Influence of varying degrees of malnutrition on IGF-I expression in the rat diaphragm.

This study evaluated the impact of varying degrees of prolonged malnutrition on the local insulin-like growth factor-I (IGF-I) system in the costal diaphragm muscle. Adult rats were provided with either 60 or 40% of usual food intake over 3 wk. Nutritionally deprived (ND) animals (i.e., ND60 and ND40) were compared with control (Ctl) rats fed ad libitum. Costal diaphragm fiber types and cross-sectional areas were determined histochemically. Costal diaphragm muscle IGF-I mRNA levels were determined by RT-PCR. Serum and muscle IGF-I peptide levels were determined by using a rat-specific radioimmunoassay. The body weights of Ctl rats increased by 5%, whereas those of ND60 and ND40 animals decreased by 16 and 26%, respectively. Diaphragm weights were reduced by 17 and 27% in ND60 and ND40 animals, respectively, compared with Ctl. Diaphragm fiber proportions were unaffected by either ND regimen. Significant atrophy of both type IIa and IIx fibers was noted in the ND60 group, whereas atrophy of all three fiber types was observed in the diaphragm of ND40 rats. Serum IGF-I levels were reduced by 62 and 79% in ND60 and ND40 rats, respectively, compared with Ctl. Diaphragm muscle IGF-I mRNA levels in both ND groups were similar to those noted in Ctl. In contrast, IGF-I concentrations were reduced by 36 and 42% in the diaphragm muscle of ND60 and ND40 groups, respectively, compared with Ctl. We conclude that the local (autocrine/paracrine) muscle IGF-I system is affected in our models of prolonged ND. We propose that this contributes to disordered muscle protein turnover and muscle cachexia with atrophy of muscle fibers. This is particularly so in view of recent data demonstrating the importance of the autocrine/paracrine system in muscle growth and maintenance of fiber size.     

Chemical Composition of Volatiles; Antimicrobial,Antioxidant and Cholinesterase Inhibitory Activity of Chaerophyllum aromaticum L. (Apiaceae) Essential Oils and Extracts

The present study reports the chemical composition of the headspace volatiles (HS) and essential oils obtained from fresh Chaerophyllum aromaticum root and aerial parts in full vegetative phase, as well as biological activities of their essential oils and MeOH extracts. In HS samples, the most dominant components were monoterpene hydrocarbons. On the other hand, the essential oils consisted mainly of sesquiterpenoids, representing 73.4% of the root and 63.4% of the aerial parts essential oil. The results of antibacterial assay showed that the aerial parts essential oil and MeOH extract have no antibacterial activity, while the root essential oil and extract showed some activity. Both of the tested essential oils exhibited anticholinesterase activity (47.65% and 50.88%, respectively); MeOH extract of the root showed only 8.40% inhibition, while aerial part extract acted as an activator of cholinesterase. Regarding the antioxidant activity, extracts were found to be more effective than the essential oils.     

Structure and dynamics of two β-peptides in solution from molecular dynamics simulations validated against experiment

We have studied two different beta-peptides in methanol using explicit solvent molecular dynamics simulations and the GROMOS 53A6 force field: a heptapeptide (peptide 1) expected to form a left-handed 3(14)-helix, and a hexapeptide (peptide 2) expected to form a beta-hairpin in solution. Our analysis has focused on identifying and analyzing the stability of the dominant secondary structure conformations adopted by the peptides, as well as on comparing the experimental NOE distance upper bounds and 3J-coupling values with their counterparts calculated on the basis of the simulated ensembles. Moreover, we have critically compared the present results with the analogous results obtained with the GROMOS 45A3 (peptide 1) and 43A1 (peptide 2) force fields. We conclude that within the limits of conformational sampling employed here, the GROMOS 53A6 force field satisfactorily reproduces experimental findings regarding the behavior of short beta-peptides, with accuracy that is comparable to but not exceeding that of the previous versions of the force field. GCE legend Conformational clustering analysis of the simulated ensemble of a ss-hexapeptide with two different simulation setups (a and b). The central members of all of the clusters populating more than 5% of all of the structures are shown, together with the most dominant hydrogen bonds and the corresponding percentages of cluster members containing them.     

