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PubMed1994

Presence of gonadotropin-releasing hormone (GnRH) binding sites and compounds with GnRH-like activity in the ovary of African catfish, Clarias gariepinus.

GnRH binding was characterized in the African catfish ovary by use of an analog of salmon GnRH (sGnRH- ; [D-Arg6, Trp7, Leu8, Pro9-NEt]-GnRH) as a labeled ligand. Binding of sGnRH-A to catfish ovarian membrane preparation was found to be saturable, displaceable, reversible, and dependent on time, temperature, and tissue concentration. Optimal binding was achieved after 70 min of incubation at room temperature (approximately 22 degrees C) at pH 7.6. Addition of unlabeled sGnRH-A displaced the bound 125I-sGnRH-A in a dose-related manner. Hill plot as well as Scatchard analysis indicated the presence of one class of high-affinity binding sites with a equilibrium dissociation constant (Kd) of 0.27 +/- 0.036 nM. Bound 125I-sGnRH-A was also found to be displaceable by catfish GnRH (cfGnRH; [His5, Leu7, Asn8]-GnRH), chicken GnRH-II (cGnRH-II; [His5, Trp7, Tyr8]-GnRH), and salmon GnRH (sGnRH; [Trp7, Leu8]-GnRH); all the peptides were found to bind with lower affinities than sGnRH-A to the catfish ovarian GnRH binding sites. Further experiments using ovarian extracts indicated the presence of compounds with GnRH-like activity in the ovary of African catfish. The crude ovarian extract was found to stimulate pituitary gonadotropin release from goldfish pituitary, as well as displacing 125I-sGnRH-A binding in the catfish ovary. HPLC analysis of the catfish ovarian extract revealed the presence of two fractions that bind specifically to the catfish ovary and release gonadotropin from cultured goldfish pituitary. These fractions include an early eluting peak that does not correspond with the retention time of known GnRH forms in addition to a fraction that co-elutes with the mammalian GnRH. Overall, the study provided characterization of GnRH binding sites in the catfish ovary, and evidence for the presence of compounds with GnRH-like activity in the catfish ovary.

Animals↗
PubMed1996

Direct relaxant effects of intravenous anesthetics on airway smooth muscle.

Ketamine, at concentrations achieved with the usual clinical doses, has a direct relaxant effect on airway smooth muscle (ASM). This study investigates the dose-dependent direct relaxation effects of midazolam and propofol on both proximal and distal ASM compared with ketamine. The proximal and distal airways were dissected from eight mongrel dogs and cut into 2-mm rings. The rings were attached to pressure transducers and equilibrated in a Krebs-Ringer bicarbonate bath kept at 37 degrees C, pH 7.4, CO2 37 mm Hg, and PaO2 > 100 mm Hg. Optimal length was determined, a dose-response curve to acetylcholine was established, and the 50% effective dose (ED50) of acetylcholine was calculated. Ketamine, midazolam, or propofol were given in random order to each ring preconstricted with ED50 of acetylcholine in cumulative log incremental doses from 10(-6) to 10(-4) M. Relaxation response was the tension during anesthetic equilibrium, expressed as a percentage of the tension from ED50 of acetylcholine. The drug vehicles were tested for their effects on the ASM. No bronchorelaxation was seen with any of the intravenous anesthetics at 10(-6) M. Ketamine 10(-5) M produced at 17.9% +/- 2.1% relaxation in the distal ASM but had no effect on the proximal ASM. Neither propofol nor midazolam affected the ASM at 10(-5) M. The distal ASM was significantly (P < 0.005) more sensitive to 10(-4) M of all three drugs compared with the proximal ASM. In the proximal ASM, 10(-4) M of ketamine, midazolam and propofol reduced ASM tension by 14.9% +/- 4.4%, 19.0% +/-8.8%, and 14.7% +/- 5.5%, respectively, versus 36.4% +/- 3.2%, 58.6% +/- 6.1%, and 64.4% +/- 9.0% in the distal ASM. The drug vehicles had no effect on the ASM. We conclude that ketamine, midazolam, and propofol have direct relaxant effects on ASM. All three intravenous anesthetics have a greater direct relaxant effect on distal ASM than on proximal ASM. Only ketamine showed significant direct bronchorelaxing effects at concentrations that are likely to be achieved with the usual clinical dosing patterns.

