Effect of tool-use observation on metric body representation and peripersonal space.

In everyday life, we constantly act and interact with objects and with others' people through our body. To properly perform actions, the representations of the dimension of body-parts (metric body representation, BR) and of the space surrounding the body (peripersonal space, PPS) need to be constantly updated. Previous evidence has shown that BR and PPS representation are highly flexible, being modulated by sensorimotor experiences, such as the active use of tools to reach objects in the far space. In this study, we investigate whether the observation of another person using a tool to interact with objects located in the far space is sufficient to influence the plasticity of BR and PPS representation in a similar way to active tool-use. With this aim, two groups of young healthy participants were asked to perform 20 min trainings based on the active use of a tool to retrieve far cubes (active tool-use) and on the first-person observation of an experimenter doing the same tool-use training (observational tool-use). Behavioural tasks adapted from literature were used to evaluate the effects of the active and observational tool-use on BR (body-landmarks localization task-group 1), and PPS (audio-tactile interaction task - group 2). Results show that after active tool-use, participants perceived the length of their arm as longer than at baseline, while no significant differences appear after observation. Similarly, significant modifications in PPS representation, with comparable multisensory facilitation on tactile responses due to near and far sounds, were seen only after active tool-use, while this did not occur after observation. Together these results suggest that a mere observational training could not be sufficient to significantly modulate BR or PPS. The dissociation found in the active and observational tool-use points out differences between action execution and action observation, by suggesting a fundamental role of the motor planning, the motor intention, and the related sensorimotor feedback in driving BR and PPS plasticity.

Performance of Laser Megajoule's x-ray streak camera.

A prototype of a picosecond x-ray streak camera has been developed and tested by Commissariat à l'Énergie Atomique et aux Énergies Alternatives to provide plasma-diagnostic support for the Laser Megajoule. We report on the measured performance of this streak camera, which almost fulfills the requirements: 50-µm spatial resolution over a 15-mm field in the photocathode plane, 17-ps temporal resolution in a 2-ns timebase, a detection threshold lower than 625 nJ/cm2 in the 0.05-15 keV spectral range, and a dynamic range greater than 100.

Picosecond X-ray streak camera dynamic range measurement.

Streak cameras are widely used to record the spatio-temporal evolution of laser-induced plasma. A prototype of picosecond X-ray streak camera has been developed and tested by Commissariat à l'Énergie Atomique et aux Énergies Alternatives to answer the Laser MegaJoule specific needs. The dynamic range of this instrument is measured with picosecond X-ray pulses generated by the interaction of a laser beam and a copper target. The required value of 100 is reached only in the configurations combining the slowest sweeping speed and optimization of the streak tube electron throughput by an appropriate choice of high voltages applied to its electrodes.

Overview of the ARGOS X-ray framing camera for Laser MegaJoule.

Commissariat à l'Énergie Atomique et aux Énergies Alternatives has developed the ARGOS X-ray framing camera to perform two-dimensional, high-timing resolution imaging of an imploding target on the French high-power laser facility Laser MegaJoule. The main features of this camera are: a microchannel plate gated X-ray detector, a spring-loaded CCD camera that maintains proximity focus in any orientation, and electronics packages that provide remotely-selectable high-voltages to modify the exposure-time of the camera. These components are integrated into an "air-box" that protects them from the harsh environmental conditions. A miniaturized X-ray generator is also part of the device for in situ self-testing purposes.

Basal autophagy is involved in the degradation of the ERAD component EDEM1.

Little is known about the fate of machinery proteins of the protein quality control and endoplasmic reticulum(ER)-associated degradation (ERAD). We investigated the degradation of the ERAD component EDEM1, which directs overexpressed misfolded glycoproteins to degradation. Endogenous EDEM1 was studied since EDEM1 overexpression not only resulted in inappropriate occurrence throughout the ER but also caused cytotoxic effects. Proteasome inhibitors had no effect on the clearance of endogenous EDEM1 in non-starved cells. However, EDEM1 could be detected by immunocytochemistry in autophagosomes and biochemically in LC3 immuno-purified autophagosomes. Furthermore, influencing the lysosome-autophagy pathway by vinblastine or pepstatin A/E64d and inhibiting autophagosome formation by 3-methyladenine or ATGs short interfering RNA knockdown stabilized EDEM1. Autophagic degradation involved removal of cytosolic Triton X-100-insoluble deglycosylated EDEM1, but not of EDEM1-containing ER cisternae. Our studies demonstrate that endogenous EDEM1 in cells not stressed by the expression of a transgenic misfolded protein reaches the cytosol and is degraded by basal autophagy.

A versatile high-resolution x-ray imager (HRXI) for laser-plasma experiments on OMEGA.

