Activity-dependent phosphorylation of SNAP-25 in hippocampal organotypic cultures.

Synaptosomal-associated protein of 25 kDa (SNAP-25) is thought to play a key role in vesicle exocytosis and in the control of transmitter release. However, the precise mechanisms of action as well as the regulation of SNAP-25 remain unclear. Here we show by immunoprecipitation that activation of protein kinase C (PKC) by phorbol esters results in an increase in SNAP-25 phosphorylation. In addition, immunochemical analysis of two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels shows that SNAP-25 focuses as three or four distinct spots in the expected range of molecular weight and isoelectric point. Changing the phosphorylation level of the protein by incubating the slices in the presence of either a PKC agonist (phorbol 12,13-dibutyrate) or antagonist (chelerythrine) modified the distribution of SNAP-25 among these spots. Phorbol 12,13-dibutyrate increased the intensity of the spots with higher molecular weight and lower isoelectric point, whereas chelerythrine produced the opposite effect. This effect was specific for regulators of PKC, as agonists of other kinases did not produce similar changes. Induction of long-term potentiation, a property involved in learning mechanisms, and production of seizures with a GABA(A) receptor antagonist also increased the intensity of the spots with higher molecular weight and lower isoelectric point. This effect was prevented by the PKC inhibitor chelerythrine. We conclude that SNAP-25 can be phosphorylated in situ by PKC in an activity-dependent manner.

Time-Temperature Strength-Reaction Product Relationships in Lime-Bentonite-Water Mixtures, HR-111, 1965

The interrelation of curing time, curing temperature, strength, and reactions in lime-bentonite-water mixtures was examined. Samples were molded at constant density and moisture content and then cured for periods of from 1 to 56 days at constant temperatures that ranged from 5C to 60C. After the appropriate curing time the samples were tested for unconfined compressive strength. The broken samples were then analyzed by x-ray diffractometer and spectrophotometer to determine the identity of the reaction products present after each curing period. It was found that the strength gain of lime-clay mixtures cured at different temperatures is due to different phases of the complex reaction, lime & clay to CSH(gel) to CSH(II) to CSH(I) to tobermorite. The farther the reaction proceeds, the higher the strength. There was also evidence of lattice substitutions in the structure of the calcium silicate hydrates at curing temperatures of 50C and higher. No consistent relationship between time, temperature, strength, and the S/A ration of reaction products existed, but in order to achieve high strengths the apparent C/S ration had to be less than two. The curing temperature had an effect on the strength developed by a given amount of reacted silica in the cured lime-clay mixture, but at a given curing temperature the cured sample that had the largest amount of reacted silica gave the highest strength. Evidence was found to indicate that during the clay reaction some calcium is indeed adsorbed onto the clay structure rather than entering into a pozzolanic reaction. Finally, it was determined that it is possible to determine the amount of silica and alumina in lime-clay reaction products by spectrophotometric analysis with sufficient accuracy for comparison purposes. The spectrophotometric analysis techniques used during the investigation were simple and were not time consuming.

Validation of real-time methylation-specific PCR to determine O6-methylguanine-DNA methyltransferase gene promoter methylation in glioma.

Epigenetic silencing of the DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT) by promoter methylation predicts successful alkylating agent therapy, such as with temozolomide, in glioblastoma patients. Stratified therapy assignment of patients in prospective clinical trials according to tumor MGMT status requires a standardized diagnostic test, suitable for high-throughput analysis of small amounts of formalin-fixed, paraffin-embedded tumor tissue. A direct, real-time methylation-specific PCR (MSP) assay was developed to determine methylation status of the MGMT gene promoter. Assay specificity was obtained by selective amplification of methylated DNA sequences of sodium bisulfite-modified DNA. The copy number of the methylated MGMT promoter, normalized to the beta-actin gene, provides a quantitative test result. We analyzed 134 clinical glioma samples, comparing the new test with the previously validated nested gel-based MSP assay, which yields a binary readout. A cut-off value for the MGMT methylation status was suggested by fitting a bimodal normal mixture model to the real-time results, supporting the hypothesis that there are two distinct populations within the test samples. Comparison of the tests showed high concordance of the results (82/91 [90%]; Cohen's kappa = 0.80; 95% confidence interval, 0.82-0.95). The direct, real-time MSP assay was highly reproducible (Pearson correlation 0.996) and showed valid test results for 93% (125/134) of samples compared with 75% (94/125) for the nested, gel-based MSP assay. This high-throughput test provides an important pharmacogenomic tool for individualized management of alkylating agent chemotherapy.

