Secretion of an endogenous subtilisin by Pichia pastoris strains GS115 and KM71.

The methylotrophic yeast Pichia pastoris is widely used for the expression of heterologous enzymes. While the purity of the desired expression product is of major importance for many applications, we found that recombinant enzymes produced in methanol medium were contaminated by a 37-kDa endogenous yeast protease. This enzyme was completely inhibited by phenylmethanesulfonyl fluoride (PMSF) but not by 1,10-phenanthroline, EDTA, and pepstatin A, suggesting the nature of a serine protease. Its secretion was abolished in P. pastoris strains GS115 and KM71 by specific mutagenesis of a subtilisin gene (SUB2) but not by inactivation of the gene encoding vacuolar proteinase B (PRB). Bioinformatic comparisons of Sub2 protein with subtilisins from other fungal genomes and phylogenetic analyses indicated that this enzyme is not an orthologue of the vacuolar protease cerevisin generally present in yeasts but is more closely related to another putative subtilisin found in a small number of yeast genomes. During growth of P. pastoris, Sub2 was produced as a secreted enzyme at a concentration of 10 microg/ml of culture supernatant after overexpression of the full-length SUB2 gene. During fermentative production of recombinant enzymes in methanol medium, 1 ml of P. pastoris culture supernatant was found to contain approximately 3 ng of Sub2, while the enzyme was not detected during growth in a medium containing glycerol as a carbon source. The mutant strain GS115-sub2 was subsequently used as a host for the production of recombinant proteases without endogenous subtilisin contamination.

Previous microvascular decompression decreases the chances of pain free in patients treated with Gamma Knife radiosurgery for TIC

Object: The authors sought to establish whether the safety-efficacy of Gamma Knife radiosurgery (GKRS) as a second treatment for intractable trigeminal neuralgia (ITN) are influenced by prior microvascular decompression (MVD) which remains, for some of the authors, the reference technique. Methods: Between July 1992 and November 2010, 737 patients have been operated with GKRS for ITN and prospectively evaluated in Timone University Hospital in Marseille, France. Among these, 54 patients had a previous MVD history. Radiosurgery using a Gamma Knife (model B or C or Perfexion) was performed relying on both MR and CT targeting. A single 4 mm isocenter was positioned in the cisternal portion of the trigeminal nerve at a median distance of 7.6 mm (range 3.9- 11.9) anteriorly to the emergence of the nerve (retrogasserian target). A median maximum dose of 85 Gy (range 70-90) was delivered. Are further analyzed only 45 patients with previous MVD and a follow-up longer than one year (the patients with megadolichobasilar artery compression and multiple sclerosis were excluded). Results: The median age in this series was 56.75 years (range 28.09-82.39). The median follow-up period was 39.48 months (range 14.10-144.65). All the patients had a past history of surgery, with at least one previous failed MVD, but also a radiofrequency lesion (RFL) in 16 (35.6%) patients, balloon microcompression in 7 (15.6%) patients and glycerol rhizotomy in 1 case (2.2%). Thirty-five patients (77.8%) were initially pain free in a median time of 14 days (range 0, 180). Patients from this group had less probability of being pain free compared to our global population of essential trigeminal neuralgia without previous MVD history (p=0.010, hazard ratio of 0.64). Their probability of remaining pain free at 3, 5, 7 and 10 years was 66.5%, 59.1%, 59.1% and 44.3%, respectively. Twelve patients (34.3%) initially pain free experienced a recurrence with a median delay of 31.21 months (range 3.40-89.93). The hypoesthesia actuarial rate at 1 year was 9.1% and remained stable till 12 years with a median delay of onset of 8 months (range 8-8). Conclusions: Retrogasserian GKRS proofed to be safe and effective on the long-term basis even after failed previous MVD. Even if the initial result of pain free was of only 77.8%, the toxicity was low with only 9.1% hypoesthesia. No patient reported a bothersome hypoesthesia. The probability of maintaining pain relief in long-term was of 44.3% at 10 years.

Activation of a HIF1alpha-PPARgamma axis underlies the integration of glycolytic and lipid anabolic pathways in pathologic cardiac hypertrophy.

