The mysid-feeding guild of demersal fishes in the brackish zone of the Westerschelde estuary

The demersal fish fauna of the mesohaline zone of the Westerschelde estuary (south-west Netherlands) was sampled intensively in the period 1990–1992. Almost 500 beam trawl samples were taken in both subtidal (330 samples) and intertidal (144 samples) habitats. These yielded 44 fish species, mostly as juveniles. The area functioned as a nursery for several demersal fish species, and harboured large populations of hyperbenthic mysids. Three gobies, three flatfish, one clupeoid and one gadoid dominated the fish fauna, while three mysid species were important components of the holohyperbenthos. From c. 1500 stomach contents of 25 fish species, 44 prey species were identified, the most abundant of which were also common in the hyperbenthal. The demersal fish community consisted of a group that foraged subtidally on fast-moving epi-and hyperbenthic prey (for example gadoids, gobies and clupeoids) and a group that foraged on slow-moving or sessile endobenthic organisms, mainly in intertidal areas (for example most flatfish species). Mysidacea occurred in >50% stomachs analysed and were taken as prey by 19 of the 25 fish species. Mysids were most important in the diets of Pomatoschistus minutus, P. lozanoi, Trisopterus luscus and Merlangius merlangus, and were present in appreciable numbers in Pleuronectes flesus, Trigla lucerna, Clupea harengus and Pleuronectes platessa. These species fed mainly on the brackish water endemic Neomysis integer. Mesopodopsis slabberi (present in 35% of the gobiid stomachs) and Gastrosaccus spinifer(present in 25% of the gadoid stomachs) were of secondary importance. P. minutus and T. luscus showed a diet shift from calanoids (Eurytemora aYnis and Temora longicornis) to mysids at L_S of 30 and 50 mm, respectively. Only 1% of the standing stocks of the N. integer and M. slabberi populations was removed by the local demersal fish community, so top–down control of mysid populations in estuaries seems unlikely.     

In VitroMeasurement of β-Lactamase-Catalyzed Ampicillin Hydrolysis by RecombinantEscherichia coliExtracts Using Quantitative High-Performance Liquid Chromatography

We report a rapid and simple protocol for measuring the β-lactamase activity from recombinantEscherichia colicells transformed with any of the common plasmid vectors that provide ampicillin resistance through constitutive expression and periplasmic localization of the β-lactamase TEM-1. The hydrolytic enzyme was extracted from theE. coliperiplasm and the β-lactamase activity determined by measuring conversion of ampicillin to aminobenzyl-penicilloic acid using quantitative high-performance liquid chromatography. Under saturating conditions thein vitroassay was linear as a function of both incubation time and enzyme concentration. Application of this assay to investigate TEM-1 expression, from two different protein expression vector systems, demonstrated the potential importance of this assay in studies of recombinant protein expression and translocation.     

Vertical forces on the horse's back in sitting and rising trot

In equestrian sports, it is generally assumed that rising and sitting trot load the horse's back differently. The objective of this study was to quantify the load on the horse's back in these riding techniques. Kinematic data of 13 riders were collected in rising and sitting trot. The time-history of the position of the rider's centre of mass (CoM) was calculated, and differentiated twice to obtain the acceleration of the CoM. The reaction force between the rider and the horse's back was calculated from the acceleration. Forces were divided by the body weight of the rider to obtain dimensionless forces. As expected, the computed average vertical force did not differ between riding techniques and was not significantly different from the body weight of the riders. At trot, two force peaks were present during one stride cycle. Both peaks in rising trot were significantly lower compared to sitting trot (peak 1: 2.54±0.30 versus 2.92±0.29; p<0.001; peak 2: 1.95±0.34 versus 3.03±0.32; p<0.001). This supports the general assumption that rising trot is less demanding for the horse than sitting trot.     

Demethylation using the epigenetic modifier, 5-azacytidine, increases the efficiency of transient transfection of macrophages

This study was aimed at developing a method for high-efficiency transient transfection of macrophages. Seven methods were evaluated for transient transfection of murine macrophage RAW 264.7 cells. The highest transfection efficiency was achieved with DEAE-dextran, although the proportion of cells expressing the reporter gene did not exceed 20%. It was subsequently found that the cytomegalovirus plasmid promoter in these cells becomes methylated. When cells were treated with the methylation inhibitor 5-azacytidine, methylation of the plasmid promoter was abolished and a dose-dependent stimulation of reporter gene expression was observed with expression achieved in more than 80% of cells. Treatment of cells with 5-azacytidine also caused increased efficiency of transfection of macrophages with plasmids driven by RSV, SV40, and EF-1alpha promoters and transient transfection of human HepG2 cells. Inhibition of methylation also increased the amount and activity of sterol 27-hydroxylase (CYP27A1) detected in RAW 264.7 cells transfected with a CYP27A1 expression plasmid. Treatment of cells with 5-azacytidine alone did not affect either cholesterol efflux from nontransfected cells or expression of ABCA1 and CYP27A1. However, transfection with CYP27A1 led to a 2- to 4-fold increase of cholesterol efflux. We conclude that treatment with 5-azacytidine can be used for high-efficiency transient transfection of macrophages.     

