Diversity and structure of AMF communities as affected by tillage in a temperate soil.

Arbuscular mycorrhizal fungi (AMF) were studied in differently tilled soils from a long-term field experiment in Switzerland. Diversity and structure of AMF communities were surveyed either directly on spores isolated from the field soil or on spores isolated from trap cultures, planted with different host plants. Single-spore cultures were established from the AMF spores obtained from trap cultures. Identification of the AMF was made by observation of spore morphology and confirmed by sequencing of ITS rDNA. At least 17 recognised AMF species were identified in samples from field and/or trap cultures, belonging to five genera of AMF--Glomus, Gigaspora, Scutellospora, Acaulospora, and Entrophospora. Tillage had a significant influence on the sporulation of some species and non- Glomus AMF tended to be more abundant in the no-tilled soil. The community structure of AMF in the field soil was significantly affected by tillage treatment. However, no significant differences in AMF diversity were detected among different soil tillage treatments. AMF community composition in trap cultures was affected much more by the species of the trap plant than by the original tillage treatment of the field soil. The use of trap cultures for fungal diversity estimation in comparison with direct observation of field samples is discussed.

Lipid-bloated subretinal microglial cells are at the origin of drusen appearance in CX3CR1-deficient mice.

Drusen, the white yellowish deposits that can be seen in funduscopy, are a hallmark of age-related macular degeneration. Histologically, drusen are believed to be dome-shaped or more confluent lipid accumulations between the retinal pigment epithelium and the choriocapillaries. Recent advances in mouse funduscopy have revealed the presence of drusen-like structures in chemokine knockout animals in the absence of sizeable dome-shaped material below the retinal pigment epithelium. We show that aged CX3CR1-/- mice present with drusen-like appearance in funduscopy that is associated with a progressive age-related microglial cell accumulation in the subretinal space. We demonstrate that the anatomical equivalent of the drusen-like appearance in these mice are lipid-bloated subretinal microglial cells rather than subretinal pigment epithelium deposits [Combadière C, et al: J Clin Invest 2007;117:2920-2928].

Antibacterial burst-release from minimal Ag-containing plasma polymer coatings.

Biomaterials releasing silver (Ag) are of interest because of their ability to inhibit pathogenic bacteria including antibiotic-resistant strains. In order to investigate the potential of nanometre-thick Ag polymer (Ag/amino-hydrocarbon) nanocomposite plasma coatings, we studied a comprehensive range of factors such as the plasma deposition process and Ag cation release as well as the antibacterial and cytocompatible properties. The nanocomposite coatings released most bound Ag within the first day of immersion in water yielding an antibacterial burst. The release kinetics correlated with the inhibitory effects on the pathogens Pseudomonas aeruginosa or Staphylococcus aureus and on animal cells that were in contact with these coatings. We identified a unique range of Ag content that provided an effective antibacterial peak release, followed by cytocompatible conditions soon thereafter. The control of the in situ growth conditions for Ag nanoparticles in the polymer matrix offers the possibility to produce customized coatings that initially release sufficient quantities of Ag ions to produce a strong adjacent antibacterial effect, and at the same time exhibit a rapidly decaying Ag content to provide surface cytocompatibility within hours/days. This approach seems to be favourable with respect to implant surfaces and possible Ag-resistance/tolerance built-up.

The making of frozen-hydrated, vitreous lamellas from cells for cryo-electron microscopy.

There has been a long standing desire to produce thick (up to 500 nm) cryo-sections of fully hydrated cells and tissue for high-resolution analysis in their natural state by cryo-transmission electron microscopy. Here, we present a method that can successfully produce sections (lamellas in FIB-SEM terminology) of fully hydrated, unstained cells from high-pressure frozen samples by focused ion beam (FIB) milling. The samples are therefore placed in thin copper tubes and vitrified by high-pressure freezing. For transfer, handling and subsequent milling, the tubes are placed in a novel connective device (ferrule) that protects the sample from devitrification and contamination and passes through all operation steps. A piezo driven sample positioning stage (cryo-nano-bench, CNB) with three degrees of freedom was additionally developed to enable accurate milling of frozen-hydrated lamellas. With the CNB, high-pressure frozen samples can be milled to produce either thin lamellas (<100 nm), for direct imaging by high-resolution cryo-TEM or thicker lamellas (300-500 nm) for cryo-electron tomography. The sample remains vitreous throughout the process by using the presented tools and methods. The results are an important step towards investigating larger cells and even tissue in there natural state which in the end will enable us to gain better insights into cellular processes.

