The potential use of polymeric, functionalized nanoparticles (NPs) as drug delivery vectors was explored. Covalent conjugation of albumin to the surface of NPs via maleimide chemistry proved problematic. However, microwave assisted synthesis of NPs was not only time efficient, but enabled the exploration of size control by changing the following parameters: temperature, microwave power, reaction time, initiator concentration, and percentage of monomer used. About 1.5 g of fluorescently-labeled, carboxylic acid-functionalized NPs (100 nm diameter) were synthesized for a total cost of less than $1. Future work will address further functionalization of the NPs for the coupling of albumin (or other targeted proteins), and tests for in vivo biodistribution.

No description available.

As one of the natural polyphenols, resveratrol possesses hydroxyl substituted trans-stilbene structure and exerts impact on health by inhibiting multiple human enzymes, such as cyclooxygenase, F1 ATPase, and tyrosinase. Resveratrol has to be bound by human serum albumin (HSA) to keep a high concentration in serum, since its solubility is low in water. To improve water solubility and bioavailability, two resveratrol aliphatic acids and their esters have been designed and synthesized. The solubilities of the resveratrol and its derivatives have been measured using a standard procedure. The two aliphatic acids showed better solubilities in pure water and phosphate buffer (pH 7). The binding affinities of resveratrol derivatives for HSA were also measured, and the drug-protein interaction mechanism was investigated using fluorescence, UV-vis, and NMR spectroscopies. Interestingly, resveratrol hexanoic acid (5) was found to be a much better ligand (Ka = (6.70 ± 0.10) × 106 M-1) for HSA than resveratrol (Ka = (1.64 ± 0.07) × 105 M-1), and there was 41-fold improvement for the binding affinity. It was the first time that the increase of fluorescence of resveratrol moiety was observed during the binding to HSA, suggesting that 5 should be bound tightly by HSA. The UV-vis absorption spectroscopy revealed a maximum absorption shift from 318 to 311 nm with decreasing intensity by 20% upon complexation, suggesting that the π-π conjugation of the stilbene structure was impaired during the binding. Although HSA was reported to have only one binding site for resveratrol, the Job's and molar ratio plots suggested that HSA should bind two molecules of 5. NMR study suggested that phenyl group (B ring) in the center of the molecule of 5 should be involved in the π-π stacking interactions with HSA aromatic amino acid residues. Molecular geometry calculation of 5 with Spartan software showed that the stilbene structure had two conformers, orthogonal and planar ones. The former (E = -1.432 KJ/mol) was more stable than the latter (E = -0.128 KJ/mol), suggesting that the former should be the conformer of 5 in the complexation with HSA.

Zinc is an essential metal ion for the activity of multiple enzymes and transcription factors. Among many other transporting proteins human serum albumin (HSA) is the main carrier of Zn(II) in the blood plasma. HSA displays multiple ligand binding sites with extraordinary binding capacity for a wide range of ions and molecules including fatty acids. Hence, HSA controls the availability and distribution of those molecules throughout the body. Previous studies have established that the existence of one zinc site with high affinity (MBS-A) that is modulated by the presence of fatty acids. Therefore, the fatty acid concentration in the blood influences zinc distribution which may result in a significant effect on both normal physiological processes and a range of diseases. Based on the current knowledge of HSA's structure and its coordination chemistry with zinc ion, here, we attempted to investigate zinc interactions and coordination with HSA and the effect of different fatty acids on the protein structure, stability and on Zn(II) binding. By NMR titration, we examine the Zn(II) binding to HSA and the spectra show distinct movements of some resonances showing a conformational change has occurred as a result of Zn(II) binding. Isothermal calorimetry titrations study was performed to evaluate zinc binding affinity to HSA in the absence and presence of fatty acids. Free HSA results indicates the existence of one high affinity site and multiple low affinity sites. Upon the binding of fatty acids to HSA, three distinct behaviors of Zn(II) affinity was observed ranging from no effect to moderate to significant depending on the FAs. By the use of circular dichroism, we investigate secondary and tertiary structure of HSA in the presence and absence of FAs and Zn(II). We found albumin is predominately α-helical and the overall conformation of the protein remains unchanged even after interacting with FAs and Zn(II) with some exception. The structural stability of HSA was evaluated by obtaining the denaturation temperature in the presence and absence of fatty acid and we found the thermal denaturation of HSA increases with the increase of amount of fatty acids.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20987号 / 医博第4333号 / 新制||医||1027(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 伊達 洋至, 教授 長船 健二, 教授 一山 智 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

The kinetics of the reaction catalyzed by acyl-CoA: sn-glycerol-3-phosphate O-acyltransferase solubilized from Euglena gracilis microsomes were examined. For myristoyl-, palmitoyl-, stearoyl-, and oleoyl-CoAs, the initial reaction rates rose with increasing substrate concentration up to an optimal concentration that varied from 18.5 to 25 μ M, well above the respective critical micelle concentrations. At higher substrate concentrations, reaction was progressively inhibited. Arachidoyl-CoA was a relatively poor substrate for the acyltransferase, and substrate inhibition was not seen with it. Km values for acyl-CoAs ranged from 13 to 20 μ M while the corresponding V values varied almost 40-fold. Bovine serum albumin, among other effects, caused a change in the kinetic pattern of the reaction acyl-CoA dependency. Both acyl-CoA micelles and albumin-bound acyl-CoA were substrates. The binding of palmitoyl- and oleoyl-CoA was 2.7 and 1.5 mol, respectively, per mol of albumin. The critical micelle concentration of palmitoyl-CoA under the reaction conditions was shown by low angle light scattering photometry to be 7.1 p.M. The sn-glycerol 3-phosphate concentration dependency of the acyltransferase initial velocity exhibited Michaelis-Menten kinetics with Km values of 1.3 and 2.9 mM in the presence of 12.5 and 25 μM palmitoyl-CoA, respectively. The substrate analogues sn-glyceraldehyde 3-phosphate and dihydroxyacetone phosphate inhibited the reaction.

