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Purpose: To determine the effects of dietary sugar or carbohydrate restriction on physical activity energy expenditure, energy intake, and physiological outcomes across 24 h. Methods: In a randomized, open-label crossover design, twenty-five healthy men (n = 10) and women (n = 15) consumed three diets over a 24-h period: moderate carbohydrate and sugar content (MODSUG = 50% carbohydrate [20% sugars], 15% protein, 35% fat); low sugar content (LOWSUG = 50% carbohydrate [< 5% sugars], 15% protein, 35% fat); and low carbohydrate content (LOWCHO = 8% carbohydrate [< 5% sugars], 15% protein, 77% fat). Postprandial metabolic responses to a prescribed breakfast (20% EI) were monitored under laboratory conditions before an ad libitum test lunch, with subsequent diet and physical activity monitoring under free-living conditions until blood sample collection the following morning. Results: The MODSUG, LOWSUG and LOWCHO diets resulted in similar mean [95%CI] rates of both physical activity energy expenditure (771 [624, 919] vs. 677 [565, 789] vs. 802 [614, 991] kcal·d−1; p = 0.29] and energy intake (2071 [1794, 2347] vs. 2195 [1918, 2473] vs. 2194 [1890, 2498] kcal·d−1; P = 0.34), respectively. The LOWCHO condition elicited the lowest glycaemic and insulinaemic responses to breakfast (P < 0.01) but the highest 24-h increase in LDL-cholesterol concentrations (P < 0.001), with no differences between the MODSUG and LOWSUG treatments. Leptin concentrations decreased over 24-h of consuming LOWCHO relative to LOWSUG (p < 0.01). Conclusion: When energy density is controlled for, restricting either sugar or total dietary carbohydrate does not modulate physical activity level or energy intake over a 24-h period (~ 19-h free-living) despite substantial metabolic changes. Clinical trials registration ID: NCT03509610, https://clinicaltrials.gov/show/NCT03509610 [ABSTRACT FROM AUTHOR]

Extraction of integral membrane proteins with poly(styrene-co-maleic acid) provides a promising alternative to detergent extraction. A major advantage of extraction using copolymers rather than detergent is the retention of the lipid bilayer around the proteins. Here we report the first functional investigation of the mammalian insulin receptor which was extracted from cell membranes using poly(styrene-co-maleic acid). We found that the copolymer efficiently extracted the insulin receptor from 3T3L1 fibroblast membranes. Surprisingly, activation of the insulin receptor and proximal downstream signalling was detected upon copolymer extraction even in the absence of insulin stimulation. Insulin receptor and IRS1 phosphorylations were above levels measured in the control extracts made with detergents. However, more distal signalling events in the insulin signalling cascade, such as the phosphorylation of Akt were not observed. Following copolymer extraction, in vitro addition of insulin had no further effect on insulin receptor or IRS1 phosphorylation. Therefore, under our experimental conditions, the insulin receptor is not functionally responsive to insulin. This study is the first to investigate receptor tyrosine kinases extracted from mammalian cells using a styrene-maleic acid copolymer and highlights the importance of thorough functional characterisation when using this method of protein extraction. [ABSTRACT FROM AUTHOR]

Our recently developed ensilication approach can physically stabilize proteins in silica without use of a pre-formed particle matrix. Stabilisation is done by tailor fitting individual proteins with a silica coat using a modified sol-gel process. Biopharmaceuticals, e.g. liquid-formulated vaccines with adjuvants, frequently have poor thermal stability; heating and/or freezing impairs their potency. As a result, there is an increase in the prevalence of vaccine-preventable diseases in low-income countries even when there are means to combat them. One of the root causes lies in the problematic vaccine 'cold chain' distribution. We believe that ensilication can improve vaccine availability by enabling transportation without refrigeration. Here, we show that ensilication stabilizes tetanus toxin C fragment (TTCF), a component of the tetanus toxoid present in the diphtheria, tetanus and pertussis (DTP) vaccine. Experimental in vivo immunization data show that the ensilicated material can be stored, transported at ambient temperatures, and even heat-treated without compromising the immunogenic properties of TTCF. To further our understanding of the ensilication process and its protective effect on proteins, we have also studied the formation of TTCF-silica nanoparticles via time-resolved Small Angle X-ray Scattering (SAXS). Our results reveal ensilication to be a staged diffusion-limited cluster aggregation (DLCA) type reaction. An early stage (tens of seconds) in which individual proteins are coated with silica is followed by a subsequent stage (several minutes) in which the protein-containing silica nanoparticles aggregate into larger clusters. Our results suggest that we could utilize this technology for vaccines, therapeutics or other biopharmaceuticals that are not compatible with lyophilization. [ABSTRACT FROM AUTHOR]