A high-resolution x-ray imager (HRXI) devoted to laser-plasma experiments combines two state-of-the-art technologies developed in France: a high-resolution x-ray microscope and a high-speed x-ray streak camera. The resulting streaked imager achieves spatial and temporal resolutions of approximately 5 microm and approximately 10 ps, respectively. The HXRI has recorded enhanced spatial and temporal resolution radiographs of indirectly driven targets on OMEGA. This paper describes the main features of the instrument and details the activation process on OMEGA (particularly the alignment). Recent results obtained on joint CEA/LLE radiographic OMEGA experiments will also be presented.

X-ray diagnostic calibration with the tabletop laser facility EQUINOX.

The broadband x-ray emission of a target irradiated by a laser can be used to check the calibration of detectors. At CEA-DIF we have a tabletop picosecond laser facility called EQUINOX with 0.3 J at 800 nm. The laser is focused inside a target chamber onto a solid target and produces bright radiation in the 100-2000 eV spectral range. The x-ray source is routinely monitored with a pinhole camera for source dimension measurement and with x-ray diodes for flux measurement. In addition an x-ray transmission grating spectrometer, a crystal spectrometer, and a single count charge coupled device camera measure the x-ray spectrum between 100 eV and 15 keV. The absolute calibration of those sets of spectrometers allows us to fully characterize x-ray emission spectra. Typical duration is less than 100 ps. The spectrum can be tuned by changing target material, pulse length, and x-ray filters. An application to checking the calibration of x-ray diodes used in the broad band spectrometer DMX with single shots will be presented.

Diagnostics hardening for harsh environment in Laser Megajoule (invited).

The diagnostic designs for the Laser Megajoule (LMJ) will require components to operate in environments far more severe than those encountered in present facilities. This harsh environment will be induced by fluxes of neutrons, gamma rays, energetic ions, electromagnetic radiations, and, in some cases, debris and shrapnel, at levels several orders of magnitude higher than those experienced today on existing facilities. The lessons learned about the vulnerabilities of present diagnostic parts fielded mainly on OMEGA for many years, have been very useful guide for the design of future LMJ diagnostics. The present and future LMJ diagnostic designs including this vulnerability approach and their main mitigation techniques will be presented together with the main characteristics of the LMJ facility that provide for diagnostic protection.

Application of a lectin from the mushroom Polysporus squamosus for the histochemical detection of the NeuAcalpha2,6Galbeta1,4Glc/GlcNAc sequence of N-linked oligosaccharides: a comparison with the Sambucus nigra lectin.

The lectin from the mushroom Polysporus squamosus (PSL) has an extended carbohydrate combining site, which exhibits a high specificity and affinity toward the NeuAc5alpha2,6Galbeta1,4Glc/GlcNAc trisaccharide sequence of asparagine-linked oligosaccharides. Therefore, PSL should be a superior reagent to the lectin from Sambucus nigra (SNA), which does not discriminate between alpha2,6-linked NeuAc5 present either in asparagine- or serine/threonine-linked oligosaccharides. We have prepared a digoxigenin-conjugated PSL and applied it for histochemistry and blotting. We observed a more restricted staining pattern by PSL as compared to SNA in paraffin sections from different rat organs. Pretreatment of sections with N-glycanase F abolished PSL staining indicating that it interacts only with asparagine-linked oligosaccharides. Furthermore, PSL staining was neuraminidase sensitive. In contrast, SNA staining was only partially sensitive to N-glycanase F pretreatment demonstrating that it was in part due to alpha2,6-linked NeuAc5 present in serine/threonine-linked oligosaccharides. The most striking observation in this regard was that PSL, in contrast to SNA, did not stain the mucus of sheep submandibular gland, which is extremely rich in serine/threonine-linked Neu5Acalpha2,6N-acetylgalactosamine. Furthermore, in some tissues neuraminidase pretreatment resulted in increased intensity of SNA staining probably due to binding to exposed terminal N-acetylgalactosamine residues. Collectively, these results indicate that PSL is a useful tool for the histochemical detection of alpha2,6-linked NeuAc5 in asparagine-linked oligosaccharides.

Immunolocalization of UDP-glucose:glycoprotein glucosyltransferase indicates involvement of pre-Golgi intermediates in protein quality control.

The UDP-glucose:glycoprotein glucosyltransferase (GT) is a protein folding sensor and glycosyltransferase that constitutes an important component of the protein quality control machinery. With the use of quantitative immunogold electron microscopy, we established the subcellular distribution of GT in rat liver and pancreas and Drosophila melanogaster salivary gland as well as cell lines and correlated it with that of glucosidase II, calreticulin, and pre-Golgi intermediate markers. Labeling for GT, as well as for glucosidase II and calreticulin, was found in the endoplasmic reticulum (ER), including nuclear envelope and pre-Golgi intermediates located between ER and Golgi apparatus, and in the cell periphery. In the rough ER, labeling for GT was inhomogeneous, with variously sized labeled and unlabeled cisternal regions alternating, indicative of a meshwork of quality control checkpoints. Notably, labeling intensity for GT was highest in pre-Golgi intermediates, corresponding to twice that of rough ER, whereas the Golgi apparatus exhibited no specific labeling. These results suggest that protein quality control is not restricted to the ER and that the pre-Golgi intermediates, by virtue of the presence of GT, glucosidase II, and calreticulin, are involved in this fundamental cellular process.