Acetylcholine↗
PubMed1989

Fusicoccin-Binding Proteins in Arabidopsis thaliana (L.) Heynh. : Characterization, Solubilization, and Photoaffinity Labeling.

Using the novel radioligand, [(3)H]-9'-nor-fusicoccin-8'-alcohol, high affinity binding sites for fusicoccin were characterized in preparations from leaves of Arabidopsis thaliana (L.) Heynh. The binding site copartitioned with the plasmalemma marker, vanadate-sensitive K(+), Mg(2+)-ATPase, when microsomal fractions were further purified by aqueous two-phase partitioning in polyethylene glycol-dextran phase systems and sedimented at an equilibrium density of 1.17 grams per cubic centimeter in continuous sucrose density gradients, as did the ATPase marker. The binding of [(3)H]-9'-nor-fusicoccin-8'-alcohol was saturable and Scatchard analysis revealed a biphasic plot with two apparent dissociation constants (K(D)), K(D1) = 1.5 nanomolar and K(D2) = 42 nanomolar, for the radioligand. Binding was optimal at pH 6, thermolabile, and was reduced by 70% when the membrane vesicles were pretreated with trypsin. The data are consistent with the presence of one or several binding proteins for fusicoccin at the plasma membrane of A. thaliana. Binding of the radioligand was unaffected by pretreatment of the sites with various alkylating and reducing agents, but was reduced by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, diethylpyrocarbonate, chloramine T, and periodate. A number of detergents were tested to find optimum conditions for solubilization. Nonanoyl-N-methylglucamide (50 millimolar) solubilized 70% of the radioligand-binding protein complex in undissociated form. Photoaffinity labeling of membrane preparations with a tritiated azido analog of fusicoccin resulted in the labeling of a 34 +/- 1 kilodalton polypeptide. Labeling of this polypeptide, presumably the fusicoccin-binding protein, was severely reduced in the presence of unlabeled fusicoccin.

Journal Article↗
PubMed1985

Signal transduction and ligand-receptor dynamics in the human neutrophil. Transient responses and occupancy-response relations at the formyl peptide receptor.

The responses of neutrophils to formyl peptides are initiated and in many cases achieve a maximal level prior to equilibrium receptor occupancy. In order to begin to understand the linkage between receptor occupancy and cell response we have used a pulsed binding procedure to analyze: 1) the number of receptors contributing to three potential signalling events and six functional responses and 2) the evolution of these responses once ligand binding is interrupted. We find that the half-optimal elevations of the potential signals are produced by less than 1% occupancy (Ca2+) or 1-3% occupancy (cAMP, membrane depolarization). In contrast, actin polymerization and a rapid light scatter response are elicited by less than 0.1% occupancy. Half-optimal elastase release and degranulation require approximately 3% occupancy. While half-optimal O2- production and aggregation require approximately 30% occupancy, the half-optimal rate of O2- production requires less than 10% occupancy. To resolve the apparent lack of correlation between the responses and the signals we examined their time courses following the pulse of stimulation. At least four responses and one signal are transient and decay while occupied receptors remain on the membrane surface. These include the Quin 2-Ca2+ signal, actin polymerization, the light scatter response, O2- generation, and aggregation. Ca2+ elevation is correlated with the responses in that: 1) each of these responses is transient unless new receptors are occupied; 2) occupancy of nearly all of the receptors contributes to the time course of these responses; 3) when binding is interrupted, the responses decay with a half-time of 15 s, following a latency of approximately 10 s or less (except for disaggregation where latency is 30-40 s). We discuss evidence in support of the hypothesis that transient cell responses arise from transient receptor activation.

