Cell-free reconstitution of vacuole membrane fragmentation reveals regulation of vacuole size and number by TORC1.

Size and copy number of organelles are influenced by an equilibrium of membrane fusion and fission. We studied this equilibrium on vacuoles-the lysosomes of yeast. Vacuole fusion can readily be reconstituted and quantified in vitro, but it had not been possible to study fission of the organelle in a similar way. Here we present a cell-free system that reconstitutes fragmentation of purified yeast vacuoles (lysosomes) into smaller vesicles. Fragmentation in vitro reproduces physiological aspects. It requires the dynamin-like GTPase Vps1p, V-ATPase pump activity, cytosolic proteins, and ATP and GTP hydrolysis. We used the in vitro system to show that the vacuole-associated TOR complex 1 (TORC1) stimulates vacuole fragmentation but not the opposing reaction of vacuole fusion. Under nutrient restriction, TORC1 is inactivated, and the continuing fusion activity then dominates the fusion/fission equilibrium, decreasing the copy number and increasing the volume of the vacuolar compartment. This result can explain why nutrient restriction not only induces autophagy and a massive buildup of vacuolar/lysosomal hydrolases, but also leads to a concomitant increase in volume of the vacuolar compartment by coalescence of the organelles into a single large compartment.

Propagation velocity kinetics and repolarization alternans in a free-behaving sheep model of pacing-induced atrial fibrillation.

AIMS: Experimental models have reported conflicting results regarding the role of dispersion of repolarization in promoting atrial fibrillation (AF). Repolarization alternans, a beat-to-beat alternation in action potential duration, enhances dispersion of repolarization when propagation velocity is involved. METHODS AND RESULTS: In this work, original electrophysiological parameters were analysed to study AF susceptibility in a chronic sheep model of pacing-induced AF. Two pacemakers were implanted, each with a single right atrial lead. Right atrial depolarization and repolarization waves were documented at 2-week intervals. A significant and gradual decrease in the propagation velocity at all pacing rates and in the right atrial effective refractory period (ERP) was observed during the weeks of burst pacing before sustained AF developed when compared with baseline conditions. Right atrial repolarization alternans was observed, but because of the development of 2/1 atrioventricular block with far-field ventricular interference, its threshold could not be precisely measured. Non-sustained AF was not observed at baseline, but appeared during the electrical remodelling in association with a decrease in both ERP and propagation velocity. CONCLUSION: We report here on the feasibility of measuring ERP, atrial repolarization alternans, and propagation velocity kinetics and their potential in predicting susceptibility to AF in a free-behaving model of pacing-induced AF using the standard pacemaker technology.

Induction of transendothelial migration in normal and malignant human T lymphocytes.

Activated CD 3+ enriched human peripheral blood T cells exhibited potent capacity for transendothelial migration through HUVEC layers in the absence of T cell ***. In contrast, malignant human T cell lines *** no or negligible ability of transendothelial migration in the absence of chemoattractants. Time lapse studies of transendothelial migration of activated CD 3+ enriched peripheral blood T cells through a HUVEC layer showed that the first T cells were detected in the lower compartment of a tissue culture insert after 1 hour and that migration increased to reach a maximum of 25 x 10(4) T cells/hr after 24 hours. Adhesion assays of human T cell lines demonstrated that all T cell lines were capable of adhesion to HUVEC and that adhesion of T cells to HUVECs was primarily mediated by CD11a/CD18 and ICAM-1 interactions. Furthermore, transendothelial migration of CD 3+ enriched human peripheral blood T cells was inhibited by pretreating the T cells with anti-CD 18 monoclonal antibodies. The inability of malignant T cells to migrate through HUVEC layers in the absence of chemoattractants was not due to poor motility per se, since both normal and malignant T cells migrated well on extracellular matrix components as determined by using Boyden chambers. Crosslinking of alpha 1 beta 2 and alpha 4 beta 1 with immobilized monoclonal antibodies induced motile behaviour in activated CD 3 enriched human peripheral blood T cells but not in malignant T cell lines. In conclusion, the differences in the ability of transendothelial migration between normal and malignant human T cells in the absence of chemoattractants is primarily due to the differences in the capacity of alpha 1 beta 2 and alpha 4 beta 1 to trigger motile behaviour in the separate cell types.

Contrast agent-enhanced, free-breathing, three-dimensional coronary magnetic resonance angiography.

