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PubMed2005

Dehydrocrotonin and its beta-cyclodextrin complex: cytotoxicity in V79 fibroblasts and rat cultured hepatocytes.

Trans-dehydrocrotonin has antiulcerogenic and antitumor activities. A complex of beta-cyclodextrin with dehydrocrotonin was developed to improve the delivery of dehydrocrotonin. Complex in solid state was evaluated using X-ray diffraction (XRD), differential scanning calorimetry (DSC), thermal gravimetric analysis (TGA) and scanning electron microscopy (SEM). X-ray diffraction and scanning electron microscopy studies showed that dehydrocrotonin exists in a semicrystalline state in the complexed form with beta-cyclodextrin. Differential scanning calorimetry studies showed the existence of a complex of dehydrocrotonin with beta-cyclodextrin. The thermal gravimetric analysis studies confirmed the differential scanning calorimetry results of the complex. Free dehydrocrotonin and the dehydrocrotonin/beta-cyclodextrin inclusion complex were assayed in freshly isolated rat hepatocytes and in V79 cells. Cytotoxicity was determined using nucleic acid content, methylthiazoletetrazolium (MTT) reduction and neutral red uptake assays. In all assays, there was a large reduction (3.5-16.1-fold) in the cytotoxicity of dehydrocrotonin in hepatocytes when complexed with beta-cyclodextrin, whereas for V79 cells the decrease in cytotoxicity was 1.7- and 1.87-fold for MTT reduction and nucleic acid content assays, respectively. The lower cytotoxicity of the dehydrocrotonin/beta-cyclodextrin complex compared to free dehydrocrotonin in rat hepatocytes and V79 cells suggests that such a complex may be useful for the administration of dehydrocrotonin in vivo.

Animals↗
PubMed2006

Calorimetric investigation of protein/amino acid interactions in the solid state.

Possible protein/amino acid interactions and the physical states of amino acids after freeze-drying have been studied using isoperibol calorimetry and differential scanning calorimetry (DSC). Good linear correlations (R(2) = 0.99) between the enthalpies of solution and the percentage of antibody in all physical mixtures, as well as unchanging melting temperatures of amino acids for physical mixtures demonstrated that there is no interaction between the antibodies and amino acids studied upon physical mixing. On the other hand, positive deviations for antibody/histidine and antibody/arginine freeze-dried samples obtained from the isoperibol calorimetry results demonstrated that molecular level interactions, such as ion-dipole or electrostatic interactions or hydrogen bonding, occur between antibodies and histidine or arginine. The values of DeltaH(interaction) for antibody/histidine (1:1, w/w) and antibody/arginine (1:1, w/w) lyophilized samples were approximately 8 kJ/mol. These interactions were also confirmed by decreased and/or the disappearance of melting temperatures of the amino acids with DSC measurements. A negative deviation from linearity was detected for antibody/aspartic acid lyophilized samples which indicated partial amorphization of aspartic acid. No deviation from linearity as well as similar melting temperatures of antibody/glycine lyophilized samples indicated the absence of interactions between the antibodies and glycine upon freeze-drying.

Amino Acids↗
PubMed2005

Comparison of predictive equations for resting metabolic rate in healthy nonobese and obese adults: a systematic review.