Development of cardiac hypertrophy and progression to heart failure entails profound changes in myocardial metabolism, characterized by a switch from fatty acid utilization to glycolysis and lipid accumulation. We report that hypoxia-inducible factor (HIF)1alpha and PPARgamma, key mediators of glycolysis and lipid anabolism, respectively, are jointly upregulated in hypertrophic cardiomyopathy and cooperate to mediate key changes in cardiac metabolism. In response to pathologic stress, HIF1alpha activates glycolytic genes and PPARgamma, whose product, in turn, activates fatty acid uptake and glycerolipid biosynthesis genes. These changes result in increased glycolytic flux and glucose-to-lipid conversion via the glycerol-3-phosphate pathway, apoptosis, and contractile dysfunction. Ventricular deletion of Hif1alpha in mice prevents hypertrophy-induced PPARgamma activation, the consequent metabolic reprogramming, and contractile dysfunction. We propose a model in which activation of the HIF1alpha-PPARgamma axis by pathologic stress underlies key changes in cell metabolism that are characteristic of and contribute to common forms of heart disease.

Enhanced EGFP Fluorescence Emission in Presence of PEG Aqueous Solutions and PIB1000-PEG6000-PIB1000 Copolymer Vesicles.

An EGFP construct interacting with the PIB1000-PEG6000-PIB1000 vesicles surface reported a ~2-fold fluorescence emission enhancement. Because of the constructs nature with the amphiphilic peptide inserted into the PIB core, EGFP is expected to experience a "pure" PEG environment. To unravel this phenomenon PEG/water solutions at different molecular weights and concentrations were used. Already at ~1 : 10 protein/PEG molar ratio the increase in fluorescence emission is observed reaching a plateau correlating with the PEG molecular weight. Parallel experiments in presence of glycerol aqueous solutions did show a slight fluorescence enhancement however starting at much higher concentrations. Molecular dynamics simulations of EGFP in neat water, glycerol, and PEG aqueous solutions were performed showing that PEG molecules tend to "wrap" the protein creating a microenvironment where the local PEG concentration is higher compared to its bulk concentration. Because the fluorescent emission can be perturbed by the refractive index surrounding the protein, the clustering of PEG molecules induces an enhanced fluorescence emission already at extremely low concentrations. These findings can be important when related to the use of EGFP as reported in molecular biology experiments.

Protein turnover, ureagenesis and gluconeogenesis.

The major processes discussed below are protein turnover (degradation and synthesis), degradation into urea, or conversion into glucose (gluconeogenesis, Figure 1). Daily protein turnover is a dynamic process characterized by a double flux of amino acids: the amino acids released by endogenous (body) protein breakdown can be reutilized and reconverted to protein synthesis, with very little loss. Daily rates of protein turnover in humans (300 to 400 g per day) are largely in excess of the level of protein intake (50 to 80 g per day). A fast growing rate, as in premature babies or in children recovering from malnutrition, leads to a high protein turnover rate and a high protein and energy requirement. Protein metabolism (synthesis and breakdown) is an energy-requiring process, dependent upon endogenous ATP supply. The contribution made by whole-body protein turnover to the resting metabolic rate is important: it represents about 20 % in adults and more in growing children. Metabolism of proteins cannot be disconnected from that of energy since energy balance influences net protein utilization, and since protein intake has an important effect on postprandial thermogenesis - more important than that of fats or carbohydrates. The metabolic need for amino acids is essentially to maintain stores of endogenous tissue proteins within an appropriate range, allowing protein homeostasis to be maintained. Thanks to a dynamic, free amino acid pool, this demand for amino acids can be continuously supplied. The size of the free amino acid pool remains limited and is regulated within narrow limits. The supply of amino acids to cover physiological needs can be derived from 3 sources: 1. Exogenous proteins that release amino acids after digestion and absorption 2. Tissue protein breakdown during protein turnover 3. De novo synthesis, including amino acids (as well as ammonia) derived from the process of urea salvage, following hydrolysis and microflora metabolism in the hind gut. When protein intake surpasses the physiological needs of amino acids, the excess amino acids are disposed of by three major processes: 1. Increased oxidation, with terminal end products such as CO₂ and ammonia 2. Enhanced ureagenesis i. e. synthesis of urea linked to protein oxidation eliminates the nitrogen radical 3. Gluconeogenesis, i. e. de novo synthesis of glucose. Most of the amino groups of the excess amino acids are converted into urea through the urea cycle, whereas their carbon skeletons are transformed into other intermediates, mostly glucose. This is one of the mechanisms, essential for life, developed by the body to maintain blood glucose within a narrow range, (i. e. glucose homeostasis). It includes the process of gluconeogenesis, i. e. de novo synthesis of glucose from non-glycogenic precursors; in particular certain specific amino acids (for example, alanine), as well as glycerol (derived from fat breakdown) and lactate (derived from muscles). The gluconeogenetic pathway progressively takes over when the supply of glucose from exogenous or endogenous sources (glycogenolysis) becomes insufficient. This process becomes vital during periods of metabolic stress, such as starvation.