Current directions in DNA gel particles

A general understanding of interactions between DNA andoppositely charged compounds forms the basis for developing novelDNA-based materials, including gel particles. The association strength,which is altered by varying the chemical structure of the cationiccosolute, determines the spatial homogeneity of the gelation process,creating DNA reservoir devices and DNA matrix devices that can bedesigned to release either single- (ssDNA) or double-stranded(dsDNA) DNA. This paper reviews the preparation of DNA gelparticles using surfactants, proteins and polysaccharides. Particlemorphology, swelling/dissolution behaviour, degree of DNAentrapment and DNA release responses as a function of the nature ofthe cationic agent used are discussed. Current directions in thehaemocompatible and cytotoxic characterization of these DNA gelparticles have been also included.

Photonic characteristics of Langmuir-Blodgett self-assembled monolayers of colloidal silica particles

Monodispersed colloidal crystals based on silica sub-micrometric particles were synthesized using the Stöber-Fink-Bohn process. The control of nucleation and coalescence result in improved characteristics such as high sphericity and very low size dispersion. The resulting silica particles show characteristics suitable for self-assembling across large areas of closely-packed 2D crystal monolayers by an accurate Langmuir-Blodgett deposition process on glass, fused silica and silicon substrates. Due to their special optical properties, colloidal films have potential applications in fields including photonics, electronics, electro-optics, medicine (detectors and sensors), membrane filters and surface devices. The deposited monolayers of silica particles were characterized by means of FESEM, AFM and optical transmittance measurements in order to analyze their specific properties and characteristics. We propose a theoretical calculation for the photonic band gaps in 2D systems using an extrapolation of the photonic behavior of the crystal from 3D to 2D. In this work we show that the methodology used and the conditions in self-assembly processes are decisive for producing high-quality two-dimensional colloidal crystals by the Langmuir-Blodgett technique.

Differential protein labeling with thiol-reactive infrared DY-680 and DY-780 maleimides and analysis by two-dimensional gel electrophoresis.

Differential protein labeling with 2-DE separation is an effective method for distinguishing differences in the protein composition of two or more protein samples. Here, we report on a sensitive infrared-based labeling procedure, adding a novel tool to the many labeling possibilities. Defined amounts of newborn and adult mouse brain proteins and tubulin were exposed to maleimide-conjugated infrared dyes DY-680 and DY-780 followed by 1- and 2-DE. The procedure allows amounts of less than 5 microg of cysteine-labeled protein mixtures to be detected (together with unlabeled proteins) in a single 2-DE step with an LOD of individual proteins in the femtogram range; however, co-migration of unlabeled proteins and subsequent general protein stains are necessary for a precise comparison. Nevertheless, the most abundant thiol-labeled proteins, such as tubulin, were identified by MS, with cysteine-containing peptides influencing the accuracy of the identification score. Unfortunately, some infrared-labeled proteins were no longer detectable by Western blots. In conclusion, differential thiol labeling with infrared dyes provides an additional tool for detection of low-abundant cysteine-containing proteins and for rapid identification of differences in the protein composition of two sets of protein samples.

In-vivo performance of high-density collagen gel tubes for urethral regeneration in a rabbit model.

