Effect of formulation factors on incorporation of the hydrophilic peptide dalargin into PLGA and mPEG-PLGA nanoparticles

The objective of this study was to examine formulation factors that influence the incorporation of the hydrophilic peptide dalargin into poly(D, L-lactic-co-glycolic acid) (PLGA) and methoxy-polyethylene glycol (mPEG)-PLGA nanoparticles. In particular, the effect of ionic additives and nanoparticle method of preparation on the incorporation of dalargin and resultant nanoparticle properties was investigated. Biodegradable nanoparticles were prepared from mPEG-PLGA and PLGA by both solvent evaporation and solvent diffusion methods with inclusion of ionic additives of dextran sulphate (DS), sulfobutyl ether-beta-cyclodextrin (SB-CD), or sodium dodecyl sulfate (SDS). The resultant nanoparticles were analyzed for their mean particle size and size distribution, zeta-potential, peptide loading, yield, and morphology. The inclusion of ionic additives in the nanoparticle formulation significantly influenced dalargin entrapment efficiency (EE). For example, with the PLGA/SDS formulation EE increased from 13.3% to 91.2% and from 4.1% to 68.6% with the solvent diffusion and evaporation methods, respectively. The inclusion of ionic surfactant SDS has also lead to the formation of smaller size of nanoparticles. Isothermal titration microcalorimetry revealed a strong interaction between dalargin and DS, medium level interaction with SDS, and weak interaction with SB-CD. The results of this study suggest that a strong ionic interaction between peptides and additives may lead to enhanced peptide incorporation but also increased particle size. Intermediate ionic interaction, especially when it is associated with the formation of reversed micelles in a hydrophobic polymer solution, could be used to enhance the incorporation of hydrophilic peptides in PLGA and mPEG-PLGA nanoparticles.     

Characterization of Antimicrobial-bearing Liposomes by ζ-Potential, Vesicle Size, and Encapsulation Efficiency

Liposome entrapment may improve activity of protein or polypeptide antimicrobials against a variety of microorganisms. In this study, ability of liposomes to withstand exposure to environmental and chemical stresses typically encountered in foods and food processing operations were tested. Liposomes consisting of distearoylphosphatidylcholine (PC) and distearoylphosphatidylglycerol (PG), with 0, 5, or 10 μg/ml of the antimicrobial peptide nisin entrapped, were exposed to elevated temperatures (25–75 °C) and a range of pH (5.5–11.0). Ability of liposomes to maintain integrity was assessed by measuring the encapsulation efficiency (EE), ζ-potential, and particle size distribution of liposomes. Distearoylphosphatidylcholine, PC/PG 8:2, and PC/PG 6:4 (mole fraction) liposomes retained between ~70–90% EE despite exposure to elevated temperature and alkaline or acidic pH. Particle size of liposomes averaged between 100 and 240 nm depending on liposome preparation. Liposomal surface charge depended primarily on phospholipid composition and changed little with inclusion of nisin. Surface charge was not affected by temperature for PC and PC/PG 8:2 but decreased for PC/PG 6:4 liposomes. Our results suggest that liposomes containing nisin may be suitable for use as antimicrobial-active ingredients in low- or high-pH foods subjected to moderate heat treatments.     

Statistical Design for Formulation Optimization of Hydrocortisone Butyrate-Loaded PLGA Nanoparticles

The aim of this investigation was to develop hydrocortisone butyrate (HB)-loaded poly(d,l-lactic-co-glycolic acid) (PLGA) nanoparticles (NP) with ideal encapsulation efficiency (EE), particle size, and drug loading (DL) under emulsion solvent evaporation technique utilizing various experimental statistical design modules. Experimental designs were used to investigate specific effects of independent variables during preparation of HB-loaded PLGA NP and corresponding responses in optimizing the formulation. Plackett–Burman design for independent variables was first conducted to prescreen various formulation and process variables during the development of NP. Selected primary variables were further optimized by central composite design. This process leads to an optimum formulation with desired EE, particle size, and DL. Contour plots and response surface curves display visual diagrammatic relationships between the experimental responses and input variables. The concentration of PLGA, drug, and polyvinyl alcohol and sonication time were the critical factors influencing the responses analyzed. Optimized formulation showed EE of 90.6%, particle size of 164.3 nm, and DL of 64.35%. This study demonstrates that statistical experimental design methodology can optimize the formulation and process variables to achieve favorable responses for HB-loaded NP.     

