Growth variations in the bivalve<Emphasis Type="Italic"> Mya truncata</Emphasis>: a tool to trace changes in the Frisian Front macrofauna (southern North Sea)?

Annual monitoring of the benthic fauna living at the Frisian Front (southern North Sea) has shown a tenfold decrease in the dominant brittlestar Amphiura filiformis in 1993–1995. In search of evidence that this decline was caused by a change in benthic food supply, we analysed variations in the shell growth of the bivalve Mya truncata from the Frisian Front during the period of interest. For this purpose, the widths of the internal growth bands in the chondrophore of M. truncata were standardised and assigned to calendar years. Averaging the yearly band width in the period 1985–2000 among 25 individuals revealed low growth rates in 1986 and 1992. Growth of M. truncata quickly recovered after 1992, while A. filiformis densities remained at low levels. Moreover, the 1986 dip in M. truncata growth had no equivalent in A. filiformis density. We conclude that there is no direct coupling between fluctuations in density of A. filiformis and variations in growth of M. truncata. The data we collected during this study on the size and spatial distribution of M. truncata are discussed in the light of plans for the protection and conservation of long-lived benthic organisms in the North Sea. Communicated by E. Rachor     

Leech responses to tissue transplantation

The aim of the present work is to describe histologically, histochemically and immunocytochemically, the sequence of events that lead to first and second set rejection of allo- or xenograft in leeches. Graft responses of leeches are comparable and are described following specific steps: inflammatory phase, rejection phase and granulation tissue formation (including re-epithelialisation, angiogenesis and fibroplasia).The responses to first and second graft in first set graft rejection as well as to the first transplant in second set graft experiments are identical and in the time span of a week all grafts are destroyed and disappear. In the second set graft rejection experiments the responses against the second transplant are markedly accelerated. The second graft shows massive structural alterations and it is rapidly rejected, within 3-4 days.Our results permit to highlight that in leeches there is a specific responsiveness of immune system similar to those described in highly divergent phyla.     

Identification of proteins undergoing glutathionylation in oxidatively stressed hepatocytes and hepatoma cells

Protein glutathionylation is a post-translational modification consisting of the formation of a mixed disulfide between protein cysteines and glutathione (GSH). To identify proteins undergoing glutathionylation in primary rat hepatocytes and in human HepG2 hepatoma cells, we radiolabeled the intracellular GSH pool with L-[(35)S] cysteine. Cells were then exposed to oxidative stress. Proteins were separated by two-dimensional gel electrophoresis under nonreducing conditions, and glutathionylated proteins were located by autoradiography and identified by mass spectrometry after tryptic digestion. Several proteins previously not known to undergo glutathionylation were thus recognized. Among the identified proteins some are the same or belong to the same functional class as those we have already identified in a previous paper on T cell blasts (actin, nucleophosmin, phosphogluconolactonase, myosin, profilin, cyclophilin A, stress 70 protein, ubiquitin in HepG2 cells and actin, peroxiredoxin 5, cytochrome C oxidase, heat shock cognate 70 in hepatocytes) while others are newly recognized (Ran specific GTPase activating protein, histidine triad nucleotide binding protein 2 in HepG2 cells and enoyl CoA hydratase in hepatocytes). The technique described proved equally applicable to a variety of cell types.     

Nematodes from the Strait of Magellan and the Beagle Channel (Chile): the genera Cervonema and Laimella (Comesomatidae: Nematoda)