Golgi apparatus immunolocalization of endomannosidase suggests post-endoplasmic reticulum glucose trimming: implications for quality control.

Trimming of N-linked oligosaccharides by endoplasmic reticulum (ER) glucosidase II is implicated in quality control of protein folding. An alternate glucosidase II-independent deglucosylation pathway exists, in which endo-alpha-mannosidase cleaves internally the glucose-substituted mannose residue of oligosaccharides. By immunogold labeling, we detected most endomannosidase in cis/medial Golgi cisternae (83.8% of immunogold labeling) and less in the intermediate compartment (15.1%), but none in the trans-Golgi apparatus and ER, including its transitional elements. This dual localization became more pronounced under 15 degrees C conditions indicative of two endomannosidase locations. Under experimental conditions when the intermediate compartment marker p58 was retained in peripheral sites, endomannosidase was redistributed to the Golgi apparatus. Double immunogold labeling established a mutually exclusive distribution of endomannosidase and glucosidase II, whereas calreticulin was observed in endomannosidase-reactive sites (17.3% in intermediate compartment, 5.7% in Golgi apparatus) in addition to the ER (77%). Our results demonstrate that glucose trimming of N-linked oligosaccharides is not limited to the ER and that protein deglucosylation by endomannosidase in the Golgi apparatus and intermediate compartment additionally ensures that processing to mature oligosaccharides can continue. Thus, endomannosidase localization suggests that a quality control of N-glycosylation exists in the Golgi apparatus.

Thomsen-Friedenreich glycotope is expressed in developing and normal kidney but not in renal neoplasms.

The Thomsen-Friedenreich glycotope (TF) is considered a general carcinoma autoantigen and is therefore of importance in cancer diagnosis and immunotherapy. We report the distribution of the TF glycotope in developing and adult human kidney and renal neoplasms. A monoclonal antibody and the lectin amaranthin were used to study the TF and its sialylated, masked form by immunohistochemistry and immunoblotting. In developing kidney, the TF was restricted to the loop of Henle, distal tubules, and peripheral collecting ducts, whereas its sialylated form was detectable in all epithelial differentiations derived from the 2 embryonic anlagen, the metanephrogenic blastema being unreactive. This pattern was essentially preserved in adult kidney, with TF labeling beginning in the thick ascending limb and extending into the collecting ducts of outer medulla. The sialylated TF glycotope was additionally observed in ascending thin limbs. The TF was exclusively expressed in the luminal cell surface and hence was inaccessible to immune reactions. Analysis of a spectrum of renal neoplasms failed to detect the TF, with the exception of occasional staining of tubules in nephroblastoma. Moreover, the sialylated TF was only detectable in oncocytoma, chromophobe renal cell carcinoma, cystic nephroma, nephroblastoma, and nephroblastomatosis complex and occasionally in type 1 papillary renal cell carcinoma. Thus, the TF and its sialylated form are expressed in normal developing and adult kidney. However, the TF does not seem to represent a tumor-associated glycotope in human kidney, nor does it appear to be of value in diagnosis and immunotherapy of renal neoplasms.

Immunohistochemical evaluation of endomannosidase distribution in rat tissues: evidence for cell type-specific expression.

Asparagine-linked oligosaccharides of glycoproteins are subject to a series of trimming reactions by glucosidases and mannosidases in the endoplasmic reticulum which result in the removal of all three glucose residues and several of the nine mannose residues. At present, endomannosidase represents the only processing enzyme which cleaves internally and provides an alternate deglucosylation pathway. However, in contrast to the endoplasmic reticulum residential proteins glucosidase I and II, endomannosidase is primarily situated in the Golgi apparatus of rat liver hepatocytes and hepatocyte cell lines. We have performed a confocal immunohistochemical study to investigate endomannosidase in various rat tissues and used a monoclonal antibody against Golgi mannosidase II as a marker for the Golgi apparatus. Although immunofluorescence for both endomannosidase and Golgi mannosidase II was detectable in the epithelia of many tissues, renal proximal tubular cells, cortex and medulla of adrenal gland, gastric mucosa, and Leydig cells of testis were unreactive for endomannosidase. Furthermore, the endothelia in all studied tissues were unreactive for endomannosidase but positive for Golgi mannosidase II. It is concluded that by immunohistochemistry endomannosidase exhibits a cell type-specific expression in rat tissues.

