Biological characterization of a novel hybrid copolymer carrier system based on glycogen.

The effective drug delivery systems for cancer treatment are currently on high demand. In this paper, biological behavior of the novel hybrid copolymers based on polysaccharide glycogen were characterized. The copolymers were modified by fluorescent dyes for flow cytometry, confocal microscopy, and in vivo fluorescence imaging. Moreover, the effect of oxazoline grafts on degradation rate was examined. Intracellular localization, cytotoxicity, and internalization route of the modified copolymers were examined on HepG2 cell line. Biodistribution of copolymers was addressed by in vivo fluorescence imaging in C57BL/6 mice. Our results indicate biocompatibility, biodegradability, and non-toxicity of the glycogen-based hybrid copolymers. Copolymers were endocyted into the cytoplasm, most probably via caveolae-mediated endocytosis. Higher content of oxazoline in polymers slowed down cellular uptake. No strong colocalization of the glycogen-based probe with lysosomes was observed; thus, it seems that the modified externally administered glycogen is degraded in the same way as an endogenous glycogen. In vivo experiment showed relatively fast biodistribution and biodegradation. In conclusion, this novel nanoprobe offers unique chemical and biological attributes for its use as a novel drug delivery system that might serve as an efficient carrier for cancer therapeutics with multimodal imaging properties.

Cationic dendritic starch as a vehicle for photodynamic therapy and siRNA co-delivery.

Cationic enzymatically synthesized glycogen (cESG) is a naturally-derived, nano-scale carbohydrate dendrite that has shown promise as a cellular delivery vehicle owing to its flexibility in chemical modifications, biocompatibility and relative low cost. In the present work, cESG was modified and evaluated as a vehicle for tetraphenylporphinesulfonate (TPPS) in order to improve cellular delivery of this photosensitizer and investigate the feasibility of co-delivery with short interfering ribonucleic acid (siRNA). TPPS was electrostatically condensed with cESG, resulting in a sub-50nm particle with a positive zeta potential of approximately 5mV. When tested in normal ovarian surface epithelial and ovarian clear cell carcinoma cell culture models, encapsulation of TPPS in cESG significantly improved cell death in response to light treatment compared to free drug alone. Dosages as low as 0.16µM TPPS resulted in cellular death upon illumination with a 4.8J/cm2 light dosage, decreasing viability by 96%. cESG-TPPS was then further evaluated as a co-delivery system with siRNA for potential combination therapy, by charge-based condensation of an siRNA directed at reducing expression of manganese superoxide dismutase (Sod2) as a proof of principle target. Simultaneous delivery of TPPS and siRNA was achieved, reducing Sod2 protein expression to 48%, while maintaining the photodynamic properties of TPPS under light exposure and maintaining low dark toxicity. This study demonstrates the versatility of cESG as a platform for dual delivery of small molecules and oligonucleotides, and the potential for further development of this system in combination therapy applications.

Immunomodulatory activity of enzymatically synthesized glycogen and its digested metabolite in a co-culture system consisting of differentiated Caco-2 cells and RAW264.7 macrophages.

Previously, we developed enzymatically synthesized glycogen (ESG) from starch, and showed its immunomodulatory and dietary fiber-like activities. In this study, we investigated the metabolism of ESG and its immunomodulatory activity using differentiated Caco-2 cells as a model of the intestinal barrier. In a co-culture system consisting of differentiated Caco-2 cells and RAW264.7 macrophages, mRNA expression of IL-6, IL-8, IL-1ß and BAFF cytokines was up-regulated in Caco-2 cells and IL-8 production in basolateral medium was induced after 24 h apical treatment with 5 mg ml(-1) of ESG. The mRNA level of iNOS was also up-regulated in RAW264.7 macrophages. After characterization of the binding of anti-glycogen monoclonal antibodies (IV58B6 and ESG1A9) to ESG and its digested metabolite resistant glycogen (RG), an enzyme-linked immunosorbent assay (ELISA) system was developed to quantify ESG and RG. Using this system, we investigated the metabolism of ESG in differentiated Caco-2 cells. When ESG (7000 kDa, 5 mg ml(-1)) was added to the apical side of Caco-2 monolayers, ESG disappeared and RG (about 3000 kDa, 3.5 mg ml(-1)) appeared in the apical solution during a 24 h incubation. Neither ESG nor RG was detected in the basolateral solution. In addition, both ESG and RG were bound to TLR2 in Caco-2 cells. In conclusion, we suggest that ESG is metabolized to a RG-like structure in the intestine, and this metabolite activates the immune system via stimulation of the intestinal epithelium, although neither ESG nor its metabolite could permeate the intestinal cells under our experimental conditions. These results provide evidence for the beneficial function of ESG as a food ingredient.