Yeast Cell Microcapsules as a Novel Carrier for Cholecalciferol Encapsulation: Development,Characterization and Release Properties

In this study Saccharomyces cerevisiae yeast cells was used as a novel vehicle for encapsulation of vitamin D₃. The effects of initial cholecalciferol concentration (100,000 and 500,000 IU/g yeast), yeast cell pretreatment (plasmolysis with NaCl) and drying method (spray or freeze drying) on microcapsules properties were investigated. It was found that the vitamin concentration and drying method had significant influence on encapsulation efficiency (EE) and size of yeast microcapsules. Furthermore, EE values were more increased by the plasmolysis treatment. The highest EE was obtained for plasmolysed and spray dried yeast cells prepared using initial cholecalciferol concentration of 2.5 mg per gram of yeast cells (76.10?±?6.92%). The values of mean particle size were 3.43–7.91 μm. The presence of cholecalciferol in yeast microcapsules was confirmed by X-ray diffraction (XRD) and Fourier transform-infrared (FT-IR) analyses. The in vitro cholecalciferol release from yeast microcapsules in phosphate buffer saline solution (PBS) followed a controlled release manner consistent with a Fickian diffusion mechanism. In addition, the release studies in simulated gastrointestinal tract showed sustained release of cholecalciferol in the stomach condition and significant release in intestinal medium.     

Monodisperse chitosan nanoparticles for mucosal drug delivery

Chitosan nanoparticles (CS NPs) of a controlled size (below 100 nm) and narrow size distribution were obtained through the process of ionic gelation between CS and sodium tripolyphosphate (TPP). A high degree of CS deacetylation and narrow polymer molecular weight distribution were demonstrated to be critical for the controlling particle size distribution. Properties of the CS NPs were examined at different temperatures, values of pH, and ratios of CS to TPP. The model protein, bovine serum albumin, was encapsulated into the NPs, and the in vitro release profiles were examined in physiologically relevant media at 37 degrees C.     

Optimization on conditions of podophyllotoxin-loaded liposomes using response surface methodology and its activity on PC3 cells

The purpose of this study was to optimize the preparation conditions of podophyllotoxin liposomes (PPT-Lips), and to investigate their effects on PC3 cells. PPT-Lips were prepared by using a thin-film dispersion method. In order to achieve maximum drug encapsulation efficiency (EE), the process and formulation variables were optimized by response surface methodology (RSM). The optimum preparation conditions were cholesterol to lecithin ratio of 3.6:40 (w/w), lipid to drug ratio of 15.8:1 (w/w), and the ultrasonic intensity of 35% (total power of 400?W). The experimental EE of PPT-Lips was 90.425%, which was consistent with the theoretically predicted value. The characterization studies showed that PPT-Lips were well-dispersible spherical particles with an average size of 106?nm and a zeta potential of –10.1?mV. A gradual and time-dependent pattern of PPT from liposomes was found in in vitro drug release with a cumulative release amount up to 70.3% in 24?h. Results of cell viability experiments on PC3 cells demonstrated that PPT-Lips exhibited more effective anticancer activity in comparison with free PPT. Therefore, PPT-Lips represent an efficient and promising drug delivery system for PPT.     

A Novel Method for Preparing Surface-Modified Fluocinolone Acetonide Loaded PLGA Nanoparticles for Ocular Use: <Emphasis Type="Italic">In Vitro</Emphasis> and <Emphasis Type="Italic">In Vivo</Emphasis> Evaluations