BACKGROUND: An assessment of energy needs is a necessary component in the development and evaluation of a nutrition care plan. The metabolic rate can be measured or estimated by equations, but estimation is by far the more common method. However, predictive equations might generate errors large enough to impact outcome. Therefore, a systematic review of the literature was undertaken to document the accuracy of predictive equations preliminary to deciding on the imperative to measure metabolic rate. METHODS: As part of a larger project to determine the role of indirect calorimetry in clinical practice, an evidence team identified published articles that examined the validity of various predictive equations for resting metabolic rate (RMR) in nonobese and obese people and also in individuals of various ethnic and age groups. Articles were accepted based on defined criteria and abstracted using evidence analysis tools developed by the American Dietetic Association. Because these equations are applied by dietetics practitioners to individuals, a key inclusion criterion was research reports of individual data. The evidence was systematically evaluated, and a conclusion statement and grade were developed. RESULTS: Four prediction equations were identified as the most commonly used in clinical practice (Harris-Benedict, Mifflin-St Jeor, Owen, and World Health Organization/Food and Agriculture Organization/United Nations University [WHO/FAO/UNU]). Of these equations, the Mifflin-St Jeor equation was the most reliable, predicting RMR within 10% of measured in more nonobese and obese individuals than any other equation, and it also had the narrowest error range. No validation work concentrating on individual errors was found for the WHO/FAO/UNU equation. Older adults and US-residing ethnic minorities were underrepresented both in the development of predictive equations and in validation studies. CONCLUSIONS: The Mifflin-St Jeor equation is more likely than the other equations tested to estimate RMR to within 10% of that measured, but noteworthy errors and limitations exist when it is applied to individuals and possibly when it is generalized to certain age and ethnic groups. RMR estimation errors would be eliminated by valid measurement of RMR with indirect calorimetry, using an evidence-based protocol to minimize measurement error. The Expert Panel advises clinical judgment regarding when to accept estimated RMR using predictive equations in any given individual. Indirect calorimetry may be an important tool when, in the judgment of the clinician, the predictive methods fail an individual in a clinically relevant way. For members of groups that are greatly underrepresented by existing validation studies of predictive equations, a high level of suspicion regarding the accuracy of the equations is warranted.

Adolescent↗
PubMed2003

Metal ion binding in the active site of the junction-resolving enzyme T7 endonuclease I in the presence and in the absence of DNA.

Endonuclease I of bacteriophage T7 is a DNA junction-resolving enzyme. We have previously used crystallography to demonstrate the binding of two manganese ions into the active site that is formed by three carboxylate (Glu 20, Asp 55 and Glu 65) and a lysine residue (Lys 67). Endonuclease I is active in the presence of magnesium, manganese, iron (II) and cobalt (II) ions, weakly active in the presence of nickel, copper (II) and zinc ions, and completely inactive in the presence of calcium ions. However, using calorimetry, we have observed the binding of two calcium ions to the free enzyme in a manner very similar to the binding of manganese ions. In the presence of iron (II) ions, we have obtained a cleavage of the continuous strands of a junction bound by endonuclease I, at sites close to (but not identical with) enzyme-induced hydrolysis. The results suggest that this arises from attack by locally generated hydroxyl radicals, arising from iron (II) ions bound into the active site. This therefore provides an indirect way of examining metal ion binding in the enzyme-junction complex. Ion binding in free protein (by calorimetry) and the enzyme-junction complex (iron-induced cleavage) have been studied in series of active-site mutants. Both confirm the importance of the three carboxylate ligands, and the lack of a requirement for Lys67 for the ion binding. Calorimetry points to particularly critical role of Asp55, as mutation completely abolishes all binding of both manganese and calcium ions.

Bacteriophage T7↗
PubMed2004

Fasting breath hydrogen concentration in short bowel syndrome patients with colon incontinuity before and after antibiotic therapy.

