux measurements were often used to assess skin integrity, recent decades have witnessed the emergence of the more rapid and user-friendly transepidermal water loss (TEWL) approach. Yet to date, the nature of the correlation between TEWL and skin barrier function in vitro has still not been comprehensively established. In this study, a novel TEWL device, operating on a cold-induced vapour sink principle, was used to probe the barrier function of full-thickness porcine skin. The method was sufficiently sensitive to show the influence of punctures on barrier function although the observed non-linear pattern suggested tissue swelling processes and/or capillary action could be occurring. The results of various surfactant application experiments strongly suggested that TEWL was indeed largely predictive of skin sample integrity. A key finding was that basal TEWL was linearly correlated with basal tritiated water flux ( r 2 = 0.80, n = 63). Thus, a dedicated TEWL method can be used as a good alternative to water flux measurements for assessing full-thickness skin barrier function.

Metabolic flux analysis of a phenol producing mutant of Pseudomonas putida S12: Verification and complementation of hypotheses derived from transcriptomics

The physiological effects of genetic and transcriptional changes observed in a phenol producing mutant of the solvent-tolerant Pseudomonas putida S12 were assessed with metabolic flux analysis. The upregulation of a malate/lactate dehydrogenase encoding gene could be connected to a flux increase from malate to oxaloacetate. A mutation in the pykA gene decreased in vitro pyruvate kinase activity, which is consistent with a lower flux from phosphoenolpyruvate to pyruvate. Changes in the oprB-1, gntP and gnuK genes, encoding a glucose-selective porin, gluconokinase and a gluconate transporter respectively, altered the substrate uptake profile. Metabolic flux analysis furthermore revealed cellular events not predicted by the transcriptome analysis. Gluconeogenic formation of glucose-6-phosphate from triose-3-phosphate was abolished, in favour of increased phosphoenolpyruvate production. An increased pentose phosphate pathway flux resulted in higher erythrose-4-phosphate production. Thus, the availability of these two central phenol precursors was improved. Furthermore, metabolic fluxes were redistributed such that the overall TCA cycle flux was unaffected and energy production increased. Engineering P. putida S12 for phenol production has yielded a strain that channels carbon fluxes to previously unfavourable routes to reconcile the drain on metabolites required for phenol production, while maintaining basal flux levels through central carbon metabolism.

Effects of spatially varying roof cooling on thermal convection at high Rayleigh number in a fluid with a strongly temperature-dependent viscosity

We investigate the effects of an insulating lid of variable spatial extent on convection in the stagnant-lid regime under thermally steady-state conditions. Using a combination of laboratory experiments, numerical simulations and scaling analyses we characterize the qualitative structure and quantitative heat transfer properties of flows in terms of the fractional extent L of an insulating lid applied at the cold boundary, the thermal resistance of the lid, the magnitude of the temperature dependence of the fluid viscosity Λ and the effective Rayleigh number Rae for the composite system. A partial insulating lid has two main effects: (i) To increase the mean interior temperature and reduce the average viscosity of the system, which enhances fluid motions, and (ii) to impart a lateral asymmetry to the thermal structure of the cold boundary that leads, in turn, to lateral temperature gradients that drive an overturning flow. Consequently, whereas flow in the uninsulated stagnant-lid regime is in the form of ‘small-scale’ rising and sinking thermals, there is an additional ‘large-scale’ circulation in the presence of partial insulation. The structure, wavelength and heat transfer properties of this large-scale stirring depends on L, Λ and Rae. For given Rae – Λ conditions we find optimal values of L at which there occur well-defined maxima in the rate of overturn, the local heat flux carried into the uninsulated part of the cold boundary and in the global average heat flux Nu carried across the system. Whereas both the rate of overturning and local heat flux are associated with the largest lateral temperature gradients, the optimal basal heat flux depends also on a tradeoff with the fractional surface area of the lid. Remarkably, maximal values of the global heat flux can significantly exceed that of the uninsulated stagnant-lid case. The occurrence of such maxima is insensitive to the mechanical boundary conditions applied and is not strongly influenced by lid shape. However, the magnitude and location of optimal heat fluxes depends in a complicated way on the lid surface area and shape, as well as the structure of the hot and cold boundary layers and the wavelength of the large-scale flow.

Novel bUT-B2 urea transporter isoform is constitutively activated

Our previous studies have detailed a novel facilitative UT-B urea transporter isoform, bUT-B2. Despite the existence of mouse and human orthologs, the functional characteristics of UT-B2 remain undefined. In this report, we produced a stable MDCK cell line that expressed bUT-B2 protein and investigated the transepithelial urea flux across cultured cell monolayers. We observed a large basal urea flux that was significantly reduced by known inhibitors of facilitative urea transporters; 1,3 dimethylurea (P < 0.001, n = 17), thionicotinamide (P < 0.05, n = 11), and phloretin (P < 0.05, n = 9). Pre-exposure for 1 h to the antidiuretic hormone vasopressin had no effect on bUT-B2-mediated urea transport (NS, n = 3). Acute vasopressin exposure for up to 30 min also failed to elicit any transient response (NS, n = 9). Further investigation confirmed that bUT-B2 function was not affected by alteration of intracellular cAMP (NS, n = 4), intracellular calcium (NS, n = 3), or protein kinase activity (NS, n = 4). Finally, immunoblot data suggested a possible role for glycosylation in regulating bUT-B2 function. In conclusion, this study showed that bUT-B2-mediated transepithelial urea transport was constitutively activated and unaffected by known regulators of renal UT-A urea transporters.