A practical method for quantification of phosphorus- and glycogen-accumulating organism populations in activated sludge systems.

Enhanced biological phosphorus removal (EBPR) from wastewater relies on the enrichment of activated sludge with phosphorus-accumulating organisms (PAOs). The presence and proliferation of glycogen-accumulating organisms (GAOs), which compete for substrate with PAOs, may be detrimental for EBPR systems, leading to deterioration and, in extreme cases, failure of the process. Therefore, from both process evaluation and modeling perspectives, the estimation of PAO and GAO populations in activated sludge systems is a relevant issue. A simple method for the quantification of PAO and GAO population fractions in activated sludge systems is presented in this paper. To develop such a method, the activity observed in anaerobic batch tests executed with different PAO/GAO ratios, by mixing highly enriched PAO and GAO cultures, was studied. Strong correlations between PAO/GAO population ratios and biomass activity were observed (R2 > 0.97). This served as a basis for the proposal of a simple and practical method to quantify the PAO and GAO populations in activated sludge systems, based on commonly measured and reliable analytical parameters (i.e., mixed liquor suspended solids, acetate, and orthophosphate) without requiring molecular techniques. This method relies on the estimation of the total active biomass population under anaerobic conditions (PAO plus GAO populations), by measuring the maximum acetate uptake rate in the presence of excess acetate. Later, the PAO and GAO populations present in the activated sludge system can be estimated, by taking into account the PAO/GAO ratio calculated on the basis of the anaerobic phosphorus release-to-acetate consumed ratio. The proposed method was evaluated using activated sludge from municipal wastewater treatment plants. The results from the quantification performed following the proposed method were compared with direct population estimations carried out with fluorescence in situ hybridization analysis (determining Candidatus Accumulibacter Phosphatis as PAO and Candidatus Competibacter Phosphatis as GAO). The method showed to be potentially suitable to estimate the PAO and GAO populations regarding the total PAO-GAO biomass. It could be used, not only to evaluate the performance of EBPR systems, but also in the calibration of potential activated sludge mathematical models, regarding the PAO-GAO coexistence.

Performance following prolonged sub-maximal cycling at optimal versus freely chosen pedal rate.

It was tested whether cyclists perform better during all-out cycling following prolonged cycling at the pedal rate resulting in minimum oxygen uptake (VO(2)), i.e. the energetically optimal pedal rate (OPR) rather than at the freely chosen pedal rate (FCPR). Nine trained cyclists cycled at 180 W to determine individual OPR and FCPR. Baseline performance was determined by measuring mean power output (W(5min)) and peak VO(2) during 5-min all-out cycling at FCPR. Subsequently, on two separate days, the cyclists cycled 2.5 h at 180 W at OPR and FCPR, with each bout followed by a 5-min all-out trial. FCPR was higher (P < 0.05) than OPR at 180 W (95 +/- 7 and 73 +/- 11 rpm, respectively). During the prolonged cycling, VO(2), heart rate (HR), and rate of perceived exertion (RPE) were 7-9% higher (P < 0.05) at FCPR than at OPR and increased (P < 0.05) 2-21% over time. During all-out cycling following prolonged cycling at OPR and FCPR, W(5min) was 8 and 10% lower (P < 0.05) than at baseline, respectively. Peak VO(2) was lower (P < 0.05) than at baseline only after FCPR. The all-out trial power output was reduced following 2.5 h of cycling at 180 W at both OPR and FCPR. However, this aspect of performance was similar between the two pedal rates, despite a higher physiological load (i.e. VO(2), HR, and RPE) at FCPR during prolonged cycling. Still, a reduced peak VO(2) only occurred after cycling at FCPR. Therefore, during prolonged sub-maximal cycling, OPR is at least as advantageous as FCPR for performance optimization in subsequent all-out cycling.