OBJECTIVE: Nutrition success in short bowel syndrome (SBS) depends on the intake nutrients and the intestinal absorption capacity. An evaluation of energy expenditure and oxidation of substrate can be obtained with indirect calorimetry by measuring O(2) and CO(2) in the respiration. Elevated colonic fermentation can occur in SBS, producing H(2) and CO(2), which can also be eliminated through respiration and as a consequence affect the results from indirect calorimetry. The objective of this study was to determine the fasting breath H(2) concentration and alterations before and after antibiotic therapy in patients with severe SBS with colon in continuity. METHODS: The study was conducted in two phases. In phase 1, the fasting breath H(2) concentrations were measured in 10 patients with severe SBS with colon incontinuity and a control group of 10 healthy volunteers. In phase 2, the fasting breath H(2) concentrations were re-evaluated after treatment for 7 d with antibiotics in six patients with high rates of H(2). The analyses were performed with a gas chromatograph (microanalyzer DP; Quintron Instruments, Milwaukee, WI, USA), with results of breath hydrogen and methane concentration expressed in parts per million (ppm). RESULTS: In phase 1, the levels of fasting breath H(2) were higher in the patients with severe SBS with colon incontinuity than in the healthy controls (32.00 +/- 17.77 versus 5.30 +/- 3.31 ppm; P < 0.001), with 7 of 10 patients presenting levels of H(2) above the normal rate (12 ppm). The presence of an ileocecal valve did not modify the results significantly. In phase 2, all six patients treated with antibiotics presented normalization in the levels of fasting breath H(2) (from 43.50 +/- 6.90 ppm to 1.33 +/- 1.03 ppm; P < 0.001) and concomitant improvement in the gastrointestinal symptoms. CONCLUSIONS: In relation to the healthy controls, patients with SBS with colon incontinuity presented higher levels of fasting breath H(2). Antibiotic therapy normalized the levels of fasting breath H(2) and improved the gastrointestinal symptoms. We suggest that the breath H(2) test may be performed routinely in patients with SBS to diagnose elevated intestinal fermentation, prevent errors in the interpretation of the indirect calorimetry, and treat eventual associated gastrointestinal symptoms.

Adult↗
PubMed2004

Immunonutrition risk factors of respiratory complications after esophagectomy.

BACKGROUND: There are many reports suggesting predictive factors for respiratory complications after esophagectomy, but few studies have focused on this problem from the aspect of immunonutrition. METHODS: A series of 45 esophageal cancer patients who underwent potentially curative resection between April 1996 and March 2001 was enrolled in this study. Preoperative and intraoperative variables were analyzed for a correlation between risk factors and respiratory complications. In this study, immunonutrition variables as assessed by ultrasonography and indirect calorimetry were used. Uni- and multivariate analyses were performed to determine the predictive factors. RESULTS: Of 45 patients, nine patients (20%) developed respiratory complications. In univariate analysis, forced expiratory volume in 1 s per body surface area (m(2)) in preoperative respiratory parameters, respiratory quotient, and caloric contributions of fat (percentage) and carbohydrate according to indirect calorimetry, and serum CH50 level significantly influenced the occurrence of postoperative respiratory complications. In multivariate analysis, the caloric contribution of fat (percentage) by indirect calorimetry and serum alpha(1)-antitrypsin (serine protease inhibitor) independently affected the occurrence of respiratory complications. CONCLUSIONS: Preoperative excessive fat oxidation (potential starvation) and alpha(1)-antitrypsin were independent predictive factors for postoperative complications. Nutrition support such as enteral feeding to improve this malnourished state would reduce the incidence of postoperative respiratory complications.

Analysis of Variance↗
PubMed1997

Involvement of water molecules in the association of monoclonal antibody HyHEL-5 with bobwhite quail lysozyme.

Fluorescence polarization spectroscopy and isothermal titration calorimetry were used to study the influence of osmolytes on the association of the anti-hen egg lysozyme (HEL) monoclonal antibody HyHEL-5 with bobwhite quail lysozyme (BWQL). BWQL is an avian species variant with an Arg-->Lys mutation in the HyHEL-5 epitope, as well as three other mutations outside the HyHEL-5 structural epitope. This mutation decreases the equilibrium association constant of HyHEL-5 for BWQL by over 1000-fold as compared to HEL. The three-dimensional structure of this complex has been obtained recently. Fluorescein-labeled BWQL, obtained by labeling at pH 7.5 and purified by hydrophobic interaction chromatograpy, bound HyHEL-5 with an equilibrium association constant close to that determined for unlabeled BWQL by isothermal titration calorimetry. Fluorescence titration, stopped-flow kinetics, and isothermal titration calorimetry experiments using various concentrations of the osmolytes glycerol, ethylene glycol, and betaine to perturb binding gave a lower limit of the uptake of approximately 6-12 water molecules upon formation of the HyHEL-5/BWQL complex.

Animals↗
PubMed1997

Total energy expenditure in patients with small-cell lung cancer: results of a validated study using the bicarbonate-urea method.