Congenital malformations or injuries of the urethra can be treated using existing autologous tissue, but these procedures are sometimes associated with severe complications. Therefore, tissue engineering may be advantageous for generating urethral grafts. We evaluated engineered high-density collagen gel tubes as urethral grafts in 16 male New Zealand white rabbits. The constructs were either acellular or seeded with autologous smooth muscle cells, isolated from an open bladder biopsy. After the formation of a urethral defect by excision, the tissue-engineered grafts were interposed between the remaining urethral ends. No catheter was placed postoperatively. The animals were evaluated at 1 or 3 months by contrast urethrography and histological examination. Comparing the graft caliber to the control urethra at 3 months, a larger caliber was found in the cell-seeded grafts (96.6% of the normal caliber) than in the acellular grafts (42.3%). Histology of acellular and cell-seeded grafts did not show any sign of inflammation, and spontaneous regrowth of urothelium could be demonstrated in all grafts. Urethral fistulae, sometimes associated with stenosis, were observed, which might be prevented by urethral catheter application. High-density collagen gel tubes may be clinically useful as an effective treatment of congenital and acquired urethral pathologies.

Two-step postmortem angiography with a modified heart-lung machine: preliminary results.

OBJECTIVE: The purpose of this study was to adapt and improve a minimally invasive two-step postmortem angiographic technique for use on human cadavers. Detailed mapping of the entire vascular system is almost impossible with conventional autopsy tools. The technique described should be valuable in the diagnosis of vascular abnormalities. MATERIALS AND METHODS: Postmortem perfusion with an oily liquid is established with a circulation machine. An oily contrast agent is introduced as a bolus injection, and radiographic imaging is performed. In this pilot study, the upper or lower extremities of four human cadavers were perfused. In two cases, the vascular system of a lower extremity was visualized with anterograde perfusion of the arteries. In the other two cases, in which the suspected cause of death was drug intoxication, the veins of an upper extremity were visualized with retrograde perfusion of the venous system. RESULTS: In each case, the vascular system was visualized up to the level of the small supplying and draining vessels. In three of the four cases, vascular abnormalities were found. In one instance, a venous injection mark engendered by the self-administration of drugs was rendered visible by exudation of the contrast agent. In the other two cases, occlusion of the arteries and veins was apparent. CONCLUSION: The method described is readily applicable to human cadavers. After establishment of postmortem perfusion with paraffin oil and injection of the oily contrast agent, the vascular system can be investigated in detail and vascular abnormalities rendered visible.

Processus de "deuil gelé" chez les femmes bosniaques, épouses de disparus

Les femmes concernées par cette recherche sont des femmes bosniaques, requérantes d'asile en Suisse, suivies au sein de l'association « Appartenances ». Elles ont quitté leur pays à la suite de la prise de la ville de Srebrenica, en juillet 1995. Lors de cet exode, elles ont été séparées de leurs maris, qui sont aujourd'hui encore portés disparus. Ces femmes partagent une expérience douloureuse qui transparaît derrière un certain nombre de symptômes physiques et psychiques. Nous avons été frappés par la répétition de pertes importantes, sans que les conditions permettant un travail de deuil soient réunies. Aucune signification ne peut être attribuée aux disparitions et aux décès, masqués par le silence politique et par l'impossibilité d'accomplir un rituel. Le deuil peut ainsi se compliquer jusqu'à devenir un deuil impossible, dont le prototype pourrait être le « deuil gelé » lors d'une disparition. D'autre part, le stress et l'attaque de l'identité occasionnés par les traumatismes et l'exil ont coupé ces femmes de leurs ressources personnelles et culturelles. La perte d'une image entière de soi est prolongée par le statut précaire de requérant d'asile et par la perte des espoirs mis dans le pays d'accueil. Devant la complexité de cette problématique, nous nous sommes demandés sur quel aspect axer la prise en charge thérapeutique. Nous avons décidé de travailler sur le vécu et les deuils actuels de ces patientes (stress post-traumatique et statut précaire en Suisse). Le concept-clé qui a étayé cette recherche s'inspire de Pollock (7): « le processus de deuil devient très significatif, dans le sens qu 'il est apparemment une des formes les plus universelles de l'adaptation et de la croissance à travers la structuration, disponible pour l'homme. » Dès lors, nous avons fait l'hypothèse que si le deuil est une forme universelle d'adaptation, l'élaboration d'un travail de deuil ou d'un processus d'adaptation quel qu'il soit, pourra servir d'apprentissage pour l'élaboration d'autres deuils. Concrètement, en travaillant sur le rétablissement de leur identité, les femmes pourront retrouver un accès à leurs ressources personnelles et culturelles, tandis que travailler sur les espoirs et déceptions face au statut en Suisse permettra de développer des moyens permettant plus de contrôle sur l'environnement. Leur autonomie rétablie, elles pourront consacrer leurs énergies à attribuer une signification à leur vécu et faire face aux autres deuils paralysant leur guérison. Nous avons assisté aux entretiens thérapeutiques, en récoltant des témoignages sur la base d'un canevas d'entretien semi-structuré, puis nous avons transposé les éléments sur une grille d'analyse regroupant les informations en items. L'analyse de ces entretiens nous a permis de préciser notre hypothèse et de développer des pistes de réflexion pour l'accompagnement thérapeutique de personnes vivant une disparition. A l'issue de ce travail, nous pouvons tirer des parallèles entre les processus d'élaboration des divers deuils. Les femmes sont entrées peu à peu dans le souvenir de la personne aimée et non plus dans le souvenir des événements traumatiques qui ont marqué sa disparition. S'appuyant alors sur la « mémoire sereine » de Kristeva (1) une recherche de signification a pu être entreprise, rejoignant ce que Métraux et Fleuiy (43) définissent comme la santé : « ... se voir créateur du sens qu'on veut donner à sa propre existence, à ses actes et à ses projets. »