Metabolic role of aquaglyceroporin 9 in astrocytes

Aim: Aquaglyceroporin-9 (AQP9) is a member of the Aquaporin channel family involved in water flux through plasma membranes and exhibits the distinctive feature of also being permeable to glycerol and monocarboxylates. AQP9 is detected in astrocytes and catecholaminergic neurons.1 However, the presence of AQP9 in the brain is now debated after a recent publication claiming that AQP9 is not expressed in the brain.2 Based on our results,3 we have evidence of the presence of AQP9 in the brain and we further hypothesize that AQP9 plays a functional role in brain energy metabolism. Methods: The presence of AQP9 in brain of OF1 mice was studied by RT-PCR and immunohistochemistry. To address the role of AQP9 in brain, we used commercial siRNA against AQP9 to knockdown its expression in 2 cultures of astrocytes from two distinct sources (from differentiated stem cells4 and primary astrocyte cultures). After assessment of the decrease of AQP9, glycerol uptake was measured using [H3]-glycerol. Then, modifications of the astrocytic energy metabolism was evaluated by measurement of glucose consumption, lactate release5 and evaluation of the mitochondrial activity by MTT staining. Results: AQP9 is expressed in astrocytes of OF1 mouse brain (mRNA and protein levels). We also showed that AQP9 mRNA and protein are present in cultured astrocytes. Four days after AQP9 siRNA application, the level of expression is significantly decreased by 76% compared to control. Astrocytes with AQP9 knockdown exhibit a 23% decrease of glycerol uptake, showing that AQP9 is a glycerol channel in cultured astrocytes. In parallel, astrocytes with AQP9 knockdown have a 155% increase of their glucose consumption without modifications of lactate release. Moreover, considering the observed glucose consumption increase and the absence of proliferation induction, the significant MTT activity increase (113%) suggests an increase of oxidative metabolism in astrocytes with AQP9 knockdown. Discussion: The involvement of AQP9 in astrocyte energy metabolism adds a new function for this channel in the brain. The determination of the role of AQP9 in astrocytes provides a new perspective on the controversial expression of AQP9 in brain. We also suggest that AQP9 may have a complementary role to monocarboxylate transporters in the regulation of brain energy metabolism.

The fasting-induced adipose factor/angiopoietin-like protein 4 is physically associated with lipoproteins and governs plasma lipid levels and adiposity.

Proteins secreted from adipose tissue are increasingly recognized to play an important role in the regulation of glucose metabolism. However, much less is known about their effect on lipid metabolism. The fasting-induced adipose factor (FIAF/angiopoietin-like protein 4/peroxisome proliferator-activated receptor gamma angiopoietin-related protein) was previously identified as a target of hypolipidemic fibrate drugs and insulin-sensitizing thiazolidinediones. Using transgenic mice that mildly overexpress FIAF in peripheral tissues we show that FIAF is an extremely powerful regulator of lipid metabolism and adiposity. FIAF overexpression caused a 50% reduction in adipose tissue weight, partly by stimulating fatty acid oxidation and uncoupling in fat. In addition, FIAF overexpression increased plasma levels of triglycerides, free fatty acids, glycerol, total cholesterol, and high density lipoprotein (HDL)-cholesterol. Functional tests indicated that FIAF overexpression severely impaired plasma triglyceride clearance but had no effect on very low density lipoprotein production. The effects of FIAF overexpression were amplified by a high fat diet, resulting in markedly elevated plasma and liver triglycerides, plasma free fatty acids, and plasma glycerol levels, and impaired glucose tolerance in FIAF transgenic mice fed a high fat diet. Remarkably, in mice the full-length form of FIAF was physically associated with HDL, whereas truncated FIAF was associated with low density lipoprotein. In human both full-length and truncated FIAF were associated with HDL. The composite data suggest that via physical association with plasma lipoproteins, FIAF acts as a powerful signal from fat and other tissues to prevent fat storage and stimulate fat mobilization. Our data indicate that disturbances in FIAF signaling might be involved in dyslipidemia.