The bicarbonate-urea method for measuring CO2 production was applied to eight free-living patients (mean age, 68 +/- 10 years; mean weight, 69 +/- 10 kg; mean height, 1.65 +/- 0.10 m) with unresectable small-cell lung cancer for a period of 1 day (n = 5) or 2 days (n = 3). The basal metabolic rate (BMR) was measured in all subjects. The technique was first validated against whole-body indirect calorimetry over an additional 24-hour period in five of these subjects. The bicarbonate-urea method predicted net CO2 production to be 102.1% +/- 3.4% of that measured by whole-body indirect calorimetry, and energy expenditure, 101.5% +/- 3.8% of the measured calorimeter value (8.1 +/- 1.6 MJ/d). The 24-hour recovery of label in CO2 excreted by the body was 95.6% +/- 0.5%. In free-living conditions, the bicarbonate-urea method predicted energy expenditure to be 9.0 +/- 2.6 MJ/d. BMR was elevated by a mean of 6% (P < .05) compared with the Schofield standards. The physical activity level ([PAL] the ratio of total energy expenditure [TEE] to BMR) was variable (1.15 to 1.87), but the mean value was only 1.36 +/- 0.22, considerably less than that of moderately active healthy subjects with estimated PAL values of 1.55 (P < .05) to 1.65 (P < .01) and the mean results obtained by doubly labeled water (previous studies) in healthy age- and sex-matched subjects. This is the first time a tracer method for measuring CO2 production and energy expenditure has been validated against whole-body 24-hour indirect calorimetry in patients with lung cancer or a systemic inflammatory reaction. The agreement between the two methods is similar to that observed in normal subjects. This is also the first time a tracer method has been used to measure energy expenditure in free-living patients with lung cancer. The results suggest that TEE and the energy requirements necessary to maintain energy balance were not increased despite basal hypermetabolism, because of the associated decrease in physical activity.

Adult↗
PubMedDate not supplied

Prospective randomized trial to assess caloric and protein needs of critically Ill, anuric, ventilated patients requiring continuous renal replacement therapy.

OBJECTIVES: We measured the energy and protein needs in 50 sequential, critically ill, ventilated patients requiring continuous renal replacement therapy (CRRT) for renal failure by using indirect calorimetry and three sequential isocaloric protein-feeding regimes of 1.5, 2.0, and 2.5 g. kg(-1). d(-1). We also assessed the compliance of actual feeding with target feeding and correlated the predictive energy requirements of the formulae with the actual energy expenditure (EE) measured by indirect calorimetry. We also determined whether these feeding regimes affected patient outcome. METHODS: The energy and protein needs of 50 consecutive, critically ill patients (31 male; age 53.3 +/- 17.4 y; Acute Physiology and Chronic Health Evaluation (APACHE II) score: 26.0 +/- 8.0; Acute Physiology and Chronic Health Evaluation score predicted risk of death: 50.0 +/- 25.0%) were assessed by using indirect calorimetry and ultrafiltrate nitrogen loss. Entry into this study was on commencement of CRRT. To eliminate any beneficial effect from the passage of time on nitrogen balance, 10 of the 50 patients were randomized to receive 2.0 g. kg(-1). d(-1) throughout the study, and the others received an escalating isocaloric feeding regime (1.5, 2.0, and 2.5 g. kg(-1). d(-1)) at 48-h intervals. Enteral feeding was preferred, but if this was not tolerated or unable to meet target, it was supplemented or replaced by a continuous infusion of total parenteral nutrition. Energy was given to meet caloric requirements as predicted by the Schofield equation corrected by stress factors or based on the metabolic cart readings of EE and was kept constant for all patients throughout the trial. Patients were stabilized on each feeding regime for at least 24 h before samples of dialysate were taken for nitrogen analysis at 8-h intervals on the second day. CRRT was performed by using a blood pump with a blood flow of 100 to 175 mL/min. Dialysate was pumped in and out counter-currently to the blood flow at 2 L/h. A biocompatible polyacrylonitrile hemofilter was used in all cases. RESULTS: EE was 2153 +/- 380 cal/d and increased by 56 +/- 24 cal/d (P < 0.0001) throughout the 6-d study period to 2431 +/- 498 cal/d. At study entry, the mean predicted (Schofield) caloric requirement was 2101 +/- 410. Patients received 99% of the predicted energy requirements. However, the mean EE was 11% higher at 2336 +/- 482 calories. This difference was not uniform. If the predicted caloric requirement was less than 2500, the EE exceeded the predicted by an average of 19%. If the predicted caloric requirement was greater than 2500, the EE on average was 6% less than predicted. This relation was significant (P = 0.025) and has not been described previously. Nitrogen balance was inversely related to EE (P = 0.05), positively related to protein intake (P = 0.0075), and more likely to be attained with protein intakes larger than 2 g. kg(-1). d(-1) (P = 0.0001). Nitrogen balance became positive in trial patients over time but were negative in control patients over time (P = 0.0001). Nitrogen balance was directly associated with hospital outcome (P = 0.03) and intensive care unit outcome (P = 0.02). For every 1-g/d increase in nitrogen balance, the probability of survival increased by 21% (P = 0.03; odds ratio, 1.211; 95% confidence limits, 1.017,1.443). Further, although enterally and parenterally fed patients had lower mortalities than predicted, the presence of enteral feeding, even after adjusting for predicted risk of death, had a statistically significant benefit to patient outcome (P = 0.04). CONCLUSIONS: This study found that a metabolic cart can improve the accuracy of energy provision and that a protein intake of 2.5 g. kg(-1). d(-1) in these patients increases the likelihood of achieving a positive nitrogen balance and improving survival. Enteral feeding is preferable, but if this is not possible or does not achieve the target, then it should be supplemented by parenteral feeding.