Simulation of folding of a small alpha-helical protein in atomistic detail using worldwide-distributed computing

By employing thousands of PCs and new worldwide-distributed computing techniques, we have simulated in atomistic detail the folding of a fast-folding 36-residue alpha-helical protein from the villin headpiece. The total simulated time exceeds 300 micros, orders of magnitude more than previous simulations of a molecule of this size. Starting from an extended state, we obtained an ensemble of folded structures, which is on average 1.7A and 1.9A away from the native state in C(alpha) distance-based root-mean-square deviation (dRMS) and C(beta) dRMS sense, respectively. The folding mechanism of villin is most consistent with the hydrophobic collapse view of folding: the molecule collapses non-specifically very quickly ( approximately 20ns), which greatly reduces the size of the conformational space that needs to be explored in search of the native state. The conformational search in the collapsed state appears to be rate-limited by the formation of the aromatic core: in a significant fraction of our simulations, the C-terminal phenylalanine residue packs improperly with the rest of the hydrophobic core. We suggest that the breaking of this interaction may be the rate-determining step in the course of folding. On the basis of our simulations we estimate the folding rate of villin to be approximately 5micros. By analyzing the average features of the folded ensemble obtained by simulation, we see that the mean folded structure is more similar to the native fold than any individual folded structure. This finding highlights the need for simulating ensembles of molecules and averaging the results in an experiment-like fashion if meaningful comparison between simulation and experiment is to be attempted. Moreover, our results demonstrate that (1) the computational methodology exists to simulate the multi-microsecond regime using distributed computing and (2) that potential sets used to describe interatomic interactions may be sufficiently accurate to reach the folded state, at least for small proteins. We conclude with a comparison between our results and current protein-folding theory.     

Crystal structure of OxyB,a cytochrome P450 implicated in an oxidative phenol coupling reaction during vancomycin biosynthesis

Gene-inactivation studies point to the involvement of OxyB in catalyzing the first oxidative phenol coupling reaction during glycopeptide antibiotic biosynthesis. The oxyB gene has been cloned and sequenced from the vancomycin producer Amycolatopsis orientalis, and the hemoprotein has been produced in Escherichia coli, crystallized, and its structure determined to 1.7-A resolution. OxyB gave UV-visible spectra characteristic of a P450-like hemoprotein in the low spin ferric state. After reduction to the ferrous state by dithionite or by spinach ferredoxin and ferredoxin reductase, the CO-ligated form gave a 450-nm peak in a UV-difference spectrum. Addition of putative heptapeptide substrates to resting OxyB produced type I changes to the UV spectrum, but no turnover was observed in the presence of ferredoxin and ferredoxin reductase, showing that either the peptides or the reduction system, or both, are insufficient to support a full catalytic cycle. OxyB exhibits the typical P450-fold, with helix L containing the signature sequence FGHGXHXCLG and Cys(347) being the proximal axial thiolate ligand of the heme iron. The structural similarity of OxyB is highest to P450nor, P450terp, CYP119, and P450eryF. In OxyB, the F and G helices are rotated out of the active site compared with P450nor, resulting in a much more open active site, consistent with the larger size of the presumed heptapeptide substrate.     

Equilibrium shapes of erythrocytes in rouleau formation

A well known physiological property of erythrocytes is that they can aggregate and form a rouleau. We present a theoretical analysis of erythrocyte shapes in a long rouleau composed of cells with identical sizes. The study is based on the area difference elasticity model of lipid membranes, and takes into consideration the adhesion of curved axisymmetric membranes. The analysis predicts that the erythrocytes in the rouleau can have either a discoid or a cup-like shape. These shapes are analogous to the discoid and stomatocyte shapes of free erythrocytes. The transitions between the discoid and cup-like shapes in the rouleau are characterized. The occurrence of these transitions depends on three model parameters: the cell relative volume, the preferred difference between the areas of the membrane bilayer leaflets, and the strength of the adhesion between the membranes. The cup-like shapes are favored at small relative volumes and small preferred area differences, and the discoid shapes are favored at large values of these parameters. Increased adhesion strength enlarges the contact area between the cells, flattens the cells, and consequently promotes the discoid shapes.     

Pathogenesis of murine cytomegalovirus infection

Infection of mice with murine cytomegalovirus (MCMV) is an established model for studying human cytomegalovirus (HCMV) infection. Similarly to HCMV infection, pathological changes and disease manifestations during MCMV infection are mainly dependent on the immune status of the mouse host. This review focuses mainly on the pathogenesis of MCMV infection in immunocompetent and immunodeficient and/or immature mice and discusses the principles of immunosurveillance of infection and the mechanisms by which this virus evades immune control.