Evaluation of the SHRP Modified Georgia Digital Faultmeter, MLR-93-01, 1993

The SHRP Modified Georgia Digital Faultmeter was loaned to the Iowa Department of Transportation in January 1993 for evaluation. A study was undertaken comparing the faultmeter to Iowa's current method of fault measurement. The following conclusions were made after comparing the faultmeter to Iowa's gauge: The faultmeter was lighter and easier to maneuver and position. The faultmeter's direct readout was quicker to read. The faultmeter has increased precision. The faultmeter gave consistently lower fault readings than the Iowa gauge.

Fusion of the extended modified liquid drop model for nucleation and dynamical nucleation theory

We present a new phenomenological approach to nucleation, based on the combination of the extended modified liquid drop model and dynamical nucleation theory. The new model proposes a new cluster definition, which properly includes the effect of fluctuations, and it is consistent both thermodynamically and kinetically. The model is able to predict successfully the free energy of formation of the critical nucleus, using only macroscopic thermodynamic properties. It also accounts for the spinodal and provides excellent agreement with the result of recent simulations.

Melt/peridotite interaction in the Southern Lanzo peridotite: Field, textural and geochemical evidence

This paper presents field, petrographic-structural and geochemical data on spinet and plagioclase peridotites from the southern domain of the Lanzo ophiolitic peridotite massif (Western Alps). Spinet lherzolites, harzburgites and dunites crop out at Mt. Arpone and Mt. Musine. Field evidence indicates that pristine porphyroclastic spinet lherzolites are transformed to coarse granular spinet harzburgites, which are in turn overprinted by plagioclase peridotites, while strongly depleted spinet harzburgite and dunite bands and bodies replace the plagioclase peridotites. On the northern flank of Mt. Arpone, deformed, porphyroclastic (lithospheric) lherzolites, with diffuse pyroxenite banding, represent the oldest spinel-facies rocks. They show microstructures of a composite subsolidus evolution, suggesting provenance from deeper (asthenospheric) mantle levels and accretion to the lithosphere. These protoliths are locally transformed to coarse granular (reactive) spinet harzburgites and dunites, which show textures reminiscent of melt/rock reaction and geochemical characteristics suggesting that they are products of peridotite interaction with reactively percolating melts. Geochemical data and modelling suggest that <1-5% fractional melting of spinel-facies DMM produced the injected melts. Plagioclase peridotites are hybrid rocks resulting from pre-existing spinet peridotites and variable enrichment of plagioclase and micro-gabbroic material by percolating melts. The impregnating melts attained silica-saturation, as testified by widespread orthopyroxene replacement of olivine, during open system migration in the lithosphere. At Mt. Musine, coarse granular spinet harzburgite and dunite bodies replace the plagioclase peridotites. Most of these replacive, refractory peridotites have interstitial magmatic clinopyroxene with trace element compositions in equilibrium with MORB, while some Cpx have REE-depleted patterns suggesting transient geochemical features of the migrating MORB-type melts, acquired by interaction with the ambient plagioclase peridotite. These replacive spinet harzburgite and dunite bodies are interpreted as channels exploited for focused and reactive migration of silica-undersaturated melts with aggregate MORB compositions. Such melts were unrelated to the silica-saturated melts that refertilized the pre-existing plagioclase peridotites. Finally, MORB melt migration occurred along open fractures, now recorded as gabbroic dikes. Our data document the complexity of rock-types and mantle processes in the South Lanzo peridotite massif and describe a composite tectonic and magmatic scenario that is not consistent with the ``asthenospheric scenario'' proposed by previous authors. We envisage a ``transitional scenario'' in which extending subcontinental lithospheric mantle was strongly modified (both depleted and refertilized) by early melts with MORB-affinity formed by decompression partial melting of the upwelling asthenosphere, during pre-oceanic rifting and lithospheric thinning in the Ligurian Tethys realm. (C) 2006 Elsevier B.V. All rights reserved.

