Approximate linear confidence and curvature of a kinetic model of dodecanedioic acid in humans

Dicarboxylic acids with an even number of carbon atoms have been proposed as an alternate energy substrate for enteral or parenteral nutrition in the acutely ill patient, due to their water solubility and their yielding TCA cycle intermediates upon beta-oxidation. In the present work, a nonlinear compartmental model of the kinetics of dodecanedioic acid is developed, and its parameters are estimated from time concentration experimental observations obtained from six healthy volunteers undergoing a per os administration of 3 g of the substance. Although the model is linear in the transfer of the free substance from plasma to the tissues, the exchange between gut and plasma compartments is represented as a saturable function. Albumin binding is then incorporated to obtain the final model in terms of the measured total concentrations. Estimates of the model's structural parameters were computed for each experimental subject, and the usual single-subject approximate confidence regions for the parameters were derived by inversion of the Hessian at the optimum. To verify the applicability of this approximation, the nonlinearity of the expectation surface at the optimum was measured by computing the normal (intrinsic) component of curvature. Because the model curvature was excessive in all subjects, the usual approximation could not be trusted to provide acceptable approximations to the parameter confidence regions. A suitable Monte Carlo simulation yielded empirical joint parameter distributions from which the approximate parameter variances could finally be obtained.     

Solution NMR structure of the iron-sulfur cluster assembly protein U (IscU) with zinc bound at the active site

IscU is a highly conserved protein that serves as the scaffold for IscS-mediated assembly of iron-sulfur ([Fe-S]) clusters. We report the NMR solution structure of monomeric Haemophilus influenzae IscU with zinc bound at the [Fe-S] cluster assembly site. The compact core of the globular structure has an alpha-beta sandwich architecture with a three-stranded antiparallel beta-sheet and four alpha-helices. A nascent helix is located N-terminal to the core structure. The zinc is ligated by three cysteine residues and one histidine residue that are located in and near conformationally dynamic loops at one end of the IscU structure. Removal of the zinc metal by chelation results in widespread loss of structure in the apo form. The zinc-bound IscU may be a good model for iron-loaded IscU and may demonstrate structural features found in the [Fe-S] cluster bound form. Structural and functional similarities, genomic context in operons containing other homologous genes, and distributions of conserved surface residues support the hypothesis that IscU protein domains are homologous (i.e. derived from a common ancestor) with the SufE/YgdK family of [Fe-S] cluster assembly proteins.     

Molecular cytogenetic study of three common Mediterranean limpets, Patella caerulea, P. rustica and P. ulyssiponensis (Archaeogastropoda, Mollusca)

The present paper shows the results of chromosome banding and rDNA-FISH study performed on several specimens of different populations of Patella caerulea, Patella rustica and Patella ulyssiponensis. The taxonomic attribution of specimens was ascertained by the molecular phylogenetic analysis of the mitochondrial 16S rRNA gene. P. caerulea and P. rustica had 2n = 18 chromosomes with first seven of biarmed pairs and the remaining two uniarmed pairs. P. ulyssiponensis had 2n = 16 with all biarmed chromosomes. Ag-NOR loci were on the short arms of the first metacentric pair in the three studied limpets, whereas they showed a different pattern of heterochromatin distribution and composition. A chromosome mosaicism was observed in several P. caerulea specimens, which exhibited an unpaired metacentric element and loss of a telocentric pair. The obtained results suggest that in the genus Patella specific diversification was accompanied by variations in heterochromatin distribution and composition and reduction of chromosome number by Robertsonian centric fusion.     