Effects of pH and substrate on the competition between glycogen and phosphorus accumulating organisms.

This article evaluates effects of pH and substrate on the competition between glycogen (GAOs) and phosphorus accumulating organisms (PAOs). A sequencing batch reactor system was operated for enhanced biological phosphorus removal (EBPR) with acetate as the sole carbon source and acetate added domestic wastewater at different influent acetate/phosphate ratios. Some batch tests were performed using acetate added domestic wastewater at different influent acetate, phosphate ratios, with different initial pH values of acetate and domestic wastewater mixture. The resulting experimental data supported the presence of GAOs for all tested HAC P ratios, especially under P limiting conditions for acetate as sole carbon source. Strong evidence is observed that acetate added domestic wastewater system had higher PAOs fraction than acetate system as sole source carbon, with using model components, namely substrate uptake, glycogen utilization and P release.

Metabolic model for acetate uptake by a mixed culture of phosphate- and glycogen-accumulating organisms under anaerobic conditions.

This paper proposes a new metabolic model for acetate uptake by a mixed culture of phosphate- and glycogen-accumulating organisms (PAOs and GAOs) under anaerobic conditions. The model uses variable overall stoichiometry based on the assumption that PAOs may have the ability of using the glyoxylate pathway to produce the required reducing power for polyhydroxyalkonate (PHA) synthesis. The proposed model was tested and verified by experimental results. A sequencing batch reactor system was operated for enhanced biological phosphorus removal (EBPR) with acetate as the sole carbon source at different influent acetate/phosphate ratios. The resulting experimental data supported the validity of the proposed model, indicating the presence of GAOs for all tested HAc/P ratios, especially under P-limiting conditions. Strong agreement is observed between experimental values and model predictions for all model components, namely, PHB production, PHA composition, glycogen utilization, and P release.

The competition between PAOs (phosphorus accumulating organisms) and GAOs (glycogen accumulating organisms) in EBPR (enhanced biological phosphorus removal) systems at different temperatures and the effects on system performance.

It is well known and firmly established that the rate of chemical and biochemical reactions slow down as temperature decreases. Nevertheless, several studies have reported that the efficiency of enhanced biological phosphorus removal (EBPR) improves as temperature decreases. However, several recent studies have reported that EBPR reaction rates decrease with temperature decrease in accordance with the Arrhenius relationship. This study was designed to more thoroughly investigate this controversy using two UCT plants fed with a synthetic wastewater consisting primarily of acetate as the COD form, and a small amount of supplemental yeast extract. Experiments were performed over temperatures ranging from 5 to 20 degrees C. The results showed that, even though the kinetic rates decrease as temperature decreases, EBPR systems perform better at colder temperatures. The reason for better system performance is apparently related to reduced competition for substrate in the non-oxic zones, which results in an increased population of PAOs and, thus, greater EBPR efficiency. The proliferation of PAOs apparently occurs because they are psychrophilic whereas their competitors are not. The experiments showed that the EBPR sludges accumulated high concentrations of both PHA and glycogen at 20 degrees C, but accumulated more PHA and much less glycogen at 5 degrees C. Although the results could be interpreted as the result of changes in the PAO-GAO competition, Mann-Whitney non-parametric comparisons of transmission electron microscopy examinations revealed no indication of the presence of GAOs population under any temperature conditions. Regardless, mass balances of the glycogen data showed that the involvement of glycogen is less at cold temperature, even though EBPR was greater. Unlike current EBPR models (e.g. Mino model), the results suggest that glycogen metabolism is not a precursor for EBPR biochemistry. The results also indicate that temperature not only may cause selective pressure on the dominant organisms, but also may force them to use a different metabolic pathway as temperature decreases.