For free-breathing, high-resolution, three-dimensional coronary magnetic resonance angiography (MRA), the use of intravascular contrast agents may be helpful for contrast enhancement between coronary blood and myocardium. In six patients, 0.1 mmol/kg of the intravascular contrast agent MS-325/AngioMARK was given intravenously followed by double-oblique, free-breathing, three-dimensional inversion-recovery coronary MRA with real-time navigator gating and motion correction. Contrast-enhanced, three-dimensional coronary MRA images were compared with images obtained with a T2 prepulse (T2Prep) without exogenous contrast. The contrast-enhanced images demonstrated a 69% improvement in the contrast-to-noise ratio (6.6 +/- 1.1 vs. 11.1 +/- 2.5; P < 0.01) compared with the T2Prep approach. By using the intravascular agent, extensive portions (> 80 mm) of the native left and right coronary system could be displayed consistently with sub-millimeter in-plane resolution. The intravascular contrast agent, MS-325/AngioMARK, leads to a considerable enhancement of the blood/muscle contrast for coronary MRA compared with T2Prep techniques. The clinical value of the agent remains to be defined in a larger patient series. J. Magn. Reson. Imaging 1999;10:790-799.

Gonadotrophin replacement for induction of fertility in hypogonadal men.

Congenital hypogonadotrophic hypogonadism (CHH) is a rare form of infertility caused by deficient secretion or action of gonadotrophin-releasing hormone. There is no consensus regarding the optimal approach to fertility treatment in CHH men. In most cases, appropriate hormonal treatment with human chorionic gonadotrophin with or without follicle stimulating hormone will induce testicular development, spermatogenesis and fertility. Recent studies have examined sequential treatment with FSH pre-treatment to optimize fertility outcomes in severely affected CHH patients. This paper reviews historical and recent literature to summarize the current evidence on therapeutic approaches for CHH men seeking fertility.

cFLIP protein prevents tumor necrosis factor-alpha-mediated induction of caspase-8-dependent apoptosis in insulin-secreting betaTc-Tet cells.

Type 1 diabetes is characterized by the infiltration of activated leukocytes within the pancreatic islets, leading to beta-cell dysfunction and destruction. The exact role played by interferon-gamma, tumor necrosis factor (TNF)-alpha, and interleukin-1beta in this pathogenic process is still only partially understood. To study cytokine action at the cellular level, we are working with the highly differentiated insulin-secreting cell line, betaTc-Tet. We previously reported that it was susceptible to apoptosis induced by TNF-alpha, in combination with interleukin-1beta and interferon-gamma. Here, we report that cytokine-induced apoptosis was correlated with the activation of caspase-8. We show that in betaTc-Tet cells, overexpression of cFLIP, the cellular FLICE (FADD-like IL-1beta-converting enzyme)-inhibitory protein, completely abolished cytokine-dependent activation of caspase-8 and protected the cells against apoptosis. Furthermore, cFLIP overexpression increased the basal and interleukin-1beta-mediated transcriptional activity of nuclear factor (NF)-kappaB, whereas it did not change cytokine-induced inducible nitric oxide synthase gene transcription and nitric oxide secretion. The presence of cFLIP prevented the weak TNF-alpha-induced reduction in cellular insulin content and secretion; however, it did not prevent the decrease in glucose-stimulated insulin secretion induced by the combined cytokines, in agreement with our previous data demonstrating that interferon-gamma alone could induce these beta-cell dysfunctions. Together, our data demonstrate that overexpression of cFLIP protects mouse beta-cells against TNF-alpha-induced caspase-8 activation and apoptosis and is correlated with enhanced NF-kappaB transcriptional activity, suggesting that cFLIP may have an impact on the outcome of death receptor-triggered responses by directing the intracellular signals from beta-cell death to beta-cell survival.

Chlamydiaceae and Chlamydia-like organisms in free-living small mammals in Europe and Afghanistan.