Loss of proline-rich tyrosine kinase 2 function induces spreading and motility of epithelial prostate cells

Although prostate carcinoma is an aggressive cancer preferentially metastasizing to the bones, many prostate tumors remain localized and confined to the prostate indefinitely. Prediction of the behavior of anatomically localized and moderately differentiated prostate tumors remains difficult because of lack of prognostic markers. Cell motility is an important step in the progression of epithelial tumor toward invasive metastatic carcinomas and changes in the expression and function of adhesion molecules contribute to the acquisition of a more malignant phenotype. Proline-rich tyrosine kinase 2 (Pyk2) is implicated in regulating the organization of actin cytoskeleton, a process critical for cell migration, mitosis, and tumor metastasis. In this report, we investigated whether Pyk2 played a role in the acquisition of an aggressive phenotype in prostate cell. Data reported here demonstrate that loss of Pyk2 kinase function results in induction of cell motility and migration in EPN cells, a line of non-transformed epithelial cells derived from human normal prostate tissue. Changes in motility and migration of prostate cells were associated with changes in the expression of several proteins involved in cell adhesion and reorganization of actin cytoskeleton. Ablation of Pyk2 kinase activity caused a dramatic decrease of the expression of E-cadherin and IRS1 and an increase of the expression of alpha5-integrin. In addition, a massive reorganization of actin cytoskeleton was observed. Our data indicate that Pyk2 plays a central role in the mechanism that regulate cell-cell and cell-substrate interaction and lack of its kinase activity induces prostate cells to acquire a malignant, migrating phenotype.     

Modeling the euglycemic hyperinsulinemic clamp by stochastic differential equations

The Euglycemic Hyperinsulinemic Clamp (EHC) is the most widely used experimental procedure for the determination of insulin sensitivity. In the present study, 16 subjects with BMI between 18.5 and 63.6 kg/m² have been studied with a long-duration (5 hours) EHC. In order to explain the oscillations of glycemia occurring in response to the hyperinsulinization and to the continuous glucose infusion at varying speeds, we first hypothesized a system of ordinary differential equations (ODEs), with limited success. We then extended the model and represented the experiment using a system of stochastic differential equations (SDEs). The latter allow for distinction between (i) random variation imputable to observation error and (ii) system noise (intrinsic variability of the metabolic system), due to a variety of influences which change over time. The stochastic model of the EHC was fitted to data and the system noise was estimated by means of a (simulated) maximum likelihood procedure, for a series of different hypothetical measurement error values. We showed that, for the whole range of reasonable measurement error values: (i) the system noise estimates are non-negligible; and (ii) these estimates are robust to changes in the likely value of the measurement error. Explicit expression of system noise is physiologically relevant in this case, since glucose uptake rate is known to be affected by a host of additive influences, usually neglected when modeling metabolism. While in some of the studied subjects system noise appeared to only marginally affect the dynamics, in others the system appeared to be driven more by the erratic oscillations in tissue glucose transport rather than by the overall glucose-insulin control system. It is possible that the quantitative relevance of the unexpressed effects (system noise) should be considered in other physiological situations, represented so far only with deterministic models.The work was supported by grants from the Danish Medical Research Council and the Lundbeck Foundation to S. Ditlevsen.     

Regio and stereoselective oxidations of unsaturated steroidal compounds with H2O2 mediated by CH3ReO3

We have investigated the oxidative behavior of sterols such as cholesteryl acetate (1), 7-dehydrocholesteryl acetate (2), ergosteryl acetate (3), cholecalciferol acetate (Vitamin D(3) acetate) (4) and ergocalciferol acetate (Vitamin D(2) acetate) (5) with the oxidant system methyltrioxorhenium/H(2)O(2)/pyridine in order to check potential parameters controlling the selectivity. The reactions, performed in CH(2)Cl(2)/H(2)O at 25 degrees C, have shown good regio- and stereoselectivity. All oxidation products were isolated by high-performance liquid chromatography (HPLC) and characterized by MS(EI) or FAB, (1)H NMR, (13)C NMR, APT, COSY, HSQC, HMBC, ROESY and NOEDS measurements. Seven new oxygenated compounds were also obtained. Under the experimental conditions adopted in this work, only the diene steroids, i.e. 7-dehydrocholesteryl acetate and ergosteryl acetate, undergo hydrolytic oxirane ring opening, whereas Vitamin D(2) and D(3) acetates, containing the triene system and cholesteryl acetate yield only epoxides. The selectivity seems to be controlled by the nucleophilicity of double bonds and by stereoelectronic and steric effects.