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Human serum albumin (HSA) binds with drugs and fatty acids (FAs). This study was initiated to elucidate the relationship between the warfarin binding affinity of HSA and the positions of bound FA molecules. Molecular dynamics simulations of 11 HSA-warfarin-myristate complexes were performed. HSA-warfarin binding free energy was then calculated for each of the complexes by the molecular mechanics-Poisson-Boltzmann surface area (MM-PBSA) method. The results indicated that the magnitude of the binding free energy was smaller in HSA-warfarin complexes that had 4 or more myristate molecules than in complexes with no myristate molecules. The unfavorable effect on the HSA-warfarin binding affinity was caused sterically by the binding of a myristate molecule to the FA binding site closest to the warfarin binding site. On the other hand, the magnitude of HSA-warfarin binding free energy was largest when 3 myristate molecules were bound to the high-affinity sites. The strongest HSA-warfarin binding was attributable to favorable entropic contribution related to larger atomic fluctuations of the amino acid residues at the warfarin binding site. In the binding of 2 myristate molecules to the sites with the highest and second-highest affinities, allosteric modulation that enhanced electrostatic interactions between warfarin and some of the amino acid residues around the warfarin binding site was observed. This study clarified the structural and energetic properties of steric/allosteric effects of FAs on the HSA-warfarin binding affinity and illustrated the approach to analyze protein-ligand interactions in situations such that multiple ligands bind to the other sites of the protein.

Human serum albumin (HSA) has seven common fatty acid (FA) binding sites. In this study, we used the molecular mechanics Poisson-Boltzmann surface area method to identify high affinity FA binding sites on HSA in terms of binding free energy. Using multiple HSA-FA (myristate, palmitate) complex models constructed by molecular dynamics simulations, two methods were performed in molecular mechanics Poisson-Boltzmann surface area, the "three-trajectory method" and the "single-trajectory method". The former, which is less precise than the latter but may be more accurate as it includes the effects of conformational change upon binding, was used to classify high and low affinity sites. As a result, Sites 2, 4, and 5 were identified as high affinity sites for both FAs. The latter method, which is precise because energies are calculated from snapshots of the same trajectory for HSA-FAcomplex, was performed to compare the magnitude of binding free energy for these sites. The order of magnitude was 5>4>2, identical to that of a previous publication by others. In this way, a combination of the two methods was effectively used to identify high affinity sites. This study therefore provides an insight into the quantitative identification of high affinity FA binding sites on HSA.

This study is aimed at constructing the neurophysiological basis for determining the characteristic features of cerebral motor disturbance in representative cerebral palsy (CP) types using topographical S-SEPs technology. Median-nerve stimulated S-SEPs (MN-SSEPs) were examined for 23 patients with four representative types of cerebral palsy: 6 athetotic (including 3 patients due to hypoxic-ischemic encephalopathy (HIE) and 3 to kernicterus), 7 hemiplegic, 5 diplegic and 5 tetraplegic types, and 13 normal controls. In HIE group of athetotic CP, frontal N30 specifically showed severe amplitude reduction or abolishment. In hemiplegic CP, both N20 and N30 on the affected cerebral side tended either to disappear or to be normally evoked at the same time, and their mean amplitudes declined severely. In diplegic CP, the amplitudes of subcortical N18 and parietal N20 were not small but significantly enlarged. N30 amplitude stayed within normal. The reason for this unexpected enlargement of N18 and N20 is unclear, but may be partly due to premature birth which caused abnormally abundant dendritic spine due to absence from perinatal normal spine elimination in the brainstem. In several quadriplegic patients, both N20 and N30 disappeared. The mean amplitude of N30 severely decreased. In conclusion, topographical results of N18, N20 and N30 may basically suggest the underlying involvement of nervous structures in CP according to their representative type.

