Influence of the conformational flexibility on the kinetics and dimerisation process of two Candida rugosa lipase isoenzymes. (1/27)

We have investigated the interfacial activation process of two isoenzymes from Candida rugosa (Lip1 and Lip3) using triacetin as substrate. Kinetics were coupled to inhibition experiments in order to analyse the transition between the open and closed conformers. This process was slow, particularly for Lip1, in the absence of an interface provided by the substrate or a detergent. Dimers of Lip3 were also purified and their catalytic action was closer to that of a typical esterase. In spite of the high sequence homology between Lip1 and Lip3, small changes enhance hydrophobicity in the binding pocket of Lip3 and increase the flexibility of its flap. We postulated that these factors account for the higher tendency of Lip3 to dimerise fixing its open conformation.  (+info)

Total parenteral nutrition with short- and long-chain triglycerides: triacetin improves nitrogen balance in rats. (2/27)

Little is known about the long-term metabolic effects of parenteral administration of short-chain triglycerides. These studies were undertaken to investigate triacetin, the water-soluble triglyceride of acetate when it is incorporated into nutritionally balanced total parenteral nutrition formulas. Male Sprague-Dawley rats (n = 22) were fed an isovolemic, isocaloric and isonitrogenous diet for 7 d. The lipid energy represented 30% of the nonprotein energy with short-chain triglycerides representing 0, 50 or 90% of the lipid energy. Plasma acetate concentration was determined as well as indicators of protein metabolism: daily and cumulative nitrogen balance, whole body leucine kinetics and rectus muscle and liver fractional protein synthetic rates. No overt toxic effects were observed at any point during the study. As the proportion of short-chain triglycerides in the diet increased from 0 to 50 or 90% of the lipid energy, cumulative nitrogen balance increased 50 or 120%, respectively (P less than 0.05). Whole-body and tissue leucine kinetics (determined during the last 2.5 h of the 7-d study) were unaffected by the lipid composition of the diet. Plasma acetate concentration was not significantly different among groups. These results indicate that incorporation of the short-chain triglyceride, triacetin, in nutritionally balanced total parenteral nutrition formulas improves nitrogen balance with no overt toxic effects. These data indicate that triacetin may have a future role as a parenteral nutrient, and that further studies of its use are warranted.  (+info)

Fluorescence spectroscopic characterization of Humicola lanuginosa lipase dissolved in its substrate. (3/27)

The conformational dynamics of Humicola lanuginosa lipases (HLL) and its three mutants were investigated by steady state and time-resolved fluorescence spectroscopy in two different media, aqueous buffer and the substrate triacetin. The fluorescence of the four Trps of the wild-type HLL (wt) reports on the global changes of the whole lipase molecule. In order to monitor conformational changes specifically in the alpha-helical surface loop, the so-called 'lid' of HLL comprised of residues 86-93, the single Trp mutant W89m (W117F, W221H, W260H) was employed. Mutants W89L and W89mN33Q (W117F, W221H, W260H, N33Q) were used to survey the impact of Trp89 and mannose residues, respectively. Based on the data obtained, the following conclusions can be drawn. (i) HLL adapts the 'open' conformation in triacetin, with the alpha-helical surface loop moving so as to expose the active site. (ii) Trp89 contained in the lid plays an unprecedently important role in the structural stability of HLL. (iii) In triacetin, but not in the buffer, the motion of the Trp89 side chain becomes distinguishable from the motion of the lid. (iv) The carbohydrate moiety at Asn33 has only minor effects on the dynamics of Trp89 in the lid as judged from the fluorescence characteristics of the latter residue.  (+info)

Parenteral nutrition with short- and long-chain triglycerides: triacetin reduces atrophy of small and large bowel mucosa and improves protein metabolism in burned rats. (4/27)

The effect of total parenteral nutrition with combinations of long-chain triglycerides (LCTs) and triacetin, the water-soluble triglyceride of acetate, on structural components of the gastrointestinal tract and protein metabolism was assessed in burned (30% body surface area) rats. Rats received isovolemic, isocaloric, and isonitrogenous diets that delivered 672 kJ.kg-1.d-1 (160 kcal.kg-1.d-1), 9.6 g amino acids.kg-1.d-1, and 30% nonprotein calories as 90% triacetin/10% LCTs, 50% triacetin/50% LCTs, or 100% LCTs for 7 d. Daily and cumulative nitrogen balances and whole-body leucine kinetics and fractional protein synthetic rates in rectus muscle and liver were determined on the last day of nutrition. DNA, protein, and total weight were determined in mucosal scrapings from segments of jejunum and colon. Plasma acetate concentrations were substantially higher in both triacetin groups. Parenteral nutrition with 50% triacetin and 50% LCTs promoted a positive nitrogen balance similar to that of 100% LCTs, increased protein in rectus muscle and liver, smaller and more numerous mucosal cells in jejunum and colon, and increased colonic mucosal weight compared with the other groups. Triacetin did not appreciably affect whole-body and tissue leucine kinetics. The equicaloric provision of triacetin and LCTs improved protein utilization and structural components of the small and large bowel and reduced the development of intestinal mucosal atrophy associated with conventional parenteral nutrition in burn injury.  (+info)