APACHE↗
PubMed1995

A calorimetric study of the thermal stability of barstar and its interaction with barnase.

The temperature-induced unfolding of single, double, and triple mutants of barstar, the specific intracellular protein inhibitor of barnase from Bacillus amyloliquefaciens, has been studied by high-sensitivity differential scanning calorimetry. The thermal unfolding of barstar mutants, where at least one of the two cysteine residues in the molecule had been replaced by alanine, follows a two-state mechanism at neutral and alkaline pH. The unfolding enthalpy and heat capacity changes are slightly lower than those accepted for highly compact, small, globular proteins. We have found that at pH 2.5, where barstar seems to be in a molten globule state, the protein has a heat capacity between that of the native and the unfolded states and shows some tendency for association. Scanning calorimetry experiments were also extended to the barstar--barnase complex in the neutral and alkaline pH range. The binding constants obtained from DSC studies are similar to those already obtained from other (kinetic) studies. The interaction of barstar and barnase was also investigated by isothermal calorimetry in various buffers within the pH range 6.0-10.0 and a temperature range of 15-35 degrees C. The favorable enthalpy contribution to the binding is about 4 times higher than the entropic one at 25 degrees C. The overall data analysis of the combined calorimetric results has led to the thermodynamic characterization of barstar unfolding and the interaction of barstar and barnase over a wide range of temperatures.

Bacterial Proteins↗
PubMed1985

1-Palmitoyl-2-pyrenedecanoyl glycerophospholipids as membrane probes: evidence for regular distribution in liquid-crystalline phosphatidylcholine bilayers.

