Development of an open-tubular trypsin reactor for on-line digestion of proteins

A study was initiated to construct a micro-reactor for protein digestion based on trypsin-coated fused-silica capillaries. Initially, surface plasmon resonance was used both for optimization of the surface chemistry applied in the preparation and for monitoring the amount of enzyme that was immobilized. The highest amount of trypsin was immobilized on dextran-coated SPR surfaces which allowed the covalent coupling of 11 ng mm−2 trypsin. Fused-silica capillaries were modified in a similar manner and the resulting open-tubular trypsin-reactors having a pH optimum of pH 8.5, display a high activity when operated at 37 °C and are stable for at least two weeks when used continuously. Trypsin auto-digestion fragments, sample carry-over, and loss of signal due to adsorption of the protein were not observed. On-line digestion without prior protein denaturation, followed by micro-LC separation and photodiode array detection, was tested with horse-heart cytochrome C and horse skeletal-muscle myoglobin. The complete digestion of 20 pmol μL−1 horse cytochrome C was observed when the average residence time of the protein sample in a 140 cm ×50 μm capillary immobilized enzyme reactor (IMER) was 165 s. Mass spectrometric identification of the injected protein on the basis of the tryptic peptides proved possible. Protein digestion was favorable with respect to reaction time and fragments formed when compared with other on-line and off-line procedures. These results and the easy preparation of this micro-reactor provide possibilities for miniaturized enzyme-reactors for on-line peptide mapping and inhibitor screening

Effects of external nutrient sources and extreme weather events on the nutrient budget of a Southern European coastal lagoon

The seasonal and annual nitrogen (N), phosphorus (P), and carbon (C) budgets of the mesotidal Ria Formosa lagoon, southern Portugal, were estimated to reveal the main inputs and outputs, the seasonal patterns, and how they may influence the ecological functioning of the system. The effects of extreme weather events such as long-lasting strong winds causing upwelling and strong rainfall were assessed. External nutrient inputs were quantified; ocean exchange was assessed in 24-h sampling campaigns, and final calculations were made using a hydrodynamic model of the lagoon. Rain and stream inputs were the main freshwater sources to the lagoon. However, wastewater treatment plant and groundwater discharges dominated nutrient input, together accounting for 98, 96, and 88 % of total C, N, and P input, respectively. Organic matter and nutrients were continuously exported to the ocean. This pattern was reversed following extreme events, such as strong winds in early summer that caused upwelling and after a period of heavy rainfall in late autumn. A principal component analysis (PCA) revealed that ammonium and organic N and C exchange were positively associated with temperature as opposed to pH and nitrate. These variables reflected mostly the benthic lagoon metabolism, whereas particulate P exchange was correlated to Chl a, indicating that this was more related to phytoplankton dynamics. The increase of stochastic events, as expected in climate change scenarios, may have strong effects on the ecological functioning of coastal lagoons, altering the C and nutrient budgets.Portuguese Science and Technology Foundation (FCT) [POCI/MAR/58427/2004, PPCDT/MAR/58427/2004]; Portuguese Science and Technology Foundation (FCT

Non-monotonic variation with salt concentration of the second virial coefficient in protein solutions

The osmotic virial coefficient $B_2$ of globular protein solutions is calculated as a function of added salt concentration at fixed pH by computer simulations of the ``primitive model''. The salt and counter-ions as well as a discrete charge pattern on the protein surface are explicitly incorporated. For parameters roughly corresponding to lysozyme, we find that $B_2$ first decreases with added salt concentration up to a threshold concentration, then increases to a maximum, and then decreases again upon further raising the ionic strength. Our studies demonstrate that the existence of a discrete charge pattern on the protein surface profoundly influences the effective interactions and that non-linear Poisson Boltzmann and Derjaguin-Landau-Verwey-Overbeek (DLVO) theory fail for large ionic strength. The observed non-monotonicity of $B_2$ is compared to experiments. Implications for protein crystallization are discussed.Comment: 43 pages, including 17 figure

Lipoproteins act as vehicles for lipid antigen delivery and activation of invariant natural killer T cells

Invariant natural killer T (iNKT) cells act at the interface between lipid metabolism and immunity because of their restriction to lipid antigens presented on CD1d by antigen-presenting cells (APCs). How foreign lipid antigens are delivered to APCs remains elusive. Since lipoproteins routinely bind glycosylceramides structurally similar to lipid antigens, we hypothesized that circulating lipoproteins form complexes with foreign lipid antigens. In this study, we used 2-color fluorescence correlation spectroscopy to show, for the first time to our knowledge, stable complex formation of lipid antigens α-galactosylceramide (αGalCer), isoglobotrihexosylceramide, and OCH, a sphingosine-truncated analog of αGalCer, with VLDL and/or LDL in vitro and in vivo. We demonstrate LDL receptor-mediated (LDLR-mediated) uptake of lipoprotein-αGalCer complexes by APCs, leading to potent complex-mediated activation of iNKT cells in vitro and in vivo. Finally, LDLR-mutant PBMCs of patients with familial hypercholesterolemia showed impaired activation and proliferation of iNKT cells upon stimulation, underscoring the relevance of lipoproteins as a lipid antigen delivery system in humans. Taken together, circulating lipoproteins form complexes with lipid antigens to facilitate their transport and uptake by APCs, leading to enhanced iNKT cell activation. This study thereby reveals a potentially novel mechanism of lipid antigen delivery to APCs and provides further insight into the immunological capacities of circulating lipoproteins