The effect of GAOs (glycogen accumulating organisms) on anaerobic carbon requirements in full-scale Australian EBPR (enhanced biological phosphorus removal) plants.

Glycogen-accumulating organisms (GAOs) were present in six full-scale plants investigated and in all but one made a significant contribution to the amount of volatile fatty acid (VFA) taken up anaerobically. While most plants surveyed contain GAOs, it was demonstrated that it is possible for a full-scale plant to operate with an insignificant GAO population. "Candidatus Accumulibacter phosphatis" were the significant polyphosphate-accumulating organisms (PAOs) in all plants surveyed. "Candidatus Competibacter phosphatis" were found in all plants along with other possible GAOs that were observed but not identified. A significant GAO population will increase the carbon requirements by removing VFA that could otherwise have been used by PAOs. Process optimization minimizing GAOs in full-scale plants would lead to a more efficient use of VFA.

Competition between polyphosphate- and glycogen-accumulating organisms in biological phosphorus removal systems--effect of temperature.

This study demonstrated that temperature is an important factor in determining the outcome of competition between polyphosphate-accumulating organisms (PAOs) and glycogen-accumulating non-poly-P organisms (GAOs) and the resultant stability of enhanced biological phosphorus removal (EBPR) systems. At 20 degrees C and a 10-day sludge age, PAOs were dominant in the anaerobic/aerobic (A/O) SBR, however, at 30 degrees C and a 10-day sludge age, GAOs were dominant in the A/O SBR. For kinetic batch studies, the anaerobic specific acetate uptake rate of GAO-dominated sludge (1.34 x 10(-3) mg C/mg VSS x minute) was higher than the rate of PAO-dominated sludge (0.89 x 10(-3) mg C/mg VSS x minute) at 30 degrees C, leading to the eventual failure of EBPR processes at high temperatures.

pH as a key factor in the competition between glycogen-accumulating organisms and phosphorus-accumulating organisms.

The effects of pH on the anaerobic metabolism of glycogen-accumulating organisms (GAOs) and phosphorus-accumulating organisms (PAOs) were compared using models for the kinetics of acetate uptake. The comparison revealed that GAOs take up acetate faster than PAOs when the pH of the anaerobic zone is less than 7.25, but that PAOs remove acetate faster than GAOs at pHs greater than 7.5. It was also found that the growth efficiencies of the two organisms are similar. Furthermore, the amount of polyhydroxy-alkanoates available after replenishment of the polymers used during acetate uptake under anaerobic conditions is similar for the two organisms, making GAOs highly competitive in nutrient removal systems. The effects of pH on the competition between the two organisms were demonstrated during the operation of a laboratory-scale sequencing batch reactor. When the overall pH of the system was low, poor phosphate removal was observed. When the pH of the system was allowed to increase to a maximum of 7.5, phosphate removal improved, but was still incomplete. Total removal was only achieved when the pH of the system was never allowed to drop lower than 7.25. After the minimum pH in the system was increased, total removal of phosphate was achieved in 14 days. The results showed that pH control is a promising strategy for minimizing the accumulation of GAOs and increasing the reliability of biological excess phosphorus removal systems.

Effect of glycogen loading on skeletal muscle cross-sectional area and T2 relaxation time.

