196,266 research outputs found
Indipendenza della magistratura in Polonia. Lo “strappo nel cielo di carta” della rule of law e l’argomento identitario
No full textChange of ganglioside accessibility at the plasma membrane surface of cultured neurons, following Protein Kinase C activation
No full textWhile the mechanism of signal transduction across the plasma membrane from the exo- to the endoplasmic side has been extensively investigated, the possible return of messages back to the outer layer is less known. We studied the effect of protein kinase C activation on the ganglioside accessibility at the exoplasmic face of intact rat cerebellar granule cells in culture, using the enzyme sialidase as the probing molecule. Under the experimental conditions (1 milliunit/mL enzyme, 2 min incubation at 37 degrees C), only GT1b and GD1a gangliosides were partially affected by the enzyme (28.6 and 25.7% hydrolysis, respectively). After cell treatment with phorbol 12-myristate 13-acetate, inducing protein kinase C activation, GT1b and GD1a ganglioside susceptibility to sialidase was strongly decreased (8.6 and 15.9% hydrolysis, respectively). A reduction of ganglioside hydrolysis was also observed when protein kinase C activation was induced by cell treatment for 15 min with 100 mu M glutamate. On the contrary, accessibility did not vary when protein kinase C translocation was not effective (either in the absence of Ca2+ in the medium or using 1 mu M glutamate) or when the kinase activity was inhibited by staurosporine. These data suggest that following PKC activation, a key step of inbound transmembrane signaling, cell may dispatch outbound messages to the plasma membrane outer layer, changing the selective recognition and crypticity of glycolipids at the cell surface, possibly through a modulation of their segregation state
Sialidase in Cerebellar Granule Cells Differentiating in Culture
No full textAbstract: The optimal conditions for the assay of sialidase in cerebellar granule cells cultivated in vitro, established using f3H]GDla and 2′‐(4‐methylumbelliferyl)‐α‐D‐N‐acetyl‐neuraminic acid (MUB‐NeuNAc) as substrates, were the following: pH optimum for both substrates, 3.9; optimal molarity of sodium acetate/acetic acid buffer, 0.05 M with [3HJGDla and 0.1 M for MUB‐NeuNAc; substrate concentration for apparent maximal activity, 0.5 mM for MUB‐NeuNAc and 0.1 mM for [3H]GDla; enzyme activity linear with time up to 30 min with MUB‐NeuNAc and up to 90 min with f3HJGDla; and enzyme activity linear with enzyme protein content up to 80 μg with MUB‐NeuNAc and up to 20 μg with f3H]GDla. The assay with [3H]GDla required the presence of Triton X‐100 in a molar ratio to GDla of 15:1. Poly‐L‐lysine, which was used for plating the cells, was capable of decreasing sialidase activity against [3H]GDla/ Triton X‐100 when added to the incubation mixture. However, it had no effect on the enzyme working on MUB‐NeuNAc. Using no more than 20 μg of cellular protein, the contamination, if any, by poly‐L‐lysine released from the dish was below the concentration limit exhibiting inhibition. Using the above optimal conditions, sialidase activity was measured during cerebellar granule cell differentiation in culture. From day 0 to day 7–8 in culture, the enzyme activity rose from 20 to 130 nmol of product released/h/mg of protein with MUB‐NeuNAc and from 1 to 100 nmol of product released/ h/mg of protein with [3H]GDla. The values of enzyme activity in differentiated granule cells are the highest ever reported for mammalian sialidases in isolated cells or tissue homogenates. In fully differentiated cells, the sialidase activity against endogenous substrates was 4.2 nmol of liberated N‐acetylneuraminic acid/h/mg of protein. The marked increase of sialidase activity in cerebellar granule cells during the process of differentiation with formation of functional synapses suggests that sialidase enrichment is a marker for the same process
Chapter 3. PROBABILISTIC TOOLS B. Bagen (Lead Editor), D. Cirio, D. Jacobson, S. Massucco, G. Migliavacca, M. Papic, A. Pitto and S. Sterpu
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Rappresentanza e mobilità degli elettori: le nuove sfide per il diritto di voto nella società in movimento
No full textCHARACTERIZATION OF CARROT NUCLEAR PROTEINS ABLE TO BIND DNA SEQUENCES WITH DIFFERENT MATHYLATION CONTEXTS
No full textSPONTANEOUS TRANSFER OF GM3 GANGLIOSIDE BETWEEN VESICLES
No full textThe spontaneous transfer between membranes of GM3, a ganglioside present in a vesicular form of aggregation instead of micellar form like the majority of gangliosides in aqueous medium, has been studied. Upon incubation of GM3 in the presence of dipalmitoylphosphatidylcholine (DPPC) large unilamellar vesicles at 50 degrees C, mixed GM3/DPPC vesicles are formed. The maximum amount of GM3 that can be inserted into vesicles is about 8%. The temperature dependence of the kinetics has been followed by the excimer formation technique, using the fluorescent analogue pyrenyldodecanoyl-GM3. The transfer of ganglioside from its vesicles to DPPC vesicles depends on the physicochemical characteristics of both the donor and of the acceptor vesicles and increases with the temperature (k = 0.006 0.012, 0.037 at 30, 41 and 50 degrees C, respectively), with a major break point at 41 degrees C and a minor one at 35 degrees C. These temperatures correspond to the gel- to liquid-crystalline transition of DPPC (T-m=41.3 degrees C), and to a temperature transition displayed by GM3 ganglioside. Similar experiments performed with erythrocyte ghosts yielded a rate constant of 0.04 at 37 degrees C. For the transfer of ganglioside from DPPC (donor) to DMPC (acceptor) the rate constants were 0 at 15 degrees C (both phospholipids in the gel phase), 0.005 at 37 degrees C (donor in the gel phase, acceptor in the fluid phase) and 0.04 at 50 degrees C (both phospholipids in the fluid phase). The fastest kinetics were observed when both donor and acceptor membranes were in the fluid state. The kinetics was not affected by the physical state and by the lipid moiety of acceptor phosphatidylcholine when the donor was in the gel phase. The data obtained suggest that GM3 transfer occurs via monomers through the aqueous medium and that the rate-limiting step is the off-rate of the ganglioside from the donor membrane
Metabolic rejuvenation of plasma membrane glycoconjugates: De-novo biosynthesis, direct glycosylation and salvage processes
No full textBiofeedback training in the treatment of anal incontinence in children. Preliminary results [Il biofeedback training nel trattamento dell'incontinenza anale in età pediatrica. Risultati preliminari]
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