Micro- and macrorheological properties of actin networks effectively cross-linked by depletion forces

The structure and rheology of cytoskeletal networks are regulated by actin binding proteins. Aside from these specific interactions, depletion forces can also alter the properties of cytoskeletal networks. Here we demonstrate that the addition of poly(ethylene glycol) (PEG) as a depletion agent results not only in severe structural changes, but also in alterations in mechanical properties of actin solutions. In the plateau of the elastic modulus two regimes can be distinguished by micro and macrorheological methods. In the first, the elastic modulus increases only slightly with increasing depletion agent, whereas above a critical concentration c*, a strong increase of cPEG6k3.5 is observed in a distinct second regime. Microrheological data and electron microscopy images show a homogenous network of actin filaments in the first regime, whereas at higher PEG concentrations a network of actin bundles is observed. The concentration dependence of the plateau modulus G0, the shift in entanglement time taue, and the nonlinear response indicate that below c* the network becomes effectively cross-linked, whereas above c* G0(cPEG6k) is primarily determined by the network of bundles that exhibits a linearly increasing bundle thickness.     

Determination of the dissociation constants of ropinirole and some impurities and their quantification using capillary zone electrophoresis

Ropinirole, 4-[2-(dipropylamino)ethyl]-1,3-dihydro-2H-indol-2-one, is a potent anti-Parkinson’s disease drug developed by SmithKline Beecham Pharmaceuticals. Capillary zone electrophoresis (CZE) was used for the determination of the dissociation constants of ropinirole and five structurally related impurities, potentially formed during its synthesis and for separation and quantification of these substances. The dissociation constants obtained from the CZE measurements were confirmed by UV spectrophotometry for some of the test compounds, obtaining a good agreement between the values. Careful optimization of the running buffer composition permitted base-line resolution of the six compounds in a borate buffer containing acetonitrile and magnesium sulfate (a 100 mM borate buffer containing 30 mM MgSO₄ and 20 vol.% of acetonitrile). It was shown that CZE can determine the level of these impurities, down to a level of 0.05% of the main component within 15 min.     

Anatomical and functional changes in the upper airways of sleep apnea patients due to mandibular repositioning: a large scale study

The obstructive sleep apnea-hypopnea syndrome (OSAHS) is a sleep related breathing disorder. A popular treatment is the use of a mandibular repositioning appliance (MRA) which advances the mandibula during the sleep and decreases the collapsibility of the upper airway. The success rate of such a device is, however, limited and very variable within a population of patients. Previous studies using computational fluid dynamics have shown that there is a decrease in upper airway resistance in patients who improve clinically due to an MRA. In this article, correlations between patient-specific anatomical and functional parameters are studied to examine how MRA induced biomechanical changes will have an impact on the upper airway resistance. Low-dose computed tomography (CT) scans are made from 143 patients suffering from OSAHS. A baseline scan and a scan after mandibular repositioning (MR) are performed in order to study variations in parameters. It is found that MR using a simulation bite is able to induce resistance changes by changing the pharyngeal lumen. The change in minimal cross-sectional area is the best parameter to predict the change in upper airway resistance. Looking at baseline values, the ideal patients for MR induced resistance decrease seem to be women with short airways, high initial resistance and no baseline occlusion.     

The newborn rat's stress system readily habituates to repeated and prolonged maternal separation, while continuing to respond to stressors in context dependent fashion

Adrenal corticosterone secretion of newborn mice rapidly desensitizes to repeated maternal absence. The present study investigated the effects of novelty exposure, maternal care and genotype on this phenomenon.Maternal separation (MS) took place on postnatal days (pnd) 3-5. In Wistar rats, the degree of novelty in the MS-environment was varied by exposing pups to: (i) “home separation”: pups remained in the home cage; (ii) “novel separation”: pups were placed individually in a novel cage. Maternal care was recorded on pnd 1 to 4. To investigate the effect of genotype, we also examined Long Evans in the “home separation” condition. Basal and stress-induced ACTH and corticosterone levels were measured. Adrenal tyrosine hydroxylase (TH) and melanocortin receptor-2 (MCR-2) proteins served as markers for adrenal function.We show, in both rat strains, that the rise in plasma corticosterone induced by a single 8 h-MS on pnd 5 was abolished, when this separation procedure had also been performed on pnd 3 and 4. Habituation to maternal absence occurred irrespective of housing conditions. However, pups in the “home separation” condition received less maternal care upon reunion than those placed in the “novel separation”. These “home separation” pups appeared more responsive to a subsequent acute novelty-stressor, and their adrenal TH and MCR-2 were higher. Long Evans rats appeared more stress responsive than the Wistars, in the home separation condition.In conclusion, separation environment, maternal care and genotype do not affect adrenal desensitization to repeated 8 h-MS itself, but may modulate the adrenal stress-responsiveness of separated pups.     