Specialized expression of simple O-glycans along the rat kidney nephron.

Glycosyltransferases can exhibit tissue-specific expression. By histochemistry glycosyltransferases and their products can be localized to specific cell types in organs of complex cellular composition. We have applied the lectin Amaranthin, having a nominal specificity for Galbeta1,3GalNAcR and Neu5Ac2,3Galbeta1, 3GalNAcalpha-R, and a monoclonal antibody raised against Galbeta1, 3GalNAcalphaR to examine the distribution of these simple O-glycans in adult rat kidney. The monoclonal antibody stained ascending thin limbs of Henle, distal convoluted tubules, and collecting ducts of cortex and outer medulla. Remarkably, the ascending thick limb of Henle, located between ascending thin limb and distal convoluted tubules, was unreactive. However, Amaranthin staining was detectable in ascending thick limbs of Henle, in addition to the structures positive with the monoclonal antibody. In kidney extracts, two bands of approximately 160 kDa and >210 kDa were reactive with both Amaranthin and the monoclonal antibody. One band at approximately 200 kDa, and a smear at approximately 100 kDa, were reactive only with Amaranthin. Our data show that in rat kidney simple O-linked glycans are expressed in a highly specialized manner along the renal tubule and can be detected only on a few glycoproteins. This may reflect a cell-type-specific expression of the corresponding glycosyltransferases.

Histochemistry and cell biology: what's in a name?

Histochemistry represents an integrative discipline aiming at the in situ detection, localization and functional characterization of cellular and extracellular components in single cells and complex organs. Therefore, the development of new methods and improvement of existing ones continues to be important. This, however, is with the declared intent for their application in the various fields of developmental and cell biology as well as pathology in order to contribute to the solution of open problems. This review summarizes recent advancements in these fields.

Lectin-Gold Histochemistry on Paraffin and Lowicryl K4M Sections Using Biotin and Digoxigenin-Conjugated Lectins.

A variety of staining reactions for the visualization of cellular and extracellular glycoconjugates at the light microscopic level are available that are based on the detection of carboxyl and sulfate groups or periodic acid reactive configurations (1,2). Starting in the late 1960s lectins have replaced many of these chemical staining reactions because of their high specificity for defined monoand oligosaccharidic sequences in both N- and o-glycosidic-linked oligosaccharide side-chains of glycoproteins and glycolipids. In order to be useful as histochemical reagents, lectins must be tagged with appropriate markers and those employed in immunocytochemistry have been used successfully (3-9) Horisberger and coworkers were the first to prepare lectins labeled with particles of colloidal gold and used them in scanning electron mrcroscopy (10) Subsequently, gold-labeled lectins were applied in transmission electron microscopy to study various aspects of cell surface expression and internalization of lectin-binding sites (5,8,11,12), as well as in postembedding labeling of Lowicryl K4M thin sections (13). Later, it was shown that gold-labeled lectins can be used to stain sections of paraffin-embedded tissues (14-16), as well as semithin sections of Epon (17) and Lowicryl K4M-embedded tissues (18,19), However, in order to achieve a visible pink staining, which is the natural color of particles of colloidal gold in transmitted visible light (20), highly concentrated lectin-gold complexes had to be used, thereby allowing the possibility of nonspecific staining. A major improvement resulted through the application of a photochemrcal silver reaction for signal amphfication (21-24), whrch permitted the use of lectins for light microscopy in concentrations as applied for electron microscopy (25,26).

Blot analysis with lectins for the evaluation of glycoproteins in cultured cells and tissues.

The carbohydrate-binding properties of plant and animal lectins have found many diverse applications in biomedical research that are comprehensively treated in the various chapters of this book. In our studies, lectins are often applied for the in situ detection of their respective binding sites in specific cells either grown in culture or present in various tissues (see Chapter 3 ). In order to complement these in situ localization studies, blots are analyzed using lectins to identify glycosylation patterns of glycoproteins that contain accessible lectin-binding sites in their oligosaccharide side-chains. The isolation and characterization of lectins which exhibit a narrow spectrum of reactivity with oligosaccharide structures has provided new possibilities for such kind of analyses. In comparison to Concanavalin A, which recognizes glucose/mannose residues in terminal nonreducing position as well as internal linkages (1), lectins such as the Bowringia milbraedii lectin (2), the Narcissus pseudonarcissus lectin (3), and Galanthus nivalis lectin (4) react with well defined processing intermediates of asparagine-linked oligosaccharides. Another example for such a high degree of specificity is provided by sialic acid-specific lectins such as the Sambucus nigra lectin I (5), the Maackia amurensis lectin (6,7), and the Amaranthus caudatus lectin (8-10). These lectins, in contrast to the general sialic acid binding Limulus polyphemus (11) and Limax flavus (12,13) lectins, discriminate terminal sialic acids present in different ketosidic linkages.