Five species of Cervonema and four species of Laimella are described from the Strait of Magellan and the Beagle Channel, Chile, six species of which are new to science. Cervonema chilensisn. sp. and Cervonema hermanin. sp. are separated from other known species of Cervonema by a short cervical region (less than one head diameter from the front end to the anterior border of the amphids). Cervonema chilensisn. sp. is characterised by a tail length of 5 anal diameters with posterior half filiform; Cervonema hermani n. sp. is characterised by a tail length of 6–9 anal diameter and posterior part (75%) cylindrical. Cervonema shiaen. sp. is characterised by the cephalic seta 4 m long, amphids 9–10 m in diameter; spicules 16 m long and 0.8–0.9 abd; tail 4.7–5.4 anal diameter and 50% posterior part filiform; 4–5 minute precloacal supplements. Laimella subterminatan. sp. is characterised by the subterminal position of the buccal cavity which separates it from the other species of the genus. Laimella annaen. sp. is characterised by the head diameter 9–11 m, cephalic setae and external labial setae 9 + 5 m long, respectively, amphids 7 m in diameter; spicules 28–30 m long; tail 14–17 anal diameter and posterior part (75%) filiform; 5 precloacal supplements. Laimella sandraen. sp. is very close to Laimella annaen.sp. in having similar cephalic sensilla, amphids and spicules. Laimella sandraen. sp., however, can be separated from L.annaen. sp. by the shape of head and the structure of sperm cells, the total body length and the cylindrical part of tail. Cervonema papillatum Jensen, 1988, C. tenuicauda (Stekhoven, 1950) and L. longicauda Cobb, 1920 are found in this area as well. The key of all known species of Cervonemaand Laimellais presented.     

Altered transarcolemmal Ca transport modifies the myofibrillar ultrastructure and protein metabolism in cultured adult ventricular cardiomyocytes

The present study was designed to investigate how prolonged (24-72 h) exposure to modifiers of Ca transarcolemmal transport affects the myofibrillar structure, protein turnover and content of myofibrillar proteins in adult guinea pig cardiomyocytes maintained beating synchronously in long-term cultures. First we established the functional responses (the contractile activity and [Ca]_i transients) of the cultured myocytes to acute exposures to several drugs used in this study. The ultrastructural characteristics of these cultures under the various treatments were determined using immunohistochemistry and confocal scanning laser microscopy, and their biochemical properties were evaluated using analysis of total cellular protein content, myofibrillar protein content and SDS-PAGE electrophoretic examination. We compared the effects of 24, 36 and 72 h-long exposures to the various specific Ca-flux modifiers. Increased Ca influx via Ca_L-channel agonist (Bay K 8644) or via the reversed- mode of the Na/Ca exchanger (veratrine) did not alter the myofibrillar structure or the specific protein profiles or proteosynthesis. However, when cytosolic Ca was increased by three different types of inhibitors of Ca extrusion from the cells via Na/Ca exchange, (Na-free solution, 5 mM NiCl₂ and 10^-6 M ouabain), very significant changes in all investigated parameters occurred almost immediately. Twenty-four h-long exposure to Na-free did not affect significantly the total cellular protein (TCP), but the protein synthesis was decreased by 87% and the total myofibrillar protein (TMP) content was decreased by 38%. The myofibrils were heavily fragmented. Similarly, 24 h-long exposure to 5 mM NiCl₂ did not affect the TCP, but it reduced protein synthesis by about 90% and decreased the total myofibrillar protein content by 30%. These effects were even more pronounced at 72 h of exposure and they were accompanied with a complete disassembly of myofilaments. Exposure to 10^-6 M ouabain over 72 h resulted in > 80% inhibition of protein synthesis, a 45% decrease in TCP content and a 53% in TMP content. In contrast, 10^-7 M ouabain did not produce any such changes. The changes produced by the Na/Ca-exchange inhibitors were accompanied by only minor changes in DNA content, indicating that the myocytes remained viable. Moreover, these effects were not due to the associated contractile arrest, since exposure to Ca_L-channel antagonists (5-20 M nifedipine or 10 M verapamil) produced only very minor changes in the myofibrillar structure and in protein profiles.Our data demonstrate that short-term (up to 72 h) increased Ca influx or contractile arrest of well-interconnected, spontaneously beating adult cardiomyocytes does not affect their ultrastructural characteristics or their myofibrillar protein turnover greatly, while any situations leading to Ca accumulation (via inhibition of Na/Ca exchange) affect cardiomyocyte function and ultrastructure almost immediately. These data are in sharp contrast to those previously reported from immature, neonatal myocytes.     