Replication Stress Shapes a Protective Chromatin Environment across Fragile Genomic Regions

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Seasonal composition and spatial distribution of hyperbenthic communities along estuarine gradients in the Westerschelde

The hyperbenthic fauna of the Westerschelde estuary was sampled in spring, summer and winter of 1990 at 14 stations along the salinity gradient. Mysids dominated the hyperbenthos in each season. Other important species, either permanently (e.g. amphipods and isopods) or temporarily (e.g. fish larvae and decapod larvae) hyperbenthic, belong to a variety of faunistic groups. Spatial structure was stable through time: the estuary could be divided in the same geographically defined zones in each season. Each zone had a characteristic fauna. Gradients in salinity, dissolved oxygen and turbidity correlated strongly with the observed variation in community structure. The spatial patterns dominated over the temporal patterns, especially in the brackish part of the estuary. In the marine part, seasonal differences in the communities were more pronounced due to the occurrence of a series of temporary hyperbenthic species in spring and summer. In each season, the upstream (brackish) communities were characterized by few species occurring in very high numbers, whereas the downstream (marine) communities were composed of many species but at lower densities.     

γ-H2AX and other histone post-translational modifications in the clinic

Chromatin is a dynamic complex of DNA and proteins that regulates the flow of information from genome to end product. The efficient recognition and faithful repair of DNA damage, particularly double-strand damage, is essential for genomic stability and cellular homeostasis. Imperfect repair of DNA double-strand breaks (DSBs) can lead to oncogenesis. The efficient repair of DSBs relies in part on the rapid formation of foci of phosphorylated histone H2AX (γ-H2AX) at each break site, and the subsequent recruitment of repair factors. These foci can be visualized with appropriate antibodies, enabling low levels of DSB damage to be measured in samples obtained from patients. Such measurements are proving useful to optimize treatments involving ionizing radiation, to assay in vivo the efficiency of various drugs to induce DNA damage, and to help diagnose patients with a variety of syndromes involving elevated levels of γ-H2AX. We will survey the state of the art of utilizing γ-H2AX in clinical settings. We will also discuss possibilities with other histone post-translational modifications. The ability to measure in vivo the responses of individual patients to particular drugs and/or radiation may help optimize treatments and improve patient care. This article is part of a Special Issue entitled: Chromatin in time and space.     

SNPs Altering Ammonium Transport Activity of Human Rhesus Factors Characterized by a Yeast-Based Functional Assay

Proteins of the conserved Mep-Amt-Rh family, including mammalian Rhesus factors, mediate transmembrane ammonium transport. Ammonium is an important nitrogen source for the biosynthesis of amino acids but is also a metabolic waste product. Its disposal in urine plays a critical role in the regulation of the acid/base homeostasis, especially with an acid diet, a trait of Western countries. Ammonium accumulation above a certain concentration is however pathologic, the cytotoxicity causing fatal cerebral paralysis in acute cases. Alteration in ammonium transport via human Rh proteins could have clinical outcomes. We used a yeast-based expression assay to characterize human Rh variants resulting from non synonymous single nucleotide polymorphisms (nsSNPs) with known or unknown clinical phenotypes and assessed their ammonium transport efficiency, protein level, localization and potential trans-dominant impact. The HsRhAG variants (I61R, F65S) associated to overhydrated hereditary stomatocytosis (OHSt), a disease affecting erythrocytes, proved affected in intrinsic bidirectional ammonium transport. Moreover, this study reveals that the R202C variant of HsRhCG, the orthologue of mouse MmRhcg required for optimal urinary ammonium excretion and blood pH control, shows an impaired inherent ammonium transport activity. Urinary ammonium excretion was RHcg gene-dose dependent in mouse, highlighting MmRhcg as a limiting factor. HsRhCG^R202C may confer susceptibility to disorders leading to metabolic acidosis for instance. Finally, the analogous R211C mutation in the yeast ScMep2 homologue also impaired intrinsic activity consistent with a conserved functional role of the preserved arginine residue. The yeast expression assay used here constitutes an inexpensive, fast and easy tool to screen nsSNPs reported by high throughput sequencing or individual cases for functional alterations in Rh factors revealing potential causal variants.     

CXCR4/CXCL12 participate in extravasation of metastasizing breast cancer cells within the liver in a rat model

Introduction

Organ-specific composition of extracellular matrix proteins (ECM) is a determinant of metastatic host organ involvement. The chemokine CXCL12 and its receptor CXCR4 play important roles in the colonization of human breast cancer cells to their metastatic target organs. In this study, we investigated the effects of chemokine stimulation on adhesion and migration of different human breast cancer cell lines in vivo and in vitro with particular focus on the liver as a major metastatic site in breast cancer.

Methods

Time lapse microscopy, in vitro adhesion and migration assays were performed under CXCL12 stimulation. Activation of small GTPases showed chemokine receptor signalling dependence from ECM components. The initial events of hepatic colonisation of MDA-MB-231 and MDA-MB-468 cells were investigated by intravital microscopy of the liver in a rat model and under shRNA inhibition of CXCR4.

Results

In vitro, stimulation with CXCL12 induced increased chemotactic cell motility (p<0.05). This effect was dependent on adhesive substrates (type I collagen, fibronectin and laminin) and induced different responses in small GTPases, such as RhoA and Rac-1 activation, and changes in cell morphology. In addition, binding to various ECM components caused redistribution of chemokine receptors at tumour cell surfaces. In vivo, blocking CXCR4 decreased extravasation of highly metastatic MDA-MB-231 cells (p<0.05), but initial cell adhesion within the liver sinusoids was not affected. In contrast, the less metastatic MDA-MB-468 cells showed reduced cell adhesion but similar migration within the hepatic microcirculation. Conclusion: Chemokine-induced extravasation of breast cancer cells along specific ECM components appears to be an important regulator but not a rate-limiting factor of their metastatic organ colonization.