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Analisi geometrica e cinematica di Face Gears ingrananti con pignoni a dentatura elicoidale
No full textThe role of Store-Operated Cyclic AMP Signalling (SOcAMPS) in cardiac physiology and pathology: an in vitro study on neonatal rat cardiomyocytes.
No full textBackground and Aims: Store-Operated Cyclic AMP Signaling (SOcAMPS) represents a recently
identified mechanism of cross-talk between Ca2+ and cAMP signals. In this process, depletion of
Ca2+ in the endoplasmic reticulum (ER) leads to increases in cAMP levels, independently of
cytosolic Ca2+ changes. Expression and functionality of STIM1 (Stromal Interaction Molecule 1), a
transmembrane ER Ca2+ sensor protein, is necessary for SOcAMPS to occur. Interestingly, recent
reports have demonstrated a critical role for STIM1 in the development of cardiac hypertrophy, a
process notoriously controlled both by Ca2+ and cAMP signaling. Here we aimed to evaluate
whether SOcAMPS was manifest in neonatal rat cardiomyocytes and its potential role in cardiac
cell hypertrophy.
Methods: To monitor changes in cAMP levels, real time imaging experiments were performed on
neonatal rat cardiomyocytes transiently transfected with an EPAC-based fluorescent probe for
[cAMP], EPAC H30. Fura-2 and Fluo-4 were used to monitor cytosolic Ca2+ levels and an ER/SR
targeted probe, D1ERcameleon, was used to measure ER [Ca2+]. Long term incubation (48h) of
cardiomyocytes with angiotensin II (1 μM) and aldosterone (1 μM) was used to induce "in vitro"
cell hypertrophy. Increases in cell size and/or sarcomere alignment were monitored microscopically
after labeling with phalloidin-TRITC.
Results: To verify the existence of SOcAMPS in neonatal rat cardiomyocytes, cells were stimulated
in Ca2+-free Ringer's solutions with the low affinity membrane permeant Ca2+ chelator TPEN
(1mM), able to induce a reduction of SR Ca2+ levels ([Ca2+]SR) without affecting cytosolic
[Ca2+]. SR Ca2+ measurements demonstrated that under these experimental conditions, 1 mM
TPEN led to a reduction in intraluminal [Ca2+] that was 50,5±2,4% (8 exp, 11 cells, p<0.001) of
the maximal store depletion. Parallel experiments performed with the EPAC H30 cAMP sensor
showed increases in [cAMP] that were 26,5±3% (13 exp, 13 cells, p<0.001) of the maximum delta
ratio. In the presence of 5 μM Forskolin (FRSK) the TPEN-induced cAMP augmentation resulted
63,7±3,9% of the maximal response (16 exp, 19 cells, p<0.001). Also depletion of SR by the Ca2+
ionophore ionomycin (10 μM) was found to induce significant cAMP increases both in the absence
and presence of FRSK. The participation of STIM1 in the observed phenomenon was proven by the
47 % reduction of the TPEN+FRSK induced [cAMP] signal after transfection of cells with a
shRNA against STIM1 (6 exp, p<0,01). To evaluate the putative role of SOcAMPS in cardiac
hypertrophy, cAMP measurements were performed on angio+aldo treated cells and compared to
control cardiomyocytes. Under these experimental conditions a 20% increase of the TPEN+FRSK
induced response was observed in hypertrophic myocytes (16 exp, p<0,01).
Conclusions: These data straightforwardly establish, for the first time, the existence of SOcAMPS
in the neonatal cardiomyocyte cell model. Also, a significantly increased SOcAMP signalling was
shown to exist in hypertrophic cardiomyocytes. Further experiments to ascertain whether a causeand-
effect relationship exists between SOcAMPS and cardiac cell hypertrophy are in progress
Flavin adenine dinucleotide metabolism in nucleus
No full textFollowing its uptake from outside, in the cell riboflavin undergoes an ATP-dependent phosphorylation catalyzed by riboflavin kinase (RFK, E.C. 2.7.1.26) to form FMN, most of which is further converted by FAD synthase (FADS, E.C. 2.7.7.2) into FAD, the cofactor for many flavoenzymes involved in several biological processes in different cellular compartments.
We previously demonstrated that beside a cytosolic enzyme (i.e. isoform 2 of human FLAD1 gene product) a mitochondrial isoform of FADS exists (isoform 1) (1, 2), presumably responsible for the biogenesis of mitochondrial flavoenzymes. Interestingly, the possibility of unexpected localizations for FADS has arisen (3) and, meanwhile, at least two new isoforms of FADS have been deposited in databases.
