Synthesis, resolution and charge-donor properties of six tryptophan analogues.

In order to investigate the special function of tryptophan in peptide hormones, six tryptophan analogues have been synthesized, in which structural resemblance has been grossly retained and potentially essential properties have only partially been varied. The L-enantiomers have been isolated after enzymatic digestion of racemic dipeptide derivatives, and charge-transfer properties of the compounds have been studied.     

Metabolic network discovery through reverse engineering of metabolome data

Reverse engineering of high-throughput omics data to infer underlying biological networks is one of the challenges in systems biology. However, applications in the field of metabolomics are rather limited. We have focused on a systematic analysis of metabolic network inference from in silico metabolome data based on statistical similarity measures. Three different data types based on biological/environmental variability around steady state were analyzed to compare the relative information content of the data types for inferring the network. Comparing the inference power of different similarity scores indicated the clear superiority of conditioning or pruning based scores as they have the ability to eliminate indirect interactions. We also show that a mathematical measure based on the Fisher information matrix gives clues on the information quality of different data types to better represent the underlying metabolic network topology. Results on several datasets of increasing complexity consistently show that metabolic variations observed at steady state, the simplest experimental analysis, are already informative to reveal the connectivity of the underlying metabolic network with a low false-positive rate when proper similarity-score approaches are employed. For experimental situations this implies that a single organism under slightly varying conditions may already generate more than enough information to rightly infer networks. Detailed examination of the strengths of interactions of the underlying metabolic networks demonstrates that the edges that cannot be captured by similarity scores mainly belong to metabolites connected with weak interaction strength.     

Reevaluation of the electrophoretic migration behavior of soluble globular proteins in the native and detergent-denatured states in polyacrylamide gels

Although sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis is widely used for estimating molecular masses of proteins, considerable uncertainty still exists both about the structure of SDS-protein complexes and about their mechanism of electrophoretic migration. In this study, soluble globular proteins, with masses of 14-200 kDa, were heat-denatured in the presence of SDS and their relative total molecular volume and net charge were estimated from Ferguson plots of electrophoretic mobility vs acrylamide concentration. Native globular protein served as standards for overall molecular size and effective radii. Results revealed at least two independent electrophoretic migration mechanisms for the SDS-protein complexes: (i) for proteins in the 14-65 kDa range at <15% acrylamide, linear Ferguson plots suggested that they migrated ideally and that their effective radii could be estimated in this manner: (ii) concave plots at higher gel concentrations, and for complexes derived from larger proteins, indicated that migration in these cases could be described by reptation theory. Migration of the large proteins at lower gel concentrations and small proteins at higher gel concentrations was not well described by either theory, representing intermediate behavior not described by these mechanisms. These data support models in which all but the smallest SDS-protein complexes adopt a necklace-like structure in which spherical micelles are distributed along the unfolded polypeptide chain. Possible relations to recent alternative models of gel electrophoresis are also discussed.     

Pigment organization of the B800–850 antenna complex of Rhodopseudomonas sphaeroides

The B800–850 antenna complex of Rhodopseudomonas sphaeroides was studied by comparing the spectral properties of several different types of complexes, isolated from chromatophores by means of the detergents lithium dodecyl sulfate (LDS) or lauryl dimethylamine N-oxide (LDAO). Fluorescence polarization spectra of the BChl 800 emission at 4 K indicated that rapid energy transfer between at least two BChl 800 molecules occurs with a rate constant of energy transfer k_ET > 3 · 10¹² s^?1. The maximal dipole-dipole distance between the two BChl 800 molecules was calculated to be 18–19 Å. The porphyrin rings of the BChl 800 molecules are oriented parallel to each other, while their Q_y transition moments are mutually perpendicular. The energy-transfer efficiency from carotenoid to bacteriochlorophyll measured in different complexes showed that two functionally different carotenoids are present associated with, respectively, BChl 800 and BChl 850. Fluorescence polarization and linear dichroism spectra revealed that these carotenoids have different absorption spectra and a different orientation with respect to the membrane. The carotenoid associated with BChl 800 absorbs some nanometers more to the red and its orientation is approximately parallel to the membrane, while the carotenoid associated with BChl 850 is oriented more or less perpendicular to the membrane. The fluorescence polarization of BChl 850 was the same for the different complexes. This indicates that the observed polarization of the fluorescence is determined by the smallest complex obtained which contains 8–10 BChl 850 molecules. The B800–850 complex isolated with LDAO thus must consist of a highly ordered array of smaller structures. On basis of these results a minimal model is proposed for the basic unit consisting of four BChl 850 and two BChl 800 and three carotenoid molecules.     