The influence of bacterial activity on phosphorite formation in the Miocene Monterey Formation, California

Authigenic phosphorites from the Miocene Monterey Formation (California) including an autochthonous phosphatic laminite were analyzed for molecular biomarkers, element content, and sulfur isotopic composition of associated pyrite and sulfate to evaluate the role of bacterial activity in the precipitation of phosphate minerals. The phosphorites formed in a depositional environment typified by upwelling with dynamic bottom currents and hardground formation. Pyrite enclosed in the phosphorites shows delta S-34 values as low as -36.5 parts per thousand VCDT, which is consistent with bacterial sulfate reduction. In a three-step extraction phosphorite dissolution extraction procedure, molecular fossils of sulfate-reducing bacteria (di-O-alkyl glycerol ethers and short-chain branched fatty acids i- and ai-C-15:0, i- and ai-C-17:0, and 10MeC(16:0)) were preferentially released from the mineral lattice. This suggests that the molecular fossils were tightly bound to carbonate fluorapatite, indicating that sulfate-reducing bacteria were involved in mineral formation. A close association of sulfate-reducing bacteria with large sulfide-oxidizing bacteria, which was previously suggested to favor carbonate fluorapatite precipitation, could neither be confirmed nor excluded for the Miocene Monterey Formation phosphorites. (C) 2012 Elsevier B.V. All rights reserved.

Induction of brain aquaporin 9 (AQP9) in catecholaminergic neurons in diabetic rats.

Aquaporin 9 facilitates the diffusion of water but also glycerol and monocarboxylates, known as brain energy substrates. AQP9 was recently observed in catecholaminergic neurons that are implicated in energy homeostasis and also possibly in neuroendocrine effects of diabetes. Recently it has been observed that the level of AQP9 expression in hepatocytes is sensitive to the blood concentration of insulin. Furthermore, insulin injection in the brain is known to be related to the energy homeostasis. Based on these observations, we investigated if the concentration of insulin affects the level of brain AQP9 expression and if so, in which cell types. This study has been carried out, in a model of the diabetic rat generated by streptozotocin injection and on brainstem slices. In diabetic rats showing a decrease in systemic insulin concentration, AQP9 is only increased in brain areas containing catecholaminergic neurons. In contrast, no significant change is detected in the cerebral cortex and the cerebellum. Using immunocytochemistry, we are able to show that the increase in AQP9 expression is specifically present in catecholaminergic neurons. In brainstem slice cultures, 2 microM insulin induces a significant decrease in AQP9 protein levels 6 h after application, suggesting that brain AQP9 is also regulated by the insulin. These results show that the level of expression of brain AQP9 is affected by variations of the concentration of insulin in a diabetic model and in vitro.

Temporal quantification of MAPK induced expression in single yeast cells.

The quantification of gene expression at the single cell level uncovers novel regulatory mechanisms obscured in measurements performed at the population level. Two methods based on microscopy and flow cytometry are presented to demonstrate how such data can be acquired. The expression of a fluorescent reporter induced upon activation of the high osmolarity glycerol MAPK pathway in yeast is used as an example. The specific advantages of each method are highlighted. Flow cytometry measures a large number of cells (10,000) and provides a direct measure of the dynamics of protein expression independent of the slow maturation kinetics of the fluorescent protein. Imaging of living cells by microscopy is by contrast limited to the measurement of the matured form of the reporter in fewer cells. However, the data sets generated by this technique can be extremely rich thanks to the combinations of multiple reporters and to the spatial and temporal information obtained from individual cells. The combination of these two measurement methods can deliver new insights on the regulation of protein expression by signaling pathways.

Sclerotherapy of telangiectasias: a painless two-step technique.