Our objective was to prepare nanoparticulate system using a simple yet attractive innovated method as an ophthalmic delivery system for fluocinolone acetonide to improve its ocular bioavailability. Poly(lactic-co-glycolic acid) (PLGA) nanoparticles were prepared by adopting thin film hydration method using PLGA/poloxamer 407 in weight ratios of 1:5 and 1:10. PLGA was used in 75/25 and 50/50 copolymer molar ratio of DL-lactide/glycolide. Results revealed that using PLGA with lower glycolic acid monomer ratio exhibited high particle size (PS), zeta potential (ZP) and drug encapsulation efficiency (EE) values with slow drug release pattern. Also, doubling the drug concentration during nanoparticles preparation ameliorated its EE to reach almost 100%. Furthermore, studies for separating the un-entrapped drug in nanoparticles using centrifugation method at 20,000 rpm for 30 min showed that the separated clear supernatant contained nanoparticles encapsulating an important drug amount. Therefore, separation of un-entrapped drug was carried out by filtrating the preparation using 20–25 μm pore size filter paper to avoid drug loss. Aiming to increase the PLGA nanoparticles mucoadhesion ability, surface modification of selected formulation was done using different amount of stearylamine and chitosan HCl. Nanoparticles coated with 0.1% w/v chitosan HCl attained most suitable results of PS, ZP and EE values as well as high drug release properties. Transmission electron microphotographs illustrated the deposition of chitosan molecules on the nanoparticles surfaces. Pharmacokinetic studies on Albino rabbit’s eyes using HPLC indicated that the prepared novel chitosan-coated PLGA nanoparticles subjected to separation by filtration showed rapid and extended drug delivery to the eye.     

Liposomal hydrogel formulation for transdermal delivery of pirfenidone

Context: Pirfenidone (PFD) is an anti-fibrotic and anti-inflammatory agent indicated for the treatment of idiopathic pulmonary fibrosis (IPF). The current oral administration of PFD has several limitations including first pass metabolism and gastrointestinal irritation.

Objective: The aim of this study is to investigate the feasibility of transdermal delivery of PFD using liposomal carrier system.

Materials and methods: PFD-loaded liposomes were prepared using soy phosphatidylcholine (SPC) and sodium cholate (SC). Encapsulation efficiency (EE) of PFD in liposomes was optimized using different preparation techniques including thin film hydration (TFH) method, direct injection method (DIM) and drug encapsulation using freeze–thaw cycles. In vitro drug release study was performed using dialysis membrane method. The skin permeation studies were performed using excised porcine ear skin model in a Franz diffusion cell apparatus.

Results and discussion: The average particle size and zeta-potential of liposomes were 191?±?4.1?nm and ?40.4?±?4.5?mV, respectively. The liposomes prepared by TFH followed by 10 freeze–thaw cycles showed the greatest EE of 22.7?±?0.63%. The optimized liposome formulation was incorporated in hydroxypropyl methyl cellulose (HPMC) hydrogel containing different permeation enhancers including oleic acid (OA), isopropyl myristate (IPM) and propylene glycol (PG). PFD-loaded liposomes incorporated in hydrogel containing OA and IPM showed the greatest flux of 10.9?±?1.04?μg/cm²/h across skin, which was 5-fold greater compared with free PFD. The cumulative amount of PFD permeated was 344?±?28.8?μg/cm² with a lag time of 2.3?±?1.3?h.

Conclusion: The hydrogel formulation containing PFD-loaded liposomes can be developed as a potential transdermal delivery system.     

Preparation of submicron liposomes exhibiting efficient entrapment of drugs by freeze-drying water-in-oil emulsions

A novel liposome preparation method is described as freeze-drying of water-in-oil emulsions containing sucrose in the aqueous phase (W) and phospholipids and poly(ethylene glycol)₁₅₀₀ (PEG) in the oil phase (O). The water-in-oil emulsions were prepared by sonication and then lyophilized to obtain dry products. Upon rehydration, the dry products formed liposomes with a size smaller than 200 nm and an encapsulation efficiency (EE) higher than 60% for model drugs. The presence of lyoprotectant and PEG was found to be a prerequisite for the formation of liposomes with desirable properties, such as a small particle size and high EE. The lyophilates were stable and could be rehydrated to form liposomes without any change in size or EE even after a storage period of 6 months. Also, the lipophilic drug-containing FWE liposomes were stable and could be stored for at least 6 months although the liposomes containing hydrophilic drugs showed significant leakage. Based on the vesicle size and EEs of the model drugs, as well as the scanning electron micrograph (SEM) and small angle X-ray scattering (SAXS) pattern of the lyophilates, a possible mechanism for the liposome formation is proposed.     