Among FAD-dependent enzymes, lysine-specific demethylase-1 (E.C. 1.14.11.B1) is a nuclear protein recently shown to regulate cellular energy balance depending on FAD availability (4).
These observation prompted us to investigate on the presence of flavin coenzymes in the nucleus and on a possible metabolism of FAD in this organelle.
To this aim HPLC analysis of acid-precipitable flavins in nuclei isolated from rat liver were performed. The observation of a time-dependent decrease of FAD suggested us the existence of a hydrolytic activity, as confirmed by spectrofluorimetric measurements revealing an increase in fluorescence due to conversion of FAD into its more fluorescent precursor FMN/riboflavin (5).
Immunoblotting experiments performed on rat liver nuclei using a home-made antibody against FADS revealed the enrichment of an immunoreactive band in respect to the other sub-cellular fractions.
Confocal microscopy on several mammalian cells confirmed the existence of FADS co-localizing with nuclear markers.
1. Barile et al., Eur J Biochem, 2000, 15, 4888-900.
2. Torchetti et al., Mitochondrion, 2010, 10, 263-73.
3. Lin et al., J Neurol, 2009, 256, 774-82.
4. Hino et al., Nat Commun, 2012, 3, 758.
5. Brizio et al., Eur J Biochem, 1997, 3, 777-85
Activation of insulin secretory granules exocytosis induces changes in local extracellular [Ca2+] in INS-1E pseudoislets
No full textThe role of Store-Operated Cyclic AMP Signalling (SOcAMPS) in cardiac physiology and pathology
No full textStore-Operated Cyclic AMP Signaling (SOcAMPS) represents a novel signaling mechanism in which depletion of Ca2+ in the endoplasmic reticulum (ER) leads to a STIM1- dependent (Stromal Interaction Molecule 1) increase in cAMP levels, independently of cytosolic Ca2+.
Here we aimed to evaluate whether SOcAMPS was manifest in neonatal rat ventricular myocytes (NRVM) and human "iCardiomyocytes" and exploit its potential role in cardiac cell hypertrophy.
cAMP levels and ER [Ca2+]were monitored by live cell fluorescence imaging after transfection with the EPAC H30 and D1ER cameleon probes, respectively.
The existence of SOcAMPS in NRVM was first assessed by using the low affinity Ca2+ chelator TPEN, able to induce a reduction of SR Ca2+ levels without affecting cytosolic [Ca2+]. TPEN (1mM) was shown to induce significant cAMP increases both in the absence and presence of 5 M Forskolin (FRSK). Depletion of SR by ionomycin (10 M) was found to exert similar effects. Similar data were obtained in human "iCardiomyocytes".
The participation of STIM1 in the observed phenomenon was proven in NRVM by the 47% reduction of the [cAMP] response obtained after shRNA-mediated knockdown of STIM1. Interestingly, a significant increase of the TPEN+FRSK induced response was found after "in vitro" induced cell hypertrophy.
These data establish, for the first time, the existence of SOcAMPS in the two cardiac cell models analyzed and suggest a potential role for this new signaling mechanism in cardiac cell hypertrophy
CDKL5 regulates the initiation of retrograde axonal transport through CLIP170-dynactin complex formation
No full textCyclin-dependent kinase-like 5 (CDKL5) is a serine-threonine kinase implicated in regulating microtubule (MT) dynamics. Mutations in CDKL5 are associated with a rare neurodevelopmental disease called CDKL5 deficiency disorder (CDD), which is characterized by early-onset seizures and intellectual disabilities. Microtubule (MT)-related functions of CDKL5 are in part correlated with its interaction with MT-associated proteins, such as CAP-Gly domain-containing linker protein 1 [CLIP1; also known as cytoplasmic linker protein 170 alpha-2 (CLIP170)]. CLIP170 is a MT plus-end tracking protein that, once activated, can bind MTs and other proteins, favoring MT dynamics. Importantly, we have previously shown that CLIP170 is inactive in the absence of CDKL5, thus hindering MT functions. One of the best-characterized interactors of CLIP170 is dynactin, a multisubunit complex that binds the motor protein dynein. In particular, in neurons, active CLIP170 localizes to MTs in the axonal periphery, where it serves as a docking site for the interaction with dynactin, which in turn recruits dynein and various cargos, favoring the initiation of retrograde transport toward the neuronal soma. Here, we demonstrated that CLIP170-dynactin complex formation is impaired in the absence of CDKL5, thus leading to defective retrograde cargo trafficking. Overall, our findings expand the knowledge on the molecular mechanisms underlying neuronal transport and provide novel information regarding the etiopathogenesis of CDD
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
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