Derivations that enable the testing of fetal urine production as a method of fetal surveillance

To calculate the measurement error of the hourly fetal urine production rate (HFUPR) and evaluate the implication of different methods for measuring the HFUPR, i.e. ellipsoid versus sum-of-cylinders method

Maternal health-related quality of life after induction of labor or expectant monitoring in pregnancy complicated by intrauterine growth retardation beyond 36 weeks

Objective: Pregnancies complicated by intrauterine growth retardation (IUGR) beyond 36 weeks of gestation are at increased risk of neonatal morbidity and mortality. Optimal treatment in IUGR at term is highly debated. Results from the multicenter DIGITAT (Disproportionate Intrauterine Growth Intervention Trial At Term) trial show that induction of labor and expectant monitoring result in equal neonatal and maternal outcomes for comparable cesarean section rates. We report the maternal health-related quality of life (HR-QoL) that was measured alongside the trial at several points in time. Methods: Both randomized and non-randomized women were asked to participate in the HR-QoL study. Women were asked to fill out written validated questionnaires, covering background characteristics, condition-specific issues and the Short Form (SF-36), European Quality of Life (EuroQoL 6D3L), Hospital Anxiety and Depression scale (HADS), and Symptom Check List (SCL-90) at baseline, 6 weeks postpartum and 6 months postpartum. We compared the difference scores of all summary measures between the two management strategies by ANOVA. A repeated measures multivariate mixed model wa

PAS-positive lymphocyte vacuoles can be used as diagnostic screening test for Pompe disease

Screening of blood films for the presence of periodic acid-Schiff (PAS)-positive lymphocyte vacuoles is sometimes used to support the diagnosis of Pompe disease, but the actual diagnostic value is still unknown. We collected peripheral blood films from 65 untreated Pompe patients and 51 controls. Lymphocyte vacuolization was quantified using three methods: percentage vacuolated lymphocytes, percentage PAS-positive lymphocytes, and a PAS score depending on staining intensity. Diagnostic accuracy of the tests was assessed using receiver operating characteristic (ROC) curves. All three methods fully discerned classic infantile patients from controls. The mean values of patients with milder forms of Pompe disease were significantly higher than those of controls, but full separation was not obtained. The area under the ROC curve was 0.98 for the percentage vacuolated lymphocytes (optimal cutoff value 3; sensitivity 91%, specificity 96%) and 0.99 for the percentage PAS-positive lymphocytes and PAS score (optimal cutoff value 9; sensitivity 100%, specificity 98%). Our data indicate that PAS-stained blood films can be used as a reliable screening tool to support a diagnosis of Pompe disease. The percentage of PAS-positive lymphocytes is convenient for use in clinical practice but should always be interpreted in combination with other clinical and laboratory parameters

An Increase in Mitochondrial DNA Promotes Nuclear DNA Replication in Yeast

Coordination between cellular metabolism and DNA replication determines when cells initiate division. It has been assumed that metabolism only plays a permissive role in cell division. While blocking metabolism arrests cell division, it is not known whether an up-regulation of metabolic reactions accelerates cell cycle transitions. Here, we show that increasing the amount of mitochondrial DNA accelerates overall cell proliferation and promotes nuclear DNA replication, in a nutrient-dependent manner. The Sir2p NAD+-dependent de-acetylase antagonizes this mitochondrial role. We found that cells with increased mitochondrial DNA have reduced Sir2p levels bound at origins of DNA replication in the nucleus, accompanied with increased levels of K9, K14-acetylated histone H3 at those origins. Our results demonstrate an active role of mitochondrial processes in the control of cell division. They also suggest that cellular metabolism may impact on chromatin modifications to regulate the activity of origins of DNA replication

Metalloporphyrin intercalation in liposome membranes: ESR study

Liposomes characterized by membranes featuring diverse fluidity (liquid-crystalline and/or gel phase), prepared from egg yolk lecithin (EYL) and dipalmitoylphosphatidylcholine (DPPC), were doped with selected metalloporphyrins and the time-related structural and dynamic changes within the lipid double layer were investigated. Porphyrin complexes of Mg(II), Mn(III), Fe(III), Co(II), Ni(II), Cu(II), Zn(II), and the metal-free base were embedded into the particular liposome systems and tested for 350 h at 24°C using the electron spin resonance (ESR) spin probe technique. 5-DOXYL, 12-DOXYL, and 16-DOXYL stearic acid methyl ester spin labels were applied to explore the interior of the lipid bilayer. Only the 16-DOXYL spin probe detected evident structural changes inside the lipid system due to porphyrin intercalation. Fluidity of the lipid system and the type of the porphyrin complex introduced significantly affected the intermolecular interactions, which in certain cases may result in self-assembly of metalloporphyrin molecules within the liposome membrane, reflected in the presence of new lines in the relevant ESR spectra. The most pronounced time-related effects were demonstrated by the EYL liposomes (liquid-crystalline phase) when doped with Mg and Co porphyrins, whereas practically no spectral changes were revealed for the metal-free base and both the Ni and Zn dopants. ESR spectra of the porphyrin-doped gel phase of DPPC liposomes did not show any extra lines; however, they indicated the formation of a more rigid lipid medium. Electronic configuration of the porphyrin’s metal center appeared crucial to the degree of molecular reorganization within the phospholipid bilayer system