This study was performed to investigate if glycogen loading of skeletal muscles, by binding water, would effect the cross-sectional area (CSA) and if an altered water content would alter the transverse relaxation time (T2) measured by magnetic resonance imaging (MRI). Five healthy volunteers participated in a programme with 4 days of extremely carbohydrate-restricted meals followed by 4 days of extremely high carbohydrate intake. The CSA and T2 of thigh and calf muscles were related to the intramuscular glycogen content evaluated at days 4 and 8. An increase in glycogen content from 281 to 634 mmol kg(-1) dry wt increased the CSA of the vastus muscles by 3.5% from 78 +/- 11 to 80 +/- 12 cm2 and the thigh circumference by 2.5% from 146 +/- 20 to 150 23 cm2. Calf circumference increased non-significantly by 4% from 78 +/- 15 to 82 +/- 19 cm2. Mono-exponential T2 decreased in m. tibialis anterior from 27.8 +/- 1.2 to 26.9 +/- 1.7 ms, did not change in m. vastus lateralis 26.5 +/- 1.9 ms/26.6 +/- 1.3 ms or in m. gastrocnemius 29.5 +/- 1.0 ms/29.8 +/- 1.9 ms. Glycogen loading increased the signal intensity mainly at different echo times (TE) 15 and 30 ms. The study shows that increased glycogen filling in the muscles increases muscle CSA and that this can be detected by MRI. The signal intensity increased the most at shorter TEs suggesting a more tight intracellular binding of water in glycogen loaded muscles.

Comparison of fatty acid composition and kinetics of phosphorus-accumulating organisms and glycogen-accumulating organisms.

It was demonstrated that glycogen-accumulating organisms (GAOs) were able to compete with phosphorus-accumulating organisms (PAOs) for acetate in a biological phosphorus removal (BPR) process, leading to a loss of BPR capability. Cellular fatty acid composition, which serves as a fingerprint for microbial identification, was used to determine microbial population change and to investigate the competition mechanisms of PAOs and GAOs. Analysis of cellular fatty acid composition indicated that PAOs grown with acetate and glucose were different species and that GAOs and PAOs grown with the same substrate were also different species. Glycogen-accumulating organisms seemed to coexist with PAOs even in a well-developed BPR process. The GAOs were able to accumulate more poly-beta-hydroxybutyrate (PHB) and glycogen than PAOs during the anaerobic stage of the BPR process. The GAOs synthesized more in-cell glycogen than PAOs. The growth rate for PAOs was always greater than that for GAOs at various acetate or glucose concentrations, while GAOs had higher acetate uptake and PHB synthesis rates than PAOs. Therefore, GAOs are thought to compete with PAOs only at long solids retention times (> or = 20 days).

Adrenaline stimulated glycogen breakdown in rat epitrochlearis muscles: fibre type specificity and relation to phosphorylase transformation.

The effect of adrenaline on glycogen breakdown in different skeletal muscle fibre types was investigated in the epitrochlearis muscle in vitro. Histochemical studies showed that adrenaline stimulated glycogen breakdown in all three major fibre types, with a higher absolute glycogen breakdown in type IIB fibres compared to type IIA and type I fibres. In biochemical studies we found that the glycogenolytic rate decreased during prolonged incubation with adrenaline, although the percentage phosphorylase in the a form and the concentration of glucose-6-phosphate (G-6-P) remained high. In the dose response studies we found an EC50 of 2.2 x 10(-8) M adrenaline for adrenaline stimulated glycogen breakdown, with an EC50 of 2.0 x 10(-7) M for adrenaline stimulated accumulation of G-6-P, excluding G-6-P as the key inhibitor of phosphorylase a activity.

Skeletal muscle ammonia production and repeated, intense exercise in humans.

We investigated the impact of repeated, high-intensity exercise on NH3 metabolism using the single-leg knee extensor model. The muscle glycogen level would be lowered by the initial exercise and low glycogen may stimulate NH3 production independent of any other effects of previous exercise. Therefore a high muscle glycogen condition was included in the protocol so that the pre-exercise glycogen concentration would be at least at a normal resting level for the second exercise. The subjects (n = 6) used previous exercise and (or) diet to begin the exercise with either normal (87.0 +/- 14.4 mmol/kg wet weight) or high (176.8 +/- 22.9 mmol/kg wet weight) glycogen (C and HG, respectively) in the quadriceps. They exercised (Ex1) one leg to exhaustion (140% leg VO2 max), rested 1 h, repeated the exercise (Ex2), and then repeated the protocol with the opposite leg. The exercise durations of Ex1 and Ex2, respectively, for C were 2.82 +/- 0.51 and 2.47 +/- 0.47 min (p < 0.05) and for HG were 2.92 +/- 0.57 and 2.77 +/- 0.50 min. The NH3 efflux was reduced (p < 0.05) from Ex1 to Ex2 in both C (516 +/- 159 and 250 +/- 69 mumol, respectively) and HG (618 +/- 233 and 275 +/- 124 mumol, respectively). While NH3 efflux was virtually identical between C and HG in both Ex1 and Ex2, HG consistently had a greater arterial NH3 concentration (p < 0.05). The decreased efflux in Ex2 compared with Ex1 was not due to greater accumulation of muscle NH3.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro influence of hormones on transport of glucose and glycogen in liver and muscle of pinealectomised pigeons, Columba livia Gmellin.