ARPE-19 retinal pigment epitheliaL CELLS are highly resistant to oxidative stress and exercise strict control over their lysosomal redox-active iron

Normal retinal pigment epithelial (RPE) cells are postmitotic, long-lived and basically not replaced. Daily, they phagocytose substantial amounts of lipid-rich material (photoreceptor outer segment discs), and they do so in the most oxygenated part of the body – the retina. One would imagine that this state of affairs should be associated with a rapid formation of the age pigment lipofuscin (LF). However, LF accumulation is slow and reaches significant amounts only late in life when, if substantial, it often coincides with or causes age-related macular degeneration. LF formation occurs inside the lysosomal compartment as a result of iron-catalyzed peroxidation and polymerization. This process requires phagocytosed or autophagocytosed material under degradation, but also the presence of redox-active low mass iron and hydrogen peroxide. To gain some information on how RPE cells are able to evade LF formation, we investigated the response of immortalized human RPE cells (ARPE-19) to oxidative stress with/without the protection of a strong iron-chelator. The cells were found to be extremely resistant to hydrogen peroxide-induced lysosomal rupture and ensuing cell death. This marked resistance to oxidative stress was not explained by enhanced degradation of hydrogen peroxide, but to a certain extent further increased by the potent lipophilic iron chelator SIH. The cells were also able to survive, and even replicate, at high concentrations of SIH and showed a high degree of basal autophagic flux. We hypothesize that RPE cells have a highly developed capacity to keep lysosomal iron in a non-redox-active form, perhaps by pronounced autophagy of iron-binding proteins in combination with an ability to rapidly relocate low mass iron from the lysosomal compartment.

Preserved endothelial-dependent and endothelial-independent skin vasodilator responses in relatives of type 1 diabetes patients

Since increased plasma and cell levels of oxidative products have been found in non diabetic relatives of type 1 diabetic patients, we hypothesized the occurrence of an endothelial dysfunction in these subjects. To verify this hypothesis we investigated the skin blood flow responses to iontophoresis of both the endothelial-dependent vasodilator acetylcholine (ACh) and the endothelial-independent vasodilator sodium nitroprusside (SNP) in 31 non diabetic healthy relatives (DR) (14 siblings, 17 parents) of 17 type 1 diabetic patients. Twenty healthy control subjects (CS) without a family history of diabetes, matched for age (± 5 years) and gender, were also investigated. DR and CS did not significantly differ either in basal skin blood flux (6.75 ± 0.72 PU and 5.78 ± 0.37 PU, respectively) or in skin vasodilator response to both ACh (728 ± 53% and 711 ± 44%, respectively) and SNP iontophoresis (758 ± 71% and 731 ± 64%, respectively). This finding is consistent with a preserved skin microvascular endothelial function in the studied subjects. However, since previous data suggest that both nitric oxide (NO) and prostacyclin released form the cutaneous vascular endothelium have an interchanging compensatory role in controlling the skin vasodilator response to ACh iontophoresis, our finding does not allow a defect in NO dependent skin vasodilatation to be excluded in the studied relatives of diabetic patients.

Interferon-γ Released by Gluten-Stimulated Celiac Disease-Specific Intestinal T Cells Enhances the Transepithelial Flux of Gluten Peptides

Celiac sprue is a T-cell-mediated enteropathy elicited in genetically susceptible individuals by dietary gluten proteins. To initiate and propagate inflammation, proteolytically resistant gluten peptides must be translocated across the small intestinal epithelium and presented to DQ2-restricted T cells, but the effectors enabling this translocation under normal and inflammatory conditions are not well understood. We demonstrate that a fluorescently labeled antigenic 33-mer gluten peptide is translocated intact across a T84 cultured epithelial cell monolayer and that preincubation of the monolayer with media from gluten-stimulated, celiac patient-derived intestinal T cells enhances the apical-to-basolateral flux of this peptide in a dose-dependent, saturable manner. The permeability-enhancing activity of activated T-cell media is inhibited by blocking antibodies against either interferon-gamma or its receptor and is recapitulated using recombinant interferon-gamma. At saturating levels of interferon-gamma, activated T-cell media does not further increase transepithelial peptide flux, indicating the primacy of interferon-gamma as an effector of increased epithelial permeability during inflammation. Reducing the assay temperature to 4 degrees C reverses the effect of interferon-gamma but does not reduce basal peptide flux occurring in the absence of interferon-gamma, suggesting active transcellular transport of intact peptides is increased during inflammation. A panel of disease-relevant gluten peptides exhibited an inverse correlation between size and transepithelial flux but no apparent sequence constraints. Anti-interferon-gamma therapy may mitigate the vicious cycle of gluten-induced interferon-gamma secretion and interferon-gamma-mediated enhancement of gluten peptide flux but is unlikely to prevent translocation of gluten peptides in the absence of inflammatory conditions.