Characterization of MAP1B heavy chain interaction with actin.

Microtubule-associated protein 1B is an essential protein during brain development and neurite outgrowth and was studied by several assays to further characterize actin as a major interacting partner. Tubulin and actin co-immunoprecipitated with MAP1B at similar ratios throughout development. Their identity was identified by mass spectrometry and was confirmed by Western blots. In contrast to previous reports, the MAP1B-actin interaction was not dependent on the MAP1B phosphorylation state, since actin was precipitated from brain tissue throughout development at similar ratios and equal amounts were precipitated before and after dephosphorylation with alkaline phosphatase. MAP1B heavy chain was able to bind actin directly and therefore the N-terminal part of MAP1B heavy chain must also contain an actin-binding site. The binding force of this interaction was measured by atomic force microscopy and values were in the same range as those of MAP1B binding to tubulin or that measured in MAP1B self-aggregation. Aggregation was confirmed by negative staining and electron microscopy. Experiments including COS-7 cells, PC12 cells, cytochalasin D and immunocytochemistry with subsequent confocal laser microscopy, suggested that MAP1B may bind to actin but has no obvious microfilament stabilizing effect. We conclude, that the MAP1B heavy chain has a microtubule-stabilization effect, and contains an actin-binding site that may play a role in the crosslinking of actin and microtubules, a function that may be important in neurite elongation.

Exovesicles from human activated dendritic cells fuse with resting dendritic cells, allowing them to present alloantigens.

Dendritic cells (DCs) can release microvesicles, but the latter's numbers, size, and fate are unclear. Fluorescently labeled DCs were visualized by laser-scanning microscopy. Using a Surpass algorithm, we were able to identify and quantify per cell several hundred microvesicles released from the surface of stimulated DCs. We show that most of these microvesicles are not of endocytic origin but result from budding of the plasma membrane, hence their name, exovesicle. Using a double vital staining, we show that exovesicles isolated from activated DCs can fuse with the membrane of resting DCs, thereby allowing them to present alloantigens to lymphocytes. We concluded that, within a few hours from their release, exovesicles may amplify local or distant adaptive immunological response.

Estrogen-dependent in vitro transcription from the vitellogenin promoter in liver nuclear extracts.

One approach to analyzing the molecular mechanisms of gene expression in vivo is to reconstitute these events in cell-free systems in vitro. Although there is some evidence for tissue-specific transcription in vitro, transcriptionally active extracts that mimic a steroid hormone-dependent enhancement of transcription have not been described. In the study reported here, nuclear extracts of liver from the frog Xenopus laevis were capable of estrogen-dependent induction of a homologous vitellogenin promoter that contained the estrogen-responsive element.

PSD-95 promotes synaptogenesis and multiinnervated spine formation through nitric oxide signaling

Postsynaptic density 95 (PSD-95) is an important regulator of synaptic structure and plasticity. However, its contribution to synapse formation and organization remains unclear. Using a combined electron microscopic, genetic, and pharmacological approach, we uncover a new mechanism through which PSD-95 regulates synaptogenesis. We find that PSD-95 overexpression affected spine morphology but also promoted the formation of multiinnervated spines (MISs) contacted by up to seven presynaptic terminals. The formation of multiple contacts was specifically prevented by deletion of the PDZ(2) domain of PSD-95, which interacts with nitric oxide (NO) synthase (NOS). Similarly, PSD-95 overexpression combined with small interfering RNA-mediated down-regulation or the pharmacological blockade of NOS prevented axon differentiation into varicosities and multisynapse formation. Conversely, treatment of hippocampal slices with an NO donor or cyclic guanosine monophosphate analogue induced MISs. NOS blockade also reduced spine and synapse density in developing hippocampal cultures. These results indicate that the postsynaptic site, through an NOS-PSD-95 interaction and NO signaling, promotes synapse formation with nearby axons.