Outcome of a Modified Broström-Gould Procedure for Lateral Ankle Instability

Introduction: Ankle sprains affect 200'000 persons/year in Switzerland. Most incidences are successfully treated by conservative measures but 20% require reconstruction for symptomatic chronic lateral ankle instability. This study evaluates the functional outcome after a modified Broström-Gould technique as measured by different clinical scores and compares the functional outcome of this technique with other surgical treatments of ankle instability. Methods: This retrospective cohort study evaluates 47 patients who underwent a modified Broström-Gould procedure using suture anchors to refix the lateral ankle capsuloligamentary structures at our institution from 2005 to 2009 with a minimum follow-up of one year (13-72 Mo). All patients were operated by one single surgeon and evaluated by an independent examiner. The function was assessed using 4 scores including: the AOFAS (American Orthopaedic Foot and Ankle Society's Score) hindfoot score; the FAAM (Foot and Ankle Ability Measurement); the CAIT (Cumberland Ankle Instability Tool); the CAIS (Chronic Ankle Instability Scale). Results: Six patients were excluded leaving 41 patients for examination. 34 patients (83%) thought that their ankle was more stable after the surgery, 7 (17%) did not feel any difference. 27 patients were very satisfied, 11 satisfied and 3 not satisfied. Reasons for non satisfaction included persistent instability and pain. Ankle mobility returned to normal in 93% of patients. Five patients had transcient hypoesthesy in the area of the superficial peroneal nerve. One patient suffered from a superficial infection treated successfully by local measures. 80% had the perception of a normal ankle, 20% thought to be below normal. At follow-up the AOFAS was 89/100 (37-100), the FAAM 85/100% (35-100%), the CAIT 20/30 (5-30), and the CAIS 74/100% (27-100%). Conclusions: The modified Broström-Gould procedure, which belongs to the anatomic ankle stabilizations is relatively simple and offers good outcome that satisfied 93% of the patients in the present study. No active stabilisator is sacrificed. Preservation of the ankle mobility is better and the complication rate is lower than after non-anatomical procedures described in the literature. The CAIT appeared as the most severe score compared to the other scales used in our study.

Resistance of genetically modified potatoes to Potato virus Y under field conditions

The objective of this work was to evaluate the resistance of genetically modified clones of potato to Potato virus Y (PVY) under field conditions. Genetically modified plants were compared with nontransformed plants of the same cultivar. The plots were flanked with potato plants infected with both PVYº and PVY N strains (spread lines), in order to provide the experimental area with the source of virus, which was naturally spread by the native aphid population. The experiment was weekly monitored by visual inspections and by DAS-Elisa in the plants produced from the harvested tubers, in order to evaluate the resistance of transgenic plants throughout the plant growth cycle. By the end of the third year, no infection symptoms were observed in the 1P clone; clone 63P showed 1% of infection, in contrast to about 90% of nontransformed plants infected. The stable expression of resistance to PVY provided by the coat protein gene was obtained in genetically modified clones of potato plants cultivar Achat under field conditions, during three consecutive years.