Interplay between Endophyte Prevalence,Effects and Transmission: Insights from a Natural Grass Population

Two main mechanisms are thought to affect the prevalence of endophyte-grass symbiosis in host populations: the mode of endophyte transmission, and the fitness differential between symbiotic and non-symbiotic plants. These mechanisms have mostly been studied in synthetic grass populations. If we are to improve our understanding of the ecological and evolutionary dynamics of such symbioses, we now need to determine the combinations of mechanisms actually operating in the wild, in populations shaped by evolutionary history. We used a demographic population modeling approach to identify the mechanisms operating in a natural stand of an intermediate population (i.e. 50% of plants symbiotic) of the native grass Festuca eskia. We recorded demographic data in the wild over a period of three years, with manipulation of the soil resources for half the population. We developed two stage-structured matrix population models. The first model concerned either symbiotic or non-symbiotic plants. The second model included both symbiotic and non-symbiotic plants and took endophyte transmission rates into account. According to our models, symbiotic had a significantly higher population growth rate than non-symbiotic plants, and endophyte prevalence was about 58%. Endophyte transmission rates were about 0.67 or 0.87, depending on the growth stage considered. In the presence of nutrient supplementation, population growth rates were still significantly higher for symbiotic than for non-symbiotic plants, but endophyte prevalence fell to 0%. At vertical transmission rates below 0.10–0.20, no symbiosis was observed. Our models showed that a positive benefit of the endophyte and vertical transmission rates of about 0.6 could lead to the coexistence of symbiotic and non-symbiotic F. eskia plants. The positive effect of the symbiont on host is not systematically associated with high transmission rates of the symbiont over short time scales, in particular following an environmental change.     

Enhanced extraction of proteins using cholinium‐based ionic liquids as phase‐forming components of aqueous biphasic systems

Aqueous biphasic systems (ABS) composed of ionic liquids (ILs) are promising platforms for the extraction and purification of proteins. In this work, a series of alternative and biocompatible ABS composed of cholinium‐based ILs and polypropylene glycol were investigated. The respective ternary phase diagrams, tie‐lines, tie‐line lengths and critical points were determined at 25°C. The extraction performance of these systems for commercial bovine serum albumin (BSA) was then evaluated. The stability of BSA at the IL‐rich phase was ascertained by size exclusion high‐performance liquid chromatography and Fourier transform infrared spectroscopy. Appropriate ILs lead to the complete extraction of BSA for the IL‐rich phase, in a single step, while maintaining the protein's native conformation. Furthermore, to evaluate the performance of these systems when applied to real matrices, the extraction of BSA from bovine serum was additionally carried out, revealing that the complete extraction of BSA was maintained and achieved in a single step. The remarkable extraction efficiencies obtained are far superior to those observed with typical polymer‐based ABS. Therefore, the proposed ABS may be envisaged as a more effective and biocompatible approach for the separation and purification of other value‐added proteins.     

Which Way In? The RalF Arf-GEF Orchestrates Rickettsia Host Cell Invasion

Bacterial Sec7-domain-containing proteins (RalF) are known only from species of Legionella and Rickettsia, which have facultative and obligate intracellular lifestyles, respectively. L. pneumophila RalF, a type IV secretion system (T4SS) effector, is a guanine nucleotide exchange factor (GEF) of ADP-ribosylation factors (Arfs), activating and recruiting host Arf1 to the Legionella-containing vacuole. In contrast, previous in vitro studies showed R. prowazekii (Typhus Group) RalF is a functional Arf-GEF that localizes to the host plasma membrane and interacts with the actin cytoskeleton via a unique C-terminal domain. As RalF is differentially encoded across Rickettsia species (e.g., pseudogenized in all Spotted Fever Group species), it may function in lineage-specific biology and pathogenicity. Herein, we demonstrate RalF of R. typhi (Typhus Group) interacts with the Rickettsia T4SS coupling protein (RvhD4) via its proximal C-terminal sequence. RalF is expressed early during infection, with its inactivation via antibody blocking significantly reducing R. typhi host cell invasion. For R. typhi and R. felis (Transitional Group), RalF ectopic expression revealed subcellular localization with the host plasma membrane and actin cytoskeleton. Remarkably, R. bellii (Ancestral Group) RalF showed perinuclear localization reminiscent of ectopically expressed Legionella RalF, for which it shares several structural features. For R. typhi, RalF co-localization with Arf6 and PI(4,5)P₂ at entry foci on the host plasma membrane was determined to be critical for invasion. Thus, we propose recruitment of PI(4,5)P₂ at entry foci, mediated by RalF activation of Arf6, initiates actin remodeling and ultimately facilitates bacterial invasion. Collectively, our characterization of RalF as an invasin suggests that, despite carrying a similar Arf-GEF unknown from other bacteria, different intracellular lifestyles across Rickettsia and Legionella species have driven divergent roles for RalF during infection. Furthermore, our identification of lineage-specific Arf-GEF utilization across some rickettsial species illustrates different pathogenicity factors that define diverse agents of rickettsial diseases.     