Human serum albumin (HSA) binds with fatty acids under normal physiologic conditions. To date, there is little published information on the tertiary structure of HSA-fatty acid complex in aqueous solution. In the present study, we used molecular dynamics (MD) simulations to elucidate possible structural changes of HSA brought about by the binding of fatty acids. Both unliganded HSA and HSA-fatty acid complex models for MD calculations were constructed based on the X‐ray crystal structures. Five myristates (MYRs) were bound in the HSA-fatty acid complex model. In the present MD study, the motion of domains I and III caused by the binding of MYR molecules increased the radius of gyration of HSA. Root‐mean‐square fluctuations from the MD simulations revealed that the atomic fluctuations of the specific amino acids at drug‐binding site I that can regulate the drug‐binding affinity were increased by the binding of MYR molecules. Primary internal motions, characterized by the first three principal components, were observed mainly at domains I and III in the principal component analysis for trajectory data. The directional motion projected on the first principal component of unliganded HSA was conserved in HSA-MYR complex as the third principal directional motion with higher frequency. However, the third principal directional motion in unliganded HSA turned into the first principal directional motion with lower frequency in the HSA-MYR complex. Thus, the present MD study provides insights into the possible conformational changes of HSA caused by the binding of fatty acids. Proteins 2006. © 2006 Wiley‐Liss, Inc.

For estimating pharmacokinetic parameters, we introduce the minimum relative entropy (MRE) method and compare its performance with least squares methods. There are several variants of least squares, such as ordinary least squares (OLS), weighted least squares, and iteratively reweighted least squares. In addition to these traditional methods, even extended least squares (ELS), a relatively new approach to nonlinear regression analysis, can be regarded as a variant of least squares. These methods are different from each other in their manner of handling weights. It has been recognized that least squares methods with an inadequate weighting scheme may cause misleading results (the "choice of weights" problem). Although least squares with uniform weights, i.e., OLS, is rarely used in pharmacokinetic analysis, it offers the principle of least squares. The objective function of OLS can be regarded as a distance between observed and theoretical pharmacokinetic values on the Euclidean space RN, where N is the number of observations. Thus OLS produces its estimates by minimizing the Euclidean distance. On the other hand, MRE works by minimizing the relative entropy which expresses discrepancy between two probability densities. Because pharmacokinetic functions are not density function in general, we use a particular form of the relative entropy whose domain is extended to the space of all positive functions. MRE never assumes any distribution of errors involved in observations. Thus, it can be a possible solution to the choice of weights problem. Moreover, since the mathematical form of the relative entropy, i.e., an expectation of the log-ratio of two probability density functions, is different from that of a usual Euclidean distance, the behavior of MRE may be different from those of least squares methods. To clarify the behavior of MRE, we have compared the performance of MRE with those of ELS and OLS by carrying out an intensive simulation study, where four pharmaco-kinetic models (mono- or biexponential, Bateman, Michaelis-Menten) and several variance models for distribution of observation errors are employed. The relative precision of each method was investigated by examining the absolute deviation of each individual parameter estimate from the known value. OLS is the best method and MRE is not a good one when the actual observation error magnitude conforms to the assumption of OLS, that is, error variance is constant, but OLS always behaves poorly with the other variance models. On the other hand, MRE performs better than ELS and OLS when the variance of observation is proportional to its mean. In contrast, ELS is superior to MRE and OLS when the standard deviation of observation is proportional to its mean. In either case the difference between MRE and ELS is relatively small. Generally, the performance of MRE is comparable to that of ELS. Thus MRE provides as reliable a method as ELS for estimating pharmacokinetic parameters.