These studies indicate that tumor necrosis factor-α (TNFα) alters albumin permeability and unlike bradykinin (BK) the increased albumin permeability lasts for the duration of the application. Neither agonist requires the presence of white blood cells or other blood-borne substances to produce this inflammatory response. These experiments were completed in the in situ, microcannulated, perfused venules of the mesentery in the anesthetized hamster. Albumin transport was measured using intravital fluorescence microscopy, TRITC-labeled albumin, and densitometric tracking. Further, by varying the intravascular pressure, the hydraulic (L(p)(1 - σ)) and diffusive permeability (P0) coefficients of these microvessels were determined. Both BK and TNFα produced an increase in albumin flux, which was dependent upon the dose and time domains. This response was present when the agonists were given by either intra- or extravascular presentation. Both hydraulic coupling and microvascular permeability were increased by BK and TNFα. TNFα increased albumin permeability rapidly and its effect lasted as long as TNFα was present, whereas the increased albumin transport by BK was biphasic. The results implicate a dynamic modification in the microvascular wall to these inflammatory agonists and the mechanism(s) for transduction in the endothelium are quite different.

The purpose of this study was to determine if post-operative serum albumin and percentage of weight loss improved in patients who received formalized pre-operative nutrition counseling. Nutrition intervention was measured quantitatively. A retrospective review of records was conducted on 77 RYGB patients (68 female subjects and 9 male subjects), ages 21-64, during January 2001 through January 2006. The results indicated that patients who received pre-operative nutrition intervention had better clinical outcomes of serum albumin than those with no nutrition intervention from a registered dietitian. However, outcomes regarding percentage of weight loss varied. Both pre-operatively and at the 3 month post-operative visit the weight of subjects who received nutrition intervention seemed to be increasing by the 6 month post-operative visit the subjects with no nutrition intervention had lost more weight.

Introduction- Exercise-induced plasma volume (PV) expansion is typically associated with an increase in plasma albumin content. Increased hepatic albumin synthesis, a transcriptionally regulated process, is thought to contribute to the increase in albumin content. Objective- We tested the hypothesis that exercise training induces an increase in albumin gene expression in relationship to the increase in PV. Methods and Results- 40 adult male Sprague-Dawley rats weighing between 245-350 grams were randomly assigned to one of four groups: cage control (CC); sham exercise 10 min/day at 48% VO2max (NE); continuous exercise training, 60 min /day at 72% VO2max (LI); and high intensity, intermittent exercise training, 8 bouts of 4 min at 98% VO2max followed by 5 min at 48% VO2max (HI). The training period lasted for two weeks with 12 training sessions with equalized training volumes in the exercise groups. 24 hours after the last training session the rats were anesthetized and a jugular catheter was placed for collecting blood samples during PV determination by a dilution of a labeled-albumin molecule (Texas Red albumin). The liver and red quadriceps (RQ) muscle tissue was then removed, flash frozen, and stored for later analysis. The training protocol produced a significant increase in RQ citrate synthase activity (p < 0.05). PV increased in proportion to the exercise intensity (p < 0.05) averaging 23.6 ± 2.7 ml•kg-1 body weight in the CC group and 26.6 ± 1.3 ml•kg-1 body weight in the HI group. Albumin mRNA expression determined by real time polymerase chain reaction (PCR) increased 2.2 ± 0.1 and 2.9 ± 0.2 fold following LI and HI exercise training, respectively. Conclusion- These data support the hypothesis that, during exercise-induced PV expansion, albumin gene expression is increased and contributes to an increase in plasma albumin content and PV.

Corrosion and metal release mechanisms of CoCrMo alloys are at human biological conditions not fully understood. The main objective of this master thesis was to investigate whether the Vroman effect influences the extent of metal release from CoCrMo alloy in mixed protein solutions. The project focuses on the corrosion properties and release of cobalt (Co), chromium (Cr) and molybdenum (Mo) from a CoCrMo alloy into simulated physiological solutions of pH 7.2-7.4 in the presence of proteins. The metal release study was performed in phosphate buffered saline (PBS) for 4 and 24 h at 37 °C with and without different concentration of proteins (bovine serum albumin-BSA and fibrinogen-Fbn from bovine plasma). In order to investigate whether any Vroman effect could affect the extent of released metals in solutions, sequential tests were performed by sampling after 1, 4, 6 and 24 h in solutions that were partially replenished after 5 h. Significant metal-induced protein aggregation and precipitation were observed in solutions of physiologically-relevant protein concentrations (40 g/L BSA and 2.67 g/L Fbn). Cr was strongly enriched in the surface oxide of CoCrMo after exposure in all solutions. This was for all solutions accompanied by metal release processes dominated by Co. Based on electrochemical investigations, the electrochemical activity did not increase, but rather decreased, in protein-containing solutions as compared to PBS alone. This could possibly be explained by blocking of cathodic areas as a result of protein adsorption.