We have synthesized 1-palmitoyl-2-pyrenedecanoyl-sn-glycero derivatives of 3-phosphatidylcholine, 3-phosphatidylethanolamine, 3-phosphatidylserine, 3-phosphatidylglycerol, 3-phosphatidylinositol, and 3-phosphatidic acid and investigated their behavior in monolayers and in neat and mixed bilayers. Fluorescence spectroscopy of neat pyrene phospholipid dispersions revealed a well-defined thermotropic transition at 13.5-19 degrees C depending on the polar head group. An endotherm coinciding with this transition was observed with differential scanning calorimetry, indicating it to be due to the melting of the lipid acyl chains. For pyrenephosphatidylethanolamine, the endotherm was observed at a much higher temperature (70 degrees C). Compression isotherms obtained at an argon/water interface revealed that the pyrene moiety somewhat increases the mean molecular area of a phospholipid molecule but does not prevent the expression of head-group-dependent packing behavior. Partition of the pyrene lipids between coexisting fluid and solid phases was investigated with fluorometry and calorimetry. Both techniques indicate that these lipids prefer the fluid phase and that this preference is independent of the head group. The rates and apparent activation energies of lateral diffusion in fluid bilayers were found to be similar for most pyrene lipids, suggesting that the lateral movement of phospholipids is not critically dependent on interactions at the head-group level. Lateral distribution of the pyrene lipids in gel and fluid phosphatidylcholine bilayers was studied with the excimer technique and calorimetry. In gel-state dipalmitoylphosphatidylcholine bilayers, the pyrene lipids form clusters. These clusters, however, do not consist of pure pyrene lipid but of aggregates (compounds) of the labeled and unlabeled lipid.(ABSTRACT TRUNCATED AT 250 WORDS)

Calorimetry, Differential Scanning↗
PubMed1987

Kinetics of the subtransition in dipalmitoylphosphatidylcholine.

The kinetics of the interconversions of the subgel and gel phases in dipalmitoylphosphatidylcholine have been studied by using differential dilatometry, differential scanning calorimetry (DSC), and neutral buoyancy centrifugation as a function of incubation temperature and deuteriation of the solvent. As seen by others, DSC scans show two peaks in the subgel transition region for incubation temperatures below 1 degree C. After incubation at 0.1 degree C, the DSC peak that occurs at the lower scanning temperature appears with an incubation half-time of 0.5 day and eventually converts into a peak at higher scanning temperature with an incubation half-time of 18 days. By varying the scanning rate, we show that these two peaks merge into one at slow scanning rates with a common equilibrium transition temperature of 13.8 degrees C, in agreement with equilibrium calorimetry and dilatometry (delta V = 0.017 +/- 0.001 mL/g). For incubation temperatures above 4.6 degrees C, only one peak appears in both scanning dilatometry and calorimetry. While the initial rate of subgel conversion is smaller at the higher incubation temperatures, after 300 h a higher percentage of the sample has converted to subgel than at the lower incubation temperatures. We suggest that higher incubation temperatures (near 5 degrees C) are preferable for forming the stable subgel phase, and we present a colliding domain picture that indicates why this may be so. Our results in D2O and the similarity of the kinetics of volume decrease with the kinetics of wide-angle diffraction lines also support the suggestion that the partial loss of interlamellar water plays a kinetic role in subgel formation.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1981

Calorimetric and fluorescence depolarization studies on the lipid phase transition of bacteriorhodopsin--dimyristoylphosphatidylcholine vesicles.

The thermotropic lipid phase transition of dimyristoylphosphatidylcholine vesicles reconstituted with bacteriorhodopsin was investigated as a function of the lipid to protein ratio by means of differential scanning calorimetry and fluorescence depolarization of the embedded probe 1,6-diphenyl-1,3,5-hexatriene. Two attractive features of this system are that the lipid phase transition induces lipid-protein segregation and that the state of aggregation of the protein is known. Above the lipid phase transition and above molar lipid to protein ratios of about 100, bacteriorhodopsin is monomeric. Well below the phase transition, bacteriorhodopsin is aggregated in a hexagonal protein lattice. With increasing amounts of incorporated bacteriorhodopsin, the calorimetric transition broadens, and a second component develops at a temperature which is lower than that of the unperturbed transition. The latter transition was assigned to the disaggregation of the bacteriorhodpsin lattice which occurs 6-7 degrees C below the phase transition of the protein-free lipids according to previous measurements. The van't Hoff enthalpy of the phase transition, as determined from the fluorescence depolarization of diphenylhexatriene, is in surprisingly good agreement with that obtained from differential scanning calorimetry over a wide range of lipid to protein ratios. The differential scanning calorimetry data can be simulated on the basis of a model which takes the protein segregation and crystallization specifically into account. The essential feature of this model is that calorimetrically detectable lipid melting occurs in the temperature region of the protein crystallization, since for the disaggregation of the close packed bacteriorhodopsin lattice additional lipids of an intermediate chain conformation are required.