Purpose: We have previously shown that 6 weeks of reduced-exertion high-intensity interval training (REHIT) improves VO2max in sedentary men and women and insulin sensitivity in men. Here, we present two studies examining the acute physiological and molecular responses to REHIT.Methods: In Study 1, five men and six women (age: 26 ± 7 year, BMI: 23 ± 3 kg m(-2), VO2max: 51 ± 11 ml kg(-1) min(-1)) performed a single 10-min REHIT cycling session (60 W and two 20-s 'all-out' sprints), with vastus lateralis biopsies taken before and 0, 30, and 180 min post-exercise for analysis of glycogen content, phosphorylation of AMPK, p38 MAPK and ACC, and gene expression of PGC1α and GLUT4. In Study 2, eight men (21 ± 2 year; 25 ± 4 kg·m(-2); 39 ± 10 ml kg(-1) min(-1)) performed three trials (REHIT, 30-min cycling at 50 % of VO2max, and a resting control condition) in a randomised cross-over design. Expired air, venous blood samples, and subjective measures of appetite and fatigue were collected before and 0, 15, 30, and 90 min post-exercise.Results: Acutely, REHIT was associated with a decrease in muscle glycogen, increased ACC phosphorylation, and activation of PGC1α. When compared to aerobic exercise, changes in VO2, RER, plasma volume, and plasma lactate and ghrelin were significantly more pronounced with REHIT, whereas plasma glucose, NEFAs, PYY, and measures of appetite were unaffected.Conclusions: Collectively, these data demonstrate that REHIT is associated with a pronounced disturbance of physiological homeostasis and associated activation of signalling pathways, which together may help explain previously observed adaptations once considered exclusive to aerobic exercise. [ABSTRACT FROM AUTHOR]

Aims/hypothesis: The glucose transporter GLUT4 is present mainly in insulin-responsive tissues of fat, heart and skeletal muscle and is translocated from intracellular membrane compartments to the plasma membrane (PM) upon insulin stimulation. The transit of GLUT4 to the PM is known to be dependent on a series of Rab proteins. However, the extent to which the activity of these Rabs is regulated by the action of insulin action is still unknown. We sought to identify insulin-activated Rab proteins and Rab effectors that facilitate GLUT4 translocation. Methods: We developed a new photoaffinity reagent (Bio-ATB-GTP) that allows GTP-binding proteomes to be explored. Using this approach we screened for insulin-responsive GTP loading of Rabs in primary rat adipocytes. Results: We identified Rab3B as a new candidate insulin-stimulated G-protein in adipocytes. Using constitutively active and dominant negative mutants and Rab3 knockdown we provide evidence that Rab3 isoforms are key regulators of GLUT4 translocation in adipocytes. Insulin-stimulated Rab3 GTP binding is associated with disruption of the interaction between Rab3 and its negative effector Noc2. Disruption of the Rab3-Noc2 complex leads to displacement of Noc2 from the PM. This relieves the inhibitory effect of Noc2, facilitating GLUT4 translocation. Conclusions/interpretation: The discovery of the involvement of Rab3 and Noc2 in an insulin-regulated step in GLUT4 translocation suggests that the control of this translocation process is unexpectedly similar to regulated secretion and particularly pancreatic insulin-vesicle release. [ABSTRACT FROM AUTHOR]