Analyse critique des résultats de biopsies musculaires et de la méthodologie utilisée dans 889 cas d'affections neuromusculaires [Critical analysis of the results of muscle biopsies and of the methods used in 889 cases of neuromuscular disorders]

The contribution of muscle biopsies to the diagnosis of neuromuscular disorders and the indications of various methods of examination are investigated by analysis of 889 biopsies from patients suffering from myopathic and/or neurogenic disorders. Histo-enzymatic studies performed on frozen material as well as immunohistochemistry and electron microscopy allowed to provide specific diagnoses in all the neurogenic disorders (polyneuropathies and motor neuron diseases), whereas one third of myopathies remained uncertain. Confrontation of neuropathological data with the clinical indications for histological investigations shows that muscle biopsies reveal the diagnosis in 25% of the cases (mainly in congenital and metabolic myopathies) and confirm and/or complete the clinical diagnosis in 50%. In the remaining cases with non specific abnormalities neuropathological investigations may help the clinician by excluding well defined neuromuscular disorders. Analysis of performed studies and results of investigations show the contribution and specificity of each method for the diagnosis. Statistical evaluation of this series indicates that cryostat sectioning for histo- and immunochemical and electron microscopy increases the rate of diagnoses of neuromuscular diseases: full investigation was necessary for the diagnosis in 30% of the cases. The interpretation of the wide range of pathological reactions in muscles requires a close cooperation with the clinician.

Predicting the mating system from phenotypic correlations between life-history and sperm quality traits in the Alpine whitefish Coregonus zugensis

The mating behavior and reproductive strategies of Alpine whitefish like Coregonus zugensis (Nusslin) are poorly understood, probably because they spawn in deep water where direct observations are difficult. In this study, we interpret life-history and sperm quality traits of fish that we caught from their spawning place. We found that males invest heavily into gonadal tissue (up to 5.6% of their body weight), which is, in comparison to other fish, consistent with external fertilization, distinct pairing and moderate to high communal spawning, or no pairing and low to moderate communal spawning. Sperm competition theory and recent experimental studies on other salmonids predict that males optimize ejaculate characteristics in relation to the costs of sperm and the level of competition they have to expect: dominant males are predicted to invest less into ejaculate quality and to have slower spermatozoa than subdominant males. We found that spermatozoa of older males are slower than those of younger males. Moreover, older males have larger breeding tubercles, a secondary sexual trait that has, in some previous studies, been found to be linked to good condition and to good genetic quality. Our results suggest that C. zugensis has age-linked reproductive strategies, that multimale spawning is common, i.e., that sperm competition plays a significant role, and that older males are on average dominant over younger males at the spawning place.

Further ultrastructural evidence that spirochaetes may play a role in the aetiology of Alzheimer's disease

Recently it was reported that, at autopsy, in neuropathologically confirmed cases of Alzheimer's disease spirochaetes were found in blood and cerebrospinal fluid using dark-field microscopy. Moreover, the spirochaetes were isolated and cultured from brain tissue. We now show, using scanning electron microscopy and atomic force microscopy that the helically shaped microorganisms isolated and cultured from the Alzheimer brains possess axial filaments. This indicates that these microorganisms taxonomically indeed belong to the order Spirochaetales. A morphometric analysis reinforces this notion.

Ultrastructural, morphometrical and immunocytochemical analyses of the exocrine pancreas in a hibernating dormouse.