Bacteriorhodopsins↗
PubMed2006

Thermodynamics of tryptophan-mediated activation of the trp RNA-binding attenuation protein.

The trp RNA-binding attenuation protein (TRAP) functions in many bacilli to control the expression of the tryptophan biosynthesis genes. Transcription of the trp operon is controlled by TRAP through an attenuation mechanism, in which competition between two alternative secondary-structural elements in the 5' leader sequence of the nascent mRNA is influenced by tryptophan-dependent binding of TRAP to the RNA. Previously, NMR studies of the undecamer (11-mer) suggested that tryptophan-dependent control of RNA binding by TRAP is accomplished through ligand-induced changes in protein dynamics. We now present further insights into this ligand-coupled event from hydrogen/deuterium (H/D) exchange analysis, differential scanning calorimetry (DSC), and isothermal titration calorimetry (ITC). Scanning calorimetry showed tryptophan dissociation to be independent of global protein unfolding, while analysis of the temperature dependence of the binding enthalpy by ITC revealed a negative heat capacity change larger than expected from surface burial, a hallmark of binding-coupled processes. Analysis of this excess heat capacity change using parameters derived from protein folding studies corresponds to the ordering of 17-24 residues per monomer of TRAP upon tryptophan binding. This result is in agreement with qualitative analysis of residue-specific broadening observed in TROSY NMR spectra of the 91 kDa oligomer. Implications for the mechanism of ligand-mediated TRAP activation through a shift in a preexisting conformational equilibrium and an induced-fit conformational change are discussed.

Allosteric Regulation↗
PubMed2003

Iminosugar glycosidase inhibitors: structural and thermodynamic dissection of the binding of isofagomine and 1-deoxynojirimycin to beta-glucosidases.

The design and synthesis of transition-state mimics reflects the growing need both to understand enzymatic catalysis and to influence strategies for therapeutic intervention. Iminosugars are among the most potent inhibitors of glycosidases. Here, the binding of 1-deoxynojirimycin and (+)-isofagomine to the "family GH-1" beta-glucosidase of Thermotoga maritima is investigated by kinetic analysis, isothermal titration calorimetry, and X-ray crystallography. The binding of both of these iminosugar inhibitors is driven by a large and favorable enthalpy. The greater inhibitory power of isofagomine, relative to 1-deoxynojirimycin, however, resides in its significantly more favorable entropy; indeed the differing thermodynamic signatures of these inhibitors are further highlighted by the markedly different heat capacity values for binding. The pH dependence of catalysis and of inhibition suggests that the inhibitory species are protonated inhibitors bound to enzymes whose acid/base and nucleophile are ionized, while calorimetry indicates that one proton is released from the enzyme upon binding at the pH optimum of catalysis (pH 5.8). Given that these results contradict earlier proposals that the binding of racemic isofagomine to sweet almond beta-glucosidase was entropically driven (Bülow, A. et al. J. Am. Chem. Soc. 2000, 122, 8567-8568), we reinvestigated the binding of 1-deoxynojirimycin and isofagomine to the sweet almond enzyme. Calorimetry confirms that the binding of isofagomine to sweet almond beta-glucosidases is, as observed for the T. maritima enzyme, driven by a large favorable enthalpy. The crystallographic structures of the native T. maritima beta-glucosidase, and its complexes with isofagomine and 1-deoxynojirimycin, all at approximately 2.1 A resolution, reveal that additional ordering of bound solvent may present an entropic penalty to 1-deoxynojirimycin binding that does not penalize isofagomine.

1-Deoxynojirimycin↗
PubMed2006

Thermodynamic insights into the binding of Triton X-100 to globular proteins: a calorimetric and spectroscopic investigation.

