Thermott: A comprehensive online tool for protein–ligand binding constant determination

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Molecular interaction of imino sugars with human α-galactosidase: Insight into the mechanism of complex formation and pharmacological chaperone action in Fabry disease

Enzyme enhancement therapy (EET) for Fabry disease involving imino sugars has been developed and attracted interest. It is thought that imino sugars act as pharmacological chaperones for wild-type and mutant α-galactosidases (GLAs) in cells, but the mechanisms underlying the molecular interactions between the imino sugars and the enzyme have not been clarified yet. We examined various kinds of imino sugars and found that galactostatin bisulfite (GBS) inhibited GLA in vitro and increased the enzyme activity in cultured Fabry fibroblasts as in the case of 1-deoxygalactonojirimycin (DGJ). Then, we analyzed the molecular interactions between the imino sugars and recombinant human GLA by means of isothermal titration calorimetry and surface plasmon resonance biosensor assays, and first determined the thermodynamic and binding-kinetics parameters of imino sugar and GLA complex formation. The results revealed that DGJ bound to the enzyme more strongly than GBS, the binding of DGJ to the enzyme protein being enthalpy-driven. In the case of GBS, the reaction was mainly enthalpy-driven, but there was a possibility that entropy-driven factors were involved in the binding. Structural analysis in silico revealed that both the chemicals fit into the active-site pocket and undergo hydrogen bonding with residues comprising the active-site pocket including the catalytic ones. The side chain of GBS was oriented towards the entrance of the active-site pocket, and thus it could be in contact with residues comprising the wall of the active-site pocket. Thermodynamic, kinetic and structural studies should provide us with a lot of information for improving EET for Fabry disease.     

Resting energy expenditure in patients with alcoholic chronic pancreatitis

The aim of this study was to assess resting energy expenditure in patients with chronic pancreatitis; 33 patients with alcohol-related chronic pancreatitis (group 1: 13 normal weight, group 2: 20 underweight) and 11 undernourished patients without identifiable disease (group 3) were studied. Body composition was determined by bioelectric impedance analysis and energy expenditure by indirect calorimetry. The percentage of body weight occupied by fat-free mass was similar among the three groups (76.4±1.5%, 78.6±1.3% and 76.8±2.1% for groups 1, 2, and 3, respectively). The measured resting energy expenditure (REE) was higher than the predicted EE (Harris and Benedict formula and Cunningham's equation) for the underweight patients with chronic pancreatitis (group 2) (P<0.05), but not for the two other groups. According to Cunningham's equation, 65% of the group 2 patients were hypermetabolic (REE>110% of predicted EE) versus 23.1% and 20% in groups 1 and 3. When adjusted for fat free mass, REE was significantly (P<0.01) higher in group 2 (35.0±0.9 kcal/kg/24 hr) than in the other two groups (30.1±0.7 kcal/kg/24 hr and 30.8±1.4 kcal/kg/24 hr in groups 1 and 3, respectively). During chronic pancreatitis, weight loss is accompanied by hypermetabolism, which should be taken into consideration during nutritional support.     

Reorganization of Lamellar Diblock Copolymer Poly(ε‐caprolactone)‐block‐poly(4‐vinylpyridine) in the Melting Temperature Range

The reorganization kinetics of the “original” lamellar diblock copolymer poly(ε‐caprolactone)‐block‐poly(4‐vinylpyridine) crystals formed at 260 K is studied in the melting region from 270 K (10 K below the onset of the melting peak of original crystals) to 310 K (the melting peak temperature) on the time scale starting from 10^?4 to 10² s by ultrafast differential scanning calorimetry. Different reorganization pathways are observed in this temperature range. Annealing at temperatures below 295 K leads to further stabilization of original crystals by secondary crystallization. At annealing temperatures higher than 295 K, crystals partially melt and the reorganization occurs via the melting–recrystallization. For even higher temperature, such as 310 K, the melting is completed within a few milliseconds and recrystallization starts from the nuclei formation. The sigmoidal recrystallization kinetics is analyzed by the Avrami equation. It is found that the copolymer experiences about one order of magnitude slower recrystallization rate and has higher melting peak temperatures of crystals formed after recrystallization than the homopolymer. The slower recrystallization kinetics in the copolymer is discussed from the viewpoint of the nanoscale spatial constraint and the intermediate state prior to the recrystallization.

     

Calendering as a direct shaping tool for the continuous production of fixed-dose combination products via co-extrusion

In this study calendering is used as a downstream technique to shape monolithic co-extruded fixed-dose combination products in a continuous way. Co-extrudates with a metoprolol tartrate-loaded sustained-release core and a hydrochlorothiazide-loaded immediate-release coat were produced and immediately shaped into a monolithic drug delivery system via calendering, using chilled rolls with tablet-shaped cavities. In vitro metoprolol tartrate release from the ethylcellulose core of the calendered tablets was prolonged in comparison with the sustained release of a multiparticulate dosage form, prepared manually by cutting co-extrudates into mini-matrices. Analysis of the dosage forms using X-ray micro-computed tomography only detected small differences between the pore structure of the core of the calendered tablet and the mini-matrices. Diffusion path length was shown to be the main mechanism behind the release kinetics. Terahertz pulsed imaging visualized that adhesion between the core and coat of the calendered tablet was not complete and a gradient in coat thickness (varying from 200 to 600 μm) was observed. Modulated differential scanning calorimetry and X-ray diffraction indicated that the solid-state properties of both drugs were not affected by the calendering procedure.     