BACKGROUND: Sclerotherapy of telangiectasias and reticular leg veins can be unpleasant and painful for some patients. OBJECTIVE: To determine pain level with two different sclerotherapy techniques in a prospective randomized trial. METHODS: Patients with symmetrical telangiectasias and reticular veins on both legs (C(1A) or (S)E(P)A(S)P(N1) were randomized to the standard (successive injections of chromated glycerin mixed with one-third lidocaine-epinephrine 1%) or two-step technique (first treating only reticular veins with a single injection at the base of each cluster of telangiectasias and then successively injecting all remaining telangiectasias a few minutes later. Pain was assessed using a 100-point visual analogue scale (0 = no pain, 100 = maximum pain). RESULTS: Data from 53 consecutive patients could be evaluated. The two-step technique was significantly less painful (28.2) than the standard technique (40.6, p < .001). CONCLUSION: The two-step technique with chromated glycerin mixed with one-third lidocaine-epinephrine 1% significantly reduces sclerotherapy pain. This may be a useful technique for patients who are particularly sensitive or afraid of sclerotherapy.

Release of vancomycin and teicoplanin from a plasticized and resorbable gelatin sponge: in vitro investigation of a new antibiotic delivery system with glycopeptides.

BACKGROUND: The aim of this study was to evaluate the efficacy of sustained release of vancomycin and teicoplanin from a resorbable gelatin glycerol sponge, in order to establish a new delivery system for local anti-infective therapy. MATERIALS AND METHODS: 60 plasticized glycerol gelatin sponges containing either 10 or 20% gelatin (w/v) were incubated in vancomycin or teicoplanin solution at 20 degrees C for either 1 or 24 h. In vitro release properties of the sponges were investigated over a period of 1 week by determining the levels of vancomycin and teicoplanin eluted in plasma using fluorescent polarization immunoassay. The rate constant and the half-life for the antibiotic release of each group were calculated by linear regression assuming first order kinetics. RESULTS: Presoaking for 24 h was associated with a significant increase in the total antibiotic release in all groups opposed to 1 h of incubation, except for the 10% sponges presoaked in teicoplanin. Doubling the gelatin content of the sponges from 10 to 20% significantly increased the total release of antibiotic load only in teicoplanin-containing sponges after 24 h incubation. In all corresponding groups investigated, release of vancomycin was more prolonged compared to teicoplanin, which allowed a gradual release beyond 5 days. The half-life (h +/- SEM) of both types of vancomycin-containing sponges was significantly prolonged by 24 h incubation in comparison to 1 h incubation (29.1 +/- 5.9 vs 5.9 +/- 1.0; p < 0.001, 30.0 +/- 2.1 vs 11.1 +/- 1.9; p < 0.001). However, neither doubling the gelatin content of the sponges nor a prolonged incubation was associated with a significantly prolonged delivery of teicoplanin. CONCLUSION: This study demonstrated a better diffusion-controlled release of vancomycin-impregnated glycerol gelatin sponges compared to those pretreated with teicoplanin. The plasticized glycerol gelatin sponge may be a promising carrier for the application of vancomycin to infected wounds for local anti-infective therapy.

Dairy calcium supplementation in overweight or obese persons: its effect on markers of fat metabolism

BACKGROUND: Dairy calcium supplementation has been proposed to increase fat oxidation and to inhibit lipogenesis. OBJECTIVE: We aimed to investigate the effects of calcium supplementation on markers of fat metabolism. DESIGN: In a placebo-controlled, crossover experiment, 10 overweight or obese subjects who were low calcium consumers received 800 mg dairy Ca/d for 5 wk. After 4 wk, adipose tissue was taken for biopsy for analysis of gene expression. Respiratory exchange, glycerol turnover, and subcutaneous adipose tissue microdialysis were performed for 7 h after consumption of 400 mg Ca or placebo, and the ingestion of either randomized slow-release caffeine (SRC; 300 mg) or lactose (500 mg). One week later, the test was repeated with the SRC or lactose crossover. RESULTS: Calcium supplementation increased urinary calcium excretion by 16% (P = 0.017) but did not alter plasma parathyroid hormone or osteocalcin concentrations. Resting energy expenditure (59.9 +/- 3.0 or 59.6 +/- 3.3 kcal/h), fat oxidation (58.4 +/- 2.5 or 53.8 +/- 2.2 mg/min), plasma free fatty acid concentrations (0.63 +/- 0.02 or 0.62 +/- 0.03 mmol/L), and glycerol turnover (3.63 +/- 0.41 or 3.70 +/- 0.38 micromol . kg(-1) . min(-1)) were similar with or without calcium, respectively. SRC significantly increased free fatty acid concentrations, resting fat oxidation, and resting energy expenditure. During microdialysis, epinephrine increased dialysate glycerol concentrations by 250% without and 254% with calcium. Expression of 7 key metabolic genes in subcutaneous adipose tissue was not affected by calcium supplementation. CONCLUSION: Dairy calcium supplementation in overweight subjects with habitually low calcium intakes failed to alter fat metabolism and energy expenditure under resting conditions and during acute stimulation by caffeine or epinephrine