Few data are available on the occurrence of chlamydial infections in wild small mammals. We investigated the significance of free-living small mammals as reservoirs or transmission hosts for microorganisms of the phylum/class Chlamydiae. We obtained 3,664 tissue samples from 911 animals in Switzerland, Germany, Austria, the Czech Republic, and Afghanistan. Samples included internal organs (n = 3,652) and feces (n = 12) from 679 rodents (order Rodentia) and 232 insectivores (order Soricomorpha) and were tested by three TaqMan® real-time PCRs specific for members of the family Chlamydiaceae and selected Chlamydia-like organisms such as Parachlamydia spp. and Waddlia spp. Only one of 911 (0.11%) animals exhibited a questionable positive result by Chlamydiaceae-specific real-time PCR. Five of 911 animals were positive by specific real-time PCR for Parachlamydia spp. but could not be confirmed by quantitative PCR targeting the Parachlamydia acanthamoebae secY gene (secY qPCR). One of 746 animals (0.13%) was positive by real-time PCR for Waddlia chondrophila. This result was confirmed by Waddlia secY qPCR. This is the first detection of Chlamydia-like organisms in small wildlife in Switzerland. Considering previous negative results for Chlamydiaceae in wild ruminant species from Switzerland, these data suggest that wild small mammals are unlikely to be important carriers or transport hosts for Chamydiaceae and Chlamydia-like organisms.

Drug uptake in a rodent sarcoma model after intravenous injection or isolated lung perfusion of free liposomal doxorubicin /

Rapport de synthèseDrug uptake in a rodent sarcoma model after intravenous injection or isolated lungperfusion of free/liposomal doxorubicinIntroductionLa distribution de doxorubicine libre et doxorubicin liposomale pegylée (Liporubicin?) a été comparée après administration intraveineuse ou application via perfusion isolée du poumon (ILP) dans le parenchyme pulmonaire et dans la tumeur des poumons de rongeurs, porteurs d'une tumeur sarcomateuse.Matériel et méthodeUne tumeur sarcomateuse unique a été générée dans le poumon gauche de 36 rongeurs (Fisher rats) suivie, 10 jours plus tard, par application de doxorubicine ou Liporubicin? soit par perfusion isolée du poumon (n = 20) ou administration intraveineuse (n = 12). Deux différentes concentrations ont été utilisées (100 μg et 400 pg) à doses équimolaires pour les deux formulations de doxorubicine. La concentration des agents cytostatiques ont été mesurées dans la tumeur et le parenchyme pulmonaire à l'aide de chromatographic (HPLC).RésultatsLes résultats indiquent que pour doxorubicine libre, le taux de concentration dans la tumeur et le parenchyme pulmonaire est 3 fois (dosage de 100 μ§) et 10 fois (dosage de 400 plus élevé après ILP par rapport à l'administration intraveineuse. En revanche, pour Liporubicin , le taux de concentration est similaire dans la tumeur et le parenchyme pulmonaire entre ILP et administration intraveineuse, pour les deux doses appliquées.ConclusionPour ILP et administration intraveineuse, le ratio entre accumulation de l'agent cytostatique dans la tumeur versus dans le parenchyme pulmonaire a été comparé pour les deux formulations de doxorubicine ainsi que pour les deux dosages. Pour les deux formulations et dosages de doxorubicine, ILP aboutit à un ratio plus élevé par rapport à l'administration intraveineuse. Cependant, pour les deux formulations et dosages de doxorubicine, ILP résulte également en une distribution de l'agent cytostatique plus hétérogène dans le parenchyme pulmonaire comparé à l'administration intraveineuse.En résumé, l'application de doxorubicine par ILP aboutit donc à une accumulation tumorale élevée et à une augmentation du ratio tumeur-parenchyme pulmonaire, mais en même temps également à une distribution plus hétérogène dans le parenchyme pulmonaire par rapport à l'application intraveineuse. Ceci a été observé pour les deux formulations de doxorubicine et pour les deux dosages appliqué.

VIKI : a semiotic-based system for multilingual knowledge retrieval

Abstract Since its creation, the Internet has permeated our daily life. The web is omnipresent for communication, research and organization. This exploitation has resulted in the rapid development of the Internet. Nowadays, the Internet is the biggest container of resources. Information databases such as Wikipedia, Dmoz and the open data available on the net are a great informational potentiality for mankind. The easy and free web access is one of the major feature characterizing the Internet culture. Ten years earlier, the web was completely dominated by English. Today, the web community is no longer only English speaking but it is becoming a genuinely multilingual community. The availability of content is intertwined with the availability of logical organizations (ontologies) for which multilinguality plays a fundamental role. In this work we introduce a very high-level logical organization fully based on semiotic assumptions. We thus present the theoretical foundations as well as the ontology itself, named Linguistic Meta-Model. The most important feature of Linguistic Meta-Model is its ability to support the representation of different knowledge sources developed according to different underlying semiotic theories. This is possible because mast knowledge representation schemata, either formal or informal, can be put into the context of the so-called semiotic triangle. In order to show the main characteristics of Linguistic Meta-Model from a practical paint of view, we developed VIKI (Virtual Intelligence for Knowledge Induction). VIKI is a work-in-progress system aiming at exploiting the Linguistic Meta-Model structure for knowledge expansion. It is a modular system in which each module accomplishes a natural language processing task, from terminology extraction to knowledge retrieval. VIKI is a supporting system to Linguistic Meta-Model and its main task is to give some empirical evidence regarding the use of Linguistic Meta-Model without claiming to be thorough.