Twenty-four-hour energy expenditure in Pima Indians with Type 2 (non-insulin-dependent) diabetes mellitus

Summary To assess the impact of Type 2 (non-insulin-dependent) diabetes mellitus on energy metabolism, 24-h energy expenditure, basal metabolic rate and sleeping metabolic rate were measured in a respiratory chamber in 151 Pima Indians, 102 with normal glucose tolerance (67 male/35 female, (mean ± SD) 28±7 years, 99±24 kg, 32±9% body fat) and in 49 with Type 2 diabetes (22 male/27 female, 35±11 years, 107±33 kg, 39±7% body fat), after at least 3 days on a weight maintaining diet. After adjustment for differences in fat-free mass, fat mass, age and sex, 24-h energy expenditure, basal metabolic rate and sleeping metabolic rate were significantly higher in diabetic patients than in control subjects (72 kcal/day, p<0.05; 99 kcal/day, p<0.005; 99 kcal/day, p<0.001 respectively). Spontaneous physical activity was similar in both groups whereas the thermic effect of food, calculated as the mean energy expenditure corrected for activity throughout the day above sleeping metabolic rate and expressed as a percentage of energy intake, was significantly lower in Type 2 diabetic patients (17.1±7.1 vs 19.8±5.6%, p<0.05). Adjusted values of 24-h energy expenditure, basal metabolic rate and sleeping metabolic rate were correlated with hepatic endogenous glucose production (r=0.22, p<0.05; r=0.22, p<0.05; r=0.31, p<0.01 respectively). Therefore, increased basal and sleeping metabolic rates, resulting in increased 24-h sedentary energy expenditure may play a role in the weight loss so often observed in Type 2 diabetic subjects in addition to the energy loss from glycosuria.     

Development of a loop-mediated isothermal amplification assay for the detection of porcine hokovirus

Hokoviruses have recently been detected as pathogens belonging to the family Parvoviridae, which comprises porcine hokovirus (PHoV) and bovine hokovirus (BHoV). In this study, we developed a loop-mediated isothermal amplification (LAMP) assay for the rapid, specific and sensitive detection of PHoV. A set of four primers specific for six regions within the PHoV VP1/2 genes was designed using online software. The reaction temperature and time were optimized at 65 °C and 60 min, respectively. LAMP products were detected by agarose gel electrophoresis or by visual inspection of a color change caused by a fluorescent dye. The method was highly specific for PHoV, and no cross-reaction was observed with porcine circovirus type 2 (PCV2), porcine parvovirus (PPV), porcine bocavirus (PBoV), pseudorabies virus (PRV), porcine reproductive and respiratory syndrome virus (PRRSV), classic swine fever virus (CSFV), or Japanese encephalitis virus (JEV). The detection limit was approximately 10 copies per reaction, which was 10 times more sensitive than conventional PCR. Furthermore, the efficiency of detection of PHoV in clinical samples was comparable to that of PCR and sequencing. These results show that the LAMP assay is a simple, rapid, sensitive and specific method for detecting PHoV. It does not require specialized equipment and can be used to detect PHoV both in the laboratory and in the field.     

Study of the water structure in poly(methyl methacrylate-block-2-hydroxyethyl methacrylate) and its relationship to platelet adhesion on the copolymer surface

The water structure and platelet compatibility of poly(methyl methacrylate (MMA)-block-2-hydroxyethyl methacrylate (HEMA)) were investigated. The molecular weight (Mn) of the polyHEMA segment was kept constant (average: 9600), while the Mn of the polyMMA segment was varied from 1340 to 7390. The equilibrium water content of the copolymers was found to be mainly governed by the HEMA content. The water structure in the copolymers was characterized in terms of the amounts of non-freezing and freezing water (abbreviated as W_nf and W_fz, respectively) using differential scanning calorimetry. It was found that the W_nf for the copolymers were higher than those estimated from the W_nf for the HEMA and MMA homopolymers and that the amount of excess non-freezing water depended on the polyMMA segment length. In addition, X-ray diffraction analysis revealed that some of the copolymers had cold-crystallizable water. These facts suggested that the polyMMA segments were involved in determining the water structures in the copolymers. Furthermore, the platelet compatibility of the copolymers was improved as compared to that of the HEMA homopolymer. It was therefore concluded that the platelet compatibility of the copolymer was related to the amount of excess non-freezing water.     

Disposable devices for the dispensing of oral drops. Study of photoprotectiveness and compatibility

in the first part of this article calorimetric studies on poly (hydroxy ethyl methacrylate) (PHEMA) are presented. In the past the irregular melting curves in this type of experiment have been interpreted as evidence for the existence of different types of water in these gels. The studies presented here demonstrate that the occurrence of a glass transition in the freezing hydrogels may be responsible for this irregular melting behavior, and that this behavior is not (necessarily) an indication for the existence of different types of water. In the second part results are shown of measurements of the mobility of water in hydrogels, made by relaxation NMR. These results indicate that very rapid interchange occurs between the water molecules, and they support the conclusion that the calorimetric data mentioned above are not indicative for the existence of different classes of water in hydrogels. These results are compared with data from other fields of science, especially from fundamental freeze drying studies, which support the alternative interpretation of the calorimetric measurements.