A phosphatidylinositol 3-kinase class III sub-complex containing VPS15, VPS34, Beclin 1, UVRAG and BIF-1 regulates cytokinesis and degradative endocytic traffic

The mammalian class III phosphatidylinositol 3-kinase (PI3K-III) complex regulates fundamental cellular functions, including growth factor receptor degradation, cytokinesis and autophagy. Recent studies suggest the existence of distinct PI3K-III sub-complexes that can potentially confer functional specificity. While a substantial body of work has focused on the roles of individual PI3K-III subunits in autophagy, functional studies on their contribution to endocytic receptor downregulation and cytokinesis are limited. We therefore sought to elucidate the specific nature of the PI3K-III complexes involved in these two processes. High-content microscopy-based assays combined with siRNA-mediated depletion of individual subunits indicated that a specific sub-complex containing VPS15, VPS34, Beclin 1, UVRAG and BIF-1 regulates both receptor degradation and cytokinesis, whereas ATG14L, a PI3K-III subunit involved in autophagy, is not required. The unanticipated role of UVRAG and BIF-1 in cytokinesis was supported by a strong localisation of these proteins to the midbody. Importantly, while the tumour suppressive functions of Beclin 1, UVRAG and BIF-1 have previously been ascribed to their roles in autophagy, these results open the possibility that they may also contribute to tumour suppression via downregulation of mitogenic signalling by growth factor receptors or preclusion of aneuploidy by ensuring faithful completion of cell division.     

High-activity radio-iodine labeling of conventional and stealth liposomes

A new method to label preformed liposomes with high activities of radiohalogenated compounds has been developed. It uses activated esters of simple synthetic molecules that may be readily halogenated, such as Bolton-Hunter reagent (BH), and arginine-containing liposomes. BH, in the form of an activated ester, crosses the liposome membrane to react with arginine inside the liposomes, as demonstrated by thin-layer chromatography and by the fact that saline-containing liposomes, or hydrolyzed BH or the water soluble sulfo-BH afforded only marginal encapsulation yields. Under optimized conditions, between 37 and 55 degrees C, 62 +/- 4% (mean +/- SD) of radiolabeled BH were consistently encapsulated in the liposomes within 30 min. In molar amounts, this corresponds to a mean of 56 nmol of BH per micromol of lipids. Based on achievable specific activity, up to 2.8 GBq of iodine-131 could be entrapped per micromol of lipids. Leakage of radioactivity was very low, with less than 5% of the encapsulated activity released within 6 days at 4 degrees C in phosphate-buffered saline and less than 50% within 24 h in human serum at 37 degrees C. The labeling stability, and the fact that both conventional and PEGylated liposomes can be readily labeled with high doses of radioactivity, will make this technique useful for in vivo targeting applications, such as tumor detection (using iodine-123 or iodine-124) or therapy (with iodine-131 or astatine-211).     

Tracking the opioid receptors on the way of desensitization

Opioid receptors belong to the super family of G-protein coupled receptors (GPCRs) and are the targets of numerous opioid analgesic drugs. Prolonged use of these drugs results in a reduction of their effectiveness in pain relief also called tolerance, a phenomenon well known by physicians. Opioid receptor desensitization is thought to play a major role in tolerance and a lot of work has been dedicated to elucidate the molecular basis of desensitization. As described for most of GPCRs, opioid receptor desensitization involves their phosphorylation by kinases and their uncoupling from G-proteins realized by arrestins. More recently, opioid receptor trafficking was shown to contribute to desensitization. In this review, our knowledge on the molecular mechanisms of desensitization and recent progress on the role of opioid receptor internalization, recycling or degradation in desensitization will be reported. A better understanding of these regulatory mechanisms would be helpful to develop new analgesic drugs or new strategies for pain treatment by limiting opioid receptor desensitization and tolerance.     

Oxylipin metabolism in soybean seeds containing different sets of lipoxygenase isozymes after homogenization

The oxylipin metabolism was analyzed in soybean homogenates containing different sets of lipoxygenase isozymes (L-1, -2, and -3); namely, Suzuyutaka (containing L-1, -2, and -3), Yumeyutaka (containing only L-1), Kanto102 (containing L-2), Kyushu119 (containing L-3), and Ichihime (lacking all three isozymes). The amount of oxidized fatty acids in the esterified form was higher than that in the free form with every cultivar. Kanto102 formed the highest amount of oxidized lipids, and Yumeyutaka and Ichihime formed the lowest. With Kanto102 and Kyushu119, high amounts of keto fatty acids were formed, while they were undetectable with Yumeyutaka and Ichihime. Due to the lack of lipoxygenases in Ichihime, an accumulation of free fatty acids was expected; however, their amount in Yumeyutaka was significantly lower than was expected. It is suggested that a pathway existed to form C6-volatiles through hydroperoxides in the esterified form.     

