Adsorption behavior and enzymatically or chemically induced cross‐linking of a mussel adhesive protein

The adsorption behavior of the mussel adhesive protein Mytilus edulis foot protein‐1 (Mefp‐1) has been investigated on a negatively charged polar SiO₂ surface and an electrically inert non‐polar CH₃‐terminated thiolated gold surface. How the structure of adsorbed Mefp‐1 is changed upon chemically and enzymatically induced cross‐linking using sodium periodate (NaIO₄) and catechol oxidase, both of which transform DOPA residues in Mefp‐1 into highly reactive o‐quinones, was also investigated. The results are compared with those resulting from addition of Cu²⁺ to adsorbed Mefp‐1, which forms complexes with and catalyses oxidation of DOPA residues, previously suggested to participate in the cohesive and adhesive properties of the byssus thread of M. edulis. By combining surface plasmon resonance (SPR) and quartz crystal microbalance/dissipation (QCM‐D) measurements, the effects of these agents were investigated with respect to changes in the amount of coupled water, the viscoelastic properties (rigidity) and the hydrodynamic thickness of the protein adlayers. The layer of Mefp‐1 formed on the bare CH₃‐terminated surface was elongated, flexible and coupled hydrodynamically a substantial amount of water, whereas Mefp‐1 formed a rigidly attached adlayer on the SiO₂ surface. Upon enzymatically and chemically induced cross‐linking of Mefp‐1 formed on the CH₃ surface, the rigidity of the adlayer(s) increased significantly. A similar increase in the rigidity was observed also upon addition of Cu²⁺, suggesting that the high level of metal ions present in the byssus thread might be essential for the cohesive and adhesive properties of this protein. For the mass‐uptake kinetics of enzymatically induced cross‐linking, three different phases were observed and are interpreted as competition between binding of protein and release of coupled water. For the reaction with NaIO₄ and Cu²⁺, only release of water affected the coupled mass. The importance of this type of information for an improved understanding of the strong adhesion and cohesive properties in marine environments is discussed.     

Using ethidium to probe nonequilibrium states of DNA condensed for gene delivery

Here we explore the use of ethidium to determine relative affinities of different gene delivery vectors for DNA and describe an improved method for studying the interaction. Specifically, we investigate the binding of poly(amidoamine) dendrimers and show that the DNA-dendrimer-ethidium system is far from thermodynamic equilibrium. Moreover, dendrimer surface modification through PEGylation appears to make the interaction with DNA more reversible, which is favorable from the perspective of vector unpacking. Probing the nonequilibrium state of DNA during condensation processes is thus important for developing novel vectors, and further, it could also be useful in the study of chromatin folding.     

Aluminium blunts the proliferative response and increases apoptosis of cultured human cells: putative relationship to Alzheimer's disease

Aluminium (Al) has been investigated as a neurotoxic substance. Al ranks among the potential environmental risk factors for Alzheimer's disease (AD). Epidemiological studies tested the relationship between Al in drinking water and AD, showing a significant correlation between elevated levels of monomeric Al in water and AD, although data to date remain inconclusive with respect to total Al. The aim of this study was to test whether or not Al exacerbates cellular toxicity mediated by the amyloid beta (Abeta) peptide. We evaluated the role of Al in modulating programmed cell death (apoptosis) in human cell cultures. We used the osteosarcoma cell line monolayer (SaOs-2) to demonstrate that treatment of SaOs-2 cultures with the Abeta peptide mid-fragment (25 to 35) at nano M, followed by co-incubation with physiological concentrations of aluminium chloride, which release monomeric Al in solution, led to marked expression of caspase 3, but not caspase 9, key markers of the apoptotic process. The same experimental conditions were shown to blunt significantly the proliferative response of normal human peripheral blood mononuclear cells (PBMC) to phytohemagglutinin (PHA) stimulation. Our observations support the hypothesis that Al significantly impairs certain cellular immune responses, and confirm that Al-mediated cell toxicity may play an important role in AD.     

Sampling genetic diversity in the sympatrically and allopatrically speciating Midas cichlid species complex over a 16 year time series

Background  

Speciation often occurs in complex or uncertain temporal and spatial contexts. Processes such as reinforcement, allopatric divergence, and assortative mating can proceed at different rates and with different strengths as populations diverge. The Central American Midas cichlid fish species complex is an important case study for understanding the processes of speciation. Previous analyses have demonstrated that allopatric processes led to species formation among the lakes of Nicaragua as well as sympatric speciation that is occurring within at least one crater lake. However, since speciation is an ongoing process and sampling genetic diversity of such lineages can be biased by collection scheme or random factors, it is important to evaluate the robustness of conclusions drawn on individual time samples.     