Effect of Formulation and Process Parameters on Chitosan Microparticles Prepared by an Emulsion Crosslinking Technique

There are many studies about the synthesis of chitosan microparticles; however, most of them have very low production rate, have wide size distribution, are difficult to reproduce, and use harsh crosslinking agents. Uniform microparticles are necessary to obtain repeatable drug release behavior. The main focus of this investigation was to study the effect of the process and formulation parameters during the preparation of chitosan microparticles in order to produce particles with narrow size distribution. The technique evaluated during this study was emulsion crosslinking technique. Chitosan is a biocompatible and biodegradable material but lacks good mechanical properties; for that reason, chitosan was ionically crosslinked with sodium tripolyphosphate (TPP) at three different ratios (32, 64, and 100%). The model drug used was acetylsalicylic acid (ASA). During the preparation of the microparticles, chitosan was first mixed with ASA and then dispersed in oil containing an emulsifier. The evaporation of the solvents hardened the hydrophilic droplets forming microparticles with spherical shape. The process and formulation parameters were varied, and the microparticles were characterized by their morphology, particle size, drug loading efficiency, and drug release behavior. The higher drug loading efficiency was achieved by using 32% mass ratio of TPP to chitosan. The average microparticle size was 18.7 μm. The optimum formulation conditions to prepare uniform spherical microparticles were determined and represented by a region in a triangular phase diagram. The drug release analyses were evaluated in phosphate buffer solution at pH 7.4 and were mainly completed at 24 h.     

Development and Statistical Optimization of Solid Lipid Nanoparticles of Simvastatin by Using 2<Superscript>3</Superscript> Full-Factorial Design

The objective of this study was to develop solid lipid nanoparticles (SLNs) of simvastatin and to optimize it for independent variables (amount of glycerol monostearate, concentration of poloxamer, and volume of isopropyl alcohol) in order to achieve desired particle size with maximum percent entrapment efficiency (% EE) and percent cumulative drug release (% CDR). To achieve our goal, eight formulations (F ₁–F ₈) of SLNs were prepared by solvent injection technique and optimized by 2³ full-factorial design. The design was validated by extra design checkpoint formulation (F ₉), and the possible interactions between independent variables were studied. The responses of the design were analyzed using Design Expert 7.1.6. (Stat-Ease, Inc, USA), and the analytical tools of software were used to draw Pareto charts and response surface plots. On the basis of software analysis, formulation F ₁₀ with a desirability factor of 0.611 was selected as optimized formulation and was evaluated for the independent parameters. Optimized formulation showed particle size of 258.5 nm, % EE of 75.81%, with of 82.67% CDR after 55 h. The release kinetics of the optimized formulation best fitted the Higuchi model, and the recrystallization index of optimized formulation was found to be 65.51%.     

Optimization on preparation condition of polyunsaturated fatty acids nanoliposome prepared by Mozafari method

This study presents the application of the response surface methodology (design) to develop an optimal preparation condition (independent variables) namely shear rate (600–1000?rpm), mixing time (30–60?min), and sonication time (10–20?min) for polyunsaturated fatty acids (docosahexaenoic acid and eicosapentaenoic acid) nanoliposomes. Fifteen lipid mixtures were generated by the Box–Behnken design and nanoliposomes were prepared by the Mozafari (direct hydration and without using organic solvents) method. Nanoliposomes were characterized with respect to entrapment efficiency (EE) and vesicle size as Y₁ and Y₂ dependent variables, respectively. The results were then applied to estimate the coefficients of response surface model and to find the optimal preparation conditions with maximum EE and minimum vesicle size. The response surface analysis exhibited that the significant (p?p?>?0.05) lack of fit for the reduced models. The response optimization of experiments was the shear rate: 795?rpm; mixing time: 60?min; and sonication time: 10?min. The optimal nanoliposome had an average diameter of 81.4?nm and EE of 100%. The experimental results of optimal nanoliposomes characterization confirmed an accurate fitness of the predicted values by reduced response surface models.     

