Dynamics of mitochondrial [Ca(2+)] measured with the low-Ca(2+)-affinity dye rhod-5N

Available methods to measure mitochondrial [Ca(2+)] ([Ca(2+)](M)) include both targeted proteins and fluorescent dyes. Targeted proteins usually report much higher [Ca(2+)](M) values than fluorescent dyes, up to two orders of magnitude. However, we show here that the low-Ca(2+)-affinity dye rhod-5N provides [Ca(2+)](M) values similar to those reported by targeted aequorin, suggesting that the discrepancies are mainly due to the higher Ca(2+)-affinity of the fluorescent dyes used. We find rhod-5N has an apparent in situ intramitochondrial Kd around 0.5mM. Addition of Ca(2+) buffers containing between 4.5 and 10μM [Ca(2+)] to permeabilized cells loaded with rhod-5N induced increases in calibrated [Ca(2+)](M) up to the 100μM-1mM range, which were dependent on mitochondrial membrane potential. Ca(2+) release from mitochondria was largely dependent on [Na(+)]. We have then used rhod-5N loaded cells to investigate the [Ca(2+)](M) response to agonist stimulation at the single-cell and subcellular level. The [Ca(2+)](M) peaks induced by histamine varied by nearly 10-fold among different cells, with a mean about 25μM. In the presence of the Ca(2+) uniporter stimulator kaempferol, the [Ca(2+)](M) peaks induced by histamine were also highly variable, and the mean [Ca(2+)](M) peak was 3-fold higher. Simultaneous measurement of cytosolic and mitochondrial [Ca(2+)] peaks showed little correlation among the heights of the peaks in both compartments. Studying the [Ca(2+)](M) peaks at the subcellular level, we found significant heterogeneities among regions in the same cell. In particular, the [Ca(2+)](M) increase in mitochondrial regions close to the nucleus was more than double that of mitochondrial regions far from the nucleus.     

Structural characterization of microcin E492 amyloid formation: Identification of the precursors

Microcin E492 is a low-molecular weight, channel-forming bacteriotoxin that generates amyloid structures. Using electron microscopy and image processing techniques several structural conformations can be observed. Prior to the conditions that induce amyloid formation and at its initial stage, microcin E492 molecules can be found in two main types of oligomers: a pentameric, pore-like structure consisting of globular monomers of ～25? diameter, and long filaments made up of stacked pentamers. The equilibrium between these structures depends on the properties of the solvent, because samples kept in methanol mainly show the pentameric structure. Amyloid induction in aqueous solvent reveals the presence, together with the above mentioned structures, of several amyloid structures such as flat and helical filaments. In addition, X-ray diffraction analysis demonstrated that the fibrils formed by microcin E492 presented cross-β structure, a distinctive property of amyloid fibrils. Based on the study of the observed structures we propose that microcin E492 has two conformations: a native one that assembles mainly into a pentameric structure, which functions as a pore, and an amyloid conformation which results in the formation of different types of amyloid filaments.     

miRandola: Extracellular Circulating MicroRNAs Database

MicroRNAs are small noncoding RNAs that play an important role in the regulation of various biological processes through their interaction with cellular messenger RNAs. They are frequently dysregulated in cancer and have shown great potential as tissue-based markers for cancer classification and prognostication. microRNAs are also present in extracellular human body fluids such as serum, plasma, saliva, and urine. Most of circulating microRNAs are present in human plasma and serum cofractionate with the Argonaute2 (Ago2) protein. However, circulating microRNAs have been also found in membrane-bound vesicles such as exosomes. Since microRNAs circulate in the bloodstream in a highly stable, extracellular form, they may be used as blood-based biomarkers for cancer and other diseases. A knowledge base of extracellular circulating miRNAs is a fundamental tool for biomedical research. In this work, we present miRandola, a comprehensive manually curated classification of extracellular circulating miRNAs. miRandola is connected to miRò, the miRNA knowledge base, allowing users to infer the potential biological functions of circulating miRNAs and their connections with phenotypes. The miRandola database contains 2132 entries, with 581 unique mature miRNAs and 21 types of samples. miRNAs are classified into four categories, based on their extracellular form: miRNA-Ago2 (173 entries), miRNA-exosome (856 entries), miRNA-HDL (20 entries) and miRNA-circulating (1083 entries). miRandola is available online at: http://atlas.dmi.unict.it/mirandola/index.html.     

Activities of glycolipid glycosyltransferases and sialidases during the early development of Xenopus laevis

The activities of glycosyltransferases and sialidases, together with the ganglioside content and distribution, have been extensively studied in mammals, while the informations on tissues of other animals, including amphibian, are scarce. In this paper we present data on the activities of SAT-1, SAT-2, SAT-4, SAT-5, GlcNAcT-1, GalNAcT-1, GalT-6, and sialidases studied in Xenopus laevis embryos at different stages of development. The highest activity was found at days 4 and 5 of embryogenesis for glycosyltransferases and sialidases respectively; a tentative correlation between the in vitro activity of these enzymes and the content of neutral and acidic glycolipids is discussed. (Mol Cell Biochem 166: 117-124, 1997)     

KU0063794, a Dual mTORC1 and mTORC2 Inhibitor,Reduces Neural Tissue Damage and Locomotor Impairment After Spinal Cord Injury in Mice

Autophagy is an intracellular catabolic mechanism for the degradation of cytoplasmic constituents in the autophagosomal–lysosomal pathway. This mechanism plays an important role in homeostasis and it is defective in certain diseases. Preceding studies have revealed that autophagy is developing as an important moderator of pathological responses associated to spinal cord injury (SCI) and plays a crucial role in secondary injury initiating a progressive degeneration of the spinal cord. Thus, based on this evidence in this study, we used two different selective inhibitors of mTOR activity to explore the functional role of autophagy in an in vivo model of SCI as well as to determine whether the autophagic process is involved in spinal cord tissue damage. We treated animals with a novel synthetic inhibitor temsirolimus and with a dual mTORC1 and mTORC2 inhibitor KU0063794 matched all with the well-known inhibitor of mTOR the rapamycin. Our results demonstrated that mTOR inhibitors could regulate the neuroinflammation associated to SCI and the results that we obtained evidently demonstrated that rapamycin and temsirolimus significantly diminished the expression of iNOS, COX2, GFAP, and re-established nNOS levels, but the administration of KU0063794 is able to blunt the neuroinflammation better than rapamycin and temsirolimus. In addition, neuronal loss and cell mortality in the spinal cord after injury were considerably reduced in the KU0063794-treated mice. Accordingly, taken together our results denote that the administration of KU0063794 produced a neuroprotective function at the lesion site following SCI, representing a novel therapeutic approach after SCI.     

Characterisation of yeast microbiota,chemical and sensory properties of organic and biodynamic Sangiovese red wines

Wine quality is closely linked to the fermentation step, which is driven by the microbial ecology of grape and the use of selected microbial strains as well. The microbial species developing during fermentation determines the type and concentration of many substances, which contribute to the sensory properties of wine and its safety. In this view, the present work aims to characterise the yeast microbiota, chemical and sensory properties of Sangiovese red wines obtained from both biodynamic and organic agriculture. The natural yeast populations of grape musts and their evolution during spontaneous were monitored and investigated. In addition, the volatile composition, physico-chemical and safety features (ethyl-carbamate) and sensory properties of wines were evaluated. The results showed that the yeast population was mostly related to the grape management, i.e. organic or biodynamic, while the wine composition was mainly affected by the winemaking process, and then by the grape management.