Amyloids of Alpha-Synuclein Affect the Structure and Dynamics of Supported Lipid Bilayers

Interactions of monomeric alpha-synuclein (αS) with lipid membranes have been suggested to play an important role in initiating aggregation of αS. We have systematically analyzed the distribution and self-assembly of monomeric αS on supported lipid bilayers. We observe that at protein/lipid ratios higher than 1:10, αS forms micrometer-sized clusters, leading to observable membrane defects and decrease in lateral diffusion of both lipids and proteins. An αS deletion mutant lacking amino-acid residues 71–82 binds to membranes, but does not observably affect membrane integrity. Although this deletion mutant cannot form amyloid, significant amyloid formation is observed in the wild-type αS clusters. These results suggest that the process of amyloid formation, rather than binding of αS on membranes, is crucial in compromising membrane integrity.     

Rat prostatic binding protein: the complete sequence of the C2 gene and its flanking regions.

The complete sequence (2879 bp) of the androgen-controlled rat prostatic binding protein C2 gene and 1023 bp of the 5'- and 2127 bp of the 3'-flanking regions have been determined. The gene contains three exons (93, 203 and 147 bp) and two introns (1630 and 806 bp). It is flanked by two homopurine-homopyrimidine stretches of 55 and 131 nucleotides respectively, located at positions -405 and 4151. These sequences are remarkably sensitive towards S1-nuclease, indicating an altered DNA conformation under superhelical stress. Several palindromes and dyad structures are observed in the 5'-upstream region of the gene and at position -457, and 80% homology to the consensus sequence of a glucocorticoid receptor binding site is found.     

Dynamic hydrochemical and vegetation gradients in fens

The mixing of groundwater, river water and precipitation was studied in a discharge and a recharge fen in the Vechtplassen area, the Netherlands. The aim of the study was to characterize relationships between vegetation in fens, the chemical composition of the fen water, and the hydrologic regimes. We were particularly interested in the influence of polluted water from the river Vecht on vegetation and nutrient dynamics. Analyses were made for Ionic Ratio (IR = 2[Ca]/(2[Ca] + [Cl]), molar concentrations) and Electrical Conductivity (EC), all indices of the relative importance of each of the three main water sources: groundwater, river water and precipitation.During winter, water in the discharge fen was strongly influenced by calcium-rich groundwater (high IR, moderate EC), while the recharge fen was entirely fed by precipitation (low IR and EC). During summer, river water with a moderate IR and high EC infiltrated the fens and caused a dramatic change in the chemical composition of the fen water. In the discharge fen, infiltration occurred predominantly as sheetflow, causing inundation of the entire fen surface. River water infiltration affected the surface peat of this fen more severely than deeper peat layers. Spatial variation in water chemistry along the transect was small, and only one type of plant community was found. In the recharge fen, river water infiltration occurred in subsurface water, and rainwater that had accumulated during the winter persisted in the central parts of the fen. Gradients in fen water chemistry were correlated with the observed distribution of three plant associations. Differences in water chemistry could also be attributed to intra-site variation in the relative importance of the three water sources. In both fens, there was considerable temporal variation in the chemical composition of the fen water, clearly related to dynamics of the hydrologic regime, particularly infiltration of river water. There are indications that characteristic mesotraphent fen plant communities are negatively affected by water from the river Vecht. River water supply should therefore be avoided as much as possible if these fen plant communities are to be maintained.     

Theory and practice of combining coagulant fixation and microwave histoprocessing

The German, F. Blum, introduced formalin as a fixative in 1893. Formalin rapidly became popular for hardening and preserving gross human and animal specimens. As a result, microscopy for diagnostic pathology by combining paraffin embedding and formalin fixation was developed. Alcohol-based fixatives have coagulation of proteins as their main preservative effect. Because there is no cross-linking, immunostaining is not compromised, and DNA and RNA is not damaged. Ethyl alcohol was used by Dutch scientists of the 18th century, but was replaced by the cheaper formalin. Addition of low molecular weight polyethylene glycol (PEG) optimized the coagulant fixative, Kryofix. The polyethylene glycol prevents excessive hardening and enhances the speed of coagulation of proteins. Kryofix was used on a large scale for skin biopsies in Leiden between 1987 and 2001. DNA preservation by the formulated coagulant fixative, BoonFix, is related to the concentration of ethyl alcohol, PEG and acetic acid. BoonFix has been used since 2004 in Leiden for over 40,000 diagnostic skin biopsies and more than 100,000 cervical samples. A literature review and three decades of experience with coagulant, formalin-free fixatives in pathology suggest that when health authorities realize that formalin invalidates expensive tests, it might eventually be eliminated legislatively from diagnostic pathology. Finally, coagulant fixation is optimal for microwave histoprocessing where ethyl alcohol is followed by isopropanol.