Ligand Binding to the FA3-FA4 Cleft Inhibits the Esterase-Like Activity of Human Serum Albumin

The hydrolysis of 4-nitrophenyl esters of hexanoate (NphOHe) and decanoate (NphODe) by human serum albumin (HSA) at Tyr411, located at the FA3-FA4 site, has been investigated between pH 5.8 and 9.5, at 22.0°C. Values of K _s, k ₊₂, and k ₊₂/K _s obtained at [HSA] ≥ 5×[NphOXx] and [NphOXx] ≥ 5×[HSA] (Xx is NphOHe or NphODe) match very well each other; moreover, the deacylation step turns out to be the rate limiting step in catalysis (i.e., k ₊₃ << k ₊₂). The pH dependence of the kinetic parameters for the hydrolysis of NphOHe and NphODe can be described by the acidic pK _a-shift of a single amino acid residue, which varies from 8.9 in the free HSA to 7.6 and 7.0 in the HSA:NphOHe and HSA:NphODe complex, respectively; the pK>_a-shift appears to be correlated to the length of the fatty acid tail of the substrate. The inhibition of the HSA-Tyr411-catalyzed hydrolysis of NphOHe, NphODe, and 4-nitrophenyl myristate (NphOMy) by five inhibitors (i.e., diazepam, diflunisal, ibuprofen, 3-indoxyl-sulfate, and propofol) has been investigated at pH 7.5 and 22.0°C, resulting competitive. The affinity of diazepam, diflunisal, ibuprofen, 3-indoxyl-sulfate, and propofol for HSA reflects the selectivity of the FA3-FA4 cleft. Under conditions where Tyr411 is not acylated, the molar fraction of diazepam, diflunisal, ibuprofen, and 3-indoxyl-sulfate bound to HSA is higher than 0.9 whereas the molar fraction of propofol bound to HSA is ca. 0.5.     

Membrane Cholesterol Modulates LOX-1 Shedding in Endothelial Cells

The lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is a scavenger receptor responsible for ox-LDL recognition, binding and internalization, which is up-regulated during atherogenesis. Its activation triggers endothelium dysfunction and induces inflammation. A soluble form of LOX-1 has been identified in the human blood and its presence considered a biomarker of cardiovascular diseases. We recently showed that cholesterol-lowering drugs inhibit ox-LDL binding and internalization, rescuing the ox-LDL induced apoptotic phenotype in primary endothelial cells. Here we have investigated the molecular bases of human LOX-1 shedding by metalloproteinases and the role of cell membrane cholesterol on the regulation of this event by modulating its level with MβCD and statins. We report that membrane cholesterol affects the release of different forms of LOX-1 in cells transiently and stably expressing human LOX-1 and in a human endothelial cell line (EA.hy926). In particular, our data show that i) cholesterol depletion triggers the release of LOX-1 in exosomes as a full-length transmembrane isoform and as a truncated ectodomain soluble fragment (sLOX-1); ii) endothelial cells secrete a soluble metalloproteinase which induces LOX-1 ectodomain shedding and iii) long term statins treatment enhances sLOX-1 proteolytic shedding.