The present study investigated prognostic indicators, including clinicopathological and preoperative hematological factors, and developed a prognostic factor-based risk stratification model in bladder cancer patients treated with radical cystectomy.Data were collected from 249 consecutive bladder cancer patients treated with radical cystectomy without neoadjuvant therapy. Prognostic values of the preoperative hematological parameters, along with the patients' clinicopathological parameters were evaluated. A risk stratification model was developed to predict disease-specific survival after radical cystectomy using the regression coefficients of multivariate analysis.In the multivariate analysis, preoperative hemoglobin and C-reactive protein levels, as well as the pathological factors of T stage, positive surgical margin and lymph node metastasis, were independently predictive of disease-specific survival. Low hemoglobin (10.5 g/dL), a high C-reactive protein (0.5 mg/dL), extravesical T stage (≥pT3a) and positive surgical margin were independent predictors of poor disease-specific survival. The risk stratification model showed significant differences in disease-specific survival between the three subgroups.This is the first report to show the significance of combining preoperative hemoglobin with the pathology of radical cystectomy specimens as an independent predictor for disease-specific survival, and it also represents the largest contemporary series to date demonstrating that two types of preoperative hematological disorders, assessed by hemoglobin and C-reactive protein, are independent predictors in bladder cancer patients treated with radical cystectomy. Our risk stratification model could provide physicians with useful prognostic information for identifying patients who might be candidates for multimodal treatments.

Objectives Despite the surgical curability of renal cell carcinoma (RCC) and upper urinary tract urothelial carcinoma (UUT-UC), post-nephrectomy chronic kidney disease (CKD) continues to be a cause of concern. We investigated the correlation between the expression of apoptotic regulatory molecules in the nephrectomized, noncancerous cortex, as well as CKD progression and CKD-related mortality. Materials and methods Fas and Bcl-2 mRNA and protein expression in surgically resected specimens from 100 patients with RCC and UUT-UC were determined. The estimated glomerular filtration rates (eGFR) were determined sequentially before surgery and up to 5 years after surgery. The relationships between CKD progression, the expression of these molecules in the renal cortex, and the clinical characteristics were analyzed. Results The mean 1-year postoperative percent eGFR decrease was 30.2 (Standard deviation [SD]: 15.2). The 1-year postoperative percent eGFR decrease greater than the approximate value of mean ± SD (45) was categorized as severe renal functional deterioration (SRFD). Glomerular Fas protein expression and a Fas/β-actin mRNA ratio >0.3 were independent predictors for SRFD. Significantly increased mortality rates due to cardiovascular events were indicated by glomerular Fas protein expression, Fas mRNA levels >0.3, and SRFD. No significant change in Bcl-2 levels was observed. Conclusions This study is the first report to demonstrate the significance of Fas expression in the nephrectomized normal cortex as a predictor of post-nephrectomy CKD progression. The results from nephrectomized kidney showed that the natural course of renal function in the remaining kidney may be affected not only by Fas-induced glomerular cell apoptosis but also by the total amount of Fas mRNA in cortical cells.

Two male patients (a child and an adult) with congenital mirror movement were studied using functional MRI (fMRI) and transcranial magnetic stimulation (TMS). Bilateral primary sensorimotor cortices were activated during unilateral hand gripping on fMRI when the child patient was 8 years old and the adult was 37 years old. Bilateral motor evoked potentials were induced from the hand and forearm muscles after TMS of each hemisphere. Bilateral motor responses were also induced from the arm muscles in the adult patient. Bilateral motor responses had short and similar latencies. Contralateral motor responses to TMS were smaller than ipsilateral ones in the hand muscles, while contralateral responses were larger than ipsilateral ones in the arm muscles. Contralateral hand motor responses reduced in amplitude or disappeared with increasing age while in the child patient, mirror movements decreased gradually. Our results suggest that bilateral activation of the primary sensorimotor cortices during intended unilateral hand movement and bilateral motor responses to TMS account, at least in part, for the pathophysiology of congenital mirror movement. Reduction of contralateral hand motor responses may be related to the decrease in mirror movements during development.

There has been a considerable increase in popularity of the NONMEM method as a technique for estimating population pharmacokinetic parameters. The authors present another approach to population pharmacokinetic analysis, the alternative two stage method (ATS). ATS uses the EM-algorithm for maximizing the likelihood of variance components. The performance of ATS was compared with the NONMEM method on a microcomputer. Simulation studies showed that the precision and accuracy of estimates obtained with ATS were comparable to the NONMEM method, however, the computation time, dependences on initial estimates and convergence properties were somewhat different. ATS could be a valuable alternative to the NONMEM method for estimating population pharmacokinetic parameters in some cases.