Phenotypic and genotypic characterization of some Moorella sp. strains isolated from canned foods

Six anaerobic thermophilic strains isolated from various spoiled cans including fish soups and cooked meats were characterized using a polyphasic approach. These strains were closely related to Moorella thermoacetica or Moorella thermoautotrophica species. Except the spacer region between the 16S and the 23S rRNA genes, which exhibited two PCR profiles distinguishing both species, the genotypic and phylogenetic analyses grouped these isolates, the type strains, and all sequences of Moorella thermoacetica and Moorella thermoautotrophica species contained in the GenBank database within a unique cluster. Moreover, all 16S rDNA sequences shared two characteristic DNA fragments, which were highly specific of Moorella thermoacetica/Moorella thermoautotrophica strains. However, taken together, the phenotypic, physiological and genotypic methods were conflicting, and did not enable affiliation of the isolates with one or the other species. To our knowledge, this study represents the first report of characterization of Moorella species isolated from spoiled cans. These results and previous work, very strongly argue in favor of questioning the taxonomic status of the two species.     

SOM230 inhibits insulin-like growth factor-I action in mammary gland development by pituitary independent mechanism: mediated through somatostatin subtype receptor 3?

Somatostatin analogs (SAs) treat acromegaly by lowering pituitary GH secretion, which, in turn, lowers systemic IGF-I. The profound systemic effect is often greater than expected in the face of only partial GH suppression. Here we report that the SA SOM230 can also act by a nonpituitary-mediated inhibition of IGF-I action. SOM230 inhibited mammary development in intact and hypophysectomized female rats, a process requiring IGF-I. IGF-I overcame this inhibition. SOM230 also inhibited other actions of IGF-I (inhibition of apoptosis, phosphorylation of insulin receptor substrate-1, and cell division). SOM230 did not reduce IGF-I mRNA abundance in mammary gland but did stimulate IGF binding protein 5 (IGFBP5). IGFBP5 was 3.75 times higher in mammary epithelium of SOM230 than in placebo animals (P < 0.001). Administration of IGFBP-5 also inhibited GH-induced mammary development (P < 0.001). Measurement of sstr(1-5) (somatostatin subtype receptor) by real-time RT-PCR revealed that the mammary glands had an abundance of sstr(3) and lower amounts of sstr(4) and sstr(5) but no sstr(1) or sstr(2.) That mammary development was also inhibited to a lesser degree than SOM230 by octreotide, whose main action is through sstr(2), strongly suggests that sstr(3) is at least in part mediating the effects of the SAs. We conclude that 1) SAs inhibit IGF-I action in the mammary gland through a novel nonpituitary mechanism; 2) IGFBP-5, here shown to inhibit pubertal mammary development, might mediate the effect; and 3) Measurement of available sstr receptors in the mammary gland suggests that sstr(3) mediates the SA activity, but sstr(5) is also a possible mediator.     

CD4 T cell-dependent autoimmunity against a melanocyte neoantigen induces spontaneous vitiligo and depends upon Fas-Fas ligand interactions

Better understanding of tolerance and autoimmunity toward melanocyte-specific Ags is needed to develop effective treatment for vitiligo and malignant melanoma; yet, a systematic assessment of these mechanisms has been hampered by the difficulty in tracking autoreactive T cells. To address this issue, we have generated transgenic mice that express hen egg lysozyme as a melanocyte-specific neoantigen. By crossing these animals to a hen egg lysozyme-specific CD4 TCR transgenic line we have been able to track autoreactive CD4+ T cells from their development in the thymus to their involvement in spontaneous autoimmune disease with striking similarity to human vitiligo vulgaris and Vogt-Koyanagi-Harada syndrome. Our findings show that CD4-dependent destruction of melanocytes is partially inhibited by blocking Fas-Fas ligand interactions and also highlights the importance of local control of autoimmunity, as vitiligo remains patchy and never proceeds to confluence even when Ag and autoreactive CD4+ T cells are abundant. Immune therapy to enhance or suppress melanocyte-specific T cells can be directed at a series of semiredundant pathways involving tolerance and cell death.