Use and abuse of the quasi-steady-state approximation

The transient kinetic behaviour of an open single enzyme, single substrate reaction is examined. The reaction follows the Van Slyke-Cullen mechanism, a spacial case of the Michaelis-Menten reaction. The analysis is performed both with and without applying the quasi-steady-state approximation. The analysis of the full system shows conditions for biochemical pathway coupling, which yield sustained oscillatory behaviour in the enzyme reaction. The reduced model does not demonstrate this behaviour. The results have important implications in the analysis of open biochemical reactions and the modelling of metabolic systems.     

Four stage liquid chromatographic selection of methionyl peptides for peptide-centric proteome analysis: the proteome of human multipotent adult progenitor cells

Serial application of strong cation-exchange and diagonal reversed-phase chromatography selecting methionyl peptides by stepwise shifting them from their reduced to their sulfoxide and sulfone forms generates a four-stage fractionation system, allowing high coverage analysis of complex proteome digests by LC-MALDI-MS/MS. Application to the proteome of a human multipotent adult progenitor cell line (MAPC) identified 2151 proteins with high confidence as on average four MS/MS-spectra were linked to each protein. Our dataset contains several novel, potential marker proteins that may be evaluated as affinity-anchors for isolating different adult stem cells in further studies. Furthermore, at least 2 tyrosine kinases that were previously linked to the self-renewal potential of stem cells were identified, validating the stemness of the analyzed cells. We also present data hinting at possible involvement of the ubiquitin/proteasome machinery in steering proliferation and/or differentiation of MAPC. Finally, following comparison of the MAPC proteome with proteomes of four human differentiated cell lines reveals differential usage of chromosomal information: compared to differentiated cells, MAPC do not appear to hold any preference for expressing genes located on specific chromosomes.     

Genetic diversity in Australian ancient asexual <Emphasis Type="Italic">Vestalenula</Emphasis> (Ostracoda,Darwinulidae): little variability down under

Darwinulid ostracods are putative ancient asexuals, and are thus assumed to be unable to purge deleterious mutations from their genomes. Some darwinulids species can be found both above (epigeic) and below ground (hypogeic). We hypothesize that surface populations carry more mutations than their below-ground counterparts, which are buffered from mutagens such as UV-B. Given the age of the investigated area, the Pilbara in Western Australia, we also expect geographic patterning of observed haplotypes. We have used DNA sequence data from the nuclear ITS and the mitochondrial COI region to investigate a (limited) data set on two Australian species, the endemic Vestalenula matildae and V. marmonieri from the Pilbara region. We do not find differences in genetic variability between specimens from subterranean habitats as compared to those from habitats above ground. There was also no congruence between hydrological basins and distribution patterns of the haplotypes identified. Although our data indicate that the two species may have split from each other ca. 70 myr ago, this has not resulted in any clear phylogeographic patterns among the analysed specimens across the regions of the Pilbara.     

Variation in ostracod (Crustacea,Ostracoda) communities in the alluvial valley of the upper Paraná River (Brazil) in relation to substrate

Large river floodplains are convenient model systems to test for variation in animal and plant community structure, as they have a variety of habitats and substrates and are generally dynamic systems through the occurrence of flood pulses with varying intensity. South American floodplain systems furthermore have unique types of substrates, in the form of root systems of floating macrophytes. Here, we investigate the variation in ostracod (small, bivalved crustaceans) communities in relation to substrates and related environmental variables. Sampling was effected in 2004 in the alluvial valley of the upper Paraná River, Brazil, in the wet and dry seasons. Five different substrates, including littoral sediment and four macrophyte species root and leaf systems, in four hydrological systems and a variety of habitat types, were sampled. Fifty-four species of Ostracoda were found. Variation partitioning analysis (RDA) showed that ostracod communities significantly differed between different substrates, mainly between the littoral and plants with small root systems (Eichhornia azurea) on the one hand, and plants with large and complex root systems on the other hand (Eichhornia crassipes and Pistia stratiotes). RDA analyses indicated that the pleuston (biotic communities associated with root systems of floating plants) of E. crassipes comprised more non-swimming species than the pleuston of the smaller roots of P. stratiotes, but species-level Kruskal–Wallis analyses could not detect significant differences between both macrophyte species. Also habitat type and hydrological systems contributed to variation amongst ostracod communities, but less so than the factor substrate. Abiotic factors also contributed to variation, but the ranges of all measured water chemistry variables were narrow. This uniformity in abiotic factors, which might be owing to the occurrence of large flooding events, unites all water bodies, even those that are generally separated.     

