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.

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.

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.

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.

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.

The relationship of polysialic acid and the neural cell adhesion molecule N-CAM in Wilms tumor and their subcellular distributions.

In previous studies we have reported that polysialic acid is an oncodevelopmental antigen in human kidney but its relationship to the neural cell adhesion molecule (N-CAM) remained undefined. In the present study, we showed by the combination of immunoprecipitation and immunoblotting that renal polysialic acid is a structural component of N-CAM polypeptide and that two highly sialylated N-CAM isoforms of approximately 120 kDa and 140 kDa existed in Wilms tumor. The presence of a cell surface coat composed of polysialic acid and N-CAM was revealed by immunoelectron microscopy, and morphological evidence for its involvement in modulating cell-cell adhesion has been provided. Furthermore, highly sialylated N-CAM was detectable extracellularly. N-CAM immunolabeling was present in compartments from the nuclear envelope to the plasma membrane. However, polysialic acid was only detectable at the cell surface suggesting that in Wilms tumor cells sialyl polymer synthesis may occur partially or exclusively at this site.

Expression patterns of the T antigen and the cryptic T antigen in rat fetuses: detection with the lectin amaranthin.

The lectin amaranthin, purified from the seeds of Amaranthus caudatus, has been shown to react specifically with the Gal beta 1,3GalNAc-alpha and the NeuAc alpha 2,3Gal beta 1,3GalNAc-alpha sequence which represent the T antigen and the cryptic T antigen, respectively. We report here the development of labeling techniques that apply amaranthin to stain paraffin sections from rat fetuses. Amaranthin staining was inhibited by pre-incubation of lectin-gold complexes with 10 mM Gal beta 1,3GalNAc-alpha-O-benzyl (synthetic T antigen) or 10 mM Gal beta 1,3GalNAc-alpha-O-aminophenylethyl-human serum albumin (T antigen neoglycoprotein), asialoglycophorin, asialofetuin, and asialomucin. The beta-elimination reaction also abolished the lectin staining demonstrating specificity for O-glycosidically linked structures. A comparison with monoclonal anti-T antigen antibody immunostaining demonstrated that amaranthin detects the T antigen and its cryptic form in tissue sections. Application of the galactose oxidase-Schiff sequence abolished amaranthin (and anti-T antibody) binding to the T antigen but not to its cryptic form, and therefore permitted their differentiation in tissue sections. Histochemical evidence was obtained indicating that amaranthin is a more specific anti-T reagent than peanut lectin. Data are presented that show the differential expression of the T antigen and the cryptic T antigen in organs and cells of rat fetuses late in gestation. Therefore, amaranthin can be used for histochemical detection of the T antigen and the cryptic T antigen, and facilitates discrimination between them.

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.

Can malignancy in insulinoma be predicted by the expression patterns of beta 1,6 branching of asparagine-linked oligosaccharides and polysialic acid of the neural cell adhesion molecule?

We analysed the value of the expression of beta 1,6 branching of asparagine-linked oligosaccharide chains and polysialic acid of the neural cell adhesion molecule (NCAM) in predicting malignant behaviour in human insulinomas, as these glycoconjugates have been associated with invasive growth and metastatic potential. Fifty-three insulinomas from patients with well-documented clinical and follow-up data were investigated. Lectin histochemical staining for beta 1,6 branches revealed that 11 (74%) of the 15 malignant insulinomas stained more strongly than normal beta cells. However, in as many as 23 (63.1%) of the 38 benign insulinomas with a disease-free follow up for 4-18 years (average 8 years), a staining intensity equivalent to that of malignant tumours was found. Two (13%) of the malignant insulinomas and 1 of the 4 liver metastases studied were unstained. None of the 53 insulinomas (and the rat RIN insulinoma) re-expressed polysialic acid as demonstrated by immunohistochemistry and Western blotting with the monoclonal antibody 735. Therefore, histochemical staining for beta 1,6 branches and immunohistochemistry for polysialic acid are unlikely to be of value as prognostic indicators for patients with insulinomas.

The ability to re-express polysialylated NCAM in soleus muscle after denervation is reduced in aged rats compared to young adult rats.

The neural cell-adhesion molecule, NCAM, contains an unusual homopolymer of sialic acid units, polysialic acid. This carbohydrate seems to be involved in neurite outgrowth, bundling and branching, processes which are important during reinnervation. In aged rats, reinnervation of denervated muscle fibres is incomplete. In this study, age-related changes in the degree of polysialylation of NCAM re-expressed after denervation were examined using a monoclonal antibody recognizing polysialic acid and a polyclonal antibody recognizing NCAM. The results show that, after denervation, the degree of polysialylation on NCAM was clearly reduced in rat soleus muscle of aged, compared to young, adult rats. This age-related change in expression of polysialic acid probably influences the reinnervation process in aged muscle.

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.

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).

[Veinous return from the inferior vena cava into the left atrium after surgical correction of Fallot's tetralogy]. / Retour veineux cave inférieure dans l'oreillette gauche après correction d'une tétralogie de Fallot.

A child of 6 presented with a syndrome of gross respiratory distress together with persistant arterial desaturation, requiring ventilation for maintenance of life, occurring after the apparently straightforward correction of a Fallot's tetralogy. Postoperative investigation on the 15th day showed the reasons for the desaturation: there was a massive right-left shunt caused by flow from the inferior vena cava into the auricle of the left atrium through a low atrial septal defect which had not been recognised. Reoperation on the 15th day to close the atrial septal defect corrected the condition satisfactorily.

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.

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.

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.

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.

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.