Experimental,computational and chemometrics studies of BSA-vitamin B6 interaction by UV–Vis,FT-IR,fluorescence spectroscopy,molecular dynamics simulation and hard-soft modeling methods

The interaction of pyridoxine (Vitamin B6) with bovine serum albumin (BSA) is investigated under pseudo-physiological conditions by UV–Vis, fluorescence and FTIR spectroscopy. The intrinsic fluorescence of BSA was quenched by VB6, which was rationalized in terms of the static quenching mechanism. According to fluorescence quenching calculations, the bimolecular quenching constant (k_q), dynamic quenching (K_SV) and static quenching (K_LB) at 310 K were obtained. The efficiency of energy transfer and the distance between the donor (BSA) and the acceptor (VB6) were calculated by Foster’s non-radiative energy transfer theory and were equal to 41.1% and 2.11 nm.The collected UV–Vis and fluorescence spectra were combined into a row-and column-wise augmented matrix and resolved by multivariate curve resolution-alternating least squares (MCR-ALS). MCR-ALS helped to estimate the stoichiometry of interactions, concentration profiles and pure spectra for three species (BSA, VB6 and VB6-BSA complex) existed in the interaction procedure. Based on the MCR-ALS results, using mass balance equations, a model was developed and binding constant of complex was calculated using non-linear least squares curve fitting. FT-IR spectra showed that the conformation of proteins was altered in presence of VB6. Finally, the combined docking and molecular dynamics (MD) simulations were used to estimate the binding affinity of VB6 to BSA. Five-nanosecond MD simulations were performed on bovine serum albumin (BSA) to study the conformational features of its ligand binding site. From MD results, eleven BSA snapshots were extracted, at every 0.5 ns, to explore the binding affinity (GOLD score) of VB6 using a docking procedure. MD simulations indicated that there is a considerable flexibility in the structure of protein that affected ligand recognition. Structural analyses and docking simulations indicated that VB6 binds to site I and GOLD score values depend on the conformations of both BSA and ligand. Molecular modeling results showed that VB6–BSA complex formed not only on the basis of electrostatic forces, but also on the basis of π–π staking and hydrogen bond. There was an excellent agreement between the experimental and computational results. The results presented in this paper, will offer a reference for detailed and systematic studies on the biological effects and action mechanism of small molecules with proteins.     

Purification of 6-phosphogluconate dehydrogenase from chicken liver and investigation of some kinetic properties

6-phosphogluconate (6PG) dehydrogenase (EC 1.1.1.44; 6PGD) was purified from chicken liver; some kinetic and characteristic properties of the enzyme were investigated. The purification procedure consisted of four steps: preparation of the hemolysate, ammonium sulfate precipitation, 2',5'-ADP Sepharose 4B affinity chromatography, and Sephadex G-200 gel filtration chromatography. Thanks to the four consecutive procedures, product having a specific activity of 61 U (mg proteins)(-1), was purified 344-fold with a yield of 5.57%. Optimum pH, stable pH, optimum temperature, and KM and Vmax values for NADP+ and 6PG substrates were determined for the enzyme. Molecular weight of the enzyme was also determined by Sephadex G-200 gel filtration chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). In addition, Ki values and inhibition types were estimated by means of Lineweaver-Burk graphs obtained for NADPH and CO2 products.     

Halobetasol propionate-loaded solid lipid nanoparticles (SLN) for skin targeting by topical delivery

The clinical use of halobetasol propionate (HP) is related to some adverse effects like irritation, pruritus and stinging. The purpose of this work was to construct HP-loaded solid lipid nanoparticles (HP-SLN) formulation with skin targeting to minimizing the adverse side effects and providing a controlled release. HP-SLN were prepared by solvent injection method and formula was optimized by the application of 3² factorial design. The nanoparticulate dispersion was evaluated for particle size and entrapment efficiency (EE). Optimized batch was characterized for differential scanning calorimetry (DSC), scanning electron microscopy, X-ray diffraction study and finally incorporated into polymeric gels of carbopol for convenient application. The nanoparticulate gels were evaluated comparatively with the commercial product with respect to ex-vivo skin permeation and deposition study on human cadaver skins and finally skin irritation study. HP-SLN showed average size between 200?nm and 84–94% EE. DSC studies revealed no drug-excipient incompatibility and amorphous dispersed of HP in SLN. Ex vivo study of HP-SLN loaded gel exhibited prolonged drug release up to 12?h where as in vitro drug deposition and skin irritation studies showed that HP-SLN formulation can avoid the systemic uptake, better accumulative uptake of the drug and nonirritant to the skin compared to marketed formulation. These results indicate that the studied HP-SLN formulation represent a promising carrier for topical delivery of HP, having controlled drug release, and potential of skin targeting with no skin irritation.     