Quantification of right ventricular afterload in patients with and without pulmonary hypertension

Right ventricular (RV) afterload is commonly defined as pulmonary vascular resistance, but this does not reflect the afterload to pulsatile flow. The purpose of this study was to quantify RV afterload more completely in patients with and without pulmonary hypertension (PH) using a three-element windkessel model. The model consists of peripheral resistance (R), pulmonary arterial compliance (C), and characteristic impedance (Z). Using pulmonary artery pressure from right-heart catheterization and pulmonary artery flow from MRI velocity quantification, we estimated the windkessel parameters in patients with chronic thromboembolic PH (CTEPH; n = 10) and idiopathic pulmonary arterial hypertension (IPAH; n = 9). Patients suspected of PH but in whom PH was not found served as controls (NONPH; n = 10). R and Z were significantly lower and C significantly higher in the NONPH group than in both the CTEPH and IPAH groups (P < 0.001). R and Z were significantly lower in the CTEPH group than in the IPAH group (P < 0.05). The parameters R and C of all patients obeyed the relationship C = 0.75/R (R(2) = 0.77), equivalent to a similar RC time in all patients. Mean pulmonary artery pressure P and C fitted well to C = 69.7/P (i.e., similar pressure dependence in all patients). Our results show that differences in RV afterload among groups with different forms of PH can be quantified with a windkessel model. Furthermore, the data suggest that the RC time and the elastic properties of the large pulmonary arteries remain unchanged in PH.     

Determination of molecular conformation and permeation in skin via IR spectroscopy, microscopy, and imaging

Skin tissue, in addition to its specific use in dermal research, provides an excellent model for developing the techniques of vibrational microscopy and imaging for biomedical applications. In addition to permitting characterization of various regions of skin, the relative paucity of major biological constituents in the stratum corneum (the outermost layer of skin), permits us to image, with microscopic resolution, conformational alterations and concentration variations in both the lipid and protein components. Thus we are able to monitor the effects of exogenous materials such as models for drug delivery agents (liposomes) and permeation enhancers (DMSO) on stratum corneum lipid organization and protein structure. In addition, we are able to monitor protein conformational changes in single corneocytes. The current article demonstrates these procedures, ranging from direct univariate measures of lipid chain conformational disorder, to factor analysis which permits us to image conformational differences between liposomes that have permeated through the stratum corneum from those which have remained on the surface in a reservoir outside the skin.     

In situ analysis of single-stranded and duplex siRNA integrity in living cells

To attain the full therapeutic promise of short interfering RNA (siRNA), it is believed that improvements such as increased biostability are critical. Regrettably, thus far, insufficient in situ data are on hand regarding the intracellular stability of siRNAs. We report on the use of an advanced fluorescence-based method to probe the nucleolytic decay of double labeled siRNAs, which are subject to fluorescence resonance energy transfer (FRET). In vitro measurements with RNAse A and cellular extracts demonstrate that the ratio of acceptor (5'-Cy5) to donor (3'-rhodamine green) fluorescence can be used to study the degradation of the labeled siRNA substrates upon donor excitation. Intracellular FRET analysis showed substantial degradation of single-stranded siRNA, whereas duplex siRNA stayed intact during the measured time period. These data underline the high intrinsic nuclease resistance of unmodified duplex siRNA and prove that cellular persistence is much more critical for the single-stranded structure. For the first time, the stability of siRNA is investigated in real-time inside living cells. The fluorescence-based method presented here is a straightforward technique to gain direct information on siRNA integrity inside living cells and provides a bright outlook to learn more about the intracellular fate of siRNA therapeutics.