Actins↗
PubMed1995

Probing the functional role of the N-terminal region of cystatins by equilibrium and kinetic studies of the binding of Gly-11 variants of recombinant human cystatin C to target proteinases.

The interaction between cystatin C variants, in which the evolutionarily conserved Gly-11 residue was substituted by Ala, Glu or Trp, and the cysteine proteinases, papain, ficin, actinidin and cathepsin B, was characterized. The substitutions reduced the affinity of binding in a manner consistent with the Gly residue of the wild-type inhibitor, allowing the N-terminal region to adopt a conformation that was optimal for interaction with target proteinases. Replacement of Gly-11 by Ala resulted in only a 5- to 100-fold reduction in binding affinity. Comparison with the affinities of wild-type cystatin C lacking the N-terminal region indicated that even this small structural change affects the conformation of this region sufficiently to largely abolish its interaction with the weakly binding proteinases, actinidin and cathepsin B. However, the substitution allows interactions of appreciable strength between the N-terminal region and the tightly binding enzymes, papain or ficin. Replacement of Gly-11 with the larger Glu and Trp residues substantially decreased the affinity of binding to all enzymes, from 10(3)- to 10(5)-fold. These substitutions further affect the conformation of the N-terminal region, so that interactions of this region with papain and ficin are also essentially eliminated. The decreased affinities of the three cystatin C variants for papain, ficin and actinidin were due exclusively to increased dissociation rate constants. In contrast, the decreased affinity between cathepsin B and the Ala-11 variant, the only one for which rate constants could be determined with this enzyme, was due almost entirely to a decreased association rate constant. This behaviour is analogous to that observed for forms of cystatin C lacking the N-terminal region and supports the conclusion that the mode of interaction of this region with target proteinases varies with the enzyme as a result of structural differences in the active-site region of the latter.

Cathepsin B↗
PubMed2006

Identifying optimal lipid raft characteristics required to promote nanoscale protein-protein interactions on the plasma membrane.

The dynamic lateral segregation of signaling proteins into microdomains is proposed to facilitate signal transduction, but the constraints on microdomain size, mobility, and diffusion that might realize this function are undefined. Here we interrogate a stochastic spatial model of the plasma membrane to determine how microdomains affect protein dynamics. Taking lipid rafts as representative microdomains, we show that reduced protein mobility in rafts segregates dynamically partitioning proteins, but the equilibrium concentration is largely independent of raft size and mobility. Rafts weakly impede small-scale protein diffusion but more strongly impede long-range protein mobility. The long-range mobility of raft-partitioning and raft-excluded proteins, however, is reduced to a similar extent. Dynamic partitioning into rafts increases specific interprotein collision rates, but to maximize this critical, biologically relevant function, rafts must be small (diameter, 6 to 14 nm) and mobile. Intermolecular collisions can also be favored by the selective capture and exclusion of proteins by rafts, although this mechanism is generally less efficient than simple dynamic partitioning. Generalizing these results, we conclude that microdomains can readily operate as protein concentrators or isolators but there appear to be significant constraints on size and mobility if microdomains are also required to function as reaction chambers that facilitate nanoscale protein-protein interactions. These results may have significant implications for the many signaling cascades that are scaffolded or assembled in plasma membrane microdomains.

Animals↗
PubMed1985

An evaluation of protein requirements in methylmalonic acidaemia.