The interaction of the nonionic surfactant Triton X-100 (TX-100) with two proteins (bovine serum albumin (BSA) and alpha-lactalbumin (alpha-LA)) has been investigated by using a combination of differential scanning calorimetry, isothermal titration calorimetry, and fluorescence and circular dichroism spectroscopies. All of the calorimetric transitions in BSA were partially reversible, while being two-state and reversible in the case of alpha-LA. TX-100 molecules do not reduce the thermal stability of the protein in the monomeric form. However, in the micellar form the protein might become thermally destabilized by the micelles depending upon the nature of the protein. Isothermal titration calorimetry has been used to demonstrate that TX-100 binds to BSA at two sets of sites with 4:1 stoichiometry in each case. The van't Hoff enthalpy calculated from the temperature dependence of the binding constant did not match with the calorimetric enthalpy indicating conformational change in the protein upon surfactant binding. The surfactant binds to alpha-LA with one class of binding site, and the thermal unfolding results indicate it to be a stronger destabilizer than BSA. The fluorescence, circular dichroism, and differential scanning calorimetric results corroborate well with each other. The effect of ionic strength on the binding parameters suggests that TX-100 can bind to the protein surface via both hydrophobic and polar interactions depending upon the nature of the protein. The physical chemistry underlying the interactions between TX-100 and proteins has been presented. The mode of interaction of TX-100 with proteins is via direct binding, which has been discussed quantitatively in this work.

Calorimetry, Differential Scanning↗
PubMed2002

Resting energy expenditure in young patients with cystic fibrosis receiving antibiotic therapy for acute respiratory exacerbations.

BACKGROUND: patients with cystic fibrosis commonly have severe malnutrition and growth retardation. Among possible causes of these manifestations are low caloric intake, loss of nutrients and increased resting energy expenditure. This study was designed to assess the influence of antibiotic therapy for infectious exacerbations on resting energy expenditure in young patients with cystic fibrosis. METHODS: We studied 17 patients with cystic fibrosis (mean age, 13.6 years). All were hospitalized to receive intravenous antibiotic therapy (mean duration, 2 weeks) for acute respiratory exacerbations. At the beginning of therapy and after it ended, all patients underwent blood chemical tests, anthropometrical measures, determination of body composition by bioelectrical impedance, spirometry, and indirect calorimetry. RESULTS: Antibiotic therapy led to a significant improvement in biochemical, spirometric variables and in estimated calorimetry measurements expressed in relation to fat-free mass. These findings suggest that infective exacerbations are among the causes of increased resting energy expenditure in young patients with cystic fibrosis. CONCLUSIONS: Indirect calorimetry may prove useful in the diagnosis of infective exacerbations and in monitoring the effect of antibiotic therapy in patients with cystic fibrosis.

Adolescent↗
PubMed1998

Evaluation of a commercial accelerometer (Tritrac-R3 D) to measure energy expenditure during ambulation.

This study evaluated the ability of a commercially available accelerometer (Tritrac-R3 D) to measure energy expenditure in 16 subjects at rest (pre- and post-exercise) and during three different intensities of steady-state exercise (40-70% of peak oxygen consumption [VO2peak]) while ambulating on a treadmill (no grade). Oxygen consumption and the respiratory exchange ratio from indirect calorimetry and the vector magnitude of triaxial accelerations were used to estimate energy expenditure using the manufacturers' equations. There was a significant relationship between indirect calorimetry-derived energy expenditure and the energy expenditure derived from the accelerometer (r=0.96). Using analysis of variance, there was no difference in the energy expenditure derived by the two methods at rest before exercise and during the three different intensities of ambulatory exercise. There was a significant difference between energy expenditure derived via indirect calorimetry and with the accelerometer during rest after exercise, probably due to the failure of the accelerometer to accurately estimate the energy expenditure associated with the progressive decline in post-exercise oxygen consumption. Thus, this commercially available accelerometer appears to provide statistically acceptable estimates of energy expenditure at rest and during zero-grade treadmill ambulation up to about 70% VO2peak. This may indicate its acceptable utility for large-scale population studies of physical activity involving this mode of movement. The failure of the accelerometer to accurately estimate energy expenditure during recovery from exercise may contribute to an underestimation of energy expenditure in some physically active individuals.

Adult↗