Ethosomes for skin delivery of ropivacaine: preparation,characterization and ex vivo penetration properties

Abstract

Ropivacaine, a novel long-acting local anesthetic, has been proved to own superior advantage. However, Naropin® Injection, the applied form in clinic, can cause patient non-convenience. The purpose of this study was to formulate ropivacaine (RPV) in ethosomes and evaluate the potential of ethosome formulation in delivering RPV transdermally. The RPV-loaded ethosomes were prepared with thin-film dispersion technique and the formulation was characterized in terms of size, zeta potential, differential scanning calorimetry (DSC) analysis and X-ray diffraction (XRD) study. The results showed that the optimized RPV-ethosomes displayed a typical lipid bilayer structure with a narrow size distribution of 73.86?±?2.40?nm and drug loading of 8.27?±?0.37%, EE of 68.92?±?0.29%. The results of DSC and XRD study indicated that RPV was in amorphous state when encapsulated into ethosomes. Furthermore, the results of ex vivo permeation study proved that RPV-ethosomes could promote the permeability in a high-efficient, rapid way (349.0?±?11.5?μg?cm^?2 at 12?h and 178.8?±?7.1?μg?cm^?2 at 0.5?h). The outcomes of histopathology study forecasted that the interaction between ethosomes and skin could loosen the tight conjugation of corneocyte layers and weaken the permeation barrier. In conclusion, RPV-ethosomes could be a promising delivery system to encapsulate RPV and deliver RPV for transdermal administration.     

Dietary verbascoside supplementation in donkeys: effects on milk fatty acid profile during lactation,and serum biochemical parameters and oxidative markers

Various uses of donkeys’ milk have been recently proposed for human consumption on the basis of its nutritional characteristics. Improvements in milk fatty acid profile and animal oxidative status can be induced through dietary supplementation of phenolic compounds. The study aimed to evaluate in donkeys the effects of dietary supplementation with verbascoside (VB) on: (i) the fatty acid profile and vitamins A and E contents of milk during a whole lactation, and (ii) blood biochemical parameters and markers of oxidative status of the animals. At foaling, 12 lactating jennies were subdivided into two groups (n 6): control, without VB supplement; VB, receiving a lipid-encapsulated VB supplement. Gross composition, fatty acid profile and vitamins A and E contents in milk were assessed monthly over the 6 months of lactation. Serum total cholesterol, high-density lipoproteins cholesterol and low-density lipoproteins cholesterol, tryglicerides, non-esterified fatty acid, bilirubin, alanine aminotransferase (ALT), aspartate aminotransferase, reactive oxygen metabolites, thiobarbituric acid reactive substances (TBARs), vitamin A and vitamin E were evaluated at 8 days after foaling (D0) and then at D90, D105 and D120 of lactation. In milk, the VB supplementation decreased the saturated fatty acids (P<0.05) and increased the monounsaturated fatty acids (P<0.05), and vitamins A and E (P<0.01) values. On the serum parameters, the VB supplementation decreased total cholesterol (P<0.01), tryglicerides, bilirubin, ALT and TBARs, and increased (P<0.01) vitamin E. In conclusion, the VB dietary supplementation affects the nutritional quality of donkey’s milk with a benefit on the oxidative status and serum lipidic profile of the animals.     