A 3-month-old girl and a 13-month-old boy with vitamin B12-unresponsive methylmalonic acidaemia were studied to determine responses to varying levels of protein intake of growth, nitrogen balance and organic acid metabolism. A linear increase in the excretion of methylmalonic acid was observed in both patients above a critical level of protein intake. The inflection point was judged to reflect a ceiling above which amino acid intake exceeded requirements and catabolism was initiated. Below this point in each infant there was a plateau of minimal excretion of methylmalonic acid. Within this plateau level a reasonable rate of growth and metabolic stability were achieved at intakes between 0.70 and 0.75 and between 0.75 and 1.17 g protein kg-1, respectively, indicating that there is a range of protein tolerance and the importance of an individual approach to the provision of protein in patients with methylmalonic acidaemia. In the 3-month-old infant, nitrogen equilibrium was achieved at protein intakes above 0.6 g kg-1 and modest nitrogen retention was attained at a protein intake of 0.75 g kg-1, a level at which the excretion of methylmalonic acid was minimal and weight gain satisfactory. A protein intake of 1.25 g kg-1 was required to achieve a level of nitrogen retention often considered optimal for normal growth; however, this infant demonstrated an elevated excretion of methylmalonic acid and was close to clinical illness at this level of protein intake. The 13-month-old infant demonstrated a normal level of nitrogen retention, minimal excretion of methylmalonic acid, and a satisfactory rate of growth at protein intakes of 1.0-1.17 g kg-1. The values should prove useful guidelines for the management of infants requiring minimal intakes of protein. In studies carried out at 18-20 months of age, supplementation of the basic diet containing 0.75 g kg protein-1 with a mixture of amino acids not containing the precursors of methylmalonic acid was associated with increase of retention of nitrogen and increased concentrations of some essential amino acids in plasma, but effects on growth and the excretion of methylmalonic acid were not significant.

Amino Acid Metabolism, Inborn Errors↗
PubMed2005

Comparison of CIS- and EOM-CCSD-calculated adiabatic excited-state structures. Changes in charge density on going to adiabatic excited states.

The CIS and EOM-CCSD adiabatic geometries for the first excited states of a set of small molecules (C2H4, C2H2, H2C=O, H2C=S, CS2, CO2, SO2, NO2) have been calculated using the 6-311++G** basis set to see if the former geometries can be good starting points for optimizations at the latter theoretical level. With most of the molecules, there is fairly good agreement between the results from the two methods, and EOM-CCSD gives good agreement with the available experimental data. A detailed discussion of the lowest-lying singlet excited states in CO2 and CS2 is presented, highlighting the pronounced differences in electronic character and equilibrium structure displayed by these isovalent species. The origins of the structural distortions that are frequently found for the adiabatic excited states are examined with the aid of deformation density plots and the electron localization function (ELF).

Journal Article↗
PubMed2002

Myocardial collagen type I and impaired left ventricular function under exercise in hypertrophic cardiomyopathy.

BACKGROUND: Transaortic subvalvular myectomy (TSM) reduces left ventricular outflow tract gradient and improves symptoms and working capacity in patients with hypertrophic obstructive cardiomyopathy (HOCM). Nevertheless, TSM does not completely restore normal ventricular function, and some patients complain of symptoms despite optimal surgical results. Abnormal myocardial collagen structure in hypertrophic cardiomyopathy might be an indicator of impaired cardiac function. METHODS: Nine patients with HOCM were investigated. Myocytic diameter, collagen volume fraction and light absorbance of immunohistochemically stained collagen subtype I and its product (Coll I(prod)) were measured quantitatively in myectomy specimens. Patients underwent symptom-limited bicycle exercise testing with equilibrium radionuclide angiocardiography to determine ejection fraction (EF). Right heart catheterization was performed simultaneously in order to measure pulmonary capillary wedge pressure (PCWP) as a parameter of global ventricular diastolic filling and cardiac index (CI) as a parameter of functional capacity. RESULTS: Postoperatively, CI increased from 3.1 +/- 0.4 to 5.7 +/- 1.3 l/min/m(2) under exercise. EF was normal at rest (64 +/- 9 %) but did not increase significantly under exercise (66 +/- 14 %). Coll I(prod) (13.62 +/- 7.35 Vv%(prod)) correlated inversely with EF under exercise (r = -0.64; p = 0.05). PCWP increased under exercise from 8 +/- 2 mmHg at rest to 22 +/- 9 mmHg (p = 0.01). Coll I(prod) correlated with PCWP under exercise (r = 0.90; p = 0.001). CONCLUSIONS: Increased collagen subtype I is a predictor of diastolic as well as systolic dysfunction under exercise in patients with HCM after successful TSM.