Cell surface expression of bacterial esterase A by Saccharomyces cerevisiae and its enhancement by constitutive activation of the cellular unfolded protein response. (5/27)

Yeast cell surface display is a powerful tool for expression and immobilization of biocatalytically active proteins on a unicellular eukaryote. Here bacterial carboxylesterase EstA from Burkholderia gladioli was covalently anchored into the cell wall of Saccharomyces cerevisiae by in-frame fusion to the endogenous yeast proteins Kre1p, Cwp2p, and Flo1p. When p-nitrophenyl acetate was used as a substrate, the esterase specific activities of yeast expressing the protein fusions were 103 mU mg(-1) protein for Kre1/EstA/Cwp2p and 72 mU mg(-1) protein for Kre1/EstA/Flo1p. In vivo cell wall targeting was confirmed by esterase solubilization after laminarinase treatment and immunofluorescence microscopy. EstA expression resulted in cell wall-associated esterase activities of 2.72 U mg(-1) protein for Kre1/EstA/Cwp2p and 1.27 U mg(-1) protein for Kre1/EstA/Flo1p. Furthermore, esterase display on the yeast cell surface enabled the cells to effectively grow on the esterase-dependent carbon source glycerol triacetate (Triacetin). In the case of Kre1/EstA/Flo1p, in vivo maturation within the yeast secretory pathway and final incorporation into the wall were further enhanced when there was constitutive activation of the unfolded protein response pathway. Our results demonstrate that esterase cell surface display in yeast, which, as shown here, is remarkably more effective than EstA surface display in Escherichia coli, can be further optimized by activating the protein folding machinery in the eukaryotic secretion pathway.  (+info)

Experimental short-bowel syndrome: effect of an elemental diet supplemented with short-chain triglycerides. (6/27)

To determine whether short-chain triglycerides (SCTs, 1:1 triacetin:tributyrin, wt:wt) enhance intestinal adaptation in short-bowel syndrome (SBS), male Sprague-Dawley rats underwent 60% distal small-bowel resection with cecectomy and received either a chemically defined diet (CD) or a CD containing 40% of nonprotein energy as either medium-chain triglycerides (MCTs) or SCTs. After 12 d the SCT group had significantly increased jejunal mucosal weight compared with the MCT and CD groups and had significantly increased segment weight and mucosal protein compared with the CD group. In the colon the SCT group had significantly increased segment and mucosal weights and mucosal protein and DNA compared with both the MCT and CD groups. Body-weight change and measurements of serum ketones, albumin, glucose, and triglycerides revealed no significant differences among groups. SCTs improved jejunal and colonic adaptive growth and maintained comparable nutritional status in SBS when compared with CD alone or CD with MCTs.  (+info)

Formulation design of taste-masked particles, including famotidine, for an oral fast-disintegrating dosage form. (7/27)

In this study, the taste-masking of famotidine, which could apply to any fast-disintegrating tablet, was investigated using the spray-dry method. The target characteristics of taste-masked particles were set as follows: the dissolution rate is not to be more than 30% at 1 min and not less than 85% at 15 min, and the particle size is not to be more than 150 microm in diameter to avoid a gritty feeling in the mouth. The target dissolution profiles of spray-dried particles consisting of Aquacoat ECD30 and Eudragit NE30D or triacetin was accomplished by the screening of formulas and the appropriate lab-scale manufacturing conditions. Lab-scale testing produced taste-masked particles that met the formulation targets. On the pilot scale, spray-dried particles with attributes, such as dissolution rate and particle size, of the same quality were produced, and reproducibility was also confirmed. This confirmed that the spray-dry method produced the most appropriate taste-masked particles for fast-disintegrating dosage forms.  (+info)

In vitro and in vivo transdermal studies of atenolol using iontophoresis. (8/27)

Matrix formulations of Eudragit E 100: NE 40D polymers (100:0, 70:30, 60:40, 50:50% w/w) with 20% w/w of triacetine and 5% w/w of atenolol were prepared by film casting method with different solvents (methanol, 2-propanol and acetone). In vitro release of atenolol from the films were studied by vertical Franz diffusion cells in HEPES buffer (pH 7.4) for 78 h. Direct currents of 0.1 and 0.5 mA/cm2 were applied for 6 h to the formulations with Ag/AgCl electrodes. Also, transdermal application for the Eudragit E 100: NE 40 D (70:30% w/w) formulation was compared by iontophoresis or oleic acid (2.5% w/v) with control group on Wistar rats. As a result, the in vitro release rate of atenolol from films were increased with iontophoresis by increasing the current density (from 0.240 to 0.424 mg/cm2 for 70:3% w/w formulation) and also increased with the amount of Eudragit NE 40D (from 0.646 to 1.30 mg/cm2 at the end of 78 h). It is obtained from the in vivo studies that oleic acid provided a higher plasma and skin concentration (0.825 mg/mL and 12.5 mg/cm2, respectively) than iontophoresis treatment (0.399 mg/mL and 1.81 mg/cm2, respectively) due to the different mechanisms. However, the results showed that iontophoresis is a good alternative for enhancing the transdermal delivery of atenolol.  (+info)

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