The phase separation of aminophospholipids in glycerophospholipid matrix and the effect of cholesterol were studied by means of fluorescence microscopy of giant unilamellar vesicles (GUV). GUVs were composed of binary mixtures, egg yolk phosphatidylcholine (eggPC)/egg yolk phosphatidylethanolamine (eggPE) and egg yolk phosphatidylcholine (eggPC)/brain phosphatidylserine (brainPS), and ternary ones with both aminophospholipids (eggPC/eggPE/brainPS). Gel/liquid-disordered phase coexistence was detected in these mixtures, where aminophospholipids segregate in gel leaf-like domains. When cholesterol (CHOL) was added, the phase separation was shifted at lower temperatures. CHOL increases miscibility of aminophospholipids in PC matrix. Addition of PE and PS to the ternary mixtures (eggPC/eggSM/CHOL) induced liquid-ordered domain formation at higher temperatures. Based on these results, one can conclude that aminophospholipids promote the formation of L domains. [ABSTRACT FROM AUTHOR]

The three-dimensional (3D) cell culture approach offers a means to study cells under conditions that mimic an in vivo environment, thus avoiding the limitations imposed by the conventional two-dimensional (2D) monolayer cell cultures. By using this approach we demonstrated significant differences in the plasma membrane phospholipid composition and susceptibility to oxidation in cells cultured in three-dimensional environment compared to conventional monolayer cultures. The plasma membrane sphingomyelin (SM), which is a functionally active membrane phospholipid, was markedly increased in plasma membranes of 3D cells. To analyze the mechanisms underlying SM accumulation, we determined the activities of sphingolipid-metabolizing enzymes like neutral sphingomyelinase and ceramidase, which are also related to cellular redox homeostasis and to oxidative stress. Fibroblasts cultured in three-dimensional environment showed different redox potential and lower lipid susceptibility to oxidative damage compared to monolayer cells. The relative content of unsaturated fatty acids, which serve as targets of oxidative attack, was observed to be higher in major phospholipids, such as phosphatidylcholine and phosphatidylethanolamine, in plasma membranes of 3D cells. The possibility that the higher level of SM, might be responsible for the lower degree of oxidation of 3D phospholipids was tested by selective reduction of SM through treatment with exogenous sphingomyelinase. The results showed that the decrease of plasma membrane SM was accompanied by an increase of the lipid peroxides in both 2D and 3D cells. We presume that culturing as a monolayer is stressful for the cells and leads to activation of certain stress-related enzymes, resulting in reduction of the SM level. Our results show that the lower content of plasma membrane SM in cells cultured as a monolayer renders the phospholipid molecules more susceptible to oxidative stress. [ABSTRACT FROM AUTHOR]

The spatial and temporal pattern of root water uptake in partially wetted soil was studied in the root zone of a 6-year-old microsprinkler-irrigated almond tree. The water balance of about one quarter of the root zone’s wetted soil volume (2.0×2.0×0.9 m3) was determined by catch cans, neutron probe and tensiometer measurements. Twenty-five neutron probe access tubes with catch cans were distributed in a square grid of 50 cm spacing. Eight pairs of tensiometers were installed at depths of 82.5 and 97.5 cm in a regular pattern between the access tubes. Neutron probe readings at 15 cm depth increments and tensiometer readings were taken at time intervals of 4–24 h. The rate of soil water depletion was calculated and used to estimate the spatial and temporal distributions of root water uptake. Soil water dynamics was studied in two stages: (1) during a week of conventional irrigation management with three irrigation events; and (2) during a period of 16 days without irrigation, after the monitored soil volume was thoroughly moistened so that soil water was easily available everywhere, initially. The zones of maximum root water uptake were the same for both stages in periods of high local rates of water application. After water applications, root water uptake occurred initially near the tree trunk and then progressed towards the root system periphery, thereby changing locations of maximum root water uptake and shifting to root zone regions with minimum soil water stress. [ABSTRACT FROM AUTHOR]