Adolescent↗
PubMed2006

Regulation of catalysis by the smallpox virus topoisomerase.

The poxvirus type IB topoisomerases catalyze relaxation of supercoiled DNA by cleaving and rejoining DNA strands via a pathway involving a covalent phosphotyrosine intermediate. Recently we determined structures of the smallpox virus topoisomerase bound to DNA in covalent and non-covalent DNA complexes using x-ray crystallography. Here we analyzed the effects of twenty-two amino acid substitutions on the topoisomerase activity in vitro in assays of DNA relaxation, single cycle cleavage, and equilibrium cleavage-religation. Alanine substitutions at 14 positions impaired topoisomerase function, marking a channel of functionally important contacts along the protein-DNA interface. Unexpectedly, alanine substitutions at two positions (D168A and E124A) accelerated the forward rate of cleavage. These findings and further analysis indicate that Asp(168) is a key regulator of the active site that maintains an optimal balance among the DNA cleavage, religation, and product release steps. Finally, we report that high level expression of the D168A topoisomerase in Escherichia coli, but not other alanine-substituted enzymes, prevented cell growth. These findings help elucidate the amino acid side chains involved in DNA binding and catalysis and provide guidance for designing topoisomerase poisons for use as smallpox antivirals.

Amino Acid Substitution↗
PubMed1997

Multiparametric continuous monitoring of brain metabolism and substrate delivery in neurosurgical patients.

Brain function and tissue integrity are highly dependent on continuous oxygen supply and clearance of CO2. Aerobic metabolism is the major energy source to normal brain, however, during hypoxia and ischemia, lactate accumulation may sometimes be seen, indicating anaerobic glycolysis after severe head injury. Current monitoring techniques often fail to detect such events which can affect substrate delivery to the injured brain. We have recently adapted a method for continuous monitoring of brain tissue pO2, pCO2, pH and temperature, using a single sensor. The multiparameter sensor is inserted into brain tissue, via a new three lumen bolt, together with a standard ventriculostomy catheter and a microdialysis probe. The system has been left in place as long as needed, but never more than 7 days. All readings were compared to clinical parameters, and outcome. Stable measurements could be obtained in the first group of 20 patients, after calibration and rigid fixation, using the new bolt. Severely head injured patients had brain oxygen levels of less than 25-30 mmHg for the first hours after injury. Thereafter two patterns could be seen. Patients with favorable outcome had a slow increase in brain oxygen, and brain CO2 decreased to normal values, as long as the cerebral perfusion pressure (CPP) was kept above 70 mmHg. However, in those patients with secondary ischemic events, and bad outcome, a further decline in brain oxygen to anaerobic levels (< 20 mmHg) was seen. For these patients, both decreased and increased brain CO2 levels could be seen. Brain CO2 levels of 90-150 mmHg were consistently seen after brain death. Brain pH was inversely related to brain CO2 for all patients. Brain glucose and lactate in patients with poor outcome were 639 microM l-1 +/- 330, and 1642 microM l-1 +/- 788, whereas patients with good outcome had brain glucose levels of 808 microM l-1 +/- 321 and lactate levels of 1001 microM l-1 +/- 417. Extended neuromonitoring using a combined sensor for brain oxygen, CO2, pH and temperature measurements, as well as a microdialysis probe for glucose and lactate analysis may optimize the management of comatose neurosurgical patients in the future, by allowing a fuller understanding of dynamic factors affecting brain metabolism.