Acute post-exercise oxygen uptake, hormone and plasma metabolite response in obese men.

This study aimed to compare oxygen uptake (  V˙O2), hormone and plasma metabolite responses during the 30 min after submaximal incremental exercise (Incr) performed at the same relative/absolute exercise intensity and duration in lean (L) and obese (O) men. Eight L and 8 O men (BMI: 22.9±0.4; 37.2±1.8 kg · m(-2)) completed Incr and were then seated for 30 min.   V˙O2 was monitored during the first 10 min and from the 25-30(th) minutes of recovery. Blood samples were drawn for the determination of hormone (catecholamines, insulin) and plasma metabolite (NEFA, glycerol) concentrations. Excess post-exercise oxygen consumption (EPOC) magnitude during the first 10 min was similar in O and in L (3.5±0.4; 3.4±0.3 liters, respectively, p=0.86). When normalized to percent change (  V˙O2END=100%), %   V˙O2END during recovery was significantly higher from 90-120 s in O than in L (p≤0.04). There were no significant differences in catecholamines (p≥0.24), whereas insulin was significantly higher in O than in L during recovery (p=0.01). The time-course of glycerol was similar from 10-30 min of recovery (-42% for L; -41% for O, p=0.85), whereas significantly different patterns of NEFA were found from 10-30 min of recovery between groups (-18% for L; +8% for O, p=0.03). Despite similar EPOC, a difference in   V˙O2 modulation between groups was observed, likely due to faster initial rates of   V˙O2 decline in L than in O. The different patterns of NEFA between groups may suggest a lower NEFA reesterification during recovery in O, which was not involved in the rapid EPOC component.

Living with an imperfect cell wall: compensation of femAB inactivation in Staphylococcus aureus.

BACKGROUND: Synthesis of the Staphylococcus aureus peptidoglycan pentaglycine interpeptide bridge is catalyzed by the nonribosomal peptidyl transferases FemX, FemA and FemB. Inactivation of the femAB operon reduces the interpeptide to a monoglycine, leading to a poorly crosslinked peptidoglycan. femAB mutants show a reduced growth rate and are hypersusceptible to virtually all antibiotics, including methicillin, making FemAB a potential target to restore beta-lactam susceptibility in methicillin-resistant S. aureus (MRSA). Cis-complementation with wild type femAB only restores synthesis of the pentaglycine interpeptide and methicillin resistance, but the growth rate remains low. This study characterizes the adaptations that ensured survival of the cells after femAB inactivation. RESULTS: In addition to slow growth, the cis-complemented femAB mutant showed temperature sensitivity and a higher methicillin resistance than the wild type. Transcriptional profiling paired with reporter metabolite analysis revealed multiple changes in the global transcriptome. A number of transporters for sugars, glycerol, and glycine betaine, some of which could serve as osmoprotectants, were upregulated. Striking differences were found in the transcription of several genes involved in nitrogen metabolism and the arginine-deiminase pathway, an alternative for ATP production. In addition, microarray data indicated enhanced expression of virulence factors that correlated with premature expression of the global regulators sae, sarA, and agr. CONCLUSION: Survival under conditions preventing normal cell wall formation triggered complex adaptations that incurred a fitness cost, showing the remarkable flexibility of S. aureus to circumvent cell wall damage. Potential FemAB inhibitors would have to be used in combination with other antibiotics to prevent selection of resistant survivors.