To substantiate the increased peripheral utilization of blood glucose by pineal in wild pigeons, an in vitro study on the ability of liver and muscle slices of intact and pinealectomised wild pigeons (C. livia) in terms of uptake and release of glucose, and deposition and depletion of glycogen, in presence of insulin, acetylcholine, glucagon and adrenaline has been undertaken. A total insensitivity of liver and muscle of pinealectomised birds for glycogen deposition and insensitivity of liver for glucose uptake has been observed. Increased glucose release from liver in response to adrenalin has been observed. The results are discussed in terms of involvement of pineal in metabolic regulation associated with breeding activities.

Storage of glycogen in rat colonic epithelium during induction of secretion and absorption in vitro.

Changes induced in the ultrastructure of the epithelium of the rat colon descendens by long-term electric field stimulation (EFS) in an Ussing chamber were investigated. The anion secretion, which was induced by EFS and was measured by the short-circuit current, fell continuously during a 5 h stimulation. At the end of the stimulation period, small particles were observed in the epithelium; these did not appear in unstimulated control tissue. They were localized predominantly in the apical part of the cell. By staining with periodic acid-thiosemicarbazide-silver proteinate and because of their sensitivity to alpha-amylase, they were identified as glycogen deposits. This storage of glycogen was time-dependent and was first visible after an EFS of 2 h. It did not appear if glucose was substituted in the bathing solution by sodium butyrate. Glycogen particles were also observed after addition of forskolin, which in contrast to EFS causes a high secretory activity that is stable over several hours. The surface cells contained significantly more glycogen than the crypt cells when secretion was stimulated by EFS or forskolin. The formation of glycogen during EFS was not prevented by tetrodotoxin (TTX). In contrast, TTX itself, which causes maximal absorptive activity by blocking secretomotor neurons, induced the appearance of glycogen in the enterocytes without EFS. However, in the presence of TTX, the amount of glycogen was the same in surface and crypt cells. The results demonstrate that the capacity to synthesize and store glycogen, which has up to now only been observed in embryonic or tumor epithelial cells, is still present in adult colonic mucosa.(ABSTRACT TRUNCATED AT 250 WORDS)

Muscle glycogen availability and temperature regulation in humans.

The effects of intramuscular glycogen availability on human temperature regulation were studied in eight seminude subjects immersed in 18 degrees C water for 90 min or until rectal temperature (Tre) decreased to 35.5 degrees C. Each subject was immersed three times over a 3-wk period. Each immersion followed 2.5 days of a specific dietary and/or exercise regimen designed to elicit low (L), normal (N), or high (H) glycogen levels in large skeletal muscle groups. Muscle glycogen concentration was determined in biopsies taken from the vastus lateralis muscle before and after each immersion. Intramuscular glycogen concentration before the immersion was significantly different among the L, N, and H trials (P less than 0.01), averaging 247 +/- 15, 406 +/- 23, and 548 +/- 42 (SE) mmol glucose units.kg dry muscle-1, respectively. The calculated metabolic heat production during the first 30 min of immersion was significantly lower during L compared with N or H (P less than 0.05). The rate at which Tre decreased was more rapid during the L immersion than either N or H (P less than 0.05), and the time during the immersion at which Tre first began to decrease also appeared sooner during L than N or H. The results suggest that low skeletal muscle glycogen levels are associated with more rapid body cooling during water immersion in humans. Higher than normal muscle glycogen levels, however, do not increase cold tolerance.