High risk of early sub-therapeutic penicillin concentrations after intramuscular benzathine penicillin G injections in Ethiopian children and adults with rheumatic heart disease

IntroductionIntramuscular benzathine penicillin G (BPG) injections are a cornerstone of secondary prophylaxis to prevent acute rheumatic fever (ARF) and rheumatic heart disease (RHD). Uncertainties regarding inter-ethnic and preparation variability, and target exposure profiles of BPG injection are key knowledge gaps for RHD control.MethodsTo evaluate BPG pharmacokinetics (PK) in patients receiving 4-weekly doses in Ethiopia, we conducted a prospective cohort study of ARF/RHD patients attending cardiology outpatient clinics. Serum samples were collected weekly for one month after injection and assayed with a liquid chromatography-mass spectroscopy assay. Concentration-time datasets for BPG were analyzed by nonlinear mixed effects modelling using NONMEM.ResultsA total of 190 penicillin concentration samples from 74 patients were included in the final PK model. The median age, weight, BMI was 21 years, 47 kg and 18 kg/m², respectively. When compared with estimates derived from Indigenous Australian patients, the estimate for median (95% confidence interval) volume of distribution (V/F) was lower (54.8 [43.9–66.3] l.70kg^-1) whilst the absorption half-life (t_1/2-abs2) was longer (12.0 [8.75–17.7] days). The median (IQR) percentage of time where the concentrations remained above 20 ng/mL and 10 ng/mL within the 28-day treatment cycle was 42.5% (27.5–60) and 73% (58.5–99), respectively.ConclusionsThe majority of Ethiopian patients receiving BPG as secondary prophylaxis to prevent RHD do not attain target concentrations for more than two weeks during each 4-weekly injection cycle, highlighting the limitations of current BPG strategies. Between-population variation, together with PK differences between different preparations may be important considerations for ARF/RHD control programs.     

Evidence for carrier-mediated transport of glucocorticoids by human placental membrane vesicles

Glucocorticoid uptake by isolated placental membrane vesicles has been studied in an attempt to identify a membrane-mediated carrier mechanism. A preliminary communication from this laboratory has reported that uptake of the glucocorticoid corticosterone by these vesicles was a time-dependent, saturable, osmotically sensitive process (Fant, M.E., Harbison, R.D. and Harrison, R.W. (1979) J. Biol. Chem. 254, 6218-6221), but did not conclusively demonstrate a carrier mechanism. Further studies of labeled corticosterone uptake by placental vesicles are described herein which indicate that steroid uptake by these vesicles is a carrier-mediated process. We found that corticosterone uptake was temperature-sensitive, and an apparent phase-transition effect on the rate of uptake was seen to occur at approximately 16 degrees C. Treatment of the vesicles with phospholipase A2 and the sulfhydryl group attacker, p-chloromercuriphenylsulfonate, inhibited corticosterone uptake. In contrast to our previous findings in intact cells, neuraminidase treatment of membranes did not inhibit steroid uptake, perhaps indicating a species variation. Lastly, it was possible to show that corticosterone movement across the membrane exhibited countertransport, a phenomenon common only to carrier-mediated transport mechanisms. These studies show that placental vesicles accumulate corticosterone by a carrier-mediated mechanism.     

The influence of contemporary and historic landscape features on the genetic structure of the sand dune endemic,Cirsium pitcheri (Asteraceae)

Narrow endemics are at risk from climate change because of their restricted habitat preferences, lower colonization ability and dispersal distances. Landscape genetics combines new tools and analyses that allow us to test how both past and present landscape features have facilitated or hindered previous range expansion and local migration patterns, and thereby identifying potential limitations to future range shifts. We have compared current and historic habitat corridors in Cirsium pitcheri, an endemic of the linear dune ecosystem of the Great Lakes, to determine the relative contributions of contemporary migration and post-glacial range expansion on genetic structure. We used seven microsatellite loci to characterize the genetic structure for 24 populations of Cirsium pitcheri, spanning the center to periphery of the range. We tested genetic distance against different measures of geographic distance and landscape permeability, based on contemporary and historic landscape features. We found moderate genetic structure (Fst=0.14), and a north–south pattern to the distribution of genetic diversity and inbreeding, with northern populations having the highest diversity and lowest levels of inbreeding. High allelic diversity, small average pairwise distances and mixed genetic clusters identified in Structure suggest that populations in the center of the range represent the point of entry to the Lake Michigan and a refugium of diversity for this species. A strong association between genetic distances and lake-level changes suggests that historic lake fluctuations best explain the broad geographic patterns, and sandy habitat best explains local patterns of movement.     

Centrosome proteins form an insoluble perinuclear matrix during muscle cell differentiation

Background  

Muscle fibres are formed by elongation and fusion of myoblasts into myotubes. During this differentiation process, the cytoskeleton is reorganized, and proteins of the centrosome re-localize to the surface of the nucleus. The exact timing of this event, and the underlying molecular mechanisms are still poorly understood.     

A new technique for staining mast cells using ferroin

We describe here a new method for specific staining of mast cells using ferroin. Different hamster tissues were fixed in 4% formalin and processed for paraffin embedding. Sections were stained with hematoxylin followed by ferroin acidified with 2.5 N sulfuric acid to pH 4.0. Mast cells stained an intense orange color that contrasted markedly with bluish violet nuclei. High contrast was also observed when ferroin colored sections were counterstained with light green instead of hematoxylin. To evaluate the specificity of the stain, hamster cheek pouch sections were stained with toluidine blue, alcian blue-safranin O, and ferroin. Quantitative evaluation of mast cells stained with the three techniques showed no statistical difference. The simplicity and selectivity of this method is sufficient for image analysis of mast cells.