Multiblock PLS analysis of an industrial pharmaceutical process

The performance of an industrial pharmaceutical process (production of an active pharmaceutical ingredient by fermentation, API) was modeled by multiblock partial least squares (MBPLS). The most important process stages are inoculum production and API production fermentation. Thirty batches (runs) were produced according to an experimental planning. Rather than merging all these data into a single block of independent variables (as in ordinary PLS), four data blocks were used separately (manipulated and quality variables for each process stage). With the multiblock approach it was possible to calculate weights and scores for each independent block. It was found that the inoculum quality variables were highly correlated with API production for nominal fermentations. For the nonnominal fermentations, the manipulations of the fermentation stage explained the amount of API obtained (especially the pH and biomass concentration). Based on the above process analysis it was possible to select a smaller set of variables with which a new model was built. The amount of variance predicted of the final API concentration (cross-validation) for this model was 82.4%. The advantage of the multiblock model over the standard PLS model is that the contributions of the two main process stages to the API volumetric productivity were determined.     

Isolation,size estimates,and spectral heterogeneity of an oligomeric series of light-harvesting 1 complexes from Rhodobacter sphaeroides

A series of light-harvesting 1 (LH1) complexes was isolated by lithium dodecyl sulfate-polyacrylamide gel electrophoresis at 4 degrees C from Rhodobacter sphaeroides M21, which lacks the peripheral light-harvesting 2 (LH2) complex. This ladder of LH1 bands was also demonstrated in the wild type, partially superimposed upon a smaller number of LH2 complexes. An assessment of electrophoretic mobility vs acrylamide concentration, in which the reaction center LM particle and annular LH1 and LH2 complexes were used as standards of known structure, indicated that the LH1 gel bands 2 to 10 represent regular oligomers of an alpha beta heterodimeric unit, that vary in size from (alpha beta)(2-3) to (alpha beta)(10-11). The isolated LH1 complexes exhibited oligomeric state dependent optical properties, characterized by red shifts in near-IR absorption and emission maxima at 77 K of approximately 6 nm as aggregate sizes increased from approximately 3 to 7-8 alpha beta-heterodimers, accompanied by shifts in highly polarized fluorescence from the blue to the red side of the absorption band. This has been explained by the oligomerization of heterodimers to form a curvilinear array of excitonically coupled chromophores, with the anisotropic long-wavelength component, designated originally as B896, corresponding to low energy excitonic transitions arising from interactions within inhomogeneous BChl clusters [Westerhuis et al. (1999) J. Phys. Chem. B 103, 7733-7742]. Differences in electrophoretic profiles of LH1 bands between strains M21 and M2192, an LH1-only strain that also lacks PufX, further suggested that the more rapidly migrating bands represent arced fragments of the curvilinear array of LH1 complexes thought to exist as a large closed circular structure only in the latter strain. The electrophoretic banding pattern also indicated that the LH1 complex may be located at the peripheries of dimeric intramembrane particle arrays seen in freeze-fracture replicas of tubular M21 membranes; the possible role for the PufX protein in the assembly of these structures is discussed.     

Systematic selection of competing metabolomics methods in a metabolite-sensory relationship study

Metabolomics - To study metabolic signatures can be used to identify predictive biomarkers for a patient's therapeutic response. We hypothesized that the characterization of a patients’...     

Prevalence and Clinical Course in Invasive Infections with Meningococcal Endotoxin Variants

Background

Meningococci produce a penta-acylated instead of hexa-acylated lipid A when their lpxL1 gene is inactivated. Meningococcal strains with such lipid A endotoxin variants have been found previously in adult meningitis patients, where they caused less blood coagulopathy because of decreased TLR4 activation.

Methods

A cohort of 448 isolates from patients with invasive meningococcal disease in the Netherlands were screened for the ability to induce IL-6 in monocytic cell Mono Mac 6 cells. The lpxL1 gene was sequenced of isolates, which show poor capacity to induce IL-6.. Clinical characteristics of patients were retrieved from hospital records.

Results

Of 448 patients, 29 (6.5%) were infected with meningococci expressing a lipid A variant strain. Lipid A variation was not associated with a specific serogroup or genotype. Infections with lipid A variants were associated with older age (19.3 vs. 5.9 (median) years, p = 0.007) and higher prevalence of underlying comorbidities (39% vs. 17%; p = 0.004) compared to wild-type strains. Patients infected with lipid A variant strains had less severe infections like meningitis or shock (OR 0.23; 95%CI 0.09–0.58) and were less often admitted to intensive care (OR 0.21; 95%CI 0.07–0.60) compared to wild-type strains, independent of age, underlying comorbidities or strain characteristics.

Conclusions

In adults with meningococcal disease lipid A variation is rather common. Infection with penta-acylated lipid A variant meningococci is associated with a less severe disease course.