Acid-Base Equilibrium↗
PubMed1997

Honeybee flight muscle phosphoglucose isomerase: matching enzyme capacities to flux requirements at a near-equilibrium reaction

In honeybee flight muscle, there are close matches between physiological flux rates and the maximal activities (Vmax; determined using crude homogenates) of key enzymes catalyzing non-equilibrium reactions in carbohydrate oxidation. In contrast, phosphoglucose isomerase (PGI), which catalyzes a reaction believed to be close to equilibrium, occurs at Vmax values greatly in excess of glycolytic flux rates. In this study, we measure the Vmax of flight muscle PGI, the kinetic parameters of the purified enzyme, the apparent equilibrium constants for the reaction and the tissue concentrations of substrate and product. Using the Haldane equation, we estimate that the forward flux capacity (Vf) for PGI required to achieve physiological glycolytic flux rates is between 800 and 1070 units ml-1 cell water, approximately 45&shy;60 % of the empirically measured Vmax of 1770 units ml-1 cell water at optimal pH (8.0) and low ionic strength (no added KCl). When measured at physiological pH (7.0) and ionic strength (120 mmol l-1 KCl) with saturating levels of substrate, PGI activity is 1130 units ml-1 cell water, a value close to the calculated Vf. These results reveal a very close match between predicted and measured PGI flux capacities, and support the concept of an economical design of muscle metabolism in systems working at very high metabolic rates.

Journal Article↗
PubMedDate not supplied

Contrast optimization and scan timing for single and multidetector-row computed tomography.

Various complex pharmacokinetic interrelationships affect the use of contrast media for computed tomography (CT) imaging. These include factors related to each patient's unique body habitus and their degree of health, such as their age, gender, height, weight, and cardiovascular status. Of equal importance are factors related to the contrast material injection. The volume, concentration, and rate of injection all affect the degree of enhancement that is achieved with an injection of contrast material. In addition, the injection technique--whether the contrast is infused at a constant injection rate (uniphasic injection) or whether the rate is altered during the injection (multiphasic injection)--also affects the magnitude and duration of contrast enhancement. In body CT imaging, the liver poses unique challenges in managing the use of intravenous contrast material because of its dual blood supply and the need to complete imaging before equilibrium occurs between the intravascular and extravascular compartments. The magnitude of hepatic enhancement that is ultimately achieved is related primarily to the amount of iodinated contrast material that accumulates in the extravascular space within the target organ, independent of the speed of the CT scanner. The key determinant of the onset of the equilibrium phase is the injection duration. Given that a high injection flow rate (4-5 ml/s) is desirable for arterial phase imaging, the injection duration is maintained by using an appropriate contrast volume. Thus, modifications of the total iodine dose are best performed by altering the contrast concentration. Use of a high contrast concentration (400 mg iodine/ml) may be advantageous in heavy patients, or in patients in whom routine imaging is married with a need for high-detail imaging of the vasculature with high resolution CT angiography. The magnitude of arterial enhancement that is achieved is related to both the concentration of contrast and the rate of administration. The speed of the scanner determines its ability to record image data during the most advantageous time period, the peak of arterial enhancement. Thus, rapid imaging is particularly advantageous for optimal contrast use in CT angiography as well as in multiphasic imaging of the parenchymal organs.

Body Weight↗
PubMed1998

Proteins from hyperthermophiles: stability and enzymatic catalysis close to the boiling point of water.