The basal flux of Akt in the mitochondria is mediated by heat shock protein 90

Akt is a known client protein of heat shock protein 90 (HSP90). We have found that HSP90 is responsible for Akt accumulation in the mitochondria in unstimulated cells. Treatment of SH-SY5Y neuroblastoma cells and human embryonic kidney cells with the HSP90 inhibitors novobiocin and geldanamycin caused substantial decreases in the level of Akt in the mitochondria without affecting the level of Akt in the cytosol. Moreover, intracerebroventricular injection of novobiocin into mice brains decreased Akt levels in cortical mitochondria. Knockdown of HSP90 expression with short interfering RNA also caused a significant decrease in Akt levels in the mitochondria without affecting total Akt levels. Using a mitochondrial import assay it was found that Akt is transported into the mitochondria. Furthermore, it was found that the mitochondrial import of Akt was independent of Akt activation as both an unmodified Akt and constitutively active mutant Akt; both readily accumulated in the mitochondria in an HSP90-dependent manner. Interestingly, incubation of isolated mitochondria with constitutively active Akt caused visible alterations in mitochondrial morphology, including pronounced remodeling of the mitochondrial matrix. This effect was blocked when Akt was mostly excluded from the mitochondria with novobiocin treatment. These results indicate that the level of Akt in the mitochondria is dependent on HSP90 chaperoning activity and that Akt import can cause dynamic changes in mitochondrial configuration.

Reduced Basal ATP Synthetic Flux of Skeletal Muscle in Patients with Previous Acromegaly

BackgroundImpaired mitochondrial function and ectopic lipid deposition in skeletal muscle and liver have been linked to decreased insulin sensitivity. As growth hormone (GH) excess can reduce insulin sensitivity, we examined the impact of previous acromegaly (AM) on glucose metabolism, lipid storage and muscular ATP turnover.Participants and MethodsSeven AM (4f/3 m, age: 46±4 years, BMI: 28±1 kg/m2) and healthy volunteers (CON: 3f/4 m, 43±4 years, 26±2 kg/m2) matched for age and body mass underwent oral glucose testing for assessment of insulin sensitivity (OGIS) and ß-cell function (adaptation index, ADAP). Whole body oxidative capacity was measured with indirect calorimetry and spiroergometry. Unidirectional ATP synthetic flux (fATP) was assessed from 31P magnetic resonance spectroscopy (MRS) of calf muscle. Lipid contents of tibialis anterior (IMCLt) and soleus muscles (IMCLs) and liver (HCL) were measured with 1H MRS.ResultsDespite comparable GH, insulin-like growth factor-1 (IGF-I) and insulin sensitivity, AM had ∼85% lower ADAP (p<0.01) and ∼21% reduced VO2max (p<0.05). fATP was similarly ∼25% lower in AM (p<0.05) and related positively to ADAP (r = 0.744, p<0.01), but negatively to BMI (r = −0.582, p<0.05). AM had ∼3fold higher HCL (p<0.05) while IMCLt and IMCLs did not differ between the groups.ConclusionsHumans with a history of acromegaly exhibit reduced insulin secretion, muscular ATP synthesis and oxidative capacity but elevated liver fat content. This suggests that alterations in ß-cell function and myocellular ATP production may persist despite normalization of GH secretion after successful treatment of acromegaly.

TNF‐α‐ and TRAIL‐resistant glioma cells undergo autophagy‐dependent cell death induced by activated microglia

The role of microglia, the brain resident macrophages, in glioma biology is still ill-defined. Despite their cytotoxic potential, these cells that significantly infiltrate the tumor mass seem to support tumor growth rather than tumor eradication. A proper activation of microglia anti-tumor activities within the tumor may provide a valuable additional arm of defense to immunotherapies against brain tumors. We herewith report a detailed characterization of (lipopolysaccharide and interferon-gamma)-induced anti-tumor activities of mouse primary microglia towards two TNF-alpha and TRAIL resistant glioma cell lines, in cell monolayer or spheroid cultures and in collagen-embedded tumor explants. Irrespective of the mouse strain, stimulated microglia secreted proteic factors that decreased proliferation and migration of these glioma cells and efficiently killed them. Death occurred specifically in glioma cells as demonstrated by the lack of toxicity of microglia supernatant towards primary cultures of astrocytes or neurons. Cell death was characterized by the early accumulation of acidic vesicles, phosphatidylserine exposure, appearance of double-membrane cytoplasmic vesicles, extensive zeiosis and a very late loss of DNA in cells that had lost membrane integrity. Inhibition of autophagosome formation efficiently protected glioma cells from death whereas caspase inhibition could only prevent DNA loss but not cytotoxicity. Death however, resulted from a blockade by microglia supernatant of the basal autophagic flux present in the glioma cells. These observations demonstrate that glioma cells resistant to apoptotic death ligands could be successfully and specifically killed through autophagy-dependent death induced by appropriately activated microglia.