Superoxide dismutase entrapped in long-circulating liposomes: formulation design and therapeutic activity in rat adjuvant arthritis

The aim of this study was to investigate whether long-circulating liposomes can improve the anti-inflammatory activity of superoxide dismutase (SOD). Small-sized poly(ethyleneglycol) (PEG)-liposomes containing SOD were prepared via different preparation protocols and characterized in terms of encapsulation efficiency (EE), size, enzymatic activity and protein structure, to establish conditions where high EE can be combined with preservation of enzyme activity and structure. It was observed that structural information from circular dichroism analyses does not correlate with data on enzyme activity. SOD-containing PEG-liposomes prepared by the dehydration-rehydration method appeared to represent the most attractive formulation for in vivo evaluation. The therapeutic potential of selected SOD-containing PEG-liposomes was established and compared with SOD entrapped in stearylamine (SA)-liposomes and ‘free’ SOD upon intravenous (i.v.) injection in an arthritic rat model. Both small PEG-liposomes and SA-liposomes showed a superior therapeutic activity compared to ‘free’ SOD, with PEG-liposomes inducing stronger anti-inflammatory effects than SA-liposomes.     

Physiological comparisons between aquatic resistance training protocols with and without equipment

The purpose of the present study was to compare the physiological responses of oxygen uptake (VO(2)) and energy expenditure (EE) in two different aquatic resistance training protocols performed with three sets of 20 seconds (3 × 20) and six sets of 10 seconds (6 × 10) and with and without Speedo Resistance Equipment. Ten young healthy women volunteers, familiar with exercises in an aquatic environment, participated in this study. The four separate protocols were randomly selected and performed at a 48-hour interval by the same instructor. The total time of the 3 × 20 protocol was 34 minutes and that of the 6 × 10 protocol was 43 minutes, and all exercises were performed at maximal speed and amplitude. Although the protocols had different total times, they included one minute of stimulus per muscle group and the same time intervals. EE(gross) and EE(net) values were higher in the 6 × 10 protocol than in the 3 × 20 one. The variables VO(2) and EE(min) did not present significant difference between the protocols. VO(2), EE(gross), EE(net) and EE(min) values were higher when the equipment was used (W situation) than when it was not (WO situation). In the postexercise analysis, the W situation also showed higher VO(2) and EE(gross) values than the WO situation. Therefore, this study suggests the use of Speedo Resistance Equipment to increase VO(2) and EE, and it also suggests lengthier aquatic resistance training to obtain greater EE values per session.     

Kinetic properties of the glucose-6-phosphate and 6-phosphogluconate dehydrogenases from Corynebacterium glutamicum and their application for predicting pentose phosphate pathway flux in vivo.

The glucose-6-phosphate (Glc6P) and 6-phosphogluconate (6PG) dehydrogenases of the amino-acid-producing bacterium Corynebacterium glutamicum were purified to homogeneity and kinetically characterized. The Glc6P dehydrogenase was a heteromultimeric complex, which consists of Zwf and OpcA subunits. The product inhibition pattern of the Glc6P dehydrogenase was consistent with an ordered bi-bi mechanism. The 6PG dehydrogenase was found to operate according to a Theorell-Chance ordered bi-ter mechanism. Both enzymes were inhibited by NADPH and the 6PG dehydrogenase additionally by ATP, fructose 1,6-bisphosphate (Fru1,6P2), D-glyceraldehyde 3-phosphate (Gra3P), erythrose 4-phosphate and ribulose 5-phosphate (Rib5P). The inhibition by NADPH was considered to be most important, with inhibition constants of around 25 microM for both enzymes. Intracellular metabolite concentrations were determined in two isogenic strains of C. glutamicum with plasmid-encoded NAD- and NADP-dependent glutamate dehydrogenases. NADP+ and NADPH levels were between 130 microM and 290 microM, which is very much higher than the respective Km and Ki values. The Glc6P concentration was around 500 microM in both strains. The in vivo fluxes through the oxidative part of the pentose phosphate pathway calculated on the basis of intracellular metabolite concentrations and the kinetic constants of the purified enzymes determined in vitro were in agreement with the same fluxes determined by NMR after 13C-labelling. From the derived kinetic model thus validated, it is concluded that the oxidative pentose phosphate pathway in C. glutamicum is mainly regulated by the ratio of NADPH and NADP+ concentrations and the specific enzyme activities of both dehydrogenases.