Current multiple myeloma treatment strategies with novel agents: a European perspective.

The treatment of multiple myeloma (MM) has undergone significant developments in recent years. The availability of the novel agents thalidomide, bortezomib, and lenalidomide has expanded treatment options and has improved the outcome of patients with MM. Following the introduction of these agents in the relapsed/refractory setting, they are also undergoing investigation in the initial treatment of MM. A number of phase III trials have demonstrated the efficacy of novel agent combinations in the transplant and nontransplant settings, and based on these results standard induction regimens are being challenged and replaced. In the transplant setting, a number of newer induction regimens are now available that have been shown to be superior to the vincristine, doxorubicin, and dexamethasone regimen. Similarly, in the front-line treatment of patients not eligible for transplantation, regimens incorporating novel agents have been found to be superior to the traditional melphalan plus prednisone regimen. Importantly, some of the novel agents appear to be active in patients with high-risk disease, such as adverse cytogenetic features, and certain comorbidities, such as renal impairment. This review presents an overview of the most recent data with these novel agents and summarizes European treatment practices incorporating the novel agents.

Social organization and mating system of free-ranging, greater white-toothed shrews, Crocidura russula

Indirect evidence from trapping suggests that Crocidura russula is less solitary and territorial than other shrews. To study the social organization and mating system, free-ranging adult and juvenile C. russula were tracked simultaneously throughout the year using a radioactive tracking technique. Coincident rest, coincident activity and home range overlap were measured. During winter, all individuals used the same communal nest and spent on average 84% of their total rest in coincident rest. This led to a large home range overlap (52% on average). Coincident activity was low (2% on average). At the onset of the reproductive season the females became, territorial and shared their nest with only one male. During pair formation, coincident activity and home range overlap were significantly greater between than within sexes. The social organization of C. russula appeared to be strongly influenced by season and differed in this respect from the other species in the genus Sorex which are territorial throughout the year.

PPARgamma but not PPARalpha ligands are potent repressors of major histocompatibility complex class II induction in atheroma-associated cells.

Peroxisome proliferator-activated receptors (PPARs) are essential in glucose and lipid metabolism and are implicated in metabolic disorders predisposing to atherosclerosis, such as diabetes and dyslipidemia. Conversely, antidiabetic glitazones and hypolipidemic fibrate drugs, known as PPARgamma and PPARalpha ligands, respectively, reduce the process of atherosclerotic lesion formation, which involves chronic immunoinflammatory processes. Major histocompatibility complex class II (MHC-II) molecules, expressed on the surface of specialized cells, are directly involved in the activation of T lymphocytes and in the control of the immune response. Interestingly, expression of MHC-II has recently been observed in atherosclerotic plaques, and it can be induced by the proinflammatory cytokine interferon-gamma (IFN-gamma) in vascular cells. To explore a possible role for PPAR ligands in the regulation of the immune response, we investigated whether PPAR activation affects MHC-II expression in atheroma-associated cells. In the present study, we demonstrate that PPARgamma but not PPARalpha ligands act as inhibitors of IFN-gamma-induced MHC-II expression and thus as repressors of MHC-II-mediated T-cell activation. All different types of PPARgamma ligands tested inhibit MHC-II. This effect of PPARgamma ligands is due to a specific inhibition of promoter IV of CIITA and does not concern constitutive expression of MHC-II. Thus, the beneficial effects of antidiabetic PPARgamma activators on atherosclerotic plaque development may be partly explained by their repression of MHC-II expression and subsequent inhibition of T-lymphocyte activation.

Wheat germ cell-free expression: Two detergents with a low critical micelle concentration allow for production of soluble HCV membrane proteins.