It has become clear since about a decade ago, that the biosphere contains a variety of microorganisms that can live and grow in extreme environments. Hyperthermophilic microorganisms, present among Archaea and Bacteria, proliferate at temperatures of around 80-100 degrees C. The majority of the genera known to date are of marine origin, however, some of them have been found in continental hot springs and solfataric fields. Metabolic processes and specific biological functions of these organisms are mediated by enzymes and proteins that function optimally under these extreme conditions. We are now only starting to understand the structural, thermodynamic and kinetic basis for function and stability under conditions of high temperature, salt and extremes of pH. Insights gained from the study of such macromolecules help to extend our understanding of protein biochemistry and -biophysics and are becoming increasingly important for the investigation of fundamental problems in structure biology such as protein stability and protein folding. Extreme conditions in the biosphere require either the adaptation of the amino acid sequence of a protein by mutations, the optimization of weak interactions within the protein and at the protein-solvent boundary, the influence of extrinsic factors such as metabolites, cofactors, compatible solutes. Furthermore folding catalysts, known as chaperones, that assist the folding of proteins may be involved or increased protein protein synthesis in order to compensate for destruction by extreme conditions. The comparison of structure and stability of homologous proteins from mesophiles and hyperthermophiles has revealed important determinants of thermal stability of proteins. Rather than being the consequence of one dominant type of interactions or of a general stabilization strategy, it appears that the adaptation to high temperatures reflects a number of subtle interactions, often characteristic for each protein species, that minimize the surface energy and the hydration of apolar surface groups while burying hydrophobic residues and maximizing packing of the core as well as the energy due to charge-charge interactions and hydrogen bonds. In this article, mechanisms of intrinsic stabilization of proteins are reviewed. These mechanisms are found on different levels of structural organization. Among the extrinsic stabilization factors, emphasis is put on archaea chaperonins and their still strongly debated function. It will be shown, that optimization of weak protein-protein and protein-solvent interactions plays a key role in gaining thermostability. The difficulties in correlating suitable optimization criteria with real thermodynamic stability measures are due to experimental difficulties in measuring stabilization energies in large proteins or protein oligomers and will be discussed. Thus small single domain proteins or isolated domains of larger proteins may serve as model systems for large or multidomain proteins which due to the complexity of their thermal unfolding transitions cannot be analyzed by equilibrium thermodynamics. The analysis of the energetics of the thermal unfolding of a small, hyperthermostable DNA binding protein from Sulfolobus has revealed that a high melting temperature is not synonymous with a larger maximum thermodynamic stability. Finally, it is now well documented, that many thermophilic and hyperthermophilic proteins show a statistically increased number of salt bridges and salt bridge networks. However their contribution to thermodynamic and functional stability is still obscure.

Amino Acid Sequence↗
PubMed1999

Thermodynamic prediction of active ingredient loading in polymeric microparticles.

The growing use of microparticles as a controlled-delivery system for pharmaceutical and non-pharmaceutical active ingredients (AIs) has prompted a costly trial-and-error development of new and effective microparticle systems. In order to facilitate a more rational design and optimization of AI loadings in microparticles, we have developed a molecular-thermodynamic theory to predict the loading of liquid AIs in polymeric microparticles that are manufactured by a solvent evaporation process. This process involves the emulsification of a liquid polymer solution (consisting of polymer and AI dissolved in a volatile solvent) in an aqueous surfactant solution. The theory describes the equilibrium distribution of the AI between the aqueous phase and the dispersed polymeric droplets. The universal functional activity coefficient (UNIFAC) and UNIFAC-Free Volume (FV) group-contribution methods are utilized to model the nonidealities in the water and polymeric droplet phases, respectively. The inputs to the theory are: (i) the chemical structures, densities and total masses of the manufacturing ingredients, (ii) the manufacturing temperature and (iii) the glass transition temperature of the polymer. Since surfactant concentrations exceeding the critical micellar concentration (CMC) are often required in order to stabilize the dispersed polymeric droplets during the emulsion manufacturing process, the theory also accounts for AI solubilization in surfactant micelles present in the manufacturing solution. To test the AI loading predictions, we compare theoretical predictions of AI loadings in poly(lactic acid), poly(methyl methacrylate) and polystyrene microparticles to experimentally measured ones for five model AIs with varying degrees of hydrophobicity (benzyl alcohol, n-octanol, geraniol, farnesol and galaxolide). We also demonstrate how the developed theory can be utilized to screen polymers with respect to their abilities to load a given AI, as well as to provide guidelines for manufacturing microparticles having the desired AI loading.

Drug Delivery Systems↗
PubMed1996

Structural changes in cytochrome P-450cam effected by the binding of the enantiomers (1R)-camphor and (1S)-camphor.