The phase behaviour of mixed molecular species of phosphatidylethanolamine, phosphatidylserine and sphingomyelin of biological origin were examined in aqueous co-dispersions using synchrotron X-ray diffraction. The co-dispersions of phospholipids studied were aimed to model the mixing of lipids populating the cytoplasmic and outer leaflets in the resting or “scrambled” activated cell membrane. Mixtures enriched with phosphatidylethanolamine and phosphatidylserine were characterized by a phase separation of non-lamellar phases (cubic and inverted hexagonal) with a lamellar gel phase comprising the most saturated molecular species. Inclusion of sphingomyelin in the mixture resulted in a suppression of the hexagonal-II phase in favour of lamellar phases at temperatures where a proportion of the phospholipid was fluid. The effect was also dependent on the total amount of sphingomyelin in ternary mixtures, and the lamellar phase dominated in mixtures containing more than 30 mol%, irrespective of the relative proportions of phosphatidylserine/sphingomyelin. A transition from gel to liquid-crystal phase was detected by wide-angle scattering during heating scans of ternary mixtures enriched in sphingomyelin and was shown by thermal cycling experiments to be coupled with a hexagonal-II phase to lamellar transition. In such samples there was evidence of a coexistence of non-lamellar phases with a lamellar gel phase. A transition of the gel phase to the fluid state on heating from 35 to 41 °C was evidenced by a progressive increase in the lamellard-spacing. The presence of calcium enhanced the phase separation of a lamellar gel phase from a hexagonal-II phase in mixtures enriched in phosphatidylserine. This effect was counteracted by charge screening with 150 mM NaCl. The effect of sphingomyelin on stabilizing the lamellar phase is discussed in the context of an altered composition in the cytoplasmic/outer leaflets of the plasma membrane resulting from scrambling of the phospholipid distribution. The results suggest that a lamellar structure can be retained by the inward translocation of sphingomyelin in biological membranes. The presence of monovalent cations serves also to stabilize the bilayer in activated cells where a translocation of aminoglycerophospholipids and an influx of calcium occur simultaneously. [ABSTRACT FROM AUTHOR]

A continuum model, based on the Poisson–Nernst–Planck (PNP) theory, is applied to simulate steady-state ion flux through protein channels. The PNP equations are modified to explicitly account (1) for the desolvation of mobile ions in the membrane pore and (2) for effects related to ion sizes. The proposed algorithm for a three-dimensional self-consistent solution of PNP equations, in which final results are refined by a focusing technique, is shown to be suitable for arbitrary channel geometry and arbitrary protein charge distribution. The role of the pore shape and protein charge distribution in formation of basic electrodiffusion properties, such as channel conductivity and selectivity, as well as concentration distributions of mobile ions in the pore region, are illustrated by simulations on model channels. The influence of the ionic strength in the bulk solution and of the externally applied electric field on channel properties are also discussed. [ABSTRACT FROM AUTHOR]

The experimental NMR data for the individual titratable groups in ribonuclease T[sub 1] presented in the preceding paper were analysed by means of a continuum dielectric model. The role of two factors, the alteration of hydrogen loci on the ionizable groups and the conformational flexibility, were analysed. It was suggested that the position of the titratable hydrogen is essential mainly for strongly interacting groups. For groups which are accessible to the solvent and whose ionization is not coupled with the ionization of neighbouring groups, this factor can be neglected. The influence of the conformational flexibility on the electrostatic interactions becomes apparent for the environment of K25. For some strongly interacting groups, non-sigmoidal ionization curves were calculated. On this basis the pH dependence of the NMR chemical shift of the [sup 13] C[sup ε2] resonance of H27, whose ionization is coupled with E82, was reproduced. [ABSTRACT FROM AUTHOR]

pK[sub a] values of amino acid side chains of ribonuclease T[sub 1] have been determined from the pH dependence of [sup 13] C and [sup 15] N resonances. It was possible to derive pK[sub a] values of single protonation or deprotonation sites of carboxylate and imidazole groups. Deviations from pK[sub a] values of free amino acids could be interpreted with electrostatic interactions of corresponding side chains with the protein environment. In particular, the interaction between H27 and E82 led to an increase of the H27 pK[sub a] and a decrease of the E82 pK[sub a] . The pK[sub a] of E28 at the C-terminal end of the α-helix was increased because of the dipolar character of the α-helix. D76 did not titrate in the investigated pH range of about 2–9. From the chemical shift value this buried side chain seems to be protonated. The pK[sub a] values of side chains in the active site deviate from a normal behaviour. The lower pK[sub a] value of E58 may be interpreted with the close proximity of this side chain with positively charged H40 and R77. A novel two-dimensional [sup 1] H([sup 13] C[sup δ] )[sup 13] C[sup γ] correlation experiment was developed to observe the pH dependence of the chemical shifts of the C[sup γ] resonances of histidine residues. From the inspection of the C[sup γ] chemical shift-pH profiles it was possible to determine the predominant tautomeric form for the histidine residues at higher pH values. [ABSTRACT FROM AUTHOR]