Membrane proteins are notoriously difficult to express in a soluble form. Here, we use wheat germ cell-free expression in the presence of various detergents to produce the non-structural membrane proteins 2, 4B and 5A of the hepatitis C virus (HCV). We show that lauryl maltose neopentyl glycol (MNG-3) and dodecyl octaethylene glycol ether (C12E8) detergents can yield essentially soluble membrane proteins at detergent concentrations that do not inhibit the cell-free reaction. This finding can be explained by the low critical micelle concentration (CMC) of these detergents, which keeps the monomer concentrations low while at the same time providing the necessary excess of detergent concentration above CMC required for full target protein solubilization. We estimate that a tenfold excess of detergent micelles with respect to the protein concentration is sufficient for solubilization, a number that we propose as a guideline for detergent screening assays.

Comparison of neoadjuvant cisplatin-based chemotherapy versus radiochemotherapy followed by resection for stage III (N2) NSCLC.

OBJECTIVE: Comparison of prospectively treated patients with neoadjuvant cisplatin-based chemotherapy vs radiochemotherapy followed by resection for mediastinoscopically proven stage III N2 non-small cell lung cancer with respect to postoperative morbidity, pathological nodal downstaging, overall and disease-free survival, and site of recurrence. METHODS: Eighty-two patients were enrolled between January 1994 to June 2003, 36 had cisplatin and doxetacel-based chemotherapy (group I) and 46 cisplatin-based radiochemotherapy up to 44 Gy (group II), either as sequential (25 patients) or concomitant (21 patients) treatment. All patients had evaluation of absence of distant metastases by bone scintigraphy, thoracoabdominal CT scan or PET scan, and brain MRI, and all underwent pre-induction mediastinoscopy, resection and mediastinal lymph node dissection by the same surgeon. RESULTS: Group I and II comprised T1/2 tumors in 47 and 28%, T3 tumors in 45 and 41%, and T4 tumors in 8 and 31% of the patients, respectively (P=0.03). There was a similar distribution of the extent of resection (lobectomy, sleeve lobectomy, left and right pneumonectomy) in both groups (P=0.9). Group I and II revealed a postoperative 90-d mortality of 3 and 4% (P=0.6), a R0-resection rate of 92 and 94% (P=0.9), and a pathological mediastinal downstaging in 61 and 78% of the patients (P<0.01), respectively. 5y-overall survival and disease-free survival of all patients were 40 and 36%, respectively, without significant difference between T1-3 and T4 tumors. There was no significant difference in overall survival rate in either induction regimens, however, radiochemotherapy was associated with a longer disease-free survival than chemotherapy (P=0.04). There was no significant difference between concurrent vs sequential radiochemotherapy with respect to postoperative morbidity, resectability, pathological nodal downstaging, survival and disease-free survival. CONCLUSIONS: Neoadjuvant cisplatin-based radiochemotherapy was associated with a similar postoperative mortality, an increased pathological nodal downstaging and a better disease-free survival as compared to cisplatin doxetacel-based chemotherapy in patients with stage III (N2) NSCLC although a higher number of T4 tumors were admitted to radiochemotherapy.

Transcriptional profiling of Gram-positive Arthrobacter in the phyllosphere: induction of pollutant degradation genes by natural plant phenolic compounds.

Arthrobacter chlorophenolicus A6 is a Gram-positive, 4-chlorophenol-degrading soil bacterium that was recently shown to be an effective colonizer of plant leaf surfaces. The genetic basis for this phyllosphere competency is unknown. In this paper, we describe the genome-wide expression profile of A.chlorophenolicus on leaves of common bean (Phaseolus vulgaris) compared with growth on agar surfaces. In phyllosphere-grown cells, we found elevated expression of several genes known to contribute to epiphytic fitness, for example those involved in nutrient acquisition, attachment, stress response and horizontal gene transfer. A surprising result was the leaf-induced expression of a subset of the so-called cph genes for the degradation of 4-chlorophenol. This subset encodes the conversion of the phenolic compound hydroquinone to 3-oxoadipate, and was shown to be induced not only by 4-chlorophenol but also hydroquinone, its glycosylated derivative arbutin, and phenol. Small amounts of hydroquinone, but not arbutin or phenol, were detected in leaf surface washes of P.vulgaris by gas chromatography-mass spectrometry. Our findings illustrate the utility of genomics approaches for exploration and improved understanding of a microbial habitat. Also, they highlight the potential for phyllosphere-based priming of bacteria to stimulate pollutant degradation, which holds promise for the application of phylloremediation.