Pancreatic acinar cells of euthermic, hibernating and arousing individuals of the hazel dormouse Muscardinus avellanarius (Gliridae) have been observed at the electron-microscopic level and analysed by means of ultrastructural morphometry and immunocytochemistry in order to investigate possible fine structural changes of cellular components during periods of strikingly different degrees of metabolic activity. During hibernation, the cisternae of the rough endoplasmic reticulum (RER) flatten assuming a parallel pattern, the Golgi apparatus is extremely reduced and the mitochondria contain many electron-dense particles. The cell nuclei appear irregularly shaped, with deep indentations containing small zymogen granules. They also contain abundant coiled bodies and unusual constituents, such as amorphous bodies and dense granular bodies. Large numbers of zymogen granules occur in all animals. However, the acinar lumina are open and filled with zymogen only in euthermic animals, whereas, in hibernating and arousing individuals, they appear to be closed. Morphometrical analyses indicate that, in pancreatic acinar cells, nuclei and zymogen granules significantly decrease in size from euthermia to hibernation, probably reflecting a drastic decrease of metabolic activities, mainly protein synthesis and processing. In all the studied animals, immunocytochemistry with specific antibodies has revealed an increasing gradient in alpha-amylase content along the RER-Golgi-zymogen granule pathway, reflecting the protein concentration along the secretory pathway. Moreover, during deep hibernation, significantly larger amounts of alpha-amylase accumulate in RER and zymogen granules in comparison to the other seasonal phases analysed. Upon arousal, all cytoplasmic and nuclear constituents restore their euthermic aspect and all morphometrical and immunocytochemical parameters exhibit the euthermic values, thereby indicating a rapid resumption of metabolic activities.

Remorin, a solanaceae protein resident in membrane rafts and plasmodesmata, impairs potato virus X movement.

Remorins (REMs) are proteins of unknown function specific to vascular plants. We have used imaging and biochemical approaches and in situ labeling to demonstrate that REM clusters at plasmodesmata and in approximately 70-nm membrane domains, similar to lipid rafts, in the cytosolic leaflet of the plasma membrane. From a manipulation of REM levels in transgenic tomato (Solanum lycopersicum) plants, we show that Potato virus X (PVX) movement is inversely related to REM accumulation. We show that REM can interact physically with the movement protein TRIPLE GENE BLOCK PROTEIN1 from PVX. Based on the localization of REM and its impact on virus macromolecular trafficking, we discuss the potential for lipid rafts to act as functional components in plasmodesmata and the plasma membrane.

Formation of knots in partially replicated DNA molecules.

Bacterial plasmids with two origins of replication in convergent orientation are frequently knotted in vivo. The knots formed are localised within the newly replicated DNA regions. Here, we analyse DNA knots tied within replication bubbles of such plasmids, and observe that the knots formed show predominantly positive signs of crossings. We propose that helical winding of replication bubbles in vivo leads to topoisomerase-mediated formation of knots on partially replicated DNA molecules.

Storage and uptake of D-serine into astrocytic synaptic-like vesicles specify gliotransmission.

Glial cells are increasingly recognized as active players that profoundly influence neuronal synaptic transmission by specialized signaling pathways. In particular, astrocytes have been shown recently to release small molecules, such as the amino acids l-glutamate and d-serine as "gliotransmitters," which directly control the efficacy of adjacent synapses. However, it is still controversial whether gliotransmitters are released from a cytosolic pool or by Ca(2+)-dependent exocytosis from secretory vesicles, i.e., by a mechanism similar to the release of synaptic vesicles in synapses. Here we report that rat cortical astrocytes contain storage vesicles that display morphological and biochemical features similar to neuronal synaptic vesicles. These vesicles share some, but not all, membrane proteins with synaptic vesicles, including the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) synaptobrevin 2, and contain both l-glutamate and d-serine. Furthermore, they show uptake of l-glutamate and d-serine that is driven by a proton electrochemical gradient. d-Serine uptake is associated with vesicle acidification and is dependent on chloride. Whereas l-serine is not transported, serine racemase, the synthesizing enzyme for d-serine, is anchored to the membrane of the vesicles, allowing local generation of d-serine. Finally, we reveal a previously unexpected mutual vesicular synergy between d-serine and l-glutamate filling in glia vesicles. We conclude that astrocytes contain vesicles capable of storing and releasing d-serine, l-glutamate, and most likely other neuromodulators in an activity-dependent manner.