Investigations were performed on the influence of the phospholipid composition and physicochemical properties of the rat liver microsomal membranes on acyl-CoA synthetase and acyl-CoA : 1-acyl- sn-glycero-3-phosphocholine O-acyltransferase activities. The phospholipid composition of the membranes was modified by incubation with different phospholipids in the presence of lipid transfer proteins or by partial delipidation with exogenous phospholipase C and subsequent enrichment with phospholipids. The results indicated that the incorporation of phosphatidylglycerol, phosphatidylserine and phosphatidylethanolamine induced a marked activation of acyl-CoA synthetase for both substrates used-palmitic and oleic acids. Sphingomyelin occurred as specific inhibitor for this activity especially for palmitic acid. Palmitoyl-CoA: and oleoyl-CoA : lacyl-sn-glycero-3-phosphocholine acyltransferase activities were found to depend on the physical state of the membrane lipids. The alterations in the membrane physical state were estimated using two different fluorescent probes-1,6-diphenyl-1,3,5-hexatriene and pyrene. In all cases of membrane fluidization this activity was elevated. On the contrary, in more rigid membranes obtained by incorporation of sphingomyelin and dipalmitoylphosphatidylcholine, acyltransferase activity was reduced for both palmitoyl-CoA and oleoyl-CoA. We suggest a certain similarity in the way of regulation of membrane-bound acyltransferase and phospholipase A which both participate in the deacylation-reacylation cycle. [ABSTRACT FROM AUTHOR]

Investigations were performed on the influence of the phospholipid composition and physicochemical properties of the rat liver microsomal membranes on acyl-CoA synthetase and acyl-CoA:1-acyl-sn-glycero-3-phosphocholine O-acyltransferase activities. The phospholipid composition of the membranes was modified by incubation with different phospholipids in the presence of lipid transfer proteins or by partial delipidation with exogenous phospholipase C and subsequent enrichment with phospholipids. The results indicated that the incorporation of phosphatidylglycerol, phosphatidylserine and phosphatidylethanolamine induced a marked activation of acyl-CoA synthetase for both substrates used--palmitic and oleic acids. Sphingomyelin occurred as specific inhibitor for this activity especially for palmitic acid. Palmitoyl-CoA: and oleoyl-CoA: 1-acyl-sn-glycero-3-phosphocholine acyltransferase activities were found to depend on the physical state of the membrane lipids. The alterations in the membrane physical state were estimated using two different fluorescent probes--1,6-diphenyl-1,3,5-hexatriene and pyrene. In all cases of membrane fluidization this activity was elevated. On the contrary, in more rigid membranes obtained by incorporation of sphingomyelin and dipalmitoylphosphatidylcholine, acyltransferase activity was reduced for both palmitoyl-CoA and oleoyl-CoA. We suggest a certain similarity in the way of regulation of membrane-bound acyltransferase and phospholipase A2 which both participate in the deacylation-reacylation cycle. [ABSTRACT FROM AUTHOR]

Biological substances based on proteins, including vaccines, antibodies, and enzymes, typically degrade at room temperature over time due to denaturation, as proteins unfold with loss of secondary and tertiary structure. Their storage and distribution therefore relies on a 'cold chain' of continuous refrigeration; this is costly and not always effective, as any break in the chain leads to rapid loss of effectiveness and potency. Efforts have been made to make vaccines thermally stable using treatments including freeze-drying (lyophilisation), biomineralisation, and encapsulation in sugar glass and organic polymers. Here for the first time we show that proteins can be enclosed in a deposited silica 'cage', rendering them stable against denaturing thermal treatment and long-term ambient-temperature storage, and subsequently released into solution with their structure and function intact. This 'ensilication' method produces a storable solid protein-loaded material without the need for desiccation or freeze-drying. Ensilication offers the prospect of a solution to the 'cold chain' problem for biological materials, in particular for vaccines. [ABSTRACT FROM AUTHOR]