A comparative study of the enantiomeric substrate [(1R)-camphor- and (1S)-camphor)-bound cytochrome P-450cam concerns the spin-state equilibrium, substrate dissociation, the thermal unfolding of the protein structure, and the subconformer equilibria observed in the infrared spectra of the carbon monoxide (CO) complex of cytochrome P-450cam. The behavior of the different conformational equilibria in dependence on temperature, pressure, pH-value, cosolvent, and cation binding led us to suggest that (1S)-camphor is more loosely and less optimally bound in the heme pocket, which facilitates the access of solvent molecules into the heme-iron environment. The spin reaction volume difference measured using the high pressure technique is smaller by 16 +/- 9 cm3/mol for (1S)-camphor-bound P-450cam compared to the (1R)-camphor-bound P-450cam, which might indicate a higher water content in the protein and in the heme environment in the (1S)-camphor complex. The half-transition temperature of the thermal unfolding of 53.8 degrees C for the (1S)-camphor-bound oxidized cytochrome P-450cam is one degree lower than the value for the (1R)-camphor-bound protein (54.8 degrees C). In the reduced, CO-bound form of cytochrome P-450cam at 290 K the (1S)-camphor complex reveals another CO stretch vibration population distribution with slightly higher frequencies [1940.2 cm-1 (major band) and 1946.3 cm-1 (minor band)] compared to the (1R)-camphor complex [1939.7 cm-1 (major band) and 1930 cm-1 (minor band)]. A loosening of the contact between the iron-bound CO ligand and amino acids of the I-helix, probably induced by compensating effects of the increased water content, is suggested. Assuming the carbon monoxide complex as a model for the dioxygen complex, the more loosened binding of (1S)-camphor, therefore the increased water accessibility, and the weaker contact of the iron ligand to the I-helix might explain the higher amount of uncoupling of the cytochrome P-450 reaction cycle compared to that when (1R)-camphor is used as substrate.

Binding Sites↗
PubMed1991

Oral nutrition in patients receiving home cyclic parenteral nutrition: pattern of substrate utilization.

Patients receiving cyclic home parenteral nutrition (PN) often have a significant oral caloric intake. This study describes the metabolic use of fuels, as assessed by indirect calorimetry, in eight stable, ambulatory, noncancerous, adult patients receiving glucose-based PN with (PN + oral) or without (PN only) a self-selected oral intake. Patients' weight was 91 +/- 2% (mean +/- SE) of ideal body weight, and fat mass was 22 +/- 5 and 31 +/- 2% of actual body weight in males and females, respectively. Under the PN-only regimen, providing 104 +/- 5% of predicted basal energy expenditure (BEE), patients were in equilibrium for energy and nitrogen balances. Oral supplementation (absorbed oral intake 80 +/- 5% of BEE) was associated with positive energy and nitrogen balances but also with nearly continuous net fat synthesis. We conclude that the glucose-based PN + mixed oral regimen enables the patients to face the increased energy requirements of everyday ambulatory life but is not associated with an optimal body composition in long-term PN patients.

Administration, Oral↗
PubMedDate not supplied

Temperature distributions in hyperthermia by electromagnetic induction: a theoretical model for the thorax.

Steady-state temperature solutions to the bioheat equation are presented for magnetic induction heating of a thoracic model consisting of a spherical tumor embedded in lung tissue which is layered by muscle and fatty tissue. Analytical solutions are presented for each of the tissue regions along with their numerical evaluations over a range of physical characteristics, including surface cooling effects. A strong dependence of tumor temperature on size and blood perfusion rate is shown to exist and can be used to optimize treatment parameters. Tendencies of the chest muscles and overlaying fatty tissue to overheat, particularly in the case of an obese patient, are discussed along with the alleviating influence of surface cooling. Healthy lung tissue, on the other hand, is shown to be safe from any significant damage in such a heating situation. Transient times required for tumors to achieve thermal equilibrium are computed and shown to depend strongly on tumor size and, to a lesser extent, on blood perfusion rate. Finally, the overall results obtained from the model are compared with available clinical